First, I would like to express my deepest gratitude to my supervisor, Dr. Francois Juge, who apart from being a brilliant geneticist, is also a very humble, calm and kind person. I thank you from the core of my heart for the knowledge and patience that you gave me from the day I joined as a Master Intern. I have learned a lot of things from, and I hope I will be able to use that to contribute to society.

I further extend my thanks to Marion, Carine, Christina, Karim, Laurent for being kind and helping Lab members. I also take an opportunity to extend my thanks to the team of Abivax who were always kind to me. I would like to thank my friend and previous Ph.D. student of the lab Mahdi who has been with me throughout the Ph.D. I would also like to thank Mickael who joined lab last year and has been a like a brother to me. I will always cherish the numerous games of Jenga that we played at Rebuffy!! It wouldn't have been possible to do this Ph.D. without the crucial help of Jamal TAZI, director of our RNA Metabolism group who not only provided my space in his laboratory and guidance and every step but also generously gave me extensions for continuing the research after my Ligue funding period was over. I look up to you as you are a very motivating and inspiring person.

I would take this opportunity to thank all the friends and colleagues from IGMM and Montpellier who made this journey of Ph.D. colorful.

Sometimes you meet people in your life, and they teach you things just by being who they are, for me that person is Dr. Pradeep Kumar from Rajeev Gandhi Centre for Biotechnology, Trivandrum-India who is not only a brilliant scientist but also a wittiest human beings on the planet…What a boss to work with! I thank you for all the help that you did just before I arrived in France.

I would not miss this opportunity to mention Riccardo Montecchi, Nicole, Mehuli, Ayush, Karim, Anishesh, Fran, Arunabh and Chandra Mauli my real selfless friends from India and Europe with whom I have some lovely memories together! I love you guys! And how can I miss Divy from this list, my dearest buddy from college days. Thank you for always being there for me! I thank all my four beautiful sisters Ritu Di, Richa Di, Shibbi Di and Nishu and who still pamper me as they did in my childhood days. I would also like to thank Alok Jiju, Rohit Jiju, Anurag Jiju and Anit Jiju for loving me and taking care of parents as their own.

Ultimately I dedicate this thesis to the jewel of my family i.e. Nikunj Kumar Srivastava who has enabled me and motivated me at every step in my life to reach wherever and to do whatever I have done with my life so far. I may never surpass you for the human being that you are but I can walk on the path that you have shown me and live for the cause of others because that is the real happiness.

I would love to thank my parents, who made it all possible. Whatever I did and whatever I achieve in this life will be smaller than the love you have showered upon me. Papa, you left me for heaven just weeks before my defense and I felt very weak, but I felt your presence around me and the strength you gave me to be able to defend my thesis. I know that you are always with me. I love you, and I owe you everything. Love

LIST OF FIGURES

Abstract

The Hippo pathway is a conserved pathway involved in tissue growth and tumor suppression. Studies have demonstrated its significance in the development of human cancers.

This cascade controls the activity of the transcription co-activator Yorkie (Yki) in flies and Yes-associated protein (YAP) in mammals. Due to Alternative Splicing (AS), both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains. Since WW domains are essential for interaction with specific partners, the alternative inclusion of this domain in Yki/YAP protein may remodel their interaction network and therefore their activity. The regulation and functional consequences of AS of yki/YAP in vivo are unknown.

In this Ph.D. project, we identified that depletion of splicing factor B52 in Drosophila lowers inclusion of the alternative exon in yki mRNAs and favors the expression of Yki1 isoform at the expense of the Yki2 isoform. B52 depletion in the wing reduces growth and Yki activity. We demonstrate that Yki1 isoform is an attenuated version of Yki protein that can compete with Yki2 isoform in the nucleus. To ascertain the role of yki AS in vivo and the importance of short isoform Yki1, we abrogated this splicing by using CRISPR/Cas9

technology and created flies that can express Yki2 isoform only. yki 2only flies are viable but display a random phenotype of asymmetric wing size. This rise in "fluctuating asymmetry" that is the consequence of subtle deviation from normal development, suggests that AS of yki is crucial for the development robustness. Taking together, these results highlight a new layer of modulation of Hippo pathway via AS of yki.

Alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. This further supports the idea that Yki1 and YAP1 isoforms have an important function in vivo and that AS of yki/YAP is a conserved mechanism of control of the Hippo pathway. This study opens up new perspectives for modulation of the Hippo pathway in cancer cells by altering YAP AS.

Résumé

La voie Hippo est une voie conservée impliquée dans la croissance des tissus et la suppression de tumeurs. Des études ont démontré son implication dans le développement des cancers chez l'homme. Cette cascade contrôle l'activité du co-activateur transcriptionnel Yorkie (Yki) chez la drosophile et de la protéine YAP (Yes Associated Protein) chez les mammifères. En raison de l'épissage alternatif de leur transcrits, les protéines Yki et YAP existent sous deux isoformes contenant un domaine WW (Yki1/YAP1) ou deux (Yki2/YAP2). Puisque les domaines WW sont essentiels pour l'interaction avec des partenaires spécifiques, l'inclusion alternative de ce domaine dans la protéine Yki/YAP peut remodeler leur réseau d'interaction et donc leur activité. La régulation et les conséquences fonctionnelles de l'épissage alternatif de yki / YAP in vivo sont inconnues.

Dans le cadre de ce doctorat, nous avons constaté que la déplétion du facteur d'épissage B52 chez la drosophile réduit l'inclusion de l'exon alternatif dans l'ARNm de yki et favorise l'expression de l'isoforme Yki1 aux dépens de l'isoforme Yki2. La déplétion en B52 dans l'aile réduit la croissance et l'activité de Yki. Nous montrons que l'isoforme Yki1 est une version atténuée de la protéine Yki qui peut entrer en concurrence avec l'isoforme Yki2 dans le noyau. Pour déterminer le rôle de l'épissage alternatif de yki in vivo et l'importance de l'isoforme courte Yki1, nous avons abrogé cet épissage en utilisant la technologie CRISPR/Cas9 et avons créé des mouches capables d'exprimer uniquement l'isoforme Yki2.

Ces mouches yki 2only sont viables mais présentent un phénotype aléatoire d'ailes asymétriques.

Cette augmentation de l'«asymétrie fluctuante», qui traduit une déviation par rapport au développement normal, suggère que l'épissage alternatif de yki est crucial pour la stabilité développementale. Ces résultats mettent en évidence un nouveau niveau de modulation de la voie Hippo via l'épissage alternatif de yki.

L'inclusion alternative du deuxième domaine WW est une caractéristique conservée entre Yki et YAP. Cela conforte l'idée que les isoformes Yki1 et YAP1 ont une fonction importante in vivo et que l'épissage alternatif de yki/YAP est un mécanisme conservé de contrôle de la voie Hippo. Cette étude ouvre de nouvelles perspectives pour la modulation de la voie Hippo dans les cellules cancéreuses en modifiant l'épissage alternatif de YAP.

Short Introduction and Objectives :

Over the last decade, the Hippo pathway has emerged as a major player in tissue size control, regeneration and cancer progression. The pathway is, in particular, regulated by mechanicals cues and allows to link growth of the cells with their environment. Humans have approximately 21000 protein-coding genes while Caenorhabditis elegans with much simpler physiology has a genome of 19 000 protein-coding genes and at the same time Oryza sativa Japonica, commonly known as rice contains approximately 35,825 proteincoding genes. For decades researchers were puzzled by the fact that the complexity of the organism does not correlate to the number of protein-coding genes they contain. This question, however, was answered by the discovery of how common and abundant posttranscriptional modifications are.

Once messenger RNA (mRNA) starts to be transcribed from DNA, the primary transcript (pre-mRNA) is generated. To be successfully exported out of the nucleus, this pre-mRNA would undergo a series of processing events, such as 5' capping, splicing, transcription termination and 3' polyadenylation [START_REF] Matlin | Understanding alternative splicing: Towards a cellular code[END_REF]. During splicing, some regions of the pre-mRNA are removed (i.e., introns), and stretches of sequence that contain the necessary information for protein synthesis (i.e., exons) are ligated together. Alternative splicing is the phenomenon where different combinations of sequences could be included or excluded in the final transcript leading to generation of structurally and functionally different mRNA variants [START_REF] Nilsen | Expansion of the eukaryotic proteome by alternative splicing[END_REF]. High frequencies of alternative splicing, posttranslational modifications even leads to increased protein diversity [START_REF] Wilhelm | Mass-spectrometry-based draft of the human proteome[END_REF].

Transcription: Where it all begins

In order to express the encoded information contained in the double-stranded molecule made of deoxyribonucleic acid (DNA), several key steps have to be completed. The genome contains this information in genes that code for proteins which in turn control nearly all functional aspects of a cell. Among the diversity of identified RNA species, messenger RNAs (mRNAs) are regarded as those that contain the necessary information for the synthesis of proteins.

During transcription, genes (stretches of DNA) are used as templates for the synthesis of complementary single-stranded RNA molecules (i.e., transcripts). In eukaryotic cells, based on the type of gene being targeted, such reaction can be catalyzed by three different enzymes RNA polymerases (I, II and III). RNA polymerase II is responsible for the synthesis of RNAs derived from the majority of genes, including those that encode for proteins. While, RNA polymerase I and III are specifically involved in the transcription of ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and several small RNAs [START_REF] Paule | Transcription by RNA polymerases I and III[END_REF].

Transcription by RNA pol II starts with the binding of several transcription factors to a regulatory region located upstream of the gene, known as the promoter [START_REF] Fuda | Defining mechanisms that regulate RNA polymerase II transcription in vivo[END_REF]. The transcription factors enable the subsequent assembly of the polymerase and the formation of the transcription initiation complex. After the assembly steps and further conformational rearrangements, RNA pol II releases from the large complex and enters the elongation phase [START_REF] Kwak | Control of Transcriptional Elongation[END_REF]. During elongation, RNA is synthesized from the transcription start site (TSS), and nucleotides are incorporated in a complimentary basis in the 5' to 3' direction. Eventually, the polymerase transcribes through the cleavage and polyadenylation signals that mark the end of the gene, and it is released from the DNA template [START_REF] Kuehner | Unravelling the means to an end: RNA polymerase II transcription termination[END_REF] 1.1.2 A short overview of mRNA processing:

All mRNA molecules undergo several modifications before they are exported to the cytosol, which includes the addition of a 5' cap, the polyadenylation of the 3' end and the removal of introns via splicing. The carboxyl-terminal domain (CTD) of the RNA polymerase II is involved in the cap formation by recruiting the capping enzymes (triphosphatase, guanylyltransferase and methyltransferase). During 5'capping a chemical group is added to the 5'end of the pre-mRNA. 5'cap is the addition of a modified guanine (5' guanine-N7 cap) nucleotide which is subsequently methylated, and it functions to prevent the novel transcript from degradation [START_REF] Mandal | Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II[END_REF]. Reviewed by [START_REF] Martinez-Rucobo | Molecular Basis of Transcription-Coupled Pre-mRNA Capping[END_REF].

Splicing is a much more complex reaction. During this process, introns are removed, and exons are ligated together. As a result, a mature mRNA product is obtained. Many proteins are involved in the splicing process, including small ribonucleoproteins (snRNPs), heterogeneous nuclear ribonucleoproteins (hnRNPs) and other additional proteins. [START_REF] Matlin | Understanding alternative splicing: Towards a cellular code[END_REF]. To finalize the processing, the 3' end of the pre-mRNA is first cleaved at a specific site. Sites of cleavage are encoded in the DNA sequence of the gene, and for the vast majority it is between the highly conserved polyadenylation signal (PAS) AAUAAA and a downstream sequence element (DSE), usually U or GU-rich [START_REF] Proudfoot | Integrating mRNA processing with transcription[END_REF] and then polyAdenosine (polyA) tail is added, which is typically 200-250 nucleotides long in mammalian cells. (Figure1).

Figure 1 Regulation of eukaryotic gene expression:

mRNA expression starts with the nuclear transcription of genes. Following several processing steps, some of which occur cotranscriptionally, the transcription products are further transformed into mature mRNAs that can then be exported to the cytosol and localized to sub-cellular compartments. mRNA export is linked to strict quality control mechanisms unprocessed RNAs degraded. Once in the cytosol, mRNAs can be recognized by ribosomes and translated into proteins or will be degraded.

Initially, mRNA processing was thought to happen post-transcriptionally. However, in reality, transcription and processing are not consecutive, but simultaneous and interdependent [START_REF] Neugebauer | On the importance of being co-transcriptional[END_REF] and reviewed in [START_REF] Bentley | Coupling mRNA processing with transcription in time and space[END_REF]. The 3' poly(A) tail prevents the degradation by 3'-to-5' exoribonucleases , facilitates the transfer from the nucleus to the cytoplasm and promotes mRNA translation. This transfer, however, depends on the correct processing and successful detection of CBC (cap binding complex) which is bound to 5'cap of the pre-mRNA. Detection of CBC facilitates mRNAs transfer via nuclear pore (J. D.

Lewis & Izaurralde, 1997).

Splicing Sites:

The process of splicing is complex, in eukaryotes it is carried by a large complex of proteins and RNAs and together they do the intricate job of removal of introns and stitching of exon together. This large complex is aptly termed as spliceosome, and it is also one of the most complicated machinery within the cell [START_REF] Nilsen | The spliceosome: The most complex macromolecular machine in the cell?[END_REF]. Recognition of and removal of introns by spliceosome is facilitated by the presence of specific sequence elements within the introns and at exon-Intron boundaries. The 5' and 3' splice sites of an intron are specific and very crucial recognition sequences that are recognized by the components of splicing machinery.

These sequences extend a few nucleotides into the flanking exon.

There are two types of introns in metazoan pre-mRNA. They are classified as minor introns also known as U12 type or major class of introns also known as U2 type. The U2 and U12 introns differ by the presence of characteristic splice site sequence, and thus they are spliced by a different spliceosomal complex.

U2 type introns form the majority of the introns present in metazoans, and they contain the canonical GT-AG intron boundaries. It must be noted that very few U2 introns also contain GC-AG boundaries. However, GC-AG introns represent only 0.82% of U2-type introns in humans or 0.45% of U2 type in Drosophila melanogaster. Overall U2 type introns account for 99% of all the introns present in humans, Drosophila melanogaster.

Minor class or U12 type introns are so rare that they amount to mere 0.4% of total human introns [START_REF] Sheth | Comprehensive splice-site analysis using comparative genomics[END_REF] while Drosophila melanogaster contains only 19 U12 type introns (C. F. [START_REF] Lin | Evolutionary dynamics of U12type spliceosomal introns[END_REF].

In humans, the majority of the Introns are U2 type, in these introns, the 5'splice sites or the splice donor site is defined by a nine nucleotide (nt contains essential adenosine that is required for the first nucleophilic attack of the splicing reaction [START_REF] Reed | Initial splice-site recognition and pairing during pre-mRNA splicing[END_REF]. BPS is followed by a polypyrimidine tract (PPT), a pyrimidine-rich motif which is a string of uracil bases is crucial for efficient BPS utilization and selectionreviewed in (Valadkhan, 2007a) (Figure 2). On the other hand, U12 type introns so not contain polypyrimidine tract upstream of 3' splice site. Reviewed in [START_REF] Sheth | Comprehensive splice-site analysis using comparative genomics[END_REF]. The spliceosome is a ribonucleoprotein complex that is involved in splicing of nuclear precursor mRNA (pre-mRNA). There are two types of Spliceosome in eukaryotes: the U2dependent spliceosome (Major Spliceosome), which catalyzes the removal of U2-type introns, and the less abundant U12-dependent spliceosome (Minor Spliceosome), which is present in only a subset of eukaryotes and splices the rare U12-type class of introns.

)
The major spliceosome undergoes significant conformational and compositional rearrangements during the several steps of the splicing reaction. It is composed of five different small nuclear RNA molecules (snRNAs: U1, U2, U4, U5and U6), as well as around 170 other splicing protein factors. Each of these RNA molecules associates with several proteins and form complexes called small nuclear ribonucleoproteins (snRNP). Each snRNP consists of an snRNA (two in the case of U4/U6), a common set of seven Sm proteins (B/B D3, D2, D1, E, F, and G) and a variable number of particle-specific proteins (Figure 3). Such snRNPs form the core of the spliceosome and are directly involved in recognition of splice sites and branch-point sequences, as well as the catalysis of the splicing reaction. [START_REF] Will | Spliceosome structure and function[END_REF]. The minor spliceosome is functionally analogous to the major spliceosome but differs in the use of snRNAs (minor snRNAs are U11, U12, U4atac/U6atac, and U5). There are many RNA binding proteins (RBPs) in the dynamic structure of the spliceosome that helps in splicing [START_REF] Matlin | Spliceosome Assembly and Composition[END_REF].

Two key steps of splicing:

Pre-mRNA splicing is achieved by two consecutive trans-esterification reactions which are based on nucleophilic attacks between RNA nucleotides. In the very first reaction, 5' exon is cleaved from the intron through a nucleophilic attack of the 2 nucleotide in the branch point sequence on the phosphate group of the GU dinucleotide at the 5' splice site, resulting in the formation of a lariat intermediate. In the second step, the free 3' hydroxyl group at 5'ss makes the second nucleophilic attack on the phosphodiester bond at 3'ss (3' exon-intron junction) (Figure 4). This results in ligation of the two exons and release of excised intron as a lariat. Reviewed in [START_REF] Valadkhan | The spliceosomal proteome: At the heart of the largest cellular ribonucleoprotein machine[END_REF] and [START_REF] Will | Spliceosome structure and function[END_REF]. During the course of the splicing reaction and before the actual intron can be removed, the active catalytic site of the spliceosome needs to be created, an event that requires many changes in its composition and conformation. Since spliceosome is both highly dynamic and flexible, it assembles and dissembles tediously to facilitate each splicing event. It undergoes both radical structural and compositional changes at every step of its assembly. The assembly of spliceosome has been studied extensively in vitro. Based on biochemical methods, six different complexes can be distinguished: the E, A, B, Bact, B*, and C complex. Reviewed in [START_REF] Shi | Mechanistic insights into precursor messenger RNA splicing by the spliceosome[END_REF].

RNA helicases from the DExD/H-box family (composed of the DEAD-box, DEAH-box, and Ski2-like helicases) facilitate the extensive structural and compositional remodeling of spliceosome assembly and transition to various steps [START_REF] Cordin | Structure, function and regulation of spliceosomal RNA helicases[END_REF].

Cyclophilins, a subfamily of peptidyl-prolyl cis-trans isomerases (PPIases), facilitate conformational changes within the spliceosome. Reviewed in [START_REF] Thapar | Roles of prolyl isomerases in RNA-mediated gene expression[END_REF]. Hereby, the major spliceosome catalyzes the splicing in the following steps (Figure 5).

Step1:

The crucial first step in the assembly of the major spliceosome is the association of U1 snRNP with the 5'ss and U2 snRNP with 3' ss via formation of protein dimer. This results in the formation of the commitment complex; also referred to as "E complex."

Step2: U2 snRNP then recognizes branch point sequence followed by interaction with U1 snRNP leading to the formation of pre-spliceosome. This complex made by the interaction between U1 snRNP and U2 snRNP spans across the exons and brings together the 5' and 3'splice sites and BPS in close vicinity. Thus this pre-spliceosome Complex A is also referred to as "Exon definition complex."

Step3: U4/U6.U5 is a large pre-assembled tri-snRNP spliceosomal complex. It contains U5 snRNA, extensively base-paired U4/U6 snRNAs in addition to over 30 proteins. Recruitment of this tri-snRNA to complex A results into formation of Complex B.

Step4: The complex B undergoes a series of conformational and compositional rearrangements resulting in a catalytically active complex called complex B act or complex B*.

Several remodeling of protein and RNA-RNA interactions facilitate the formation of U2-U6 snRNA structure that brings the 5'splice site and BPS in close proximity and forms the catalytic core. The activation of complex B leads to unwinding of U4 and U6 snRNA and expulsion of U4 and U1 from the complex.

Step5: The activated B complex (B act ) catalyzes the first step of splicing. This leads to the formation of complex C, which contains Intron-lariat intermediate.

Step6: Complex C catalyzes the second step of splicing after it undergoes further conformational rearrangements.

Step7: After this second transesterification reaction, the ligated exons and a lariat intron are released. The intron lariat structure is degraded. This is followed by the release of U2, U5 and U6 snRNP and which are recycled for further rounds of splicing.

However, the mRNA is just not yet free in the nucleus. A complex of proteins is then deposited to the new exon junction (thus termed as exon junction complex, EJC). This marks the completion of splicing and allows the mRNP particle to travel to the cytoplasm. Reviewed in [START_REF] Black | Mechanisms of Alternative Pre-Messenger RNA Splicing[END_REF] [START_REF] Valadkhan | The spliceosome: caught in a web of shifting interactions[END_REF]) [START_REF] Wahl | The Spliceosome: Design Principles of a Dynamic RNP Machine[END_REF] [START_REF] Will | Spliceosome structure and function[END_REF].

Alternative Splicing: Harbinger of complexity!

Constitutive splicing refers to the type of splicing in which introns are systematically removed, and the exons are ligated together to generate a final mRNA. However, splicing is also regulated alternatively, i.e., particular exon might be entirely or partially spliced out, or introns might be retained, or the splicing machinery could make the choice of the different 5' or 3' splice sites. Such processing of pre mRNA transcript which results in the inclusion of different part of a transcript into the final mRNA product is called Alternative splicing (AS).

It leads to the formation of alternative mRNA products from a given gene locus. According to (Q. [START_REF] Pan | Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[END_REF]) (E. T. [START_REF] Wang | Alternative isoform regulation in human tissue transcriptomes[END_REF], almost 95% of multiexonic genes in humans are regulated by produce several RNAs by alternative splicing.

Interestingly, proteins resulting from splicing variants often have distinct molecular functions.

AS can result in the generation of isoforms of proteins with different biological function, structure, localization and interaction capabilities [START_REF] Keren | Alternative splicing and evolution: Diversification, exon definition and function[END_REF]) [START_REF] Nilsen | Expansion of the eukaryotic proteome by alternative splicing[END_REF]. For example, the two variants of survivin have opposite functions: one has pro-apoptotic while the other has anti-apoptotic properties [START_REF] Végran | Association of p53 gene alterations with the expression of antiapoptotic survivin splice variants in breast cancer[END_REF]. It has been estimated that on average, each gene generates around ten mRNA isoforms (Z. [START_REF] Hu | Revealing Missing Human Protein Isoforms Based on Ab Initio Prediction[END_REF]. Around 40% of Drosophila genes contain one or more alternative exon (Q. [START_REF] Pan | Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[END_REF] Alternative Splicing plays a crucial role in the expansion of the coding capacity of eukaryotic genomes by giving rise to several structurally and functionally different protein isoforms from a single gene locus. Thus, it fills the gap between the total number of protein-coding genes (< 20,000) [START_REF] Ezkurdia | Multiple evidence strands suggest that theremay be as few as 19 000 human proteincoding genes[END_REF] compared to the overall number of proteins (> 100,000) and also imparts to the complexity of the organism [START_REF] Nilsen | Expansion of the eukaryotic proteome by alternative splicing[END_REF].. In unicellular cells, alternative splicing is absent or very rare, and one gene provides one protein product [START_REF] Ast | How did alternative splicing evolve?[END_REF].

One of the extreme examples of such expansion of protein-coding capacity via alternative

splicing is dscam gene in Drosophila. It can produce a whopping 38,016 different mRNA isoforms via alternative splicing in four different regions of its pre-mRNA [START_REF] Black | Protein diversity from alternative splicing: A challenge for bioinformatics and post-genome biology[END_REF] (Graveley, 2001) [START_REF] Schmucker | Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity[END_REF].

Types of Alternative Splicing:

In eukaryotes, alternative splicing can result via employing one of the following mechanisms: exon skipping or inclusion, choice of alternative 3' or 5' splices site selection, intron retention, the inclusion of mutually exclusive exon and alternative polyadenylation (J. [START_REF] Chen | Alternative splicing in cancer: Implications for biology and therapy[END_REF]. The most common type of alternative splicing is a cassette type alternative exon, i.e., Exon skipping in vertebrates and invertebrates. While in lower metazoans Intron retention is more frequent (Figure 6). Which splicing event takes place is often determined by the contributions and activity of the different splicing activators or repressors in the tissue or during a developmental stage. Alternative splicing has a central regulatory role in the gene expression pathway. It dictates several biological functions through the entire lifespan of an organism. It has been shown that the higher eukaryotes display the higher proportion of alternatively spliced genes indicating that alternative splicing is an indispensable feature of genomic evolution. Several splicing events are conserved among different species along with many splicing variants which are species-specific indicating that alternative splicing plays a significant role in species differentiation along with genomic evolution.

Regulators of Alternative Splicing:

1.

Exon and Intron definition:

The spliceosome directs both constitutive and alternating splicing with high fidelity. The average exon size is small in higher eukaryotes (approx 170nt on an average) while Introns could be up to tens of thousands of nucleotides long and harbor several "cryptic splice" sites.

It is, therefore, a huge task for spliceosome to identify bona fide sites from the pseudo or cryptic ones. An understanding of how spliceosome accomplishes such task comes from the following models.

The exon definition model postulates that that exon, rather than introns are the basic unit of recognition (Exon definition may facilitate splice site selection in RNAs with multiple exons), and the spliceosome assembles across the exon. In contrast, in lower eukaryotes such as yeast and fly where the introns are much smaller, early spliceosome assembly is centered around the introns, which is referred to as "intron definition" [START_REF] Talerico | Intron definition in splicing of small Drosophila introns[END_REF]. The intron definition model postulates that components of the spliceosome assemble and interact across small introns, where the 5' and 3' splice sites are close to each other. Transfection splicing assays have shown that as intron length rises above 250 nucleotides, splicing becomes quite inefficient [START_REF] Fox-Walsh | The architecture of pre-mRNAs affects mechanisms of splice-site pairing[END_REF]. In intron definition, the spliceosome recognizes the splice sites across the intron, provided that this intron is not too large (e.g., <200-250 nt) and beyond this length exon definition prevails [START_REF] Fox-Walsh | The architecture of pre-mRNAs affects mechanisms of splice-site pairing[END_REF]) (Figure 7). Furthermore, the authors of this report found that the inclusion of an RNA cis-element known as an exonic splice enhancer (ESE) could dramatically increase splicing efficiency of even longer introns.

While, on the other hand, Intron definition mediated splicing is an exception in mammals, it is a rule in plants, fungi, and invertebrates [START_REF] Talerico | Intron definition in splicing of small Drosophila introns[END_REF]) (X. [START_REF] Xiao | Coevolutionary networks of splicing cisregulatory elements[END_REF]. The mammalian junctions that define an exon are weakly conserved and more degenerate with respect to yeast canonical cis-elements. These elements are necessary but not sufficient to define exon/intron junctions. Internal exonic sequences far from the 5' and 3' splice sites were essential for exon recognition. These additional elements are called splicing regulatory elements (SREs), cis-acting elements which are present in the pre-mRNA and varies in terms of location and effect. They are known as ESE (exonic splicing enhancers), ESS (exonic splicing silencers), ISE (intronic splicing enhancers) and ISS (intronic splicing silencers).In general, SREs recruit trans-acting splicing factors that can act as repressor or activators of splicing. As described earlier, Introns possess several sequence elements required for pre-mRNA splicing: 5' and 3' splice sites, BP and PPT. These splice sites are often termed as ''weak'' or ''strong'' splice sites. Splice site strength depends on the complementarity between splice site sequences and U1 and U2 snRNPs binding to them. The more the degree of similarity the more the strength of a splice site (Figure 8) [START_REF] Roca | Determinants of the inherent strength of human 5 RNA[END_REF]. (De Conti, Baralle, & Buratti, 2013) Generally, constitutively spliced exons possess stronger splice sites whereas alternatively spliced exons have weak splice sites; thus these splice sites are recognized less efficiently [START_REF] Hertel | Combinatorial control of exon recognition[END_REF]. It has been shown that In general, splicing rate is modulated by the strength of splice sites and presence or absence of splicing enhancers [START_REF] Hertel | The Function of Multisite Splicing Enhancers[END_REF]) [START_REF] Erkelenz | Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms[END_REF] In addition to strength, the relative position of splice sites also affects the splicing outcome.

Closer splice sites are both favored and paired quicker [START_REF] Nogués | Influence of Polymerase II Processivity on Alternative Splicing Depends on Splice Site Strength[END_REF].

However, often very close splice sites prevent the neighbor exons from being spliced together favoring mutually exclusive exon splicing (Smith & Nadal-Ginard, 1989).

The SREs also regulate alternative splicing in addition to constitutive splicing by specifying the constitutively or alternatively spliced exon. stimulate their binding to U1 snRNP and splicing. Splicing can also be activated when regulatory proteins interfere with intronic or exonic silencer elements dependent [START_REF] Witten | Understanding splicing regulation through RNA splicing maps[END_REF].

On the other hand, Exonic splicing silencers (ESSs) and intronic splicing silencers (ISSs) are repressors of splicing and are recognized by hnRNPs. One proposed mechanism through which hnRNPs repress splicing is to prevent the assembly of general splicing factors and SR proteins. In particular, protein multimerization along an exon is initiated by the binding of an hnRNP to a strong ESS that subsequently recruits more hnRNPs to adjacent weak ESSs [START_REF] Martinez-Contreras | hnRNP proteins and splicing control[END_REF] [START_REF] Zhu | Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins[END_REF]. Alternatively, hnRNPs can also repress splicing by looping out entire exons blocking the recruitment snRNPs [START_REF] Nasim | High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5 support of a common looping out and repression mechanism[END_REF] [START_REF] Damgaard | HnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure[END_REF]. Initially identified as positive and negative regulators of splicing, respectively, it is now known that their effect on a particular splicing event is heavily context-dependent [START_REF] Witten | Understanding splicing regulation through RNA splicing maps[END_REF].

Heterogeneous ribonuclear proteins (hnRNPs)

HnRNPs represent a large family of RBPs that contribute to multiple aspects during mRNA processing post-transcription. A typical hnRNP usually contain one or more RNA binding domains(RNA recognition motif, i.e., RRM domain), a qRRM (quasi-RNA recognition motif), a glycine-rich domain constituting an RGG box (Arg-Gly-Gly repeats) and a Khomology (KH) domain [START_REF] Geuens | The hnRNP family: insights into their role in health and disease[END_REF].

The hnRNP protein family consists of at least 20 proteins in humans that have been characterized as components of protein complexes bound to pre-mRNA (hnRNP complexes) [START_REF] Dreyfuss | hnRNP Proteins and the Biogenesis of mRNA[END_REF] [START_REF] Cartegni | Listening to silence and understanding nonsense: exonic mutations that affect splicing[END_REF](Figure 10). The molecular weight of the hnRNP family ranges from 34 to 120 kDa, and they have been named alphabetically from A to U. [START_REF] Piñol-Roma | Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm[END_REF]. Although hnRNPs are expressed in all tissues, but the relative amount of different hnRNPs varies among cell types and exhibit stage-specific expression patterns, for an example, some hnRNPs are extremely abundant (~100 million copies per nucleus), while others are present in a lower amount. Together, hnRNPs are similar in abundance to histones in growing cells [START_REF] Dreyfuss | hnRNP Proteins and the Biogenesis of mRNA[END_REF] [START_REF] Kamma | Cell Type-Specific Expression of hnRNP Proteins[END_REF] Studies have uncovered a variety of ways in which hnRNPs can modulate splicing.

Mechanisms that repress exon usage involve binding that competes with the recruitment of positive splicing factors (for an example SR protein), as well as the inhibition of protein interactions involved in exon or intron definition. This has been extensively demonstrated for hnRNPA1, A2/B1, and hnRNP. A multitude of biochemical studies too have demonstrated that hnRNP binding can promote interactions that facilitate exon inclusion, highlighting their potential to positively or negatively regulate exon usage [START_REF] Martinez-Contreras | hnRNP proteins and splicing control[END_REF]). It appears that the ability to regulate exon usage both positively and negatively is a general feature of hnRNPs. [START_REF] Huelga | Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins[END_REF] reported that hnRNPs from diverse families can positively and negatively regulate thousands of exons by binding to proximal introns by using splicing-sensitive microarrays coupled with CLIP. They reported that hnRNPs are similar and cooperative in the roles that they play in the the regulation of AS and their RNA targets overlap. The negative of positive regulation of AS could be attributed to the binding of hnRNP in different positions relative to the exon, a phenomenon already reported for SR and others RBPs [START_REF] Witten | Understanding splicing regulation through RNA splicing maps[END_REF]. Position-dependent AS regulation by hnRNPs could be via looping out the intervening RNA [START_REF] Blanchette | Modulation of exon skipping by high-affinity hnRNP A1binding sites and by intron elements that repress splice site utilization[END_REF]. Indeed it has been shown that hnRNP A1 and hnRNP H collaborate to create a RNA loop so as to repress the internal splice sites and while activating external splice sites which are brought in a closer proximity.

The authors used bioluminescence resonance energy transfer (BRET) technology to show the homotypic and heterotypic interactions between these two hnRNPs in live cells [START_REF] Fisette | hnRNP A1 and hnRNP H can collaborate to modulate 5 RNA[END_REF]. The binding on opposite sides of an intron represses exon usage while looping events within an intron can bring together pairs of splice sites to promote spliceosomal interactions across an intron [START_REF] Chabot | My road to alternative splicing control: from simple paths to loops and interconnections[END_REF].

Besides splicing family of hnRNPs have been thoroughly documented to play several roles such as HnRNP A1 and A2/B1 play a role oligodendrocytic and neuronal mRNA trafficking [START_REF] Shan | A molecular mechanism for mRNA trafficking in neuronal dendrites[END_REF]. [START_REF] Villarroya-Beltri | Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs[END_REF] 1992). SR proteins are ubiquitous in higher organisms and present in many other diverse species, such as plants and fungi, reflecting an early emergence in evolutionary history [START_REF] Twyffels | Shuttling SR proteins: More than splicing factors[END_REF]. Budding yeast is devoid of "true" SR proteins but has three SR-like proteins: Npl3, Gbp2 and Hrb1, which likely represents the ancestral basis from which SR proteins subsequently evolved [START_REF] Busch | Evolution of SR protein and hnRNP splicing regulatory factors[END_REF].

SR proteins are defined by the presence of one or two RNA recognition motifs and RRM homolog (RRMH) (a more degenerate pseudo RRM) at the N terminus and an RS domain (arginine/serine-rich dipeptide domain) at the C terminus. RRM domains are widespread among RNA binding proteins, and their flexibility permits diverse modes of binding between proteins [START_REF] Maris | The RNA recognition motif, a plastic RNAbinding platform to regulate post-transcriptional gene expression[END_REF]. In humans, Twelve canonical members of the SR protein family share this characteristic domain structure. RRMs ascertain RNA-binding specificity, whereas the RS domain functions as a protein-protein interaction module by recruiting components of the core splicing apparatus to promote splice site pairing (Jane Y. [START_REF] Wu | Specific interactions between proteins implicated in splice site selection and regulated alternative splicing[END_REF]. There are 7 homologs of these SR proteins in Drosophila. These essential splicing factors can bind RNA directly, and their RS and RRM domains act to promote protein-protein and protein-RNA interactions, facilitating the recruitment and assembly of the spliceosome (S. H. [START_REF] Xiao | Phosphorylation of the ASF/SF2 RS domain affects both proteinprotein and protein-RNA interactions and is necessary for splicing[END_REF].

SR-

repetitive characteristic of true SR proteins or an RRM motif, although many of these are able to bind RNA via different domains and mechanisms [START_REF] Long | The SR protein family of splicing factors: master regulators of gene expression[END_REF]. Genome-wide analyses revealed that RS-domain containing proteins participate in numerous aspects of gene expression regulation, including those involved in the cell cycle [START_REF] Boucher | A genome-wide survey of RS domain proteins[END_REF] The SR family nomenclature is historically diverse but in the beginning of decade an agreement was reached to have consensus terminology to identify them [START_REF] Twyffels | Shuttling SR proteins: More than splicing factors[END_REF]) [START_REF] Manley | A rational nomenclature for serine/arginine-rich protein splicing factors (SR proteins)[END_REF], thus they have several aliases, however it is not surprising to find researchers using old style names even now.

Techniques to identify bindings of SR protein:

The search for RNA target sequences of SR proteins in genes known to be regulated by SR proteins first led to the discovery of exonic regulatory sequences of the Drosophila doublesex gene [START_REF] Tian | A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA[END_REF], the mouse gene [START_REF] Watakabe | The role of exon sequences in splice site selection[END_REF], the bovine growth hormone gene (Q. [START_REF] Sun | In vitro analysis of bovine growth hormone pre-mRNA alternative splicing. Involvement of exon sequences and trans-acting factor(s)[END_REF] and the fibronectin gene [START_REF] Lavigueur | A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding[END_REF].

The identification of many ESE sequences of individual SR proteins was done via systematic evolution of ligands through exponential enrichment (SELEX) protocols [START_REF] Tuerk | Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[END_REF]) (Figure 12).

The binding SELEX consists of binding high-affinity RNA targets from a pool of RNAs with 20 random nucleotides between PCR amplifiable sequences to the immobilized SR protein.

RT-PCR then amplifies the bound RNAs. This cycle is repeated at least five times before the remaining RNAs are sequenced. The idea behind the binding SELEX is that high-affinity sequences are also strong exonic splicing enhancers. The binding SELEX generally yields one or two similar high-affinity sequences [START_REF] Bourgeois | Broad Specificity of SR (Serine{plus 45 degree rule}Arginine) Proteins in the Regulation of Alternative Splicing of Pre-Messenger RNA[END_REF].

Functional SELEX is an updated and modified version of SELEX that has been developed to identify the SREs in the cells. Random RNA sequences are added at the site of enhancer sequences. Exons included after the splicing in vivo or in a cell extract are RT-PCR amplified.

After multiple selection cycles in the presence of one particular SR protein, the amplified ESEs are sequenced [START_REF] Bourgeois | Broad Specificity of SR (Serine{plus 45 degree rule}Arginine) Proteins in the Regulation of Alternative Splicing of Pre-Messenger RNA[END_REF].

However, despite the advent of SELEX it is challenging to identify the new SRE sequences and identify the RBPs and their contribution in splicing. Thus, to identify the RBP-RNA map on a global scale, CLIP (cross-linking and immunoprecipitation) analysis has been developed.

In the CLIP, UV radiation is utilized to cross-link direct RBP-RNA interactions within a very short distance (~1 Å). Further, a CLIP-Seq also referred to as HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation) is carried out to identify the RBP-RNA interactions precisely. In order to increase the efficiency of the cross-linking of RBP-RNA interaction PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was developed. Also, iCLIP (individual-nucleotide resolution CLIP) was developed that allows single-nucleotide resolution of RBP binding sites. Reviewed in [START_REF] Jeong | SR Proteins: Binders, Regulators, and Connectors of RNA[END_REF]) iCLIP is CLIP combined with high-throughput sequencing has been used to identify transcriptome-wide binding maps of several RNA-binding proteins such as by [START_REF] Ule | CLIP Identifies Nova-Regulated RNA Networks in the Brain[END_REF]. However the method identification of binding sites with CLIP has some disadvantages;

for an example, it relies on the analysis of overlapping sequence clusters and distances of less than 30 nucleotides are not resolved. Further, the requirement of reverse transcription to pass over residual amino acids that remain covalently attached to the RNA at the cross-link site is also a disadvantage. Primer extension studies have shown that the significant number of cDNAs prematurely truncate immediately before the 'cross-link nucleotide' [START_REF] Urlaub | A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles[END_REF]. iCLIP, however, employs a different cDNA cloning protocol which allows identification of the cDNAs that truncate at the cross-link sites therefore, the position of cDNA truncation allows iCLIP to identify the cross-link sites and provide nucleotide-resolution precise protein -RNA interaction map [START_REF] König | ICLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF].

In both CLIP and iCLIP, protein and RNA are crosslinked via UV, and then the protein of interest is purified, ligate the 3' adaptor, purify the protein-RNA complex, and digest the protein by proteinase K. However, In CLIP, a 5' adapter is ligated to the RNA before reverse transcription. Therefore, CLIP can only amplify cDNAs that read through the cross-link site.

However, since the cross-linked nucleotides are covalently bound to the amino acid residue, a proportion of cDNAs truncate at the cross-link site. In iCLIP, truncated cDNAs are captured by circularization and subsequent linearization. Thus iCLIP is capable of studying a more extensive repertoire of RBPs (Figure 13). UV irradiation to covalently cross-link in vivo protein-RNA complexes followed by Cell lysis and partial RNA digestion (step 1). After lysis, the crosslinked RNA is fragmented by the limited concentration of RNase I and RNA fragments are then co-immunoprecipitated with the RBP (step 2), followed by ligation of a 3 -PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments (step 5). Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). The peptide that is on the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the cross-link site. Therefore, two types of cDNAs are generated: truncated cDNAs and readthrough cDNAs. In iCLIP, the cDNA library is prepared in such a way that both truncated and readthrough cDNAs are amplified (step 7). After PCR amplification and sequencing (step 8), both truncated and readthrough cDNAs are present. Figure adapted and modified from [START_REF] Zünd | Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3 Nature Structural and Molecular Biology[END_REF] However [START_REF] Maticzka | UvCLAP is a fast and nonradioactive method to identify in vivo targets of RNA-binding proteins[END_REF] developed uvCLAP (ultraviolet cross-linking and affinity purification ) method which they describe as a fast, robust technique to identify and characterize targets of RBPs in vivo without having to resort to labor-intensive techniques that use radioactive substances. Their technique uses a tandem affinity purification tag that consists of two 6 × His tags and an in vivo biotinylation signal peptide, which allowed tagged RBPs to be sequentially purified by nickel and streptavidin beads. More recently, (Ya [START_REF] Zhao | SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy[END_REF] have developed a technique called spyCLIP that is a comparatively faster way to detect RBP-RNA interactions by further eliminating the need of SDS PAGE separation, membrane transfer and RNA labeling for visualization thus limiting the loss of RNA during purifications steps. They do so by using a SpyTag (a 13 aa peptide) fused to RBP and a SpyCatcher system. The covalent binding of SpyTag-Spy-catcher allows harshest washing steps which could not be done with CLIP, iCLIP or uvCLAP methods. Overall, these newly developed techniques will further allow understanding the RBP interactions and help to elucidate their role on a genome-wide scale.

Role of SR in constitutive splicing and alternative splicing:

SR proteins contribute to the dynamic sequential process of spliceosome assembly via recognition of ESEs. During the 1st step of splicing, i.e., the formation of commitment complex (E complex) SR protein regulate the recruitment of U1 snRNP at 5' splice site and small subunit U2AF at 3' splice site (S. [START_REF] Cho | Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly[END_REF].

Further, in A complex SR proteins facilitate the binding of U2 snRNP at branchpoint sequence by neutralizing the negative charge at phosphodiester backbone (H. [START_REF] Shen | Arginine-Serine-Rich Domains Bound at Splicing Enhancers Contact the Branchpoint to Promote Prespliceosome Assembly[END_REF])At the next step, SR proteins help in the recruitment of the U4/U6.U5 tri-snRNP [START_REF] Roscigno | SR proteins escort the U4/U6•U5 tri-snRNP to the spliceosome. RNA. -Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis[END_REF]. Additionally, RS domains of SR proteins are hypothesized to associate with the phosphodiester backbone near the 5 U6 binding (H. Shen & Green, 2004). Finally, molecular rearrangements coupled with dephosphorylation of SR proteins leads to the formation of catalytically active C complex and eventually results in splicing of introns and ligation of exons together (W. [START_REF] Cao | Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro[END_REF]. In addition, SR proteins can mediate splicing of minor U12-introns by functioning analogously to recruit the machinery of the minor spliceosome through interactions with the U5 snRNP (H. [START_REF] Shen | RS domain-splicing signal interactions in splicing of U12-type and U2-type introns[END_REF].

Besides playing a role in constitutive splicing, SR proteins mediate complex alternative splicing regulation. SR promotes the inclusion of the alternative exons which have characteristics short length and weak splice site to be adequately recognized by splicing machinery. The role of RBPs in AS is defined by the context and location (X. D. [START_REF] Fu | Context-dependent control of alternative splicing by RNA-binding proteins[END_REF], for an example, SR proteins associated with exonic sequences mainly act as enhancers, but SR proteins associated with intronic sequences can act as suppressors [START_REF] Shen | Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position[END_REF]. SR proteins have also shown to associate with constitutive exon adjacent to the alternative exon in order to mediate splicing and. For an example, SRSF1 has been shown to highlighting the association of SR protein with constitutive exon can affect AS of alternative exon upstream or downstream (J. Han et al., 2010).

As said earlier, SR proteins can also act to inhibit splicing. SRSF9 has been shown to induce skipping of exon 7B in hnRNNPA1 pre-mRNA [START_REF] Simard | SRp30c Is a Repressor of 3' Splice Site Utilization[END_REF]. Similarly, it has also been shown that SRSF11 binds to exonic splicing silencer to promote skipping of exon 10 in tau pre-mRNA (J. Y. [START_REF] Wu | SRp54 (SFRS11), a Regulator for tau Exon 10 Alternative Splicing Identified by an Expression Cloning Strategy[END_REF]. A few years ago [START_REF] Pandit | Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing[END_REF] showed that SR proteins not only promote but can also repress the alternative splicing of cassette exons.

Role of SR protein besides splicing:

Its indicated that mRNA processing events are indeed coupled with transcription [START_REF] Bentley | Coupling mRNA processing with transcription in time and space[END_REF]. SR proteins co-localize with RNA polymerase II in nuclear speckles, an interaction mediated by the Pol II C-terminal domain (CTD) in a serine phosphorylation-dependent manner [START_REF] Misteli | Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo[END_REF] [START_REF] Sapra | SR Protein Family Members Display Diverse Activities in the Formation of Nascent and Mature mRNPs In Vivo[END_REF]. It has been shown by [START_REF] De La Mata | RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20[END_REF] that CDT is absolutely required to recruit SRSF3 for exon exclusion.

Additionally, SRSF1 and SRSF3 have been shown to interact with H3 and associate with chromatin thus giving an insight into the regulation of SR protein's function via histone modification and nucleosome occupancy [START_REF] Luco | Epigenetics in alternative pre-mRNA splicing[END_REF].

SR proteins can also directly regulate the elongation rate of RNA polymerase II. It has been shown that depletion of SRSF1 or SRSF2 can induce the accumulation of RNA polymerase II at gene loci and attenuate elongation (S. [START_REF] Lin | The splicing factor SC35 has an active role in transcriptional elongation[END_REF]. SRSF2 which is an essential non-shuttling SR protein has been shown to be a part of the 7SK RNP complex that facilitates the release of paused Pol II which often is paused near gene promoter [START_REF] Ji | SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase[END_REF].

SR proteins have been shown to shuttle between nucleus and cytoplasm and they play a role even beyond splicing [START_REF] Cáceres | A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm[END_REF]. Indeed SRSF3 and SRSF7 are shown to be involved in mRNA export and by recent CLIP analysis some SR proteins (SRSF1 to SRSF7) are shown to interact with export receptor Nuclear export factor 1 (NXF1/TAP) highlighting a role of SR proteins in export of specific mRNAs from nucleus to cytoplasm [START_REF] Hargous | Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8[END_REF][START_REF] Müller-Mcnicoll | SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export[END_REF].

SR protein is also linked with mRNA translation as SRSF1 is detected in polysomal fractions of HeLa cells cytoplasm [START_REF] Sanford | A novel role for shuttling SR proteins in mRNA translation[END_REF]. Further, [START_REF] Michlewski | The Splicing Factor SF2/ASF Regulates Translation Initiation by Enhancing Phosphorylation of 4E-BP1[END_REF] have demonstrated that SRSF1 initiates translation via interactions with components of mTOR pathway. They suggest that SRSF1 recruits mTOR kinase to the mRNA targets to suppress the activity of 4EBP (a competitive inhibitor of cap-dependent translation) in a phosphorylation-dependent manner. This allows the release of eIF4E, a cytoplasmic cap-binding protein and thus initiating the translation. Further, [START_REF] Maslon | The translational landscape of the splicing factor SRSF1 and its role in mitosis[END_REF] have identified more than 500 mRNA translational targets of SRSF1 via high-throughput deep sequencing. It has also been shown that SRSF3 regulates Internal Ribosomal Entry Site (IRES)-mediated translation initiation [START_REF] Bedard | A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation[END_REF] and SRSF7 is involved in translation of unspliced viral RNA that contains containing Constitutive Transport Element (CTE) [START_REF] Swartz | The shuttling SR protein 9G8 plays a role in translation of unspliced mRNA containing a constitutive transport element[END_REF]. Similarly, [START_REF] Swanson | SRp40 and SRp55 Promote the Translation of Unspliced Human Immunodeficiency Virus Type 1 RNA[END_REF] have shown that SRSF5 and SRSF6 promote the translation of gag protein. SR protein also plays a role in mRNA stability for an example; SRSF1 regulates the stability of PKCI- [START_REF] Lemaire | Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2: A novel function for SR proteins[END_REF]. (H. [START_REF] Wu | A Splicing-Independent Function of SF2/ASF in MicroRNA Processing[END_REF] have described an association of SR protein with microRNA and their role in microRNA biogenesis. (Bradley, Cook, & Blanchette, 2015) have shown that SR proteins could bind to a diverse set of RNAs including long ncRNA suggesting that SR proteins can regulate gene expression via regulating ncRNA. These reports suggest that SR proteins play various functions ranging from coordination of splicing to transcription and post-transcriptional event (Figure 14). throughout the RS domain [START_REF] Gui | A serine kinase regulates intracellular localization of splicing factors in the cell cycle[END_REF]) [START_REF] Colwill | The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution[END_REF]. It is postulated that phosphorylation of RS domain increases the RNA binding capacity as well as is important for protein-protein interaction (R. Tacke, Chen, & Manley, 2002) (S. H. [START_REF] Xiao | Phosphorylation of the ASF/SF2 RS domain affects both proteinprotein and protein-RNA interactions and is necessary for splicing[END_REF] The localization of SR protein depends upon the phosphorylation as Clk/Sty mediate phosphorylation facilitates the release of SR proteins from nuclear speckles [START_REF] Colwill | The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution[END_REF]. After the spliceosomal assembly, some SR proteins are re-phosphorylated so as to participate in an additional round of mRNA splicing (i.e. they are recycled), and a few dephosphorylated SR proteins associate with mRNA to signal that mRNA is ready to be exported to the nucleus. Once the export to nucleus and translation is complete, thereby SR proteins are re-phosphorylated by SRPK so as to interact with transportin-Sr and be imported back in the nucleus [START_REF] Kataoka | Transportin-SR, a nuclear import receptor for SR proteins[END_REF] SRSF2 occurs in response to genotoxic stress [START_REF] Edmond | Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin[END_REF]. However, the results of post-translational modification besides phosphorylation are not clearly understood, but they seem to be essential and contribute to the regulation of SR proteins.

Targets of SR proteins:

So far, we have seen the wide range of roles that SR proteins play in the process of RNA metabolism, from export to mRNA decay. In order to further elucidate the implications of SR in AS and reveal how splicing is regulated it is vital to investigate the targets of RBPs (not only SR and hnRNPs). Thanks to RNA seq strategies, now it is possible to identify genomewide AS events and immunoprecipitation approaches such as iCLIP (Please refer to the section: 1.2.2.3.2.1 ) to identify targets of RBPs [START_REF] Huelga | Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins[END_REF] [START_REF] König | ICLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF]) [START_REF] Brooks | Conservation of an RNA regulatory map between Drosophila and mammals[END_REF]. (Änkö et al., 2012) used iCLIP technique and reported that two SR proteins SRSF3 and SRSF4 bind to a small subset of targets but their target rarely overlap with each other, implicating that most of SR proteins targets do not overlap. While [START_REF] Pandit | Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing[END_REF] doubted the conclusions and suggested that the sensitivity of the experiments might not have been enough to capture all the SR proteins bound to the transcripts. Thus, they used CLIPseq in combination with splicing-sensitive arrays to reveal that indeed the targets of SRSF1 and SRSF2 extensively overlap and also found that depletion of either of these SR proteins lead to both inclusion and exclusion of exon, reasserting that SR proteins play a role in both enhancing and suppression of splicing.

Further, (Bradley et al., 2015) 15a).

The criteria used for assuming if an AS event is altered they used a "percentage spliced in" value (PSI or ) 10%. (PSI is generally used to determine the extent of splicing change. The percent here indicates the efficiency of splicing a specific exon into the transcript population of a gene. A PSI of 100% indicates constitutive exons that are included in all transcripts and never removed from expressed isoforms. PSI values below 100% imply reduced inclusion of alternative exons and denote the percentage of isoforms that contain the exon compared to the total transcript population) [START_REF] Schafer | Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI)[END_REF].

They detected 561 events (representing 405genes) that were altered by the depletion of 8 SR proteins, which constitute to around >10% of the simple AS events (5472) that are readily detected in Drosophila S2 cells. The detected that each SR protein affected a different number of AS events from B52 affecting 253 while Rbp1 altering just 24 events. Additionally, they saw that each SR protein affect all types of AS in a similar proportion (Figure 15). Study by (Bradley et al., 2015) Their data reflected that SR proteins are both activators and repressors or AS events, they even found that among the 8 SR proteins 6 repressed more than half of their targets. Further, they showed that not only multiple SR proteins can affect the same AS event, but they can also have an antagonistic function in the same region. They also revealed that every AS event was affected by more than one SR protein with the only exception of B52 which seemed to regulate most of its targets (74%) individually. Moreover, on the other hand, Rsf1 regulated almost 82% of its targets in a combinatorial fashion. Finally, they show that all of the 8 SR proteins regulate the promotor selection of multiple targets and poly(A) site selection thus revealing much widespread role of SR protein in gene expression by directly affecting transcription start site and 3'end processing site choices.

Another study which got published a little later in 2015 done by [START_REF] Brooks | Regulation of alternative splicing in Drosophila by 56 RNA binding proteins[END_REF] complements the above-described study by (Bradley et al., 2015) in several ways. Here, the authors took a total of 56 RBPs including SR and hnRNPs and individually depleted them in Drosophila S2 DRSC cells by using RNAi. Followed to it, they did RNAseq on the polyA and RNA isolated from these 56 samples. They looked at three more types of AS events in addition to 5 that was criteria for study by (Bradley et al., 2015). To confidently assume that Basing analysis on these criteria, they detected around 2876 AS events that were affected (out of 23,079AS events) by one or more of these 56 RNA binding proteins. They found a large number of events affected by depletion of each SR protein as over 400 events alone were affected by the depletion of SR protein B52.

Similar to the works of [START_REF] Huelga | Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins[END_REF] who showed that hnRNPs too have overlaps in their targets & (Bradley et al., 2015) they reasserted the fact that SR proteins and hnRNPs promote exclusion and skipping and not work each time antagonistically according to the traditional assumption.

SR Protein B52

In Drosophila, B52 is ubiquitously expressed throughout all developmental stages, and its concentration is vital to the viability and development of the organism [START_REF] Kraus | The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development[END_REF].

This 52kDa protein was first characterized because it localized to heat shock puffs upon heat shock (puffs are the regions of visibly decondensed chromatin) on the polytene chromosome [START_REF] Champlin | Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin[END_REF].

Homozygous B52 mutants (B52 28 /B52 28 ) is lethal at the second-instar larval stage [START_REF] Ring | The SR protein B52/SRp55 is essential for Drosophila development[END_REF] and overexpression of B52 leads to lethality and various defects such as reduced salivary glands and curled wings depending on the tissue where B52 is overexpressed [START_REF] Kraus | The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development[END_REF]. In addition, they also determined that the level of B52 suddenly decreases during the first instar stage, after which it remains only 15%-20% compared to that in the embryo. From here onwards, the level of B52 remains constant for the rest of the later development highlighting its role in early development and development throughout the course of life. They also showed that B52's expression level is tissue-specific, with the highest in adult ovaries and lowest expression in the larval intestine while the imaginal disc, the brain, and ventral ganglion all have significant levels of B52. (Fic, Juge, Soret, & Tazi, 2007) showed that overexpression of B52 by using eyeless-GAL4

(ey-GAL4) driver in the eye leads to severe defects of eye development while overexpression of another SR protein, dASF did not prompt any such phenotype.

Further, they also showed that B52 regulates the RNA splicing of eyeless (ey) which is a master regulator of eye morphogenesis. In another publication [START_REF] Gabut | The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets[END_REF] showed that expression of GFP-B52 via GMR-Gal4 results in disorganization of rod and cone cells in eye imaginal discs in the 3rd-instar larvae. These authors have also identified several B52-interacting mRNAs by performing co-immunoprecipitation of B52 proteins, using GFP overexpression as a control. Some of these targets are in agreement with the result of genomic SELEX where full-length Baculovirus-expressed B52, is used as a bait to attract potential RNA targets that have a high affinity for B52 (S. [START_REF] Kim | Specific SR protein-dependent splicing substrates identified through genomic SELEX[END_REF]. [START_REF] Juge | The SR Protein B52/SRp55 Is Required for DNA Topoisomerase I Recruitment to Chromatin, mRNA Release and Transcription Shutdown[END_REF] showed that in addition to its mRNA-splicing function, B52 is also involved in the recruitment of Topoisomerase I to the chromatin. This mechanism SR protein recruiting topoisomerase 1 is conserved in humans as recently, [START_REF] Girstun | Effects of SRSF1 on subnuclear localization of topoisomerase I[END_REF] have shown that SRSF1, thanks to its distinct RS domain can recruit topoisomerase 1 to the nucleoplasm in HeLa cells. While they observed SRSF2, SRSF3, SRSF6, and partly SRSF4 exhibited an opposite effect instead. However, the mechanism of this action of SRSF1 is not yet known.

B52 has been shown, by both microarray analysis and genomic SELEX, to target a transcription factor gene BTB/POZ transcription factor longitudinal lacking, (lola), which controls axon extension via the transcription of Spire, an actin nucleation protein. In addition to lola, B52 has been shown to bind to the RNA transcripts of Syndecan (Sdc) and

RhoGAP16F [START_REF] Gabut | The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets[END_REF] (S. [START_REF] Kim | Specific SR protein-dependent splicing substrates identified through genomic SELEX[END_REF]. These findings highlight the importance of B52 in cellular regulatory networks.

SR proteins are frequently observed to be upregulated in tumors suggesting that SR protein helps in the tumor initiation and growth and in Drosophila overexpression leads to severe developmental defects. (Fernando, Audibert, Simon, Tazi, & Juge, 2015) used Drosophila bristle cell lineage as a model to study the consequences of B52 overexpression on development and to identify factors that could rescue this phenotype as it could be of therapeutic interest. By using SOP-GAL4 and neuralized P72 -GAL4 (neur P72 -GAL4) drivers they overexpressed B52 in the bristle lineage and by immunostaining at the pupal stage and upon observation of pharate adults, they showed that overexpression of B52 lead to partial to complete loss of microchaetes and macrochaetes (short and long bristles) (Figure 16). Reported in (Fernando et al., 2015) To understand the role of B52 at the cellular level, they did B52 staining at the pupal stage in the above experiment and revealed that overexpression does not affect the identity of the cells, but it increases the size of the cells which eventually induces cell death in bristle They also reported that B52 overexpression increased dMYC promoter activity measured by dMYC-lacZ reporter activity and enrichment of phosphorylated Pol II across the dMYC promoter. Finally, by using a genetic screen they identified several factors that could rescue the phenotype induced by B52 overexpression including brain tumor (brat) which is a negative regulator of growth [START_REF] Frank | The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis[END_REF] and lilliputian (lilli) which is a positive regulator of growth [START_REF] Wittwer | Lilliputian: an AF4/FMR2-related protein that controls cell identity and cell growth. Development[END_REF])(Figure 18). The authors further showed that mechanistically an antagonism between B52 and Brat exists as in B52 overexpression clones, overexpression of Brat reduces the transcription of myc to rescue the overexpression phenotype. Besides revealing the role of B52 and providing a mechanistic link between B52 and myc transcription this study opens a new perspective of identifying proteins that could repress the ill-effects of SR protein overexpression in mammals. [START_REF] Zhang | The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila[END_REF]. Further studies will be needed to elucidate the complete B52 splicing program and provide an understanding of the role that B52 plays in growth and other processes.

Additional RNA Binding Proteins that regulate slicing:

In addition to above described RBPs (SR and hnRNPs) that are ubiquitously expressed across several tissues, several reports have described some tissue-specific RBPs such as RBFOX, MBNL, PTBP2 (nPTB) and NOVA family proteins are essential regulators of tissue or cellspecific splicing (X. D. [START_REF] Fu | Context-dependent control of alternative splicing by RNA-binding proteins[END_REF]. NOVA proteins are the class of RBPs that are specific to neuronal tissue, and by using CLIPseq techniques, it has been shown that NOVA regulates AS in brain [START_REF] Ule | CLIP Identifies Nova-Regulated RNA Networks in the Brain[END_REF]. Further CLIP seq analysis revealed the positiondependent splicing program of NOVA, i.e., binding to downstream intron or upstream intron promotes exon inclusion or exon skipping. Mechanistically, NOVA binds to an ESS and blocks binding of U1 snRNP which inhibits the formation of E complex and promotes exon skipping. While on the other hand, when NOVA binds to ISE downstream of an intron, it promotes the formation of A complex and complexes that follow and thus it promotes exon inclusion [START_REF] Ule | An RNA map predicting Nova-dependent splicing regulation[END_REF]. Other RBPs such as RBFOX, MBNL, ESRP1/2 and Quaking too facilitate a position-dependent splicing program. However, the underlying mechanism of the position-dependent regulation is mostly unknown.

Additional RBPs such as RBM5 regulate the splicing by inhibiting the formation of B complex in splicing assembly while RBM17 (or SPF45) inhibits splicing at the second catalytic step during the splicing reaction [START_REF] Lallena | Splicing regulation at the second catalytic step by Sex-lethal involves 3 Cell[END_REF]) [START_REF] Bonnal | RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition[END_REF].

The combinatorial regulation of AS program is also reported, as is the case of splicing of exon 6 of human Fas, which encodes for a type 1 transmembrane protein. TIA-1 binding to ISE in intron 6 promotes the inclusion of exon 6 via U1 snRNP recruitment and produces an isoform that promotes apoptosis while binding of another RBP, PTBP1 to an ESS in exon 6 to inhibits the binding of U2AF65, leads to production of an isoform that lacks exon 6 and does not promote apoptosis [START_REF] Izquierdo | Regulation of fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition[END_REF].

Recent studies have estimated the number of proteins encoded by humans that are capable to bind directly to RNA to be close to being in thousands [START_REF] Castello | Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins[END_REF]) [START_REF] Castello | Comprehensive Identification of RNA-Binding Domains in Human Cells[END_REF]. Thus the repertoire of RBPs that regulate splicing is vastly unmapped.

Other factors affecting Alternative splicing:

Secondary structure of RNA plays an essential role in defining interactive and functioning properties of the RNA molecule. Even though RNA is a single-stranded molecule, but it can fold back against itself to produce secondary and tertiary structures [START_REF] Wan | Understanding the transcriptome through RNA structure[END_REF]. RNA secondary structures have been reported to interfere with the interactions of splicing enhancers or silencers with proteins (Blencowe, 2000) [START_REF] Smith | Alternative pre-mRNA splicing: the logic of combinatorial control[END_REF]. Splicing changes occur due to RNA secondary structure formation. [START_REF] Shepard | Conserved RNA secondary structures promote alternative splicing[END_REF] suggested that 4 % of conserved alternative splicing are associated with secondary structure. For an example, stem-loop structures affect the 5 SMN2's exon 7 and exon 10 of Tau (N. N. [START_REF] Singh | Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes[END_REF]) [START_REF] Hutton | Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17[END_REF]. RNA secondary structures can even bring together distant RNA elements together. For example, secondary structure in FGFR2 pre-mRNA brings together two elements so as to activate exon IIIb [START_REF] Baraniak | A Stem Structure in Fibroblast Growth Factor Receptor 2 Transcripts Mediates Cell-Type-Specific Splicing by Approximating Intronic Control Elements[END_REF]. In the Drosophila Dscam gene, that can form up to 38 000 different isoforms alternative cassette selection occurs after the formation of secondary structure between docking and selector sequences surrounding alternative cassette exons. This mechanism ensures that only one of the many alternative cassette exons is included in the mRNA [START_REF] Graveley | Mutually exclusive splicing of the insect Dscam Pre-mRNA directed by competing intronic RNA secondary structures[END_REF]. In other instances, intronic secondary structure elements may even function as riboswitches that bind to small molecule metabolites and activate or repress splicing in response to the concentration of the metabolite. [START_REF] Cheah | Control of alternative RNA splicing and gene expression by eukaryotic riboswitches[END_REF] Additionally, a substantial amount of evidence has accumulated demonstrating that transcription rate and chromatin structure can affect alternative splicing.

Quality control of splicing:

Although "Split gene" architecture in eukaryotes is clearly advantageous such as it expands the coding capacity, but the same division of genes into exons and introns introduces a requirement of an intricate splicing regulatory network that is composed of RNA regulatory sequences, RNA protein complex, and splicing factors.

A ''mistake'' or ''error'' can be referred to the generation of any non-canonical molecular product, which either result in a nonfunctional protein product, or which induces quality control mechanisms to take action. To maintain the fidelity of gene expression cells evolved several mRNA surveillance systems (also called as quality control systems) in both the nucleus and the cytoplasm. These mRNA surveillance pathways detect not only the aberrant transcripts that may lead to toxic protein but also regulate the wild-type mRNAs [START_REF] Kervestin | NMD: A multifaceted response to premature translational termination[END_REF]. In the last decades, much advancement has been made to an understanding of these mechanisms.

Nuclear mRNA surveillance mechanisms are mainly studied in Saccharomyces cerevisiae [START_REF] Garneau | The highways and byways of mRNA decay[END_REF]. Before mRNAs are exported from nucleus to cytoplasm, the aberrant synthesis of mRNAs (incorrect or defective splicing, cap structure, cleavage or polyadenylation at 3' end) or the retention of mRNAs in the nucleus leads to activate different surveillance mechanisms. The nuclear retention of an mRNA that should be exported to the cytoplasm leads to decay by nuclear surveillance systems [START_REF] Vasudevan | Nuclear mRNA surveillance[END_REF]. One of the key components of nuclear surveillance is the nuclear exosome. The exosome is distributed in both nucleus and cytoplasm. To promote RNA degradation, two 3'-5'

exonucleases interact with the core exosome. The nuclear exosome can lead the aberrant transcripts to accumulate at the site of transcription for degradation [START_REF] Torchet | Processing of 3 -extended read-through transcripts by the exosome can generate functional mRNAs[END_REF].

Cytoplasmic mRNA surveillance contains nonsense-mediated mRNA decay (NMD), no-go mRNA decay (NGD), non-stop mRNA decay (NSD) )(Figure 19) The nonsense-mediated mRNA decay (NMD) pathway is a cytoplasmic surveillance mechanism that targets and degrades transcripts containing a PTC (premature termination codon) [START_REF] Leeds | The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon[END_REF]) [START_REF] Peltz | mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one transacting factor[END_REF]. PTCs may arise from several sources, including DNA mutations, inaccurate transcription leading to frameshifts or single nucleotide nonsense mutations, or from alternative splicing that puts stop codons "in frame" in an inappropriate position.

A multi-subunit complex known as EJC (Exon Junction Complex) is a critical element of NMD. The EJC is essential for several cellular processes, such as nuclear export and subcellular localization [START_REF] Schell | Minireview Integration of splicing , transport and translation to achieve mRNA quality control by the nonsense-mediated decay pathway[END_REF] as well as translation [START_REF] Nott | Splicing enhances translation in mammalian cells: An additional function of the exon junction complex[END_REF]) [START_REF] Wiegand | Exon junction complexes mediate the enhancing effect of splicing on mRNA expression[END_REF]. After splicing, the exon-exon junctions formed are bound by EJC. During the pioneer round of translation, the ribosome displaces each EJC as it moves along the mRNA. Translation terminates at the natural stop-codon, which is typically located in the terminal exon and therefore does not have any downstream bound EJC. However, if an mRNA contains a PTC within the 50 nucleotides or greater upstream of the last exon-exon junction, the ribosome stalls at the PTC, giving it the chance to interact with the downstream bound EJC. This triggers the NMD process, which leads to degradation of the PTC-containing mRNA. When the ribosome recognizes a stop-codon during translation, it recruits release factors, such as eRF1 and eRF3, which are associated with the NMD-associated protein UPF1. Recognition of PTC leads to a stable interaction between UPF1 and UPF2, bridging UPF1 with the EJC. This, in turn, leads to phosphorylation of UPF1 and recruitment of either SMG6, an endonuclease that cleaves the NMD targeted RNA, or the SMG5-SMG7 complex that is associated with uncapping and deadenylation of RNAs. Either of these pathways leads to rapid degradation of the targeted RNA.

Further, SRSF1 has been shown to stimulate NMD for mRNAs with PTC (Zuo [START_REF] Zhang | Involvement of SR proteins in mRNA surveillance[END_REF] Over-expression of SRSF1 greatly enhanced NMD. This activity of SRSF1 is independent of its shuttling ability but requires an intact RS domain. Recently the same laboratory pinpointed the mechanism by which SRSF1 enhances NMD. By tethering SRSF1

to transcript at the various position, they obtained evidence that SRSF1 enhances UPF1 binding to mRNA in nucleus and the increased presence of UPF1 may accelerate the NMD process [START_REF] Aznarez | Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1[END_REF].

The link between NMD and AS was first seen in a study of expressed sequence tags (ESTs)

where, (B. P. [START_REF] Lewis | Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans[END_REF] showed that several alternatively spliced genes were found to have isoforms that would be predicted to be subject to NMD. These authors termed this phenomenon regulated unproductive splicing and translations (RUST) and this mechanism is conserved from humans to yeast [START_REF] Soergel | Regulation of Gene Expression by Coupling of Alternative Splicing and NMD. In Nonsense-mediated mRNA Decay[END_REF]. Some splicing factors and all SR proteins autoregulate the level of their own expression through alternative splicing coupled to NMD (AS-NMD or unproductive splicing) [START_REF] Lareau | Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements[END_REF]) [START_REF] Ni | Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay[END_REF]. For example, SRSF1 promotes inclusion of an intron after the canonical stop codon, which marks it as a premature termination codon (PTC) to elicit NMD of its own mRNA. These "unproductive splicing" events are highly conserved between evolutionary kingdoms. For a detailed review of NMD and mechanism it employs, one could read the following reviews by [START_REF] Hug | Mechanism and regulation of the nonsense-mediated decay pathway[END_REF], (Lykke-Andersen & Jensen, 2015) [START_REF] Brogna | The Meaning of NMD: Translate or Perish[END_REF] and also [START_REF] Popp | Nonsense-mediated mRNA Decay and Cancer[END_REF].

The NMD pathway is involved in many biological processes, including cell proliferation and growth [START_REF] Weischenfeldt | NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements[END_REF]

The No-go decay (NGD)

The No-go decay (NGD) is triggered when the elongation complex is stalled during translation. The stalling could be due to the presence of rare codons or secondary structure or poly-Lys or poly-Arg tracts, or frameshift sites in mRNA. It detects such mRNA and directs them for endonucleolytic cleavage, and subsequently, the resulting mRNA fragments are degraded by the exosome (which is a conserved multiprotein complex that degrades RNAs in -to--translational mRNA decay following the last translating ribosome) [START_REF] Doma | Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation[END_REF]) [START_REF] Passos | Analysis of Dom34 and Its Function in No-Go Decay[END_REF] [START_REF] Pelechano | Widespread co-translational RNA decay reveals ribosome dynamics[END_REF].

Non-stop decay (NSD)

Non-stop decay (NSD) is an mRNA surveillance mechanism that identifies transcripts that does not possess in-frame stop codons. These mRNAs could arise via premature polyadenylation or point mutations that disrupt stop codon and interestingly are widespread mistakes in the pathway of gene expression [START_REF] Frischmeyer | An mRNA surveillance mechanism that eliminates transcripts lacking termination codons[END_REF] [START_REF] Vasudevan | Non-stop decay -A new mRNA surveillance pathway[END_REF]) [START_REF] Cui | In Vivo Evidence that Defects in the Transcriptional Elongation Factors RPB2, TFIIS, and SPT5 Enhance Upstream Poly(A) Site Utilization[END_REF]. Because mRNAs are short of a stop codon, it leads to ribosomes to cross the poly (A) tail and remove the poly (A)-binding protein (PABP) and stall at the 3' end of the mRNA. The deed of the degradation is carried out by cytoplasmic exosome and Ski complex (Ski2p, Ski3p, and Ski8p) and Ski7p. Ski7p detects the transcript lacking stop codon and recruits the Ski complex and exosome. Once recruited, the ubiquitinproteasome pathway degrades the protein product generated from a non-stop transcript.

Alternative splicing in diseases and available therapy

Aberrant splicing is implicated in a high number of dysfunction and human diseases (R. K. [START_REF] Singh | Pre-mRNA splicing in disease and therapeutics[END_REF]. AS has been implicated in nearly all aspects of cancer development, and therefore, is a central participant in the disease [START_REF] David | Alternative pre-mRNA splicing regulation in cancer: Pathways and programs unhinged[END_REF]) [START_REF] Chabot | Defective control of pre-messenger RNA splicing in human disease[END_REF]) [START_REF] Scotti | RNA mis-splicing in disease[END_REF]. Alternative splicing is altered in a genome-wide manner in cancer cells and is now considered a "novel hallmark of cancer" as every common hallmark of cancer including metastasis is affected by dysregulation of splicing.

In their book, Genetics in medicine, (Nussbaum, Robert L; McInnes, Roderick R; Huntington, 2016) described three groups of splice defects that differ depending on their intronic or exonic location. The first group consists of splice junction mutations such as mutations at the 5'

donor or 3' acceptor sites or in the consensus sequences surrounding these donor or acceptor sites. If the normal acceptor site is inactivated, then other intronic of exonic cryptic splice sites may elicit a splicing reaction. The second group of splicing defects consists of mutations in intronic cryptic sites that make it more favorable for splicing in comparison to the normal splice site. These "activated" cryptic site mutations are often referred to as "leaky" as the normal splice site is still active and produces normal gene product, thus leading to a less severe phenotype. The third group of mutations consists of mutations in the ORF that activate a cryptic splice site in an exon without altering the encoded amino acid (i.e., point mutations that become cryptic splice sites). It has been estimated that such cis splicing mutations constitute 15%-60% of human disease-causing mutations (G. S. [START_REF] Wang | Splicing in disease: Disruption of the splicing code and the decoding machinery[END_REF]. Additionally, mutations disrupting trans-acting splicing regulators cause a wide spectrum of diseases by globally compromising the splicing of many downstream target genes [START_REF] Osborne | RNA-dominant diseases[END_REF].

Examples of disease caused by a point mutation in the exonic regulatory element:

SMA (Spinal muscular atrophy) is a neurodegenerative disease that is a leading genetic cause of mortality in children [START_REF] Arnold | Spinal muscular atrophy[END_REF]. SMA is a result of deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. However, humans have a centromeric copy of this gene called SMN2, which is identical to SMN1 except for a single nucleotide difference in exon 7. Thus, SMN2 cannot compensate for the loss of SMN1 as most mRNA produced from SMN2 lacks exon 7 (almost 90%). SMN2 thus encodes a truncated protein with reduced functionality. The single nucleotide mutation on exon 7, generates the binding site for hnRNP A1 which inhibits exon7 inclusion (Figure 20). Reviewed in (Sunghee [START_REF] Cho | 3 Sequences of Spinal Muscular Atrophy Related SMN2 Pre-mRNA Include Enhancers for Nearby Exons[END_REF] Antisense Example of a disease caused by an intronic mutation that activates a cryptic splice site:

It has been shown that mutations in Lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), a dominant disorder characterized by the dramatic, rapid appearance of aging beginning in childhood due to translation of ageing associated protein progerin [START_REF] Eriksson | Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Sandre-Giovannoli | Lamin A truncation in Hutchinson-Gilford progeria[END_REF]. Most of the typical Hutchinson-Gilford progeria cases are due to a recurrent, de novo point mutation in LMNA exon 11. This mutation resides in a splicing enhancer sequence which as a result leads to activation of a cryptic splicing site resulting in the truncated Lamin A protein.

The use of antisense oligonucleotides to sterically block the aberrant LMNA splicing site in progerin production has been shown in vitro on human HGPS patient's cells and in vivo in the mouse model [START_REF] Osorio | Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging[END_REF].

Example of disease caused by a mutation in core components of the spliceosome:

Retinitis pigmentosa (RP) is inherited a degenerative retinal disorder that is caused by germline mutations in core spliceosomal proteins. More than 3,000 mutations in about 80

genes have been identified to be associated with RP Reviewed by (M. M. [START_REF] Liu | Alternative splicing and retinal degeneration[END_REF].

Moreover, mutations in RNU4ATAC gene which encodes U4atac, a small nuclear RNA that is a crucial component of the minor spliceosome leads to Microcephalic osteodysplastic primordial dwarfism type 1 (MOPD I). Reviewed in [START_REF] Nagy | Microcephalic osteodysplastic primordial dwarfism type I with biallelic mutations in the RNU4ATAC gene[END_REF] Although there is currently no therapy for the treatment of RP, [START_REF] Nakamura | Small molecule Photoregulin3 prevents retinal degeneration in the RhoP23H mouse model of retinitis pigmentosa[END_REF] reported the discovery of a small drug-like molecule, called Photoregulin3, which alters the activity of a transcription factor that regulates rod genes. So far, it has been successfully tested in the mice model for RP. However, clinical trains in humans are underway. Further, a new strategy referred to as SMaRT TM (Spliceosome-mediated RNA trans-splicing as a tool for gene therapy)is developed, it is a system used to correct a part of the mutant transcript instead of full-length cDNA. [START_REF] Puttaraju | Spliceosome-mediated RNA trans-splicing as a tool for gene therapy[END_REF]. Recently [START_REF] Dooley | Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA[END_REF] have developed a Spliceosome-mediated RNA trans-splicing strategy to edit and rescue the expression of CEP290 mRNA which is associated with Leber congenital amaurosis type 10 (LCA10)( The visual impairment) and Joubert Syndrome (JS) (a rare, autosomal recessive disorder characterized by episodes of hyperpnea, developmental delays, ataxia, and abnormal eye movements).

Hippo pathway and its discovery:

Researchers around the globe have a long-standing quest to underpin the exact mechanism of organ size control. Indeed it is quite intriguing to know what precise and complex set of mechanisms control the cell number during development and regeneration. Many studies have pointed towards the fact that growth to the right size and patterning to an exact shape is intrinsic information that is in possession of the organ. For example, In the Salamander, when eyes or limbs are exchanged between different species with different organ size, they reach a size characteristic of the donor [START_REF] Twitty | The growth of eyes and limbs transplanted heteroplastically between two species of Amblystoma[END_REF]. A similar experiment in Drosophila showed that immature imaginal discs, when transplanted in a growth-permissive environment such as the abdomen of an adult female, grow at their normal rate until they reach their final size [START_REF] Bryant | of B JOLOGY REVIEW[END_REF].

During the course of development, an increase in cell number is required for the growth of the organ and body size. During the normal function of an organ, processes such as continuous cell turnover, apoptosis and occasional differentiation of adult stem cells to replace dysfunctional cells are required to maintain the functionality of the organ. During an injury or pathology, the process of healing accompanied by cell division and regeneration is required to compensate for the lost cells and restore the standard functionality. Although the mechanism of proliferation, apoptosis, regeneration are extensively studied, the coordinated between these events are poorly understood to this date and mechanisms underlying these molecular

events are yet to be elucidated. However, the discovery and expansion of Hippo pathway have proven to be an entry point for the researcher in a quest to answering this fundamental question as Hippo signaling pathway has been shown to play a critical role in controlling organ size by regulating both cell proliferation and apoptosis. (W. [START_REF] Kim | The history and regulatory mechanism of the Hippo pathway[END_REF].

As of now, the Hippo Pathway has the status of a highly evolutionary conserved regulator of tissue growth and cell fate. Since its discovery, it has panned out as an integrator of signals from a vast network of proteins and signaling pathways and serves as an essential regulator of critical cellular processes such as organ size regulation, cell proliferation, and stem cell regulation [START_REF] Harvey | The Hippo pathway and human cancer[END_REF]) (Figure 21). Within the last 25 years, the field of the Hippo pathway has rapidly emerged as one of the most popular pathways due to its widespread roles in processes vital to the development. Thus it has been on the radar of researches around the globe in a race to identify its components, regulators and downstream effectors. There are a plethora of reviews with new insights and advancements in the field every year which are rapidly growing every year (Figure 22) [START_REF] Goulev | SCALLOPED Interacts with YORKIE, the Nuclear Effector of the Hippo Tumor-Suppressor Pathway in Drosophila[END_REF].

As shown in (Figure 24), In Drosophila When Hippo Pathway is in ON state, i.e., activated, Hpo forms a complex with Sav. This complex phosphorylates Wts which is in complex with its co-factor Mats. This phosphorylation activates Wts/Mats kinase which subsequently phosphorylates Yki as three separate phosphorylation sites, S111, S168 and S250.

Phosphorylation of Yki at S168 site leads to its cytoplasmic retention as it binds to 14 Therefore, Yki, the effector of the Hippo pathway functions to promote growth and proliferation whereas the kinase of the pathway acts as tumor suppressors as they function to suppress the activity of the Yki.

The downstream target genes of the Hippo pathway are essential in the pathway feedback. For instance, Ex, a cytoskeletal protein for cell morphology, is not only a transcriptional target of Hippo pathway but also an upstream regulator of the pathway, therefore forming a negative feedback loop in growth control regulation [START_REF] Jukam | Opposite feedbacks in the Hippo pathway for growth control and neural fate[END_REF].

Regulation by Fat: Planar cell polarity

The Fat-Dachsous signaling pathway is a cell-contact-dependent mechanism that regulates the Hippo pathway. Fat (Ft) large protocadherin (transmembrane protein) with a molecular weight of 560kDa (P. A. [START_REF] Mahoney | The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily[END_REF]. Ft is the receptor for Hippo Signaling pathway as it genetically interacts with the pathway (F. C. [START_REF] Bennett | Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway[END_REF]) (E. [START_REF] Cho | Delineation of a Fat tumor suppressor pathway[END_REF]) [START_REF] Silva | The Tumor-Suppressor Gene fat Controls Tissue Growth Upstream of Expanded in the Hippo Signaling Pathway[END_REF]) [START_REF] Willecke | The Fat Cadherin Acts through the Hippo Tumor-Suppressor Pathway to Regulate Tissue Size[END_REF].

Dachsous (Ds) another protocadherin acts as the ligand for Ft [START_REF] Clark | Dachsous encodes a member of the cadherin superfamily that controls imaginai disc morphogenesis in Drosophila[END_REF] Fat signaling pathway has a well-characterized role in regulating planar cell polarity (PCP) in epithelial tissue [START_REF] Reddy | The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation[END_REF]. Although, PCP itself does not regulate the Hippo Hippo pathway that regulate Yki activity through the core kinase cascade [START_REF] Baumgartner | The WW Domain Protein Kibra Acts Upstream of Hippo in Drosophila[END_REF]Genevet, Wehr, Brain, Thompson, & Tapon, 2010a;[START_REF] Hamaratoglu | The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis[END_REF][START_REF] Mccartney | The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation[END_REF]J. Yu et al., 2010) The general role of FERM domain proteins is to link the F-actin cytoskeleton to cell membranes and membrane proteins as described by [START_REF] Mangeat | ERM proteins in cell adhesion and membrane dynamics[END_REF]. Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib) are the basolateral polarity protein genes which were first described as "neoplastic" tumor-suppressors as depletion of any of these genes in resulted in multilayered and invasive tumors in Drosophila imaginal discs [START_REF] Bilder | Epithelial polarity and proliferation control: Links from the Drosophila neoplastictumor suppressors[END_REF]. As studies linked overgrowth phenotype to over-activation of Yki, these and stabilizes and therefore inactivates Yki activity (T. [START_REF] Yue | The Cell Adhesion Molecule Echinoid Functions as a Tumor Suppressor and Upstream Regulator of the Hippo Signaling Pathway[END_REF]. Echinoid (Ed) is a known receptor of the EGFR pathway [START_REF] Bai | The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway[END_REF].

The upstream regulators of the Hippo pathway include the actin cytoskeleton beneath the plasma membrane. Indeed, cytoskeleton network F-actin has been demonstrated to be a negative regulator of the Hippo pathway. Ectopic accumulation of F-actin caused by Depletion of Capping proteins Capulet (an actin-binding protein that inhibits polymerization), or constitutive expression of an activated version Diaphanous Dia; induces actin polymerization resulted in increased Yki activity and quite a substantial overgrowth [START_REF] Fernandez | Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila[END_REF][START_REF] Sansores-Garcia | Modulating F-actin organization induces organ growth by affecting the Hippo pathway[END_REF]. It has also been reported that mutations in Hippo pathway genes ex, mer, hpo, sav, wts, and mats leads to an increased Factin accumulation. This could mean that F-actin and Hippo pathway can mutually suppress each other suggesting that in normal conditions, levels of F-actin levels sustain the Hippo pathway as the activation of the Hippo pathway will suppress its negative regulator F-actin.

However, regulation of the Hippo pathway by F-actin remain poorly understood to this date especially in Drosophila. Further, it has been observed that tension regulates the Hippo pathway in Drosophila.

Spectrin is a vital cytoskeleton protein that supports the integrity of cell structures. Drosophila -Heavy ---Spec is localized along the apical--H -Spec is localized at the basolateral and apical membrane, respectively. Spectrin forms hexagonal networks at the cell cortex in all animal cells and has been reported to have mechanosensory properties. Spectrin functions in parallel to the Jub-Wts pathway and loss of spectrin result in Yki activation due to increased tension mediated by activation of the actomyosin network [START_REF] Deng | Spectrin regulates Hippo signaling by modulating cortical actomyosin activity[END_REF] [START_REF] Fletcher | Positive feedback and mutual antagonism combine to polarize crumbs in the drosophila follicle cell epithelium[END_REF].This indicates that Spectrin can relay mechanical cues to the Hippo pathway.

In their review [START_REF] Hariharan | Organ Size Control: Lessons from Drosophila[END_REF] further postulated on how tension can regulate the pathway.

During growth Drosophila imaginal discs contains high levels of growth promoting morphogens in the center. One could imagine that due to this, higher growth proliferation would be focussed at the center but on the contrary uniform growth is observed in the disc, suggesting that levels of morphogen alone do not dictate the growth in the wings. As the growth takes place in the center of growing tissue, it also stretches the periphery. The resulting tension could activate Yki to promote growth. Thus, Yki activation at the periphery could compensate for non-uniform morphogen levels across tissues and promote uniform tissue growth [START_REF] Hariharan | Organ Size Control: Lessons from Drosophila[END_REF] A hippo signaling pathway is highly conserved between Drosophila and Humans. In order to ease further understanding, I am at this moment, giving a table to highlight the core components of the Hippo pathway in Drosophila and their homologs in Mammalian Pathway. Mothers against DPP (MAD).

Hippo Pathway in Mammals

Several components of the mammalian Hippo pathway were known prior to the formal discovery of the Hippo pathway in Drosophila. For example, before MOB1 was recognized as a tumor suppressor in the eukaryotes, it was known in yeast to be essential for mitosis and maintenance of the ploidy [START_REF] Luca | MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy[END_REF]. Similarly, before the identification of MST1 as a tumor suppressor, in yeast, it was known to be activated by caspases and mediate cell death. Mst1 codes for the mammalian Ste20 kinase and its interaction with MAPK, p38, MKK6, MK7, and SAPK were also reported [START_REF] Creasy | Cloning and characterization of a human protein kinase with homology to Ste20[END_REF] YAP and TAZ are homologs of the Drosophila Hippo pathway effector Yki. In ON state, i.e., when the Hippo pathway is active, SAV1 interacts with MST1/2 to form a complex in order to stabilize MST1/2 which leads to autophosphorylation and thus activation of MST1/2 [START_REF] Callus | Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation[END_REF])(Figure 27). LATS kinase which also exists in two isoforms LATS1 and LATS2 respectively interacts with adapter protein MOB1 to form a complex.

This complex is eventually phosphorylated by phosphorylation of serine 909/872 within the LATS1/2 activation loop and the hydrophobic motif (LATS1 T1079 and LATS T1041).

LATS1/2-MOB1 further phosphorylates transcriptional coactivator Yes-associated protein (Galli, Carrara, et al., 2015) [START_REF] Zanconato | Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth[END_REF]. YAP/TAZ associate with a plethora of transcription factors in addition to TEAD1-4 including YAP binding partners SMAD1/2/3/7 [START_REF] Ferrigno | Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-Oncogene[END_REF] [START_REF] Kurisaki | Transforming Growth Factor-Induces Nuclear Import of Smad3 in an Importin-1 and Ran-dependent Manner[END_REF]) [START_REF] Alarcón | Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-Cell[END_REF]) [START_REF] Varelas | TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal[END_REF], Runt-Related Transcription Factor (RUNX) [START_REF] Zaidi | Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription[END_REF] Erb-B2 Receptor Tyrosine Kinase 4 (ErbB4) (Komuro, Nagai, Navin, & Sudol, 2003), p73 [START_REF] Strano | From p63 to p53 across p73[END_REF] and TAZ binding partners RUNX, Peroxisome ProliferatorActivated (or) NCOA6 (a histone methyltransferase protein) is recruited by YAP to stimulate TEAD activity and subsequently act as co-activators of gene expression (Qing et al., 2014a) [START_REF] Skibinski | The Hippo Transducer TAZ Interacts with the SWI/SNF Complex to Regulate Breast Epithelial Lineage Commitment[END_REF].

On the other hand, the YAP/TEAD can also act as repressors by engaging deacetylase complexes [START_REF] Valencia-Sama | Hippo component TAZ functions as a cothrough TEA domain (TEAD) transcription factor[END_REF] [START_REF] Kim | Transcriptional co-repressor function of the hippo pathway transducers YAP and TAZ[END_REF]. Multiple transcriptional downstream target genes of YAP/TAZ has been identified, such as connective tissue growth factor (CTGF), cysteine-rich 61 (Cyr61),fibroblast growth factor (FGF1),the Ft4 shows maximum homology to Drosophila Ft. Ft4, and Hchs1 mutants in mice show only planar cell polarity defects but do not show YAP or LATS related defect [START_REF] Mao | Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila[END_REF]. Ft1 loss in Zebrafish activates YAP through Scrib [START_REF] Skouloudaki | Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development[END_REF]. However, Dachs is not conserved in mammals. [START_REF] Wang | Angiomotin-like proteins associate with and negatively regulate YAP1[END_REF]). This mechanism is not conserved in Drosophila. AMOTs have also been identified as direct substrates of LATS1/2 kinases and LATS-mediated phosphorylation has been shown to promote stabilization and abundance of AMOTs, thereby restricting YAP/TAZ activity in a feed-forward mechanism [START_REF] Adler | Serum deprivation inhibits the transcriptional co-activator YAP and cell growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases[END_REF]. Mutations in E-cadherin, another protein located at the adherens junction, can lead to the loss of proper cell-cell contact, and promote Epithelial-mesenchymal Transition (EMT)

EGF
which is a hallmark of cancer [START_REF] Hansen | YAP and TAZ: A nexus for Hippo signaling and beyond[END_REF] [START_REF] Harvey | The Hippo pathway and human cancer[END_REF]. Other adherens junction proteins such as Protein Tyrosine Phosphatase 14 (PTPN14) are also able to bind to and sequester YAP in the cytoplasm. The ability of these proteins to sequester phosphorylated YAP in the cytoplasm prevents transcription of YAP/TAZ target genes, and thus they are essential regulators of the Hippo pathway (F. X. Yu & Guan, 2013) (W. [START_REF] Wang | PTPN14 is required for the density-dependent control of YAP1[END_REF].

Scribble (SCRIB), a basolateral protein SCRIB responds to changes in cell density by promoting the assembly of the Crb complex at the apical border of the cell at tight junctions that promotes YAP/TAZ phosphorylation by LATS1/2 and subsequent cytoplasmic retention.

Mechanotransduction:

The process by which cells sense external forces and spatial constraints and translate them to biochemical signals that elicit an adaptive cellular response is called mechanotransduction.

The mechanical forces generated from cell-cell contacts, cell-ECM (extracellular matrix) interaction, and the microenvironment have been shown to regulate gene expression; thus they participate in growth, proliferation, and apoptosis. Evidence of YAP/TAZ as two critical mechanoregulated transcriptional effectors are mounting up. Environmental/physical cues, such as cell contact, surface texture, and mechanical forces also affect the transcriptional activities of YAP/TAZ. It has been shown that cells cultured on soft matrices or substrates with limited surface area lose their actin stress fibers which result in cytoplasmic localization of YAP/TAZ and transcriptional inactivation [START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF]) [START_REF] Wada | Hippo pathway regulation by cell morphology and stress fibers[END_REF]. The key player that relays the information about ECM stiffness, cell-cell contact, tension, cell shape is RhoGTPase. Mechanistically active RhoA promotes actin polymerization and stress fiber formation to promote nuclear localization of YAP/TAZ (Figure 28). Therefore, disruption of actin stress fiber using small molecules that Moreover, the microtubule cytoskeleton was revealed to be required for detachment-induced YAP phosphorylation [START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF]) (Wada et al., 2011) (B. Zhao et al., 2012) Further, the spectrin network is also linked as an upstream regulator of the Hippo pathway mammalian along with Drosophila pathway [START_REF] Deng | Spectrin regulates Hippo signaling by modulating cortical actomyosin activity[END_REF] [START_REF] Fletcher | The Spectrin cytoskeleton regulates the Hippo signalling pathway[END_REF])(Wong et al., 2015).

These findings revealed an essential role for cell architecture and mechanical forces in regulation of the Hippo pathway signaling. Cell morphology mediated mechanism potentially co-acts with a cell-cell contact mediated regulation to encourage density-dependent control of cell proliferation intermediated by the Hippo pathway. This regulation may play a fundamental role in normal developmental processes as well as in the progression of diseases such as cancer.

Soluble factors as upstream signals:

YAP/TAZ have been shown to be regulated by extracellular soluble factors. This breakthrough was based on speculation that extracellular molecules such as growth factors or hormones might contribute to regulating hippo signaling in order to control tissue growth and homeostasis. Growth factors mitigate the regulation of the pathway via G-protein coupled receptors (GPCRs) [START_REF] Miller | Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP[END_REF](F. X. [START_REF] Yu | Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling[END_REF]. These two independent studies have confirmed that two serum components, lysophosphatidic acid (LPA) and Serum-borne sphingosine-1-Phosphate (S1P) can inhibit LATS1/2 via activating Rho GTPases and eventually activating YAP/TAZ activity. (X. Zhou et al., 2015) showed that estrogens could hyperactivate YAP via activating specific GPCRs. Glucogen, epinephrine has been shown to inhibit YAP via PKA mediated LATS1/2 activation. The omega-3 polyunsaturated fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) too are found to inhibit YAP/TAZ via specific GPCRs (K. [START_REF] Zhang | G-protein-coupled receptors mediate omega-3 PUFAs-inhibited colorectal cancer by activating the Hippo pathway[END_REF]. Wnt ligands also have been shown to regulate YAP activity via GPCRs binding to Frizzled receptors (H. W. [START_REF] Park | HHS Public Access[END_REF].

13--

provides an exciting gateway to manipulate the Hippo pathway signaling with small diffusible molecules since GPCRs are highly druggable. However, to this date, the mechanism by which these extracellular signals and cell surface receptors mitigate and regulate the Hippo pathway remains elusive.

Components of the cell cycle as upstream regulators:

LATS1/2 is shown to be phosphorylated in a cell cycle-dependent manner. Although the precise links are yet to be shed light upon, it has been demonstrated that CDK1 and Aurora A phosphorylate LATS1/2 during mitosis checkpoint [START_REF] Toji | The centrosomal protein Lats2 is a phosphorylation target of Aurora-A kinase[END_REF]) [START_REF] Morisaki | WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis[END_REF]) [START_REF] Yabuta | Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts[END_REF] (L. Zhang et al., 2015). It was also shown by [START_REF] Ganem | Cytokinesis failure triggers hippo tumor suppressor pathway activation[END_REF] that the presence of extra centrosome leads to activation of LATS2 and subsequently inhibiting YAP/TAZ transcriptional activity and stabilization of p53.

Furthermore, (S. [START_REF] Yang | CDK1 phosphorylation of YAP promotes mitotic defects and cell motility and is essential for neoplastic transformation[END_REF] [START_REF] Lehtinen | A Conserved MST-FOXO Signaling Pathway Mediates Oxidative-Stress Responses and Extends Life Span[END_REF]) [START_REF] Geng | Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity[END_REF]. YAP interacts with FoxO1 (forkhead box proteins) and activates transcription of manganese superoxide dismutase (MnSOD) and eventually help in reducing oxidative stress and ischaemia/reperfusion (I/R)-induced injury in the heart [START_REF] Shao | A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response[END_REF].

Cells depend upon carbohydrates for metabolism. Recently, it has been demonstrated that nutritional/energy stress caused by glucose deprivation leads to inhibition of YAP/TAZ activity via LATS1/2 and AMP-activated protein kinase (AMPK) (W. [START_REF] Wang | AMPK modulates Hippo pathway activity to regulate energy homeostasis[END_REF] [START_REF] Mo | Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway[END_REF]. [START_REF] Deran | Energy stress regulates Hippo-YAP signaling involving AMPK-mediated regulation of angiomotin-like 1 protein[END_REF] showed that this inhibition is enhanced by phosphorylation of AMOTL1 by AMPK which can regulate LATS1/2 and YAP simultaneously. It was also demonstrated that this phosphorylation could further interfere with the interaction between YAP and TEAD and eventually inhibiting TEAD mediated transcription activation program.

Finally, it was also described that during the state of hypoxia, E3 ubiquitin ligase SIAH2 inhibits LATS2 which in turn promotes YAP onco-activity. Loss of SIAH2 in tumor cells was shown to restore the tumor suppressor function of a Lats2 kinase (B. [START_REF] Ma | Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase[END_REF].

Collectively, these studies established a previously unknown role of the Hippo signaling in glucose metabolism.

Crosstalk with other pathways:

In addition, to the discussed upstream regulators, the Hippo pathway has been shown to cross talk with Wingless/Ints (Wnt), bone morphogenetic proteins (BMPs), Notch, and Hedgehog (Hh), epidermal growth factor (EGF), as these signals have been shown to control the activity of YAP/TAZ (Figure 30). processes as well as in the progression of diseases such as cancer.

A subtle difference between regulation of the Hippo pathway in

Drosophila and Mammals:

Recently, in studies in Mammals lead to the identification of integrin signaling as an underlying mechanism for Hippo pathway regulation via mechanical cues such as cell morphology and cell-ECM contact area (N. G. [START_REF] Kim | Adhesion to fibronectin regulates Hippo signaling via the FAK-Src-PI3K pathway[END_REF] [START_REF] Si | Src Inhibits the hippo tumor suppressor pathway through tyrosine phosphorylation of Lats1[END_REF]) [START_REF] Elbediwy | Integrin signalling regulates YAP and TAZ to control skin homeostasis[END_REF].

Although Integrin and Src homologs are present in Drosophila, Yki protein does not respond to integrin stimulation, while in mammals integrin signaling promotes YAP/TAZ activity. One possible explanation for this different behavior may be that the N-terminus of Yki is missing a domain necessary to bind PDZ-containing proteins, which is found in its human counterpart YAP and is necessary for the activation of the integrin-Src adhesion branch of the pathway [START_REF] Elbediwy | Evolution of mechanotransduction via YAP/TAZ in animal epithelia[END_REF].

However, [START_REF] Elbediwy | Evolution of mechanotransduction via YAP/TAZ in animal epithelia[END_REF] also proposed another exciting explanation for this difference. They draw their conclusions based on a comparative analysis of the Yki protein and the evolution of the different epithelia: In mammals, the epidermis lacks the apical domain so it must sense the basal integrin to regulate the cell shape/growth (Refer to the diagram). While in the case of Drosophila, the epithelial cells are columnar and they can sense the apical signals, this makes the intigrins unnecessary for the activation of the Hippo pathway (Figure 31). . The isoform with a single WW domain is called often just referred to as YAP1, and the isoform with two WW domains is referred to as YAP2. These two isoforms are consequences of Alternative Splicing. In total, there are eight isoforms of YAP which are named as YAP1--Alternatively, in a letter also suggested the nomenclature based on the number of amino acids by directly mentioning the number of amino acids next to YAP 1 or YAP2 in the format YAP1-XXX (XXX = number of aa) (Figure 32) YAP was initially identified in 1994 by [START_REF] Fang | A transmembrane protein-tyrosine phosphatase contains spectrin-like repeats in its extracellular domain[END_REF] as a mammalian homolog of Yki. It was YAP's accumulation in the cytoplasm as a complex with 14-3-3 protein led to the assumption that even Yki might interact with 14-3-3. The WW domain has two highly conserved tryptophans separated by 20-23 amino acids [START_REF] Salah | WW domain-containing proteins: Retrospectives and the future[END_REF], which mediated the protein-protein interaction at N terminus. The WW domain recognizes a PPxY motif (proline/proline/any amino acid/tyrosine) that is found in a variety of proteins, many of which control TAZ/YAP localization and activity (Figure 33).

The YAP proline-rich domain facilitates the interaction with RNA and DNA-binding protein HNRNPU in the nucleus [START_REF] Howell | Heterogeneous nuclear ribonuclear protein U associates with YAP and regulates its co-activation of bax transcription[END_REF]. The YAP SH3-binding motif (PKQPPPLAP) also facilitates interactions with other kinases and adaptor proteins such as SRC and NCK1/2, respectively [START_REF] Sudol | Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product[END_REF]. Depending upon the Size of the isoforms, YAP could range from 450aa to 508aa. YAP also contains a C-terminal PDZ-binding motif, which mediates interactions with PDZ domains. PDZ domains are 80-90 amino acid proteininteraction domains that are found in several proteins, many of which are transmembrane or cytoskeleton-associated [START_REF] Ye | Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures[END_REF]. YAP has been shown to interact with promotors and distal enhancers in order to promote gene regulation via modulation chromatin looping and Pol II pausing release [START_REF] Lian | The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation[END_REF]) [START_REF] Galli | YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers[END_REF](C. [START_REF] Stein | YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers[END_REF]) (Zanconato, Cordenonsi, & Piccolo, 2016b).

In addition, TAZ is 395aa (50KDa) protein that is one and only paralog of YAP. It shares close to 50% sequence identity with YAP. The WWTR1 gene encodes the TAZ protein, and it contains a single WW domain along with a transactivation domain and a PDZ binding motif in its C terminus and a coiled-coil region. TAZ actually resembles the YAP1 as it contains only one WW domain, This is the reason why YAP/TAZ often have similar function such as both of these coactivators interact with RUNT family protein RUNX2 (J. H. [START_REF] Hong | TAZ, a transcriptional modulator of mesenchymal stem cell differentiation[END_REF] and with well studied TEAD family proteins (TEAD1-4) [START_REF] Vassilev | TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm[END_REF](W. M. [START_REF] Mahoney | The transcriptional coactivator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members[END_REF].

However, there are slight differences in the YAP/TAZ such as YAP can interact with the SH3 domain of YES protein and has a proline-rich region while TAZ lacks this feature [START_REF] Kanai | TAZ: A novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins[END_REF]. TAZ binds SMAD transcriptional modulators via a C-terminal coiled-coil domain, thereby facilitating TGF signaling by promoting SMAD nuclear accumulation [START_REF] Varelas | TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal[END_REF] while YAP interacts with inhibitory Smad7 [START_REF] Ferrigno | Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-Oncogene[END_REF] and BMPregulated Smad1 [START_REF] Alarcón | Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-Cell[END_REF]. YAP but not TAZ was shown to interact with p73 to induce pro-apoptotic transcriptional program. Similarly, TAZ but not YAP binds to Pax3, which is involved in the embryo limb formation (Masao [START_REF] Murakami | Transcriptional activity of Pax3 is co-activated by TAZ[END_REF].

Structurally, Yki (Drosophila homolog of YAP) is closely related to mammalian YAP [START_REF] Sudol | Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product[END_REF] (Figure 33). The Yki peptide shares 31% homology with YAP. Similar to YAP, Yki consist of WW domain that allows it to interact with the PPxY (Where P stands for Proline, X is any amino acid and T is Tyrosine) motifs of the partners. Notably, the corresponding SH3 binding region is absent in the Drosophila Yki protein.

Although initially restricted to Sd the number of partners of Yki are increasingly growing in numbers, recently [START_REF] Bohère | Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis[END_REF] showed that transcription factor Shavenbaby (Svb)

interacts with Yki to activate the expression of Diap1. Thus, the various protein binding domains present in Yki/YAP/TAZ provide a mechanism for the regulation of their localization and activity. Proline Rich domain: The N terminus of YAP1/2 consists of a proline-rich domain which facilitates the interaction with HNRNPU which is the only known partner to utilize this domain for interaction. TAZ lacks this domain TEAD Binding domain: This domain is conserved in both YAP and TAZ. YAP/TAZ bind TEAD (TEA domain) transcription factors via TEAD factor-binding domain located at N-terminal region (at amino-terminal region located between amino acid 47-154). Although TEAD4 requires similar TEAD C-terminal sites to interact but it requires different residues on YAP and TAZ.

WW Domains: The WW domain facilitates protein-protein interactions via recognition of proline-rich peptide motifs (PRM) and phosphorylated serine/threonine-proline sites. Aptly named, WW domain consists of 35-40 amino acid residues, including two highly conserved tryptophan (W) residues separated by 20-23 amino acids in the polypeptide chain. WW domain allows YAP/TAZ to bind to PPxY motifs, where P is proline, x is any amino acid and Y is tyrosine.

SH3 domain: YAP was originally identified via a screen for proteins that bind the SH3 (Src homology domain 3) domain of the non-receptor tyrosine kinase YES1. The YAP SH3-binding motif (PKQPPPLAP) facilitates interactions with other kinases and adaptor proteins such as SRC and NCK1/2, respectively. This domain is not present in TAZ.

Coiled-coil domain: This domain is present in C-terminal of both YAP and TAZ but not partners of YAP binding via this domain is known yet. TAZ utilizes this domain in heterodimer formation with TEAD. TAZ binds SMAD transcriptional modulators too via this domain to facilitate TGF-14-3-3-binding domain: TAZ was identified in a screen for 14-3-3 interactors. YAP and TAZ interact with 14-3-3 when they are phosphorylated at Ser127 and Ser 89 respectively. Interaction with 14-3-3 sequester YAP/TAZ in cytoplasm.

Transcriptional activation domain: The YAP/TAZ C-terminal region is rich in serine, threonine and acidic amino acids. This domain has strong intrinsic transcription stimulation activity.

PDZ-binding domain: Both YAP and TAZ consist of a small PDZ (Postsynaptic density 95/Disc large/Zonula occludens-1) at extreme C terminal region. PDZ binding domain is important for subcellular localization of YAP and TAZ.

Hippo pathway in diseases:

Hippo pathway has vital roles to play in both organism development and progression of the disease. As we have already seen, Hippo signaling determines organ size, regulates cell number via proliferation and apoptosis, plays a role in embryogenesis, cell fate specification after mitosis [START_REF] Jukam | Opposite feedbacks in the Hippo pathway for growth control and neural fate[END_REF], tissue-specific regeneration of progenitor cells [START_REF] Wang | The Hippo pathway in tissue homeostasis and regeneration[END_REF] mechanotransduction (Dupont, Enzo, et al., 2011) Along with cancer, perturbed hippo pathway is implicated in several non-cancer disease processes. I will, however, give a short introduction to this topic. For more extensive knowledge one can refer to the following reviews by (Plouffe, Hong, & Guan, 2015) (Zanconato et al., 2016b) (Xiaodong Zhang et al., 2018) (Figure 35).

Implications of the Hippo pathway in cancer:

Due to its complexity and range of upstream signals, the Hippo pathway could be dysregulated by a range of mechanisms. The core of the pathway plays a tumor suppressor role and the effector of the pathway, i.e., transcription coactivator is responsible for the oncogenic events. Mechanisms such as deletion or mutation of an upstream signal such as GPCR's, aberrant YAP/TAZ amplification or even by a cross-talk with any of the known (such as Wnt) or unknown (yet to be linked) signaling pathways could lead to dysregulation of growth, proliferation, and inhibition of apoptosis. Normal functioning cells depend upon aerobic oxidation of glucose as their primary source of energy, but cancerous cells switch to anaerobic glycolysis to grow and survive, even in the presence of sufficient oxygen, which is known as the "Warburg effect" and is a prominent hallmark of cancer. Reviewed in [START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF]. Indeed, [START_REF] Enzo | Aerobic glycolysis tunes YAP/TAZ transcriptional activity[END_REF] showed that a critical enzyme of glycolysis, phosphofructokinase 1 (PFK1) interacts with transcription factor TEAD1 so as to regulate the activity of YAP/TAZ providing new insight into how YAP/TAZ pro-oncogenic activity be controlled by glycolysis. Many such mechanisms could lead to the onset of disease and cancer of which several are identified to this date. A few of these are described below: the EHE cases, the oncogenic mechanism of this fusion is yet to be elucidated, but it is a strong indication that dysregulated YAP/TAZ fusion proteins may act as cancer drivers [START_REF] Tanas | Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma[END_REF][START_REF] Flucke | Epithelioid Hemangioendothelioma: Clinicopathologic, immunhistochemical, and molecular genetic analysis of 39 cases[END_REF].

YAP/TAZ activity has been correlated with an increased danger of metastasis and reduced survival across all human breast cancer subtypes [START_REF] Cordenonsi | The hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells[END_REF]. Studies done on breast cancer tissues have suggested that LATS1/2 mRNA is often poorly expressed and eventually leads to increased tumor size followed by lymph node metastasis [START_REF] Visser | LATS tumor suppressor: A new governor of cellular homeostasis[END_REF]) TAZ has been observed to be highly expressed in invasive breast cancer cell lines and primary breast cancers. It has been demonstrated that TAZ overexpression is sufficient to induce cell proliferation, transformation, and epithelial-mesenchymal transition in breast cancer cell lines [START_REF] Lei | TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway[END_REF]) [START_REF] Siew | A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells[END_REF]. YAP, however, is shown to impart metastatic abilities to benign tumors in mammary tumor cell lines in the mouse as shown by (H. [START_REF] Liu | The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain[END_REF]. On the other hand, (Q. Chen et al., 2014) demonstrated that hyperactivating YAP alone leads to differentiation of secretory cells during lactation, but it cannot induce mammary tumorigenesis in vivo. They speculated that additional mutations would be required for ultimate tumor-inducing capabilities of YAP. So more investigative studies are required to decide whether these clashing observations are due to cell type-specific differences.

Lung cancer, accounting for 12.3% of all tumor prevailing in humans is by far the most prevalent form of cancers (as of 2018), and it also ranks up in cancer with meager survival rate. WHO has categorized lung cancer in two categories: non-small cell lung cancer (NSCLC) (accounting for 85%) and Small-cell lung cancer (NSCLC) (accounting for 15%) [START_REF] Oser | Transformation from nonsmall-cell lung cancer to small-cell lung cancer: Molecular drivers and cells of origin[END_REF]. Lung adenocarcinoma (LAC), Lung squamous cell carcinoma (LSCC), and Large-cell carcinoma, all belong to the NSCLCs [START_REF] Yokota | Molecular footprints of human lung cancer progression[END_REF]. At the start of this decade, [START_REF] Li | Structural insights into the YAP and TEAD complex[END_REF])and (Z. [START_REF] Zhou | TAZ is a novel oncogene in non-small cell lung cancer[END_REF] showed that YAP/TAZ are highly expressed in non-small cell lung cancer (NSCLC), and either of their knockdown in NSCLC cells is adequate to suppress proliferation, tumor growth and metastasis in the mouse model. It has also been demonstrated that hyperactivating YAP is sufficient to drive lung cancer progression in vivo [START_REF] Lau | Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis[END_REF]. Similar to studies in breast cancer, (Wenjing Zhang et al., 2015) described that YAP/TAZ activation alone might not be sufficient to drive NSCLC genesis, but they showed that ectopic expression of YAP/TAZ could transform small adenomas to high-grade LAC in the mouse model.

Malignant pleural mesothelioma (MPM) cancer of the mesothelium is one of the rarest cancers, but it is an aggressive one which does not respond to current therapy thus survival rate is lower in patients detected with this form of cancer. It is cancer associated with the pleura, the lining which covers many of the body's internal organs. This cancer is associated with exposure to asbestos, and recently identified germline mutation is also said to predispose patients to MPM [START_REF] Takahashi | The Global Health Dimensions of Asbestos and Asbestos-Related Diseases[END_REF]) [START_REF] Cheung | Further evidence for germline BAP1 mutations predisposing to melanoma and malignant mesothelioma[END_REF]. Homozygous deletion or inactivating mutations in NF2, SAV1, or LATS2 are often observed in human malignant mesothelioma tissues and cell lines (H. [START_REF] Murakami | LATS2 is a tumor suppressor gene of malignant mesothelioma[END_REF]. [START_REF] Sekido | Neurofibromatosis Type 2 (NF2) Gene Is Somatically Mutated in Mesothelioma but not in Lung Cancer[END_REF] have

shown that nearly 50% of all mesothelioma tumors have inactivation mutation in NF2 gene.

Further, Ajuba LIM protein (AJUBA) can inactivate YAP through signaling via LATS kinase, and down-regulation of AJUBA led to induction of malignant mesothelioma in MM line [START_REF] Tanaka | LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade[END_REF]. The downregulation of AJUBA subsequently activates YAP which regulates the transcription of cell cycle promoting gene CCND1(G1/S-Specific Cyclin-D1) and FOXM1 (Forkhead Box M1; a transcription factor targeting both G1/S and G2/M progression regulators) to induce malignant mesothelioma in MM cell lines [START_REF] Mizuno | YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cyclepromoting genes[END_REF]. Thus, the Hippo pathway has been strongly implicated in cancers of mesothelioma, and further research could provide a way to be able to develop a therapy to which this type of cancer can respond to.

Pancreatic ductal adenocarcinoma (PDAC) is classified as a very aggressive form of cancer that is often detected only in a very advanced stage as the patient does not experience any noticeable symptoms until it is too late. PDAC arises from the progression of pancreatic intraepithelial neoplasias (PanINs) which are precursor lesions in fully grown metastatic cancer [START_REF] Hruban | Progression model for pancreatic cancer[END_REF]. Mutations in KRAS genes are know to be one of the initiating factors for PDACs as studies have shown that KRAS mutation is present in 95% of PDACs [START_REF] Biankin | Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes[END_REF]. Biopsies from patients with PDAC revealed an increased YAP level and higher nuclear localization. Moreover, YAP knockdown in PDAC cells resulted in reduced proliferation and reduced anchorage-independent growth, suggesting YAPs role in PDAC (Weiying Zhang et al., 2014).

Kaposi sarcoma (KS) is cancer that appears on the outer skin, lymph node or some other organs as cell masses. It is caused by the infection of Kaposi sarcoma-associated herpesvirus (KSHV) also called as HHV-8 in immunodeficient patients, such as patients with AIDS. (G. [START_REF] Liu | Kaposi sarcoma-associated herpesvirus promotes tumorigenesis by modulating the Hippo pathway[END_REF] observed elevated levels of YAP/TAZ as KSHV encodes a vGPCR to inhibit LATS1/2 and subsequently suppress the Hippo pathway. Although the implementation of antiretroviral therapy has led to increased patient survival but still this form of cancer remains to be a killer, and YAP/TAZ can be a future target for therapies [START_REF] Ledergerber | AIDS-related opportunistic illnesses occurring after initiation of potent antiretroviral therapy: The Swiss HIV cohort study[END_REF].

Colorectal carcinoma (CRC) is a cancer of the digestive system. Although CRC responds to the therapy, this cancer is still a morbid one due to the frequency of reoccurrence; thus long term therapies needs to improve and develop. YAP in often found overexpressed and YAP/TAZ activity is associated with poor patient outcome (F.-X. [START_REF] Yu | Hippo Pathway Regulation of Gastrointestinal Tissues[END_REF]. Hypermethylation of LATS1 has been shown as a cause of CRC which can explain the elevated YAP activity [START_REF] Wierzbicki | Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation[END_REF].

YAP has also been implicated in renal cell carcinoma (RCC) which is one of the most common cancers of the urinary tract. This cancer is distinct from other cancers as it is highly vascularized and resistant to conventional chemo or radiotherapy. Elevated YAP levels are associated with RCC too. Recently it was shown that LATS1 promoter is hypermethylated in RCC and therefore reducing the LATS and activating YAP (K. H. Chen et al., 2014) [START_REF] Rybarczyk | Overexpression of the YAP1 oncogene in clear cell renal cell carcinoma is associated with poor outcome[END_REF]. (J. J. [START_REF] Cao | YAP is overexpressed in clear cell renal cell carcinoma and its knockdown reduces cell proliferation and induces cell cycle arrest and apoptosis[END_REF] supported the role of YAP in RCC by

showing that knockdown of YAP in ccRCC (clear cell Renal Cell Carcinoma) cell lines inhibits proliferation and initiate apoptosis.

Most of Gastric cancer (GCs) are gastric adenocarcinomas (GAC) that derive from the glandular epithelium of the stomach. It is the fifth most common cancer and the third leading cause of deaths worldwide due to cancer [START_REF] Garattini | Molecular classifications of gastric cancers: Novel insights and possible future applications[END_REF]. The dysregulation of Hippo signaling is associated with GAC from its initiation to metastasis [START_REF] Ramos | The Hippo signaling pathway and stem cell biology[END_REF].

Elevated YAP activity and nuclear localization of observed in all kinds of gastric cancers (X.

Hu, Xin, [START_REF] Hu | Overexpression of YAP1 is Correlated with Progression, Metastasis and Poor Prognosis in Patients with Gastric Carcinoma[END_REF]. TAZ too is highly expressed and nuclear-localized in human GAC lines further supporting the YAP/TAZ association in GACs (G. [START_REF] Yue | TAZ is highly expressed in gastric signet ring cell carcinoma[END_REF] Apart from the above-described cancers overexpression of TAZ is observed in high-grade brain tumors, i.e., Glioblastoma multiforme (T. [START_REF] Tian | TAZ promotes temozolomide resistance by upregulating MCL-1 in human glioma cells[END_REF]. Immuno histo-chemistry studies on patients samples revealed high levels of YAP in both low and high grades of gliomas. This associate with shorter survival of patients with gliomas [START_REF] Orr | Yes-associated protein 1 is widely expressed in human brain tumors and promotes glioblastoma growth[END_REF].

Several studies have already described how mutations in RTK-PI3K-PTEN and ARF-MDM2-p53 INK4a-RB pathways contribute to the strengthening of the oncogenic capacity of advanced gliomas [START_REF] Zheng | p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation[END_REF]. Recently [START_REF] Rivas | WIP-YAP/TAZ as a new pro-oncogenic pathway in glioma[END_REF] have shown an actin cytoskeleton-associated protein WIP (WASP-interacting protein) as a driver of cancer progression via stabilizing YAP/TAZ.

Ovarian cancer ranks third in the list of most common malignancies in the female reproductive system [START_REF] Tworoger | Ovarian cancer[END_REF]. 5-8% of all ovarian tumors are a result of Granulosa cell tumors (GST). In a study published in 2014, (D. [START_REF] Fu | YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors[END_REF] reported that GST's show significantly higher levels of YAP when compared to healthy ovarian tissue. Although at this point the mechanism by which YAP regulates the proliferation of granulosa cell tumor is unknown.

So far we have seen that YAP/TAZ acts as oncogenes in the majority of cancers. It is worth mentioning that the Hippo Pathway plays a crucial role in lymphocyte apoptosis and YAP plays the role of tumor suppressor in hematological cancer such as multiple myeloma, lymphoma, and leukemia. It has emerged that YAP/TAZ plays a context-dependent role in hematopoietic cancers. It has been observed that YAP if often deleted or down-regulated in patients samples suffering from early to advanced stages of MM [START_REF] Cottini | Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers[END_REF]. On the one hand, YAP acts as a tumor suppressor by regulating DNA damage response via interacting with ABL1 to induce p53-independent apoptosis while on the other hand, (T. S. [START_REF] Kim | Mammalian sterile 20-like kinase 1 suppresses lymphoma development by promoting faithful chromosome segregation[END_REF] and [START_REF] Hartmann | Pathway discovery in mantle cell lymphoma by integrated analysis of high-resolution gene expression and copy number profiling[END_REF] have reported the loss of upstream components of Hippo in leukemia. Recently, TEADs too have been shown to activate the transcription of the oncogenic program by reprogramming enhancers to drive B cell transformation in leukemia (Y. [START_REF] Hu | Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia[END_REF]. These reports have now cautioned scientist to look at YAP/TAZ in a context-dependent manner and consider a possibility of a tumor suppressor role as the current paradigm is that YAP/TAZ are pro-oncogenes in all cancer-related malignancies.

Hippo pathway in non-cancer diseases:

Given the broad range implications of the Hippo pathway, it is not surprising to find its role in diseases beyond cancers too. Thus it is essential for current researchers to study the various mechanistic involvements in such diseases in order to open the possibility of treating such diseases via therapy based on the input signals or the components of this pathway.

Patients suffering from a rare genetic disorder Neurofibromatosis 2 (NF2) are predisposed to an additional risk of developing cataracts, and retinal abnormalities and about half of them proceed to develop them [START_REF] Parry | Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities[END_REF] [START_REF] Parry | Neurofibromatosis 2 (NF2): Clinical characteristics of 63 affected individuals and clinical evidence for heterogeneity[END_REF]) [START_REF] Parry | Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities[END_REF]. YAP is required for normal ocular development (J. Y. [START_REF] Kim | Yap is essential for retinal progenitor cell cycle progression and RPE cell fate acquisition in the developing mouse eye[END_REF]. Association of Hippo pathway has been made in ocular abnormalities such as ocular colobomas optic fissure closure defect [START_REF] Oatts | Novel heterozygous mutation in YAP1 in a family with isolated ocular colobomas[END_REF] [START_REF] Williamson | Heterozygous loss-of-function mutations in YAP1 cause both isolated and syndromic optic fissure closure defects[END_REF]) , Sveinsson's chorioretinal atrophy [START_REF] Kitagawa | A Sveinsson's chorioretinal atrophy-associated missense mutation in mouse Tead1 affects its interaction with the co-factors YAP and TAZ[END_REF] [START_REF] Fossdal | A novel TEAD1 mutation is the causative allele in Sveinsson's chorioretinal atrophy (helicoid peripapillary chorioretinal degeneration)[END_REF] and retinal degeneration [START_REF] Hamon | Retinal degeneration triggers the activation of YAP/TEAD in reactive Müller cells[END_REF].

Sveinsson's chorioretinal atrophy (SCRA) is an autosomal dominant disease linked to the eye that is described to be caused by the mutation in TEAD1. It is a rare condition characterized by choroid and retinal degeneration in humans. The study done [START_REF] Fossdal | A novel TEAD1 mutation is the causative allele in Sveinsson's chorioretinal atrophy (helicoid peripapillary chorioretinal degeneration)[END_REF]) by using genome-wide linkage analysis describes a missense mutation in the TEAD-1 gene that results in the substitution of tyrosine with a histidine (Tyr421) in the C-terminal of TEAD1 protein in Icelandic patients. This mutation limits the TEAD-YAP complex formation which results in SCRA.

In a study from 2013, [START_REF] Enger | The hippo signaling pathway is required for salivary gland development and its dysregulation is associated with sjogren's syndrome[END_REF] have shown evidence that Hippo pathway is implicated in Sjogren syndrome (SS), a chronic autoimmune disease which causes hyposalivation and ocular dryness in salivary and lacrimal glands respectively. In a study using non-obese diabetic (NOD) mice (a mouse model for SS), they showed that salivary glands of NOD mice phenocopies that salivary glands with LATS 2 inhibition. The IHC in salivary glands of human Sjogren patients exhibits nuclear staining of TAZ along with upregulation of transcriptional target of TAZ.

Hippo pathway is also associated with cardiac disease. Studies on the sample from patients with hypertrophic cardiomyopathy and transverse aortic constriction (TAC) in mice, have

shown elevated levels of YAP and decreased phosphorylation of Ser127 suggesting that nuclear YAP is implicated in the hypertrophic heart (P. Wang et al., 2014). In the same year (Z. [START_REF] Lin | Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model[END_REF] showed elevated YAP levels and phosphorylated LATS in the heart samples from patients with ischaemic or nonischemic heart failure suggesting increased hippo activity.

According to [START_REF] Basson | Mutations in human cause limb and cardiac malformation in Holt-Oram syndrome[END_REF] by [START_REF] Murakami | A WW domain protein TAZ is a critical coactivator for TBX5, a transcription factor implicated in Holt-Oram syndrome[END_REF] that mutation in TBX5 prevents it from binding to TAZ which subsequently leads to Holt-Oram syndrome.

Apart from the above described mammalian pathologies, components of the Hippo pathway are also involved in hepatomegaly, neurogenic muscle atrophy, Amyotrophic lateral sclerosis (ALS), Immunodeficiency, etc. however, a lot of these studies are only done on mice model, and thus it is always to kept in mind that animal model such as mice tends to develop different range of disease subtypes. Thus in the coming years, it is necessary to study the association of these pathway components in a wide range of pathologies in humans.

Hippo pathway as a therapeutic target

What we have seen so far is that in several of pathologies, deregulation of the Hippo signaling pathway occurs and eventually elevated YAP/TAZ activity induces expansion of tissuespecific stem/progenitor cells. This often leads to the development of tumors, and thus for future cancer therapy, it is essential to shed light on the physiological role of the Hippo signaling pathway and more specifically YAP/TAZ in tissue homeostasis and cancer stem cells.

We know that the association between cancer and Hippo pathway is very tissue or cell typespecific thanks to studies which demonstrated that skin-specific deletion of MST1/2 or LATS1/2 leads to no abnormalities in the epidermis or MST1/2 or SAV1 deletion does not lead to elevated YAP phosphorylation of Kidney abnormalities (Schlegelmilch et al., 2011) [START_REF] Reginensi | Yap-and Cdc42-Dependent Nephrogenesis and Morphogenesis during Mouse Kidney Development[END_REF].On the other hand, we have seen so far how dysregulation of YAP is observed in numerous forms of tumors and diseases. However, if we look the data presented in [START_REF] Harvey | The Hippo pathway and human cancer[END_REF] from the COSMIC (Catalogue of somatic mutations in cancer)

database, it shows that so far only NF2 and TAZ are classified at cancer genes out of all the genes in the pathway. Indeed, they postulate that this information might change in the future as the volume of the genomic data increases due to the global cancer-sequencing efforts.

According to most current studies, YAP, LAST1/2, GPCRs, NF2, and TAZ are related to the development of cancer. One possible explanation to the rarity of mutations in genes of Hippo pathway is so far partially due to the fact that YAP/TAZ is often affected by other pathways as we have already seen such as Wnt signaling, TGF--BMP, NOTCH, EFR, and GPCR, etc.

Thus it is critical to investigate and classify all the underlying causes of YAP/TAZ upregulation in tumors and paint a much larger picture with the mutations in Hippo genes and crosstalk with aberrant signaling pathways that can lead to tumor formation. Hereby, I will discuss the status of targeting hippo pathway components against cancer and the strategies of the therapy.

YAP/TAZ activators in Regenerative medicine:

Elevated YAP/TAZ incites cell proliferation in tumors, and it is obvious to target inhibition of YAP/TAZ to control the tumor. However, the cell division and proliferation also corresponds to the growth of animal tissues, and it is required for tissues to be maintained and repaired.

Thus, it is not surprising to see why scientists emphasize on developing YAP/TAZ activators in the field of regenerative medicine. Now there are ample amount on studies which shows that YAP1 activators could be potentially used to promote tissue repairs after injuries. For example: (Q. [START_REF] Zhou | The hippo pathway in heart development, regeneration, and diseases[END_REF] demonstrate that inhibition of the Hippo pathway improves cardiac function after infarction in adult mice. There are also reports of YAP1 activation after hepatectomy, and its deletion leads to necrosis. Deletion of YAP1 also leads to impairment of intestinal regeneration and wound healing. TAZ is also shown to be activated right after muscle injuries [START_REF] Hwang | TAZ as a novel enhancer of MyoD-mediated myogenic differentiation[END_REF]. Using the strategy of TAZ activation, [START_REF] Hata | Screening with a Novel Cell-Based Assay for TAZ Activators Identifies a Compound That Enhances Myogenesis in C2C12 Cells and Facilitates Muscle Repair in a Muscle Injury Model[END_REF] As seen in the plethora of studies, YAP/TAZ activity is high in several cancers. This is a reason why many studies are aiming to target the kinases of the Hippo signaling as it is often seen that MST1/2 and LATS1/2 activities are lowered in many cancers. Therefore enhancing kinase activity is an attractive therapeutic target for many involved in this research.

It has been shown that YAP /TAZ confer some degree of chemotherapy resistance to cells.

We know that resistance to RAF and MEK inhibitor therapy is a major challenge in patients undergoing chemotherapy. Recently, (L. [START_REF] Lin | The Hippo effector YAP promotes resistance to RAF-and MEK-targeted cancer therapies[END_REF] identified YAP as a culprit that promotes the resistance to REF and MEK inhibitors. Thus they have opened a possibility of combined suppression of YAP and RAF or MEK as an improved therapy for enhanced treatment response and survival of patients. [START_REF] Liu-Chittenden | Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP[END_REF] first promoted the idea of targeting YAP-TEAD interactions using verteporfin (Visudyne) after screening approximately 3000 FDA approved drugs in a screen to look for small molecules that can inhibit YAP/TEAD complex. Verteporfin (VP) is a heme analog approved for clinical use as a photosensitizer in patients with neurovascular macular degeneration. In murine models of hepatocellular carcinoma with YAPoverexpression and in Yki-overexpressing Drosophila S2 cells, VP was shown to inhibit YAP-Tead2/Yorkie-Sd complex formation through selective binding to YAP/Yorkie.

-, liver growth was substantially decreased, and mRNA expression of diap1 was significantly reduced in S2 cells with 200nM VP treatment. Work by [START_REF] Jiao | A Peptide Mimicking VGLL4 Function Acts as a YAP Antagonist Therapy against Gastric Cancer[END_REF] and (Wenjing Zhang et al., 2014) has also suggested that VGLL4, a transcription co-factor protein, can act as an inhibitor of YAP activity by competing with YAP for TEAD binding. These studies show VGLL4 expression in-vitro and in-vivo decreased tumor formation, growth, and cell proliferation in both gastric and lung cancers. Additionally, [START_REF] Wei | Verteporfin suppresses cell survival, angiogenesis and vasculogenic mimicry of pancreatic ductal adenocarcinoma via disrupting the YAP-TEAD complex[END_REF] have shown that verteporfin can suppress YAP activity in PDAC cells via inhibiting YAP and TEAD interaction and opened a possibility of using it as an anti-tumor drug in the treatment of pancreatic cancer (Figure36).

Also , Several studies have indicated that studies have shown that knockdown of YAP or TAZ genetically by RNA interference (RNAi) in various cancer cells could improve immune response to a wide variety of chemotherapeutic drugs such as, cisplatin, anti-tubulin drugs,EGFR inhibitors ,c-Abl inhibitor, RAF and MEK inhibitors [START_REF] Lee | Hippo pathway effector YAP inhibition restores the sensitivity of EGFR-TKI in lung adenocarcinoma having primary or acquired EGFR-TKI resistance[END_REF][START_REF] Lin | The Hippo effector YAP promotes resistance to RAF-and MEK-targeted cancer therapies[END_REF][START_REF] Lai | Taxol resistance in breast cancer cells is mediated by the hippo pathway component TAZ and its downstream transcriptional targets Cyr61 and CTGF[END_REF]) (Yulei Zhao & Yang, 2015) [START_REF] Nishio | Dysregulated YAP1/TAZ and TGF-deficient mice[END_REF] found that ivermectin, an antiparasitic drug, and its derivative, milbemycin D, inhibits YAP1/TAZ activity and suppress tumor growth in MOB1-deficient mice. Further, researchers wish to find compounds that can allow them to play with the subcellular localization of YAP/TAZ since the phosphorylated YAP/TAZ and translocated to the nucleus, and unphosphorylated YAP/TAZ are degraded in cytoplasm. In 2011, [START_REF] Bao | A cell-based assay to screen stimulators of the Hippo pathway reveals the inhibitory effect of dobutamine on the YAP-dependent gene transcription[END_REF] described that dobutamine, a class of drug used in patients who recently suffered from a cardiogenic shock can reabsorb YAP from the nucleus to cytoplasm. This opens a perspective of investigating and identifying the mechanism of inhibition of YAP-dependent gene transcription by dobutamine.

Similarly, in 2014 [START_REF] Sorrentino | Metabolic control of YAP and TAZ by the mevalonate pathway[END_REF] and (Z. Wang et al., 2014) described how statin (a class of drug to treat hypercholesterolemia) indirectly inhibit YAP and TAZ by reducing the downstream synthesis of geranylgeranyl pyrophosphate, which is required for Rho GTPases to inhibit LATS kinase activity. They have also shown that statin treatment of breast cancer cells resulted in lower CSC properties such as self-renewal and inhibited in vitro and in vivo growth.

Studies performed by [START_REF] Moroishi | The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity[END_REF] have shown that deletion of LATS1/2 enhances the antitumor immune response. Via High throughput screen [START_REF] Fan | Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration[END_REF] have identified a compound named XMU-MP-1 that can inhibit kinase MST1/2. They have demonstrated that this molecule can ameliorate chronic liver injury by enhancing tissue repair and regeneration.

However very recently, [START_REF] Helena | The hippo pathway component taz promotes immune evasion in human cancer through PD-L1[END_REF]have published results in contrast with [START_REF] Moroishi | The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity[END_REF] and affirmed that TAZ promotes human cancer evasion via inducing aberrant expression of PD-L1 on cancer cells which subsequently evade the antitumor immune response. The authors speculated that this conflict might arise because Hippo pathway gene regulation may differ from species to species. therapeutic heart regeneration. However, prolonged inhibition of kinase could also prove to be harmful to the immune system as increased YAP/TAZ activity result in abnormalities in various human organs. Thus, it is a must to be extremely cautious while manipulating Hippo pathway for the therapeutic benefit so that the drug does not significantly alter vital organs/tissues or pools of progenitors and any undesirable side effects are avoided.

However, right now the future for the therapies does not look so bleak as this is the exciting time when the Hippo field is expanding tremendously with every passing year. Further knowledge on the signals that regulate the pathway will prove to be beneficial for the development of therapies based on modulation of YAP/TAZ and prevention of cancer and other non-cancer diseases. RNA binding proteins of the SR family are conserved proteins that play a major role in AS regulation. Drosophila B52 is an essential protein that belongs to this family of splicing factors. Our previous work identified a positive link between B52 and cell growth 2 but the underlying molecular mechanisms were not characterized. We reasoned that B52's effect on growth might be due to AS modulation of one or several genes controlling growth. To identify AS events that are robustly affected by B52 depletion, we crossed two previously published RNAseq datasets corresponding to RNAi-mediated depletion of B52 in S2 cells compared to control cells 3,4 . We used MAJIQ (Modeling Alternative Junction Inclusion Quantification) 5 to identify Local Splicing Variations (LSV) that vary more that 20% between control and B52 RNAi conditions, in both datasets (see Methods). This identified 119 AS events in 119 genes (Suppl. Fig. 1a and Suppl. Table 1). Strikingly, GO term enrichment analysis of these genes places regulation of growth at the top of the list (Suppl. Fig. 1b).

RESULTS

Article in preparation

Interestingly, three genes linked to Hippo pathway were identified: yorkie (yki), WW domain binding protein 2 (Wbp2) and Zyxin (Zyx). RNAseq data indicate that B52 depletion promotes skipping of exon 3 in yki mRNA, the inclusion of exons 6 and 7 in wbp2 and the retention of last intron in Zyx (Suppl. Fig. 1c). The functional consequences of these AS events are unknown.

Hippo signaling is an evolutionary conserved pathway that regulates cell fate and cell proliferation to control organ growth and regeneration 6 . The core of the Hippo pathway in Drosophila consists of two kinases, Hippo (Hpo) and Warts (Wts), and their adaptor proteins, which phosphorylate the transcriptional coactivator Yorkie (Yki) to sequester it in the cytoplasm through binding to 14.3.3 proteins. Upon inactivation of the pathway, unphosphorylated Yki translocates in the nucleus where it binds to transcription factors such as Scalloped (Sd) and recruits partners such as NcoA6 or Wbp2 to activate transcription of its target genes.

yki gene contains an alternative exon (exon 3) that encodes one of the two WW domains of the protein, thus producing two isoforms that we named Yki2 and Yki1 according to their number of WW domains (Fig. 1a). We confirmed by RT-PCR that RNAi-induced depletion of B52 in S2R+ cells or in wing discs induces skipping of yki exon 3 and increases expression of Yki1 at the expense of Yki2 isoform (Fig. 1b,c). To test whether B52 depletion affects Yki activity, we monitored expression of two reporter genes, ex-lacZ and diap-lacZ, which are direct targets of Yki. Expression of B52 RNAi in the in the posterior domain of the wing disc decreases expression of the two reporter genes in this domain reflecting a reduction of Yki activity (Fig. 1d). This goes along with a reduction of posterior domain size. These flies are poorly viable at 25° but are viable at 18° and show a net reduction of wing posterior domain size (Fig. 1e). Using this phenotype, we tested whether B52 interacts genetically with the Hippo pathway. Overexpression of Yki (UAS-Yki-V5, corresponding to long isoform 7 ), as well as depletion of the kinases Hpo or Wts by RNAi, partially rescue the growth defect induced by B52 depletion (Fig 1f). Therefore increasing Yki activity antagonizes the effect of B52 depletion. Nevertheless, posterior domain size in these contexts remain smaller than the corresponding controls (overexpression of Yki or depletion of Wts or Hpo in the absence of B52 depletion, Fig. 1e and Suppl. Fig. 2). This suggests that B52 depletion affects other genes or pathways necessary for efficient growth. Moreover, the strong overgrowth induced by Yki overexpression, or by Hpo or Wts depletion, may compensate a growth defect in B52 depleted cells, unrelated to Hippo pathway. This is unlikely because depletion of Tgi, a transcriptional repressor interacting with Sd, which induces a very mild increase of posterior domain size on its own, also rescues the growth defect due to B52 depletion (Fig. 1e).

similarly with the transcription factors Sd, MAD, Cbt and Hth, the chromatin-associated factors GAF and Mor, and with 14.3.3 protein (Fig. 3c). These results show that interactions with these partners are not influenced by the domains encoded by yki exon 3. On the other hand, we saw that interaction with Wts, Ex, NcoA6, Wbp2 and Tgi is reduced two to four times for Yki1 compared to Yki2 (Fig. 3c). All these proteins contain multiple PPxY motifs.

Interestingly, we did not detect significant difference between Yki isoforms for the binding to MAD and Hpo which both contain a single PPxY motif. This is in agreement with in vitro studies reporting cooperative binding between multiple PPxY motifs and tandem WW domains of YAP and Yki 17,18 . Altogether, these results suggest that Yki isoforms interact similarly with TF but that Yki1 cannot efficiently recruit co-activators like NcoA6 or Wbp2 to stimulate transcription.

In addition to increasing the binding of proteins with multiple PPxY motifs, the presence of two WW domains may also allow Yki2 to interact simultaneously with two PPxY-containing proteins. To test this hypothesis we compared the ability of Yki isoforms to bridge different proteins, by monitoring their co-IP using the DULIP assay in the presence or absence of Yki. Previous reports showed that Yki2 binds to Sd through its N-terminal part and to Tgi via the WW domains 14,15,19,20 . Moreover Sd and Tgi were shown to interact directly 14,15 . Yki was shown to compete with Tgi for Sd binding 14,15 , but could also be found in a trimeric complex with Sd and Tgi 14 . We, therefore, monitored Sd/Tgi interaction in absence or presence of Yki isoforms in transfected cells (Fig. 3d). Using Firefly-Sd as bait, we detected co-IP between Firefly-Sd and Renilla-Tgi in absence of Yki as previously reported. Both Yki1 and Yki2 isoforms enhanced the co-IP between Sd and Tgi, with Yki2 having a stronger effect. Point mutations in the two WW domains of Yki2 abolish this interaction. These results are in agreement with the existence of a trimeric complex between Sd-Tgi and one isoform of Yki.

To further test Yki bridging activity, we analyzed the co-IP between Firefly-Sd and Renilla-Wbp2 by the same approach. Co-transfection of Yki1 or Yki2 increases the pull-down between Sd and Wbp2, with Yki2 having a stronger effect. Mutation of both WW domains in Yki2 abrogates this effect (Fig. 3e). These results indicate that, in interaction with Sd, both Yki1 and Yki2 isoforms participate in the recruitment of Tgi or Wbp2, with Yki2 being more efficient owing to its two WW domains. Finally, we tested if the two WW domains of Yki2 can interact with two different proteins carrying PPxY motifs. To this end we analyzed the co-IP between Tgi and Wbp2 using Firefly-Tgi as bait and Renilla-Wbp2 as prey. Remarkably, Yki2 isoform allowed to pull-down Wbp2 with Tgi, whereas Yki1 isoform did not. As expected, mutation of the two WW domains in Yki2 abrogates the co-IP (Fig. 3f).

Taken together our results show that Yki isoforms interact similarly with TF but differ by their capacity to bind and bridge PPxY-containing proteins. We propose the following model (Fig. 4a). Upon repression of Hippo pathway, unphosphorylated Yki isoforms enter the nucleus. Yki2 being more abundant, joins the transcriptionally repressed Tgi-Sd complex.

Within this trimeric complex, Yki2 may engage only one WW domain thus being able to recruit through the second WW domain another partner such as Wbp2 or NcoA6, which finally may or not displace Tgi. This could reconcile the observation that Tgi enhances Sd-Yki interaction but decreases distance between Sd and Yki as measured by FRET 14 suggesting remodeling of the complex. In the B52 depleted situation, Yki1 isoform level is increased and this isoform substitute to Yki2. In a complex with Sd and Tgi, Yki1 would be unable to recruit additional partners and activate transcription.

One prediction of this model is that Yki1 should be able to compete with Yki2 for binding to transcription factors in vivo. To test this hypothesis we used activated forms of Yki1 and Yki2 carrying the mutation S168A that kills a Wts phosphorylation site and lowers Yki cytoplasmic retention by 14.3.3 proteins 21 . We verified that this mutation favors nuclear accumulation of both Yki1 S168A and Yki2 S168A isoforms (Suppl. Fig. 4) and increases the overgrowth phenotypes induced by overexpression of these isoforms in the eye (Suppl. Fig. 3). Of note activated Yki1 S168A induces dramatically weaker overgrowth compared to Yki2 S168A confirming its reduced transcriptional activity (Suppl. Fig. 3). Overexpression of Yki2 S168A in the eye induces strong over-growth and remarkably, co-overexpression of activated Yki1 S168A , but not non-activated form Yki1, reduces this phenotype, suggesting that Yki1 can compete with Yki2 in the nucleus (Fig. 4b). These results are in agreement with a competition model between a less-active isoform Yki1 and a fully active form Yki2. We propose that modification of the balance between the two isoforms, such as created by B52 depletion, participate in the regulation of Yki activity.

To evaluate if this balance has a developmental role, we abrogated AS of yki exon3 by creating flies producing exclusively Yki2 isoform. We edited endogenous yki locus to replace the central part of yki locus by a portion of yki2 cDNA, therefore eliminating the introns surrounding exon 3 (See strategy in Suppl. Fig. 5). This allele, denoted yki 2only , carries exon2 fused to 4 and do not contain exogenous sequence (Fig. 4c). We confirmed by western blotting that flies no more produce Yki1 isoform (Fig. 4d). These flies are viable and fertile.

Interestingly we noticed that several flies in the population display asymmetric wings (Fig. 4e). The intra-individual variation between the size of right and left wings, called Fluctuating Asymmetry (FA), reflects a developmental instability. We measured wild type and yki 2only mutant wing areas and quantified the fluctuating asymmetry index FA10 (ref) which takes into account measurement errors (Suppl. Fig. 6). yki 2only flies display higher FA in both males and females (Fig. 4f), reflecting increased developmental variability upon abrogation of Yki AS.

We propose that yki AS represents an additional layer of modulation of Yki activity that participates in buffering developmental noise. Recently it has been shown that, in addition to its intrinsic effect on tissue growth, Yki is involved in systemic growth by interacting with ecdysone signaling. Yki is involved in basal expression of ecdysone 22 , interacts with the ecdysone receptor coactivator Taiman 23 and controls the expression of dilp8 which is involved in inter-organ coordination of growth 24 . Significantly, genomic deletion of dilp8 Hippo Responsive Element is sufficient to increases FA in flies 24 indicating that Yki's control on dilp8 is required to minimize developmental variability. It will be important to determine to which extent AS of yki is dynamically regulated during normal growth and regeneration, and which signals are involved.

Our identification of B52 as a modulator of yki AS is a first step in the characterization of this regulation. Finally it is worth noting that alternative inclusion of the second WW domain is a conserved feature between Yki and it human homolog YAP. Moreover, YAP1

isoform containing a single WW domain is a weaker transcriptional activator that YAP2 25 .

Therefore modulation of YAP AS could be a new strategy to decrease YAP activity in cancer cells.

Methods

RNAseq analysis

We compare them between replicates. To make a selection of best candidate genes we used a lists for each dataset were compared to select common events between them. This identified 119 AS events in 119 genes that show reproducible change of AS upon B52 depletion. GO term analysis was performed with PANTHER.

Fly strains and Genetics

Drosophila were maintained on standard cornmeal-yeast medium. Experiments were performed at 25°C, except for the analysis of wing phenotype of flies expressing B52 RNAi under the control of HH-Gal4 that were performed at 18°C, as mentioned in figure legends.

Inducible RNAi lines used were UAS-IR-B52 (GD8690), UAS-IR-Wts (GD1563 and (kindly provided by N. Tapon). NcoA6 was amplified from a plasmid kindly provided by K.

Irvine.

For DUAL-luciferase co-IP, we developed three destination vectors for the Gateway system: pAct-Flag-Firefly-RfA, pAct-HA-Renilla-RfA, pAct-RfB-Renilla-HA. The luciferase-gateway cassettes from mammalian vectors pcDNA-Flag-Firefly-RfA, pcDNA5-HA-Renilla-RfA and pcDNA5-RfB-Renilla-HA (kindly provided by E. Bertrand) were cloned into pAFW backbone. cDNAs cloned into pENTRD/Topo were transferred to the appropriate destination vector by LR recombination (Invitrogen).

Cell culture and co-immunoprecipitations

Drosophila S2R+ cells were maintained in Schneider's medium (Invitrogen) supplemented with 10% fet (Invitrogen) at 27°C. For B52 depletion, S2R+ cells were treated with a mix of two dsRNA (produced by in vitro transcription) targeting exon 2 and exon 9 of B52, for 72H. RNAs were extracted with trizol (Sigma). Proteins were prepared in urea buffer. Antibodies used for western were: rabbit anti-Yki 26 , Rabbit anti-B52 27 , Mouse anti-actin (DSHB).

For dual-luciferase co-IP, S2R+ cells were transfected with two plasmids (0.15µg each) in quadruplicate in 24-wells plates (400000 cells/well) using effectene (Qiagen).

Typically, 4 plates were handle at the same time to perform 24 co-IPs in quadruplicate. After 48h of transfection, cells were washed with PBS and lysed with 250µl HNTG buffer (20mM Hepes pH7.9, 150 mM NaCl, 1mM MgCl 2 , 1 mM EDTA, 1% triton, 10% glycerol) supplemented with proteases inhibitors (Halt inhibitor cocktail, Thermo Scientific).

Immunoprecipitation were performed on each lysate in 96-wells Neutravidin plates (Termo Scientific) previously coated for 2h with biotinylated anti-Flag antibody (Bio-M2, Sigma) in HNTG (2 µg antibody/well). IP were performed with 100µl of lysate/well and incubated overnight at 4°. IP were washed 5 times with 200 µl HNTG/well for 5 min at 20° on a thermomixer (Eppendorf) with intermittent shacking. Firefly and Renilla activities were then quantified with DUAL luciferase reporter assay (Promega) using 50µl of reagents/well and an InfiniteF200 reader (TECAN). To quantify input, 10µl of each lysate were transferred in a white 96 plate and quantified as the same time as IP plate with the same procedure. The level of co-IP is quantified by calculating the level of co-IP normalized to the efficiency of IP:

(Renilla IP /Renilla Input )/(Firefly IP /Firefly Input ).

For bridging experiments between a Firefly-tagged bait and a Renilla tagged prey in absence or presence of Yki isoforms, the same procedure was used with a co-transfection of 0.15µg of Firefly plasmid, 0.15µg Renilla plasmid and 0.3µg pAct-Myc-Yki plasmid (or without Yki for control). Transfection and IP were performed in quadruplicate as described above.

Wing measurements

Young flies (1-3 days) of the appropriate genotypes were stored in isopropanol. Wings were mounted in Euparal (Roth, germany) on glass slide with coverslip and baked overnight at 65°.

For measurements of posterior vs total area (experiments with HH-Gal4 driver), pictures were acquired on a Leica M80 stereomicroscope equipped with a Leica IC80 HD camera using LAS software. Quantification was performed with Omero (www.openmicroscopy.org).

For quantification of Fluctuating Asymmetry (FA), left and right wings were mounted as pairs. Slides were digitalized using Nanozoomer (Hamamatsu). Quantification of wing size was performed with ImageJ. Each wing was measured twice in two independent sessions, by one or two persons. In rare case where the variation between replicate measurements was superior to 0.5% of total wing size, the wing was quantified again to minimize measurement error. The FA10 index 28 was used to estimate FA, i.e. FA corrected for measurement error, directional asymmetry and inter-individual variation. For all genotypes, the interaction individual/side was significant, indicating that FA was larger than measurement error.

Conventional two-way mixed model ANOVAs were applied to area data using Prism

Software. These values were used to calculate FA10 index. To compare FA10 values between genotypes, we used F-test to compare variance of the samples.

FIGURES FOR THE ARTICLE

3.0DISCUSSION AND PERSPECTIVES

Alternative splicing (AS) of mRNA precursors is a crucial step in the regulation of the gene expression pathway. The SR proteins constitute a family of multifunctional RNA binding proteins that play a significant role in alternative splicing regulation. Several genetic studies have reported that SR proteins are essential for animal development [START_REF] Ding | Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart[END_REF]) [START_REF] Feng | SRp38 Regulates Alternative Splicing and Is Required for Ca2+ Handling in the Embryonic Heart[END_REF] [START_REF] Jumaa | Blastocyst formation is blocked in mouse embryos lacking the splicing factor SRp20[END_REF]) (H. Y. [START_REF] Wang | SC35 plays a role in T cell development and alternative splicing of CD45[END_REF].

In addition to their role in splicing, several SRSF proteins have been shown to regulate transcription elongation, RNA export, decay, translation [START_REF] Zhong | SR Proteins in Vertical Integration of Gene Expression from Transcription to RNA Processing to Translation[END_REF] and for B52, transcriptional regulation [START_REF] Juge | The SR Protein B52/SRp55 Is Required for DNA Topoisomerase I Recruitment to Chromatin, mRNA Release and Transcription Shutdown[END_REF].

Our group has been studying the functions and regulations of SR proteins by using mammalian cells and Drosophila as models, and previously showed that modulation of protein levels of B52 (which is the closest Drosophila orthologue to human SRSF6) can affect cell growth in Drosophila (Fernando et al., 2015). The study did not characterize the underline mechanism by which B52 effects growth. However, they speculated that B52 could alter the AS of genes involved in the control of growth. Indeed (Fic et al., 2007) and [START_REF] Gabut | The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets[END_REF] previously showed that B52 overexpression could alter the AS of genes involved in development.

Capitalizing on this, in my Ph.D., I aimed to explore the mechanistic link between B52 and how it can regulate growth.

AS program of B52:

Thanks to the advances in the ability that allows rapid characterization of genome-wide AS events by RNA sequencing and immunoprecipitation techniques, several groups attempted to reveal the precise map of several RBPs binding on the transcriptome. Two such studies were highily interesting for us. In studies by [START_REF] Brooks | Regulation of alternative splicing in Drosophila by 56 RNA binding proteins[END_REF] and (Bradley et al., 2015) where, among other SR proteins, B52 was depleted in Drosophila S2 cells and RNA seq was performed to check the affected AS events.

We decided to merge the two available datasets order to identify AS events reproducibly affected by B52 depletion. With our strategy, we identified several AS events modulated by B52 level in genes involved in cell growth, including three genes linked to the Hippo (Hpo) pathway. Among these three genes, we also found yorkie (yki), which encodes the central effector of the Hippo pathway. The second candidate gene was wbp2 that encodes for Wbp2 protein which has been shown to bind Yki and enhance its intrinsic transcriptional coactivator activity (X. Zhang, Milton, Poon, Hong, & Harvey, 2011). The other candidate gene was zyxin (zyx) that encodes for Zyxin (Zyx) which has been shown to acts as a negative regulator of the Hippo pathway as it is required for the activity of Yki [START_REF] Rauskolb | Zyxin links fat signaling to the hippo pathway[END_REF][START_REF] Gaspar | Zyxin antagonizes the FERM protein expanded to couple f-actin and yorkie-dependent organ growth[END_REF]. RNAseq data indicate that B52 depletion promotes skipping of exon 3 in yki mRNA, the inclusion of exons 6 and 7 in wbp2 and the retention of the last intron in zyx. The functional consequences of all the identified AS are uncharacterized including that of yki. Although the canonical isoform of Yki (Yki2) has been characterized and has been described

B52 favors

in plethora of research papers, the presence of short isoform (Yki1) has been largely neglected since the very first paper described Yki (J. Huang et al., 2005). Thus in this field of Hippo pathway, it is not known yet if the short isoform of Yki is functional or redundant.

We believed that elucidation of the role of this isoform could better allow us to understand better how B52 controls growth via the Hippo pathway. To further test the effect of B52 on the growth we depleted it in the posterior part of Drosophila wing disc and observed that it leads to a reduction of the posterior domain and in discs and in adult wings. In addition, we saw the depletion of B52 leads to down-regulation of two well-established reporter of Yki activity, diap-lacZ and Ex-lacZ .

Taking hints from this result, we hypothesized that B52 reduces growth by reducing Yki activity and if this is true then these phenotypes should be rescued by overexpression of Yki and by inactivation of the kinases Warts (Wts) or Hippo (Hpo) which phosphorylates to regulate Yki negatively. Indeed we observed the rescue of the phenotype by overexpressing Yki and depleting Wts and Hpo, but we did not attain overgrowth as observed by overexpression of Yki or RNAi Wts/Hpo in the presence of wild type level of B52, suggesting that B52 is involved in other modulation of other genes and (or) pathways controlling growth.

Given the results that we obtained one could also argue that partial rescue that we see could correspond to the overcompensatory effect of Yki overexpression of Wts/Hpo depletion of defect induced by B52 depletion acting independently of Hippo pathway. To this end, we also depleted transcription factor Tgi that binds to Sd to repress the pathway in normal state and saw that its depletion alone induces a mild overgrowth, but it too rescues the growth defect inflicted by depletion of B52. However, we did not attain overgrowth as observed in wild type lever of B52. Altogether our result allows postulating that although B52 is involved with other genes and pathway in the regulation of growth, it also regulates growth upto significant extent via regulating Yki activity by modulating yki AS.

Yki 1 is a weaker isoform compared to Yki2:

At this juncture, we observed that B52 depletion favors the expression of Yki1, decreases Yki target genes and decreases growth partially through decreased Yki activity. This suggests that Yki 1 isoform could be less efficient when compared to the canonical Yki2 isoform. Indeed by overexpression assays, we observed that Yki 1 induces a weaker phenotype when compared to Yki2 in the eyes of adult Drosophila.

We observed that expression of the Yki reporter transgenes ex-lacZ, diap1-lacZ and bantam microRNA are weakly activated by Yki1 compared to Yki2. Further, we could extend these experiments by checking the upregulation of another well-established target of Yki, cell-cycle regulator CycE [START_REF] Tapon | salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines[END_REF].

Furthermore we directly compared the ability of Yki1 and Yki2 to activate transcription in luciferase assays in Drosophila S2 cells. Since Yki does not bind DNA directly, we fused Yki isoforms to the DNA binding domain of GAL4 and tested their activity on a UAS-luciferase reporter. Our results confirm that Yk1 is a weak transcription activator compared to Yki2.

Lack of second WW domain reduces several interactions of Yki

The only difference between Yki1 and Yki2 is the absence of the second WW domain in Yki1. Several studies have highlighted that the WW domain is indispensable for Yki's interaction with its specific partners that contain PPxY motifs (Hyangyee [START_REF] Oh | Yorkie: the final destination of Hippo signaling[END_REF].

It has been shown that mutation of these domains abolishes Yki activity (H. Oh & Irvine, 2009). It is thus apparent to assume that the lack of other WW domain in Yki1 can directly reduce its interaction with several above mentioned partners. Moreover, the lack of 2 nd WW domain could hypothetically modify protein structure in such a way that partners of Yki that interact via its N terminal domain, i.e. WW-PPxY independent interactions could also be affected thus we decided to investigate this untested assumption.

In addition to kinase Wts/Hpo and transcription factor Sd, Yki has been shown to interact with Mad (Mothers against Dpp) which is an effector of Dpp signaling (Decapentaplegic) and Hth (Hemothorax) and Tsh (Teashirt) [START_REF] Alarcón | Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-Cell[END_REF] [START_REF] Peng | Transcription factor choice in the Hippo signaling pathway: Homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc[END_REF] Oh, Reddy, & Irvine, 2009). Yki also interacts with several chromatin-modifying proteins including GAGA factor (GAF) and a subunit of the Trithorax-related (Trr) histone H3 lysine 4 methyltransferase complex, Nuclear receptor coactivator 6 (Ncoa6) [START_REF] Qing | The Hippo effector Yorkie activates transcription by interacting with a histone methyltransferase complex through Ncoa6[END_REF] (Hyangyee [START_REF] Oh | Genomewide Association of Yorkie with Chromatin and Chromatin-Remodeling Complexes[END_REF].

To ascertain the role of second WW domain and to understand the consequence of its alternative inclusion on Yki protein's function, we compared the interaction of each Yki isoform with several partners, by a semi-quantitative co-immunoprecipitation (Co-IP) after transfections of luciferase-tagged proteins in S2 cells (Refer to methods). We observed that indeed Yki 1 interacts weakly with Wts, Ex, NcoA6, Wbp2, and Tgi when compared to Yki2.

All these partners contain PPxY motifs. However, both the isoforms interact similarly with partners that contain single PPxY motif such as Mad or Hpo. Our results are in agreement with the in vitro studies performed by (Nyarko, 2018) and [START_REF] Webb | Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the hippo pathway[END_REF] who have shown cooperative binding between multiple PPxY motifs and tandem WW domains of YAP and Yki.

We also observed similar interaction of both isoforms with transcription factors Sd, MAD, Cbt and Hth, and the chromatin-associated factors GAF Mor and 14-3-3 proteins. We were expecting such an observation for Sd, Mor and 14-3-3 which have been described to interact with Yki via its N terminal domain [START_REF] Goulev | SCALLOPED Interacts with YORKIE, the Nuclear Effector of the Hippo Tumor-Suppressor Pathway in Drosophila[END_REF]) (Hyangyee Oh et al., 2013) [START_REF] Dong | Elucidation of a universal size-control mechanism in Drosophila and mammals[END_REF]. However, if Cbt associates with Yki directly or via other transcription factors, is yet to be elucidated [START_REF] Ruiz-Romero | Cabut/dTIEG associates with the transcription factor Yorkie for growth control[END_REF], the authors also showed the interaction of GAF but were unable to map the binding domain precisely.

Same goes for Hth which is known to physically interacts with Yki but the domain of

interaction is yet to be elucidated [START_REF] Peng | Transcription factor choice in the Hippo signaling pathway: Homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc[END_REF]. However, this analysis should be extended to partners such as Dalao (another component of BRM complex), MED23, MED15, MED31, MED19, and MED1 (subunits of Mediator complex) which have been shown to associate with Yki via mass spectroscopy studies (Hyangyee [START_REF] Oh | Genomewide Association of Yorkie with Chromatin and Chromatin-Remodeling Complexes[END_REF].

These results allowed us to verify our hypothesis that Yki1's lack of second WW domain reduces its interactions with several partners, but we saw similar interactions with partners containing single PPxY motif and transcription factors which bind to Yki via its N terminal domain. One curious point arose from the observation of these results is the fact that Yki1 isoform interacts weakly even with kinase Wts that functions to retain Yki in the cytoplasm.

If weaker interaction of Yki1 results in lower phosphorylation then it should be accumulated in nucleus very easily even Yki1 binds weakly with Ex that has been shown to directly sequester Yki at the apical junction region to prevent it from entering the nucleus, thereby inhibiting its activation [START_REF] Badouel | The FERM-Domain Protein Expanded Regulates Hippo Pathway Activity via Direct Interactions with the Transcriptional Activator Yorkie[END_REF]. We did not yet realize experiments to determine the phosphorylation state of the two isoforms of Yki. One counter explanation to this observation could be the fact that in the normal state the expression level of Yki1 isoform is extremely low.

Further, we speculated that besides strengthening the binding of Yki2 with partners containing multiple PPxY motifs, the presence of the second domain would allow Yki2 to interact with another PPxY containing coactivator simultaneously. This would explain the lower activity of Yki1. Inside the nucleus, Yki2 binds to transcription factor Scalloped (Sd) via its N terminal domain and via WW domain to repressor Tondu domain-containing growth inhibitor (Tgi). Studies by [START_REF] Koontz | The Hippo Effector Yorkie Controls Normal Tissue Growth by Antagonizing Scalloped-Mediated Default Repression[END_REF] and [START_REF] Guo | A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity[END_REF] have shown an interaction between Sd and Tgi. However, these two studies slightly differ from each other in terms of explaining the aftermath of Yki's recruitment. According to [START_REF] Koontz | The Hippo Effector Yorkie Controls Normal Tissue Growth by Antagonizing Scalloped-Mediated Default Repression[END_REF] Yki displaces the complex between Tgi and Sd and converts Sd into a transcription activator while on the other hand, [START_REF] Guo | A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity[END_REF] reported that Tgi bind to Sd and Yki simultaneously to forms an inactive ternary complex that is transcriptionally inactive.

To this end, our Co-IP verified the interaction between Sd and Tgi. In addition, we observed that both Yki 1 and Yki2 isoforms could enhance this interaction between Sd and Tgi with Yki2 being more efficient, suggesting that both Yki isoforms can form a trimeric complex with Sd and Tgi in agreement with the study by [START_REF] Guo | A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity[END_REF].

Studies in mammalian Hippo pathway showed binding of a protein called Wbp2 to interact with YAP (Mammalian counterpart of Yki). In Drosophila, (X. Zhang et al., 2011) have described, WW-PPxY dependent association between Yki and Wbp2 and they further reported that this association enhances Yki's ability to drive transcription. Taking clues from this study, one way to explain the weaker activity of Yki1 could due to the fact that it lacks the second WW domain and thus it cannot recruit other transcriptional activators such as

Wbp2. So we extended our Co-IP experiments between Sd and Wbp2 in the presence of Yki1 or Yki2 and observed that both the isoforms could increase this Co-IP although Yki1

increases it weakly when compared to Yki2. However, in this experiment, only Wbp2 interacts via WW-PPxY motif but the other interaction was not dependent on WW-PPxY domain.

We suspected that our observations would not be the same if we check Co-IP between Tgi and Wbp2 in the presence of Yki2 and Yki1 as both of these partners realize interaction with Yki in WW-PPxY dependent manner. Indeed, we observed that Yki1 could not Co-IP Tgi and Wbp2 together whereas Yki2 does and point mutations in the two WW domains of Yki abolish these interactions.

In Drosophila, six isoforms of Wbp2 have been described (source: FlyBase), and indeed from our screening, we observed that AS of wbp2 is affected by the depletion of B52. It will be interesting to extend our Co-IP to all these isoforms of Wbp2 and check the interaction of both isoforms of Yki with individual Wbp2 isoforms because B52 could co-regulate the AS of yki and wbp2 and more effectively control the regulation of Yki activity.

Model of competition:

Based on our observations so far, Yki isoforms interact similarly with transcription factors but differ by their capacity to bind and bridge PPxY-containing proteins. We propose that both the isoforms of Yki might compete with each other for binding to the transcription factors, when the Hippo pathway is OFF the canonical isoform Yki2 joins the transcriptionally repressed Tgi-Sd complex via one of its WW domain and could recruit transcription co-activators with the other WW domain such as PPxY containing Wbp2 or NcOA6 and finally may or may not displace Tgi. However, in B52 depleted state when there is more abundant Yki1, it successfully binds to the trimeric complex between Tgi and Sd but it cannot recruit Wbp2 or NcOA6 and might not displace Tgi so as to initiate the transcription effectively. This could reconcile with [START_REF] Guo | A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity[END_REF]'s observation that Tgi enhances Sd-Yki interaction but decreases the distance between Sd and Yki as measured by FRET suggesting the remodeling of the complex.

To test this hypothesis of competition, we made use of activated forms of Yki which was first described by [START_REF] Dong | Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals[END_REF], the activated forms lack the necessary phosphorylation site that is phosphorylated by Wts, more specifically Ser168 is mutated preventing Yki's binding to 14-3-3 proteins and thus it cannot be retained in cytoplasm but accumulated in nucleus.

Overexpression of activated Yki2 S168A isoform led to a very strong overgrowth phenotype in the Drosophila eye and this was expected as it has been shown by (H. Oh & Irvine, 2009). In addition we observed a weaker overgrowth phenotype induced by activated Yki1 S168A isoform too. To answer if there is a competition between the two isoforms, we cooverexpressed the Yki1 isoforms and remarkably only activated Yki1 S168A could partially rescue this phenotype suggesting that in the nucleus there is a competition between two isoforms. This result is in perfect agreement of our proposed model of competition.

Role yki AS in development:

Although the process of AS is vastly studied, and several isoforms that are switched at either development or in disease are reported but only a very few studies have focussed to dissect function of gene as isoform level, such is not only the case of yki/YAP AS but implies to vast number of genes with known isoforms. For example, it is recently reported that the presence or absence of exon7 in two splicing isoforms of MBNL1 conveys opposite phenotypical implications of cancer [START_REF] Tabaglio | MBNL1 alternative splicing isoforms play opposing roles in cancer[END_REF].

Similarly, in AS is also regulated for development, for an example, ApoER2 which is a protein encoded by the LRP8 gene also undergoes alternative splicing. The exon 19containing domain of ApoER2 is essential for synapse formation while its inclusion is reduced in the brain of Alzheimer's patients [START_REF] Hinrich | Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides[END_REF]. Thus it is now imperative to focus on the gene function at isoform level because it could be a key in understanding the complex regulation like AS and that is why we do not undermine the role of short isoform in the yki AS. [START_REF] Yeo | Variation in alternative splicing across human tissues[END_REF] highlighted that levels of AS usually differs from one tissue to another. So it is a possibility that AS of yki can differ from one tissue to another.

This could be checked by doing western on tissues at various stages of development.

However, to ultimately, reveal the role of the yki AS, we aimed to abrogate this in vivo, i.e.

to generate flies expressing exclusively Yki2 (yki2-only) or exclusively Yki1 (yki1-only), by editing the endogenous yki gene using the CRISPR/Cas9 system. We employed a different strategy at first and failed (Please refer to Appendix Figure 37), but later we employed a second strategy and managed to create flies expressing Yki2 isoform only. We observed that flies were homozygous viable, but we noticed a phenotype in the population of these flies.

We noticed an intra variation between the size of the right and left wings. This phenotype of Fluctuating asymmetry (FA) and it is the consequence of subtle deviation from normal development. The phenotype of asymmetry cannot be blamed on the environmental or genetic variations; it arises stochastically during the development. The fact that we observe a consistent and significant FA in our analysis of Yki2only population compared to control suggests that indeed the yki AS plays a role in development robustness. Further, we would like to generate heterozygous yki2only flies so as to check if we rescue the phenotype of FA.

Recently, [START_REF] Boone | The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression[END_REF] have shown that Yki controls the expression of dilp8 during normal development. They showed that a mutation Hporesponsive element (HRE) within the dilp8 promoter is enough to result in increased FA. One way to test our theory of yki AS a mean to maintain robust development would be via extending our analysis by testing for genetic interaction between dilp8 mutant and our yki 2only mutant. We expect that the FA will further rise in this population due to the lack of endogenous Yki1 isoform.

Yki has been implicated in the control of expression of hormone ecdysone (20hydroxyecdysone) and couples insulin signaling with ecdysone production. (C. Zhang et al., 2015) have further shown that Yki interacts with Taiman, ecdysone pathway transcription factor to control the gene expression of some targets such as piwi, nanos, dilp8. We could further advance our isoform comparison studies on these targets of Yki2.

One key experiment that is currently underway with our yki 2only flies is to investigate the effect of B52 depletion on the size of the posterior domain.We depleted B52 in the posterior domain of the wing in our yki 2only flies and plan to measure the effect on growth. Upon comparing with it with the overgrowth induced by B52 depletion in the posterior domain of wild-type flies if we observe that the overgrowth phenotype exceeds in yki 2only flies, then it would mean that indeed Yki isoform functions to buffer the activity of Yki2 isoform in development.

Concluding Remarks:

Finally, it is worth noting that this AS is conserved, In mammals, yki homolog, YAP, is alternatively spliced and is expressed as eight protein isoforms that fall into two categories, YAP1 and YAP2, which respectively contain one or two WW domain(s) [START_REF] Sudol | YAP1 oncogene and its eight isoforms[END_REF].

Therefore alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. Moreover (Komuro et al., 2003) reported that YAP2 is more efficient isoform than YAP1 in their studies where they co-precipitated ErbB-4 an epidermal growth receptor. This further supports the idea that Yki1 and YAP1 isoforms have important function in vivo and that alternative splicing of Yki/YAP is a conserved mechanism of control of the Hippo pathway.

There a are plethora of reports showing YAP is amplified and overexpressed in various tumors types where YAP protein relocalizes in the nucleus. YAP knockdown in many human cancer cell lines, such as prostate cancer or malignant mesothelioma cell lines, suppresses their growth and ability to trigger tumor formation. As of now, the focus of therapeutic research studies is towards targeting YAP, which indeed is a promising way to hamper tumor cell growth. Recently, molecules targeting YAP, such as verteporfin that inhibits the formation of the YAP-TEAD complex, have been described to block tumor growth in mice [START_REF] Liu-Chittenden | Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP[END_REF]. Identification of such new modulators of this pathway can, therefore, open new therapeutic opportunities for cancer treatment. However, to this date, none of the studies differentiate between the various isoforms of YAP. We wonder if the AS of YAP in cancer cells allows the production of more YAP2 instead of YAP1 which could even be a hallmark of tumorigenesis. One amazing future perspective of this study would be to study the extent of YAP splicing and the ration of isoforms in various tumor samples. As of now we have tools to analyze the expression of the two isoforms of dlg and STAT92E in B52 mutant clones hopefully we will be able to show if that it is B52 that affects the splicing of these genes, and thus it plays a role in cell competition.

  you, Papa & Mummy! ESS: Exonic Splicing Silencers ................................................................................................ ESTs: expressed sequence tags ................................................................................................ Ex : Expanded .......................................................................................................................... ey-GAL4: eyeless-GAL4 ......................................................................................................... FD: Familial dysautonomia ...................................................................................................... FERM: Band Four-point-one, Ezrin, Radixin and Moesin ...................................................... FoxO1: forkhead box proteins .................................................................................................. GAF: GAGA factor ................................................................................................................ GCs: Gastric cancer .................................................................................................................. HITS-CLIP : high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation ............................................................................................................ Hpo: Hippo ............................................................................................................................... HSCs : hematopoietic stem cell ............................................................................................... iCLIP : individual nucleotide-resolution cross-linking and immunoprecipitation ................... IKBKAP: i-kappa-B kinase complex-associated protein ......................................................... IRES : Internal Ribosomal Entry Site ...................................................................................... Jub : Ajuba LIM protein ........................................................................................................... KH: K-homology ...................................................................................................................... KS: Kaposi sarcoma ................................................................................................................. LATS : Large tumor suppressor Kinase ................................................................................... lgl: Drosophila lethal giant larvae ........................................................................................... lncRNAs : long noncoding RNAs ............................................................................................ M: Minute gene ...................................................................................................................... MAD: Mothers against DPP .................................................................................................... MAJIQ: Modeling Alternative Junction Inclusion Quantification ......................................... MALAT1: metastasis-associated lung adenocarcinoma transcript 1 ....................................... Mats : MOB as tumor supressors ............................................................................................. Mer: Merlin .............................................................................................................................. MOB1A/MOB1B : Mps one binder kinase activator-like 1A/1B ............................................ MOPD I: Microcephalic osteodysplastic primordial dwarfism type 1 .................................... MPM: Malignant pleural mesothelioma................................................................................... mRNA : messenger RNA ......................................................................................................... Ncoa6: Nuclear receptor coactivator 6 .................................................................................... NF2 : Neurofibromin ................................................................................................................ NGD: no-go mRNA decay ....................................................................................................... NMD: nonsense-mediated mRNA decay ................................................................................. NSCLC: Small-cell lung cancer ............................................................................................... NSD: non-stop mRNA decay ................................................................................................... PDAC: Pancreatic ductal adenocarcinoma ............................................................................... PPT: polypyrimidine tract (PPT).............................................................................................. PSI or ................................................................................................ PTC: premature termination codons ........................................................................................ PTPN14: Protein Tyrosine Phosphatase 14.............................................................................. qRRM: quasi-RNA recognition motif ...................................................................................... RBP : RNA binding proteins RBPs.......................................................................................... RCC: renal cell carcinoma .......................................................................................................RhoA: Ras homolog gene family, member A .......................................................................... RP: Retinitis pigmentosa .......................................................................................................... rRNAs : Ribosomal RNA ......................................................................................................... RUNX: Runt-Related Transcription Factor ............................................................................. Sav: Salvador ............................................................................................................................ SCRA: Sveinsson's chorioretinal atrophy ................................................................................ Scrib : Scribble ......................................................................................................................... Sd: Scalloped ............................................................................................................................ SdBP : Sd-Binding-Protein ...................................................................................................... Sdc: Syndecan .......................................................................................................................... Ser: Serine ................................................................................................................................ SMA : Spinal muscular atrophy ............................................................................................... snRNP : small nuclear ribonucleoproteins ............................................................................... snRNPs : small ribonucleoproteins .......................................................................................... SR: serine-arginine rich ............................................................................................................ SRE: splicing regulatory elements ...........................................................................................SRPK : SR-specific protein kinase........................................................................................... SRSF : serine/arginine-rich protein specific kinase ................................................................. ss : splice sites .......................................................................................................................... TAZ : Transcriptional co-activator with PDZ binding motif ................................................... TERT : transcription of telomerase reverse transcriptase ........................................................ Tgi: Tondu-domain-Containing growth inhibitor .................................................................... Trr : Trithorax-related............................................................................................................... Tsh: T shirt ............................................................................................................................... Vam: Vamana ........................................................................................................................... VGLL4: vestigial like family member 4 .................................................................................. VP: Verteporfin ........................................................................................................................ Wnt: Wingless/integrated ......................................................................................................... YAP: Yes-associated protein ................................................................................................... Yki: Yorkie ............................................................................................................................... Zyx: Zyxin ................................................................................................................................

Figure 2

 2 Figure 2 Conserved sequence elements recognized by the major spliceosome: ......................

Figure 3

 3 Figure 3 Protein composition and snRNA secondary structures of the major human spliceosomal snRNPs: ..............................................................................................................

Figure 4

 4 Figure 4 The two steps of splicing catalysis: ...........................................................................

Figure 5

 5 Figure 5 Splicing assembly: .....................................................................................................

Figure 6

 6 Figure 6 Seven types of alternative splicing: ...........................................................................

Figure 7

 7 Figure 7 Exon and Intron definition models: ............................................................................

Figure 8

 8 Figure 8 Splicing regulatory elements: ....................................................................................

Figure 9

 9 Figure 9 SR proteins and hnRNPs: ..........................................................................................

Figure 10

 10 Figure 10 The hnRNP family: ..................................................................................................

Figure 11

 11 Figure 11 Domain configuration of human SR proteins: .........................................................

Figure 12

 12 Figure 12 Techniques to identify binding of SR proteins: .......................................................

Figure 13 A

 13 Figure 13 A simplified schematic of the iCLIP protocol: ........................................................

Figure 14

 14 Figure 14 Multiple roles of SR proteins: ..................................................................................

Figure 15 AS

 15 Figure 15 AS affected by individual SR proteins: ...................................................................

Figure 16

 16 Figure 16 Bristle phenotype: ....................................................................................................

Figure 17

 17 Figure 17 Effect of B52 on growth: .........................................................................................

Figure 18

 18 Figure 18 Rescue of B52: .........................................................................................................

Figure 19

 19 Figure 19 The three primary mRNA surveillance mechanisms: ..............................................

Figure 20 SMA

 20 Figure 20 SMA and its treatment by ASO: ..............................................................................

Figure 21

 21 Figure 21 Implications of the Hippo pathway in cell biology: ...............................................

Figure 22

 22 Figure 22 The rise of Hippo pathway: .....................................................................................

Figure 23

 23 Figure 23 Hippo mutant phenotypes in flies: ...........................................................................

Figure 24

 24 Figure 24 Schematics of the Hippo pathway in Drosophila melanogaster: ............................

Figure 25

 25 Figure 25 Upstream regulators of Hippo pathway in Drosophila: ..........................................

Figure 26 (

 26 Figure 26 (A) Effect on growth due to MST knockout : ..........................................................

Figure 27 Mammalian

 27 Figure 27 Mammalian Hippo Pathway: ...................................................................................

Figure 28

 28 Figure 28 Mechanotransduction: ..............................................................................................

Figure 29

 29 Figure 29 Localization of YAP/TAZ at lower vs higher cell density: .....................................

Figure 30

 30 Figure 30 Crosstalk: .................................................................................................................

Figure 31

 31 Figure 31 Apical versus basal mechanotransduction via Yki or YAP in different animals: ...

Figure 32

 32 Figure 32 The eight described isoforms of YAP: ....................................................................

Figure 33

 33 Figure 33 Schematic diagram of YAP1 and 2, TAZ, and Yki: ................................................

Figure 34

 34 Figure 34 Cellular function of YAP/TAZ: ...............................................................................

Figure 35 :

 35 Figure 35 : Implications of the Hippo pathway in various cancers and non-cancer diseases: .

Figure 36

 36 Figure 36 Schematic demonstration of the small-molecule modulators of YAP: .................... Appendix Figure 37 : CRISPR and screening strategy: ...........................................................

  First discovered inDrosophila, this conserved signaling pathway regulates the activity of a transcription coactivator called Yorkie (Yki) in flies and YAP (Yes-Associated Protein) in human. The core of the Hippo pathway is composed by the kinase Hippo (MST1/2 in mammals) which activates the kinase Warts (LATS1/2 in mammals) that phosphorylates the transcription coactivator Yki in Drosophila and YAP in mammals. Phosphorylated Yki/YAP is sequestered in the cytoplasm through interactions with 14-3-3 proteins. Inactivation of the pathway leads to translocation of unphosphorylated Yki/YAP in the nucleus and activation of its target genes.Yki/YAP does not bind DNA directly but interacts with several transcription factors, mainly members of the TEAD family such as Scalloped (Sd) in flies and TEAD1-4 in mammals.Yki/YAP bound to Sd/TEAD recruits cofactors and chromatin remodeling complexes to activate transcription of its target genes, which promote cell proliferation and inhibit apoptosis. Overexpression of Yki/YAP, or inactivation of the Hippo pathway, induces tissue overgrowth both in Drosophila and mouse models. Studies of mouse models and clinical samples have now demonstrated the significance of Hippo pathway for the development of human cancers.Our lab is interested in the mechanism of Alternative Splicing (AS), that allows producing multiple mRNAs from a single gene, by modulating inclusion or exclusion of alternative exons for example, therefore encoding different protein isoforms. By studying the function of the RNA binding protein B52 in Drosophila, a splicing factor of the SR protein family, the lab previously showed that overexpression of B52 increases cell growth, whereas B52 depletion reduces it. By the use of RNAseq data, they identified several AS events modulated by B52 level in genes involved in cell growth, including several genes linked to the Hippo pathway and especially yki.They observed that depletion of the B52 splicing factor favors skipping of yki exon 3, which encodes one of the two WW domains of Yki protein. Thus, Yki exists as two isoforms containing one or two WW domain(s), respectively called Yki1 and Yki2. WW domains are protein-protein interaction modules that bind proline-rich motifs (PPxY) present in several proteins of the pathway such as the kinase Warts, the cytoskeleton-associated protein Expanded (Ex) and the transcription co-activator Wbp2, as examples. Therefore, modulating the inclusion/exclusion of one WW domain could change Yki phosphorylation, localization or co-activator activity. The expression and function of the short Yki1 isoform has never been described. Remarkably a similar AS event exists in the human YAP homolog: AS of exon 4gives rise to two isoforms, YAP1 and YAP2, which respectively contain one or two WW domain(s). Therefore, alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. Nevertheless, the signaling differences among YAP splicing variants remain to be elucidated, as is the regulation of YAP AS.The objectives of my Ph.D. were to investigate and reveal the role of B52-triggered yki AS and to determine if this AS constitutes a new level of modulation of Hippo pathway in Drosophila. Our results show that B52 depletion favors expression of the Yki1 isoform carrying a single WW domain, and reduces growth in part through modulation of yki AS.Compared to the canonical Yki2 isoform containing two WW domains, Yki1 has reduced transcriptional and growth-promoting activities, decreased binding to PPxY motifs-containing proteins, and an inability to bridge two proteins containing PPxY motifs. Nevertheless, Yki1 and Yki2 interact similarly with transcription factors and thus compete in vivo. Flies in which the yki AS has been abrogated, thereby expressing only the Yki2 isoform, exhibit increased fluctuating wing asymmetry, a signal of increased developmental noise. These results show that yki AS represents an additional layer of modulation of Yki activity that unexpectedly participates in buffering developmental noise, and provide the first experimental evidence that AS participates in developmental robustness. I hereby introduce the process of splicing, key components that facilitate splicing and alternative splicing in general introduction. In a second part I cover in my introduction the Hippo pathway and its key components in Drosophila and mammals, starting from its discovery to most recently described key processes that regulate the pathway. The results obtained during in the thesis are presented in the form of pre manuscript followed by

  consensus sequence, YAG/GURAGU (where Y is a pyrimidine, R is a purine. Even though the consensus exists, splice site sequences are degenerate. The 3' splice site is comprised of three elements, placed within around 40 nucleotides upstream the intron/exon junction. The 3' splice site itself has a consensus YAG/N, where Y is a pyrimidine (uracil or cytosine). The spliceosome also recognizes another sequence motif called Branch Point Sequence (BPS) which is located at 20-40 nt upstream of the 3´ splice site. The BPS YNYURAY (where Y is C or U nucleotide)

Figure 2

 2 Figure 2 Conserved sequence elements recognized by the major spliceosome: This schematic shows four consensus elements required for intron recognition: the 5' splice site, branch point, polypyrimidine tract, and the 3' splice site. R corresponds to purine, Y -to pyrimidine (C or U nucleotide) and N to any nucleotide. Image is adapted from (Wahl, Will, & Lührmann, 2009) 1.1.4 Spliceosome: The machinery of splicing

Figure 3

 3 Figure 3 Protein composition and snRNA secondary structures of the major human spliceosomal snRNPs: All seven Sm proteins (B/B', D3, D2, D1, E, F, and G) or LSm proteins (Lsm2-8) are indicated by "Sm" or "LSm" at the top of the boxes showing the proteins associated with each snRNP. The U4/U6.U5 tri-snRNP contains two sets of Sm proteins and one set of LSm proteins Adapted from[START_REF] Will | Spliceosome structure and function[END_REF]

Figure 4 1 . 1 . 6

 4116 Figure 4 The two steps of splicing catalysis:During the first step, the 5'ss and branch adenosine are joined by a 2'-5' nucleophilic attack, leading to the formation of a lariat structure and a free upstream exon. The second step consists of the 3'-5' nucleophilic bond between the free exon and the 3'ss. Adapted from[START_REF] Scotti | RNA mis-splicing in disease[END_REF]

Figure 5

 5 Figure 5 Splicing assembly:Schematic representing the key steps of assembly and activation of the yeast spliceosome and the complete splicing-reaction cycle. Adapted from[START_REF] Shi | Mechanistic insights into precursor messenger RNA splicing by the spliceosome[END_REF].

Figure 6

 6 Figure 6 Seven types of alternative splicing: (From Top to bottom) Seven types of alternative splicing. Exon skipping, Mutually exclusive exons, Alternative 3' splicing, Alternative 5' splicing, Intron retention, choice of an alternative promoter, Alternative Polyadenylation. Intron retention is the major alternative splicing event in rice, whereas exon skipping is the most frequent alternative splicing in humans.

Figure 7

 7 Figure 7 Exon and Intron definition models: The left panel depicts the Intron definition model according to which pairing between the splice sites takes place across an intron when long exons are separated by short (<250 bp) introns. On the other hand, the right panel shows the Exon definition model where the splice site communication occurs across exons when they are separated by long (>250 bp) introns. Adapted from (De Conti, Baralle, & Buratti, 2013)

Figure 8

 8 Figure 8 Splicing regulatory elements: SREs can enhance and repress the splice site selection by the spliceosome. Splicing is governed by cis-regulatory sequences in the pre-mRNA; exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs)

Figure 9 SR

 9 Figure 9 SR proteins and hnRNPs:These are two major families of alternative splicing regulatory proteins, which are recruited by splicing enhancers and silencers. These regulatory proteins target components of the spliceosome (shown in green) that associate with both the 5 and the 3 lice sites flanking the alternative exon and can have either activating or inhibitory effects on the recognition and use of that site. In addition, interactions among components of the spliceosome that are recruited to the 3 can mediate exon definition. Adapted from[START_REF] Kornblihtt | Alternative splicing: A pivotal step between eukaryotic transcription and translation[END_REF]

Figure 10

 10 Figure 10 The hnRNP family:The hnRNPs are named alphabetically from hnRNP A1 to hnRNP U. The members of the hnRNP family are built up of four unique RNA-binding domains (RBDs): RRM RNA recognition motif, qRRM quasi-RNA recognition motif, and KH Khomology domain, RGG RNA-binding domain consisting of Arg-Gly-Gly repeats. Image adapted from :[START_REF] Geuens | The hnRNP family: insights into their role in health and disease[END_REF]

  . The function of hnRNPs depends upon their localization and upon undergoing PTMs such as methylation, phosphorylation, ubiquitination and sumoylation they can change their subcellular localization and thus function (S. P. Han, Tang, & Smith, 2010) (Chaudhury, Chander, & Howe, 2010) hnRNPs play an essential role in both constitutive and alternative splicing. hnRNPs generally repress splicing through binding to the silencer sequences (ESSs or ISSs) in the pre-mRNA and suppress splicing.

Figure 11

 11 Figure 11 Domain configuration of human SR proteins: SRSF1-12 are members of the canonical SR protein splicing family that is defined by N-terminal RRMs followed by a downstream RS domain. The RRM is responsible for RNA binding, while the RS domain mediates protein/protein interactions. Left: Aliases of the SR proteins. Right: Drosophila homologs.

Figure 12

 12 Figure 12 Techniques to identify binding of SR proteins: (A) SELEX (systematic evolution of ligands by exponential enrichment) for in vitro identification of SR-binding RNA motifs. A random RNA library was used for the selection of binding RNA sequences. (B) Functional SELEX. Reporterbased in vitro and in vivo identification of splicing regulatory elements. ESEs (Exonic Splicing Enhancers) can be selected in the reporter as shown here. (C) CLIP-Seq (Cross-linking and immunoprecipitation-sequencing) for the global identification of SR-binding motifs in target RNAs

Figure 13 A

 13 Figure 13 A simplified schematic of the iCLIP protocol:UV irradiation to covalently cross-link in vivo protein-RNA complexes followed by Cell lysis and partial RNA digestion (step 1). After lysis, the crosslinked RNA is fragmented by the limited concentration of RNase I and RNA fragments are then co-immunoprecipitated with the RBP (step 2), followed by ligation of a 3 -PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments (step 5). Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). The peptide that is on the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the cross-link site. Therefore, two types of cDNAs are generated: truncated cDNAs and readthrough cDNAs. In iCLIP, the cDNA library is prepared in such a way that both truncated and readthrough cDNAs are amplified (step 7). After PCR amplification and sequencing (step 8), both truncated and readthrough cDNAs are present. Figure adapted and modified from[START_REF] Zünd | Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3 Nature Structural and Molecular Biology[END_REF]

Figure 14 1 . 3 . 3 . 2 . 4

 1413324 Figure 14 Multiple roles of SR proteins: During gene expression from the nucleus to the cytoplasm

  [START_REF] Lai | A human importinprotein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins[END_REF] SR proteins also undergo other post-translational modification such as methylation and acetylation. It has been shown that blocking methylation can misregulate the localization of SR proteins and thus, it can affect alternative splicing, translation, and mRNA decay[START_REF] Bressan | Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRp30C[END_REF] [START_REF] Sinha | Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF[END_REF] (Y.-C. Chen et al., 2010) On the other hand acetylation of

Figure 15 AS

 15 Figure 15 AS affected by individual SR proteins: (a.) Types of AS considered in this study. (b.) The total number of AS events regulated by individual SR proteins separated by causing more inclusion (inc.) or exclusion (exc.) of the alternative region represented individually for each AS type. (c.) Pie charts of the distribution of simple AS types differentially regulated by individual SR proteins detected by RNA-Seq.Study by(Bradley et al., 2015)

Figure 16

 16 Figure 16 Bristle phenotype: (a.) Wild type fly consists of eight macrochaetes on thorax which are indicated by arrows. (b.) Upon down-regulation of B52 in bristle cell lineage, there is a variable loss of macrochaetes as in this fly only one of the eight macrochaetes is remaining. Reported in(Fernando et al., 2015)

  lineage. The authors further generated B52 homozygous mutant clones by somatic recombination and revealed that B52 mutant clones were smaller and eventually gave rise to smaller bristles in adults (Figure 17 b & c). They also observed that knockdown of B52 lead to delay in the development of bristle lineage and by Immunostaining, they revealed that B52 knockdown decreases Myc expression (Figure 17 a).

Figure 17

 17 Figure 17 Effect of B52 on growth: (a.) wildtype clone vs Increased cell size of clone overexpressing B52. (b.) Loss-of-function of B52 in the bristle lineage decreases cell growth (c.) bristle arising from wild-type clones vs from B52 homozygous mutants. Reported in(Fernando et al., 2015)

Figure 18

 18 Figure 18 Rescue of B52: (a.) Overexpression of B52 in the eye (b.) Co-overexpression of B52 and Brat. Reported in (Fernando et al., 2015) Recently, (Boyin Liu & Bossing, 2016) have shown the requirement of B52 in the splicing of Choline acetyltransferase (ChAT the neurotransmitter acetylcholine. Moreover, B52 has also been shown to regulate the midday siesta in flies by regulating the splicing of period gene suggesting that this SR protein can participate/contribute in natural sleep behavior as reported in a recent study by (Zhichao

1. 4 . 1

 41 Nonsense-mediated decay (NMD)One of the thoroughly-investigated RNA surveillance pathways is the Nonsense-mediated decay (NMD) pathway. It was identified in S. cerevisiae but has been shown to exist in all eukaryotes that have been investigated and is evolutionary conserved from yeast to humans (Y. H.[START_REF] Chen | UPF1, a conserved nonsense-mmediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia[END_REF] [START_REF] Delhi | Is there a classical nonsense-mediated decay pathway in trypanosomes?[END_REF])[START_REF] Schweingruber | Nonsense-mediated mRNA decay -Mechanisms of substrate mRNA recognition and degradation in mammalian cells[END_REF] [START_REF] He | Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story[END_REF].

(Figure 19

 19 Figure 19The three primary mRNA surveillance mechanisms: Adapted from[START_REF] Roy | The intimate relationships of mRNA decay and translation[END_REF]

  oligonucleotides (ASOs) are a new class of drugs that are short(15-25 nt), synthetic, single-stranded oligodeoxynucleotides that can bind to complementary target RNA and thus function to alter, reduce, restore, or modify protein expression through several distinct mechanisms. They can alter pre-mRNA splicing by sterically blocking splicing factors, or they can block mRNA translation by preventing ribosome recruitment (C. F.Bennett & Swayze, 2010) SMA is now treated by Nusinersen which is a 2'-OMe phosphorothioate ASO that can target exon 7 which in SMN2 pre-mRNA. This facilitates exon7's inclusion and leads to the production of functional protein[START_REF] Wertz | Developing therapies for spinal muscular atrophy[END_REF].Finally, in 2016, FDA (U.S. Food and drug administration) approved Nusinersen as an ASO to be used in SMA patients, and so far it remains as the only FDA approved the medication for SMA (Aartsma-Rus, 2017).

Figure 20 SMA

 20 Figure 20 SMA and its treatment by ASO:

1. 5 . 1

 51 Conclusion:Breakthroughs in high-throughput sequencing technologies promise to identify and link many more human mutations to disease. Moreover, small-molecule compounds, antisense oligonucleotides, and genome-editing approaches hold promise for correcting the effects of these mutations common in human genetic variation and disease. ASO especially have a high potential in future therapy, for an example Eteplirsen, an ASO is now being used to treat DMD, ASOs to treat ALS are already being investigated and show promising therapeutic potential (C. A.[START_REF] Stein | FDA-Approved Oligonucleotide Therapies in 2017[END_REF].[START_REF] Le | Antisense Oligonucleotides Targeting Angiogenic Factors as Potential Cancer Therapeutics[END_REF] have described the scope of ASOs that can target angiogenic factors and act as therapeutic molecules for the treatment of cancer. Small molecules such as a family of spliceostatins (derived from Pseudomonas) and herboxidienes and pladienolides (derived from Streptomyces) have been shown to target core spliceosome machinery and mediate anticancer properties. Reviewed in (J. C.[START_REF] Lin | Therapeutic applications of targeted alternative splicing to cancer treatment[END_REF].

Figure 21

 21 Figure 21 Implications of the Hippo pathway in cell biology:The Hippo pathway modulates cell proliferation, differentiation, growth, and death. The coordination of different cellular processes by the Hippo pathway may contribute to various physiological and pathological conditions such as development, tissue homeostasis, and tumorigenesis.

Figure 22 1 . 7

 2217 Figure 22 The rise of Hippo pathway: The rising number of papers being published every year in the field of the Hippo Pathway.The Hippo pathway transduces signals from the plasma membrane to the nucleus and subsequently activates the transcription of genes involved in cell proliferation, anti-apoptosis

Figure 23

 23 Figure 23 Hippo mutant phenotypes in flies:(A,B) Scanning electron micrographs of (A) a wild-type fly and (B) a fly with clones of cells homozygous mutant for hippo that exhibit overgrowth of the adult cuticle Adapted from (G.[START_REF] Halder | Hippo signaling: growth control and beyond[END_REF]

  -3-3, therefore, inhibiting its transcriptional activation role (H.Oh & Irvine, 2009)(H.Oh & Irvine, 2008) Recently, Misshapen (Msn) and Ste-20 kinase Happyhour (Hppy) kinases were shown to act in parallel to Hpo to phosphorylate Wts (Q.Li et al., 2014)[START_REF] Meng | MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway[END_REF](Pan et al., 2015). Two kinases Tao and Par-1 directly regulate the activity of Hpo via phosphorylation. Tao has been shown to phosphorylate the activation loop of Hpo to promote its activity[START_REF] Boggiano | Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway[END_REF] [START_REF] Poon | The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway[END_REF].While on the other hand, polarity kinase Par-1 restricts Hpo by promoting its phosphorylation at a different site to restrict its activity. Further, Par-1 inhibits the interaction between Hpo and Sav to promote Sav dephosphorylation and destabilization, further downregulating Hippo pathway activity (H. L.[START_REF] Huang | Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association[END_REF] Recently, (X.[START_REF] Sun | Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie[END_REF] showed that deubiquitinase Usp7 positively regulates Yki transcriptional program by deubiquitinating Yki. Further, they showed that Hpo or warts inhibit the Usp7-Yki interaction not only to promote Yki's nuclear accumulation but also to promote Yki nuclear degradation. This study provides new insight into how the Hippo pathway negatively regulates the activity of Yki.

Figure 24

 24 Figure 24 Schematics of the Hippo pathway in Drosophila melanogaster:

1. 7 . 1

 71 Upstream regulators of Drosophila Hippo Pathway: Core components of the Hippo pathway have been studied extensively to this date. Unlike typical signaling cascades, the Hippo pathway is not regulated by a defined set of ligandreceptor interactions, but it is rather modulated by a diverse set of signals from upstream constituents. However, what precise signals activate the kinase cascade to suppress the activity of transcriptional co-activators is not yet completely understood. Thus, several researchers were focused on to elucidate the regulators of the Hippo pathway. So far, The upstream signals that are known to regulate the Hippo pathway such as transmembrane receptors atypical cadherins Fat(Ft) and Dachous1/2 (Dchs), Kibra-Expanded/Merlin complex and apicobasal polarity complex, i.e., members of crumbs polarity complex play roles in maintaining the cell architecture such as cell polarity and cytoskeleton. The expansion of such knowledge can provide insight into what signals direct cells to adhere to a pattern of growth and when to inhibit the growth (Figure 25).

Figure 25

 25 Figure 25 Upstream regulators of Hippo pathway in Drosophila: It is regulated by various factors including the following aPKC, atypical protein kinase C; Crb, Crumbs; D, Dachs; Dlg, Discs large; Ds, Dachsous; Ed, Echinoid; Ex, Expanded; Ft, Fat; Hth, Homothorax; Jub, Ajuba LIM protein; Lgl, Lethal giant larvae; Mad, Mothers against decapentaplegic (Dpp); Mer, Merlin; Scrib, Scribble; Sd, Scalloped; Tgi, Tondu domaincontaining growth inhibitor; Tsh, Teashirt; Zyx, Zyxin. Growth-promoting proteins are depicted in brown and growth repressors in green. These proteins are described in text. AJ, adherens junction; SJ, septate junction. Adapted from[START_REF] Irvine | Control of organ growth by patterning and hippo signaling in drosophila[END_REF]

1 . 7 . 1 . 2

 1712 pathway, but loss of Fat leads to overgrowth phenotype and up-regulation of Hippo pathway target genes such as cycE and diap1, as has been shown for multiple Hippo pathway components. Mechanistic studies have shown that Fat signaling can positively regulate the Hippo pathway via localizing Ex, an upstream regulator of the pathway to apical junction region (F. C.[START_REF] Bennett | Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway[END_REF]) (E.[START_REF] Cho | Delineation of a Fat tumor suppressor pathway[END_REF] [START_REF] Silva | The Tumor-Suppressor Gene fat Controls Tissue Growth Upstream of Expanded in the Hippo Signaling Pathway[END_REF] (Willecke et al., 2006). Another mechanism of positive regulation of Hippo signaling by fat signaling is via inhibition of Wts degradation thus promoting abundance of Wts mediated by the atypical myosin Dachs (E.[START_REF] Cho | Delineation of a Fat tumor suppressor pathway[END_REF] [START_REF] Rodrigues-Campos | The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila[END_REF] [START_REF] Rauskolb | Zyxin links fat signaling to the hippo pathway[END_REF] [START_REF] Bosch | The Drosophila F-box protein Fbxl7 binds to the protocadherin Fat and regulates Dachs localization and Hippo signaling[END_REF] In addition,[START_REF] Vrabioiu | Fat/Dachsous Signaling Promotes Drosophila Wing Growth by Regulating the Conformational State of the NDR Kinase Warts[END_REF] showed that Fat signaling acts via Dachs to inhibit Wts from acquiring Mats-dependent active conformation to promote growth in Drosophila wing.[START_REF] Misra | Vamana Couples Fat Signaling to the Hippo Pathway[END_REF] identified Vamana (Vam) to influence Dachs membrane localization and most recently same authors described Early girl as an essential component of Fat signaling that the levels of localization of Daschs and Vamana (Misra & Irvine, 2019). Regulation by Ex/Mer/Kibra: The FERM (N-Terminal Globular Domain [Band Four-point-one, Ezrin, Radixin and Moesin]) domain-containing protein Ex and Merlin (Mer) along with the C2 and WW domain-containing protein Kibra (Kib) are three important apical membrane proteins of the

Fehon

  Ex and Mer have been shown to physically interact with each other and loss of function of any of the two genes leads to overgrowth phenotype and deletion of Ex and Mer together results into even severe overgrowth phenotype due to upregulation of Hippo pathway target genes as they interact with hippo pathway components[START_REF] Mccartney | The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation[END_REF] ([START_REF] Hamaratoglu | The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis[END_REF]. Similar to ex and mer, kib mutations too leads to overgrowth phenotype and up-regulation of Hippo pathway targets. Kib protein physically associates with both Mer and Ex and the three of them can co-localize at the apical junctional region, suggesting that these three proteins can function together to regulate the Hippo pathway.Interestingly Ex, Mer, and Kib can each function independently of each other in the regulation of the Hippo pathway as revealed by the genetic analysis studies[START_REF] Hamaratoglu | The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis[END_REF];[START_REF] Baumgartner | The WW Domain Protein Kibra Acts Upstream of Hippo in Drosophila[END_REF] (J.[START_REF] Yu | Kibra Functions as a Tumor Suppressor Protein that Regulates Hippo Signaling in Conjunction with Merlin and Expanded[END_REF]. Studies have revealed that Ex, Mer, and Kib regulate the Hippo pathway positively by recruiting the pathway components to specific cortical domains to promote their activation.[START_REF] Yin | XSpatial organization of hippo signaling at the plasma membrane mediated by the tumor suppressor merlin/NF2[END_REF] demonstrated that Mer could also directly recruit Wts to the plasma membrane to activate Hippo signaling, independently of Hpo kinase activity. Generally, Ex can function as a scaffold that recruits Wts to the apical junctional region from its inhibitor Jub where it interacts Hpo and gets activated (S.[START_REF] Sun | Localization of Hippo signalling complexes and Warts activation in vivo[END_REF].[START_REF] Su | Kibra and Merlin Activate the Hippo Pathway Spatially Distinct from and Independent of Expanded[END_REF] have demonstrated that Mer and Kib can activate Hippo signaling in parallel to Ex at a spatially distinct cellular domain, the apical cortex. Together, Ex, Mer, and Kibra may activate Hippo pathway at distinct subcellular locations, possibly mediating different inputs of the Hippo pathway. Alternatively, Ex can also regulate the pathway by physically binding with Yki and sequestering it outside of the nucleus acting independently of kinase cascade[START_REF] Badouel | The FERM-Domain Protein Expanded Regulates Hippo Pathway Activity via Direct Interactions with the Transcriptional Activator Yorkie[END_REF]. In addition to acting as upstream regulators of the Hippo pathway, Ex, Mer, and Kib are transcriptional targets of the Hippo pathway too[START_REF] Hamaratoglu | The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis[END_REF] (Genevet, Wehr, Brain, Thompson, & Tapon, 2010b). This suggests the existence of a negative feedback loop in which Yki activation turns on the expression of positive regulators of the Hippo pathway(Boggiano & Apical polarity proteins are located in the apical cell cortex. They have been shown to regulate the Hippo pathway. Crumbs (Crb) was the first apical polarity protein found to be involved in Hippo pathway regulation. In Drosophila embryos, Crb is localized at the subapical plasma membrane and plays a vital role in organizing apical-basal polarity[START_REF] Tepass | crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia[END_REF]. Both loss of Crb and constitutive expression leads to Yki activation, up-regulated Hippo target genes, and overgrowth. Crb has been shown to interact with Ex via its FERM domain and recruits Ex to the apical junctional (AJs) region to promote Hippo pathway activity (C.-L.Chen et al., 2010) [START_REF] Ling | The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded[END_REF] [START_REF] Robinson | Crumbs Regulates Salvador/Warts/Hippo Signaling in Drosophila via the FERM-Domain Protein Expanded[END_REF] [START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF]. On the other hand, it has also been demonstrated that Crb promotes ubiquitin-mediated degradation of Ex via E3 ubiquitin ligase F--TrCP (Slmb) suggesting that Crb allows precise tuning of Hippo pathway activity through its dual regulation of Ex[START_REF] Ribeiro | Crumbs promotes expanded recognition and degradation by the SCFSlimb/ -TrCP ubiquitin ligase[END_REF].The Par complex consists of atypical protein kinase C (aPKC), its regulatory subunit Par6, the Rho-GTPase Cdc42, and the scaffolding protein Bazooka (Baz; Drosophila homolog of the C.elegans and mammalian Par3). aPKC is involved in the regulation of the Hippo pathway.Expression of a dominant-negative aPKC transgene suppresses Hippo pathway inactivation in Drosophila lethal giant larvae (lgl) clones[START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF]. Overexpression of aPKC can cause mislocalization of Hpo, activation of Yki, and up-regulation of Hippo pathway target genes[START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF]) (G.[START_REF] Sun | Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors[END_REF]

1 . 7 . 1 . 4

 1714 three basolateral polarity proteins are regarded as positive regulators of the Hippo pathway[START_REF] Richardson | Tissue growth and tumorigenesis in Drosophila: cell polarity and the Hippo pathway[END_REF][START_REF] Enomoto | Deciphering tumor-suppressor signaling in flies: Genetic link between Scribble/Dlg/Lgl and the Hippo pathways[END_REF].Loss of Lgl in the eye of Drosophila does not disrupt cell polarity or activate Jun kinase signaling but mislocalizes Hpo, suggesting Lgl participates in localization of Hpo[START_REF] Bogoyevitch | c-Jun Nterminal kinase (JNK) signaling: Recent advances and challenges[END_REF] [START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF]. While on the other hand, loss of Lgl via RNAi in wing imaginal disc, disrupts cell polarity and causes activation of JNK signaling leading to Yki activation suggesting the regulation of Yki activity via Lgl is context dependent (G.[START_REF] Sun | Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors[END_REF]. Further, the same authors suggested that JNK activates the Wts inhibitor Jub via phosphorylation providing a link between basolateral polarity, JNK signaling and Yki activity (G.[START_REF] Sun | Ajuba family proteins link JNK to hippo signaling[END_REF].scrib mutant clones are eliminated by cell competition, a phenomenon of removal of "weak cells" by surrounding "fit cells"[START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF][START_REF] Chen | Tumor suppression by cell competition through regulation of the Hippo pathway[END_REF].Further,[START_REF] Grzeschik | Lgl, aPKC, and Crumbs Regulate the Salvador/Warts/Hippo Pathway through Two Distinct Mechanisms[END_REF] showed that loss of Scrib via RNAi leads to down-regulation of Hippo pathway target diap1 suggesting that Yki is inactivated in scrib mutatnt clones. However when (C.-L.Chen et al., 2012), blocked JNK signaling in scrib mutant clones they observed Yki activation and a severe overgrowth phenotype, suggesting JNK signaling is responsible for repressing Yki activity in scrib clones and this effect outweighs the activation of Yki by loss of Scrib. Although, the mechanism of repression of Yki by JNK signaling in this scenario is unknown,[START_REF] Enomoto | Deciphering tumor-suppressor signaling in flies: Genetic link between Scribble/Dlg/Lgl and the Hippo pathways[END_REF] suggests that JNK can inhibit Yki activity via Wts. Cell-Cell adhesion and junctional proteins Cell-cell adhesion and junctional proteins such as Ajuba LIM protein (Jub), Zyxin, and Echinoid regulate the Hippo pathway in Drosophila. Jub is localized at the apical junctions stabilizes preassembled cadherin complexes[START_REF] Nola | Ajuba is required for Rac activation and maintenance of E-cadherin adhesion[END_REF]. Down-regulation of Jub resembles Yki down-regulation phenotypes. For example, it leads to down-regulation of Jub Hippo pathway target genes such as diap1 and cycE resulting in tissue undergrowth. Also, jub has been shown to genetically interact with the Hippo pathway components[START_REF] Das Thakur | Ajuba LIM Proteins Are Negative Regulators of the Hippo Signaling Pathway[END_REF].[START_REF] Rauskolb | Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex[END_REF] showed that Jub sequesters and inhibits Wts at the adherens junctions (AJs) to inhibit hippo pathway activity. Zyxin (Zyx) which is another LIM domain protein, acts as a negative regulator of the Hippo pathway as it is required for the activity of Yki[START_REF] Rauskolb | Zyxin links fat signaling to the hippo pathway[END_REF] [START_REF] Gaspar | Zyxin antagonizes the FERM protein expanded to couple f-actin and yorkie-dependent organ growth[END_REF]. Depletion of Zyx leads to reduced Yorkie activity and undergrowth.[START_REF] Rauskolb | Zyxin links fat signaling to the hippo pathway[END_REF] also reported that at apical junctional region Zyx cooperates with Dachs to promote Wts degradation thus inhibiting Hippo pathway activity linking Zyx to Fat signaling branch of Hippo pathway regulation.[START_REF] Gaspar | Zyxin antagonizes the FERM protein expanded to couple f-actin and yorkie-dependent organ growth[END_REF] demonstrated that Yki activity and growth promoting ability of Zyx depends upon its interaction with the actin-associated protein Enabled (Ena). More importantly, Ena promotes F-actin polymerization linking Zyx with F-acitn branch of the Hippo pathway.[START_REF] Hirata | Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner[END_REF] showed that Zyx is associated with mechanical-force-dependent facilitation of actin polymerization at cell adhesive structures; thus this group was the first to hint at the possibility of mechanical cues regulating the Hippo pathway. Echinoid (Ed) is an upstream regulator that is required for proper sub-apical localization of Sav (T. Yue, Tian, & Jiang, 2012). Ed physically interacts with Ex, Yki, Hpo, Kibra, and Mer. However, Ed only affects Sav localization without affecting Ex or Mer localization. Depletion of Ed activates Yki, and subsequently upregulating Hippo pathway target gene expression and tissue overgrowth. Its demonstrated in Drosophila that Ed physically interacts

  [START_REF] Aegerter-Wilmsen | Model for the regulation of size in the wing imaginal disc of Drosophila[END_REF][START_REF] Aegerter-Wilmsen | Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size[END_REF]. Another study showed that artificially increased growth in clones of wing imaginal discs reduces cytoskeletal tension, which in turn decreases the activation of Yki. This study suggests mechanical feedback in which increased Yki activation and cell proliferation reduces tension and deactivates Yki. Such feedback could contribute to evenly distributed growth (Y.[START_REF] Pan | Differential growth triggers mechanical feedback that elevates Hippo signaling[END_REF]

  . The Lats1 kinase was known as NDR kinase in yeast cells and was known to function in cell division and morphology maintenance (T.[START_REF] Millward | Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase[END_REF][START_REF] Millward | Calcium regulation of NDR protein kinase mediated by S100 calcium-binding proteins[END_REF]. However, the role of these proteins in the Hippo pathway was unknown. Soon after the Hippo pathway was discovered in Drosophila, studies in mammals lead to the identification of orthologs of fly Hippo pathway components leading to the establishment of the mammalian Hippo pathway soon after. In 2003, MST was identified as a core kinase of the Hippo pathway in mammals[START_REF] Harvey | The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis[END_REF]) (S.[START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF]. By 2008 when[START_REF] Hao | Tumor suppressor LATS1 is a negative regulator of oncogene YAP[END_REF]) (B.[START_REF] Zhao | Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control[END_REF] showed that LATS1/2 Kinase negatively regulates YAP/TAZ core of Hippo pathway was considered to be complete in mammals.

Figure 26 (

 26 Figure 26 (A) Effect on growth due to MST knockout :(A) A mouse liver at two months of age from a wild-type animal and a liver at two months of age from a mouse mutant in which both Mst1/2 have been inactivated. The double null Mst1/2 mutant liver is overgrown owing to an increase in cell numbers. Adapted from (G.[START_REF] Halder | Hippo signaling: growth control and beyond[END_REF]

(

  YAP; homologous to Yki in Drosophila) and TAZ (transcriptional coactivator with PDZbinding motif, WWTR1). This phosphorylation takes place on 4 and 5 HXRXXS motifs of TAZ and YAP respectively. Once phosphorylated, YAP/TAZ interacts with 14-3-3 protein gets sequestered in the cytoplasm or degraded by ubiquitination-dependent proteasomal Phosphorylation of YAP at Ser381 and TAZ at Ser311 has been linked to regulation of YAP/TAZ protein stability, as it primes subsequent phosphorylation in a phosphodegron by -TrCP E3 ligase, leading to ubiquitination and degradation (C. Y. Liu et al., 2010) (B. Zhao, Li, Tumaneng, Wang, & Guan, 2010a)[START_REF] Schäfer | The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1[END_REF][START_REF] Dong | Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals[END_REF][START_REF] Hirabayashi | Threonine 74 of MOB1 is a putative key phosphorylation site by MST2 to form the scaffold to activate nuclear Dbf2-related kinase 1[END_REF][START_REF] Lei | TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway[END_REF][START_REF] Oka | Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP)[END_REF][START_REF] Siew | A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells[END_REF]. Many components of the Hippo pathway have already been shown to be degraded by ubiquitination such as YAP, LATS1/2; Expanded(Ex) (B. Zhao, Li, Tumaneng, Wang, & Guan, 2010b) (X. Yang et al., 2011)(Salah, Melino, & Aqeilan, 2011)(B. Ma et al., 2015) (W. Li et al., 2014) (X. Ma, Richardson, Xue, Xu, & Guo, 2018).Similar to Yki, YAP/TAZ transcriptional activators lack the DNA binding domain; thus inside nucleus they interact with transcription factors (Such as TEAD1-4) via its TEAD binding domain to be bought to the gene promoter(B.Zhao et al., 2008)(H.[START_REF] Zhang | TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition[END_REF][START_REF] Chan | TEADs mediate nuclear retention of TAZ to promote oncogenic transformation[END_REF]. So, In the scenario when Hippo pathway is OFF, the unphosphorylated YAP/TAZ gets translocated to the nucleus where it competes with VGLL4 (Vestigial Like Family Member 4; homologous to Tgi in Drosophila) to interact with TEAD transcription factor so as to induce the transcriptional activation of several sets of gene which play crucial role in cell growth, differentiation invasion/migration and survival. Reviewed by[START_REF] Meng | Mechanisms of Hippo pathway regulation[END_REF]. The C-t --helices on one side, while the N-terminal domain of YAP wraps around TEAD, forming extensive interactions (L. Chen et al., 2010)(Z.[START_REF] Li | Structural insights into the YAP and TEAD complex[END_REF] (W.[START_REF] Tian | Structural and functional analysis of the YAP-binding domain of human TEAD2[END_REF]

Figure 27 Mammalian

 27 Figure 27 Mammalian Hippo Pathway: Hippo pathway is highly conserved from Drosophila Melanogaster to human, schematic representing ON and OFF state the pathway in mammals YAP interacts with promoters and distal enhancers to regulate target gene expression, through mechanisms of modulating chromatin looping and Pol II pausing release[START_REF] Lian | The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation[END_REF]

(

  J. H.[START_REF] Hong | TAZ, a transcriptional modulator of mesenchymal stem cell differentiation[END_REF], Paired Box 3 (Pax3) (Masao[START_REF] Murakami | Transcriptional activity of Pax3 is co-activated by TAZ[END_REF], T-Box Transcription Factor 5 (TBX5)[START_REF] Murakami | A WW domain protein TAZ is a critical coactivator for TBX5, a transcription factor implicated in Holt-Oram syndrome[END_REF] and Thyroid Transcription Factor 1 (TTF-1) (K. S.[START_REF] Park | TAZ Interacts with TTF-1 and Regulates Expression of Surfactant Protein-C[END_REF] Further, It has been shown that the SWI/SNF (a chromatin remodeling protein complex) and

  family member amphiregulin (AREG), IAP family member BIRC5 , antiapoptotic gene BCL2L1, ABCB1(ATP Binding Cassette Subfamily B Member 1), ANKRD (ankyrin repeat domain-containing protein), CAT (catalase), GPATCH4 (G-Patch Domain Containing 4), LMN-B2 (Lamin-B2), PTGS2 (Prostaglandin-endoperoxide synthase 2), WSB2 (WD repeat and SOCS box-containing protein 2), and TXN(Thioredoxin)[START_REF] Koontz | The Hippo Effector Yorkie Controls Normal Tissue Growth by Antagonizing Scalloped-Mediated Default Repression[END_REF])(Rosenbluh et al., 2012) and described in[START_REF] Venkataramani | CD31 expression determines redox status and chemoresistance in human angiosarcomas[END_REF]. Most of the identified targets of YAP/TAZ are indispensable to growth, proliferation, control cell migration, cell lineage commitment viability, tissue specification, morphogenesis and angiogenesis embryonic development, tissue turnover, and injury repair.A variety of upstream signals that regulate the YAP/TAZ have been identified as we have seen in the case of Drosophila Hippo pathway. Also, signals that affect levels of TEAD also affect the functional output of the Hippo pathway (K. C.[START_REF] Lin | Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation[END_REF] Unlike in Drosophila, The Ft branch of the mammalian Hippo pathway is not well very understood. Mammals have four Ft orthologs; Fat1-4 and two Dachous orthologs; Hchs1-2.

1. 8 . 1 . 2

 812 Cellular polarity, Cell-cell contact, and morphology:Cellular polarity is important in maintaining the structure and function of epithelial cells. ABCP (apicobasal cell polarity) proteins define the polarity of the cell. Proteins that establish cell polarity in epithelial cells at cell-cell junctions can activate the core Hippo cascade and thus play an important regulatory role(Hansen, Moroishi, & Guan, 2015) (F. X.[START_REF] Yu | The Hippo pathway: Regulators and regulations[END_REF]. Similarly, proper cellular adhesion is important in maintaining structure and function, and loss of proper connectivity in either type can cause cellular distress and promote tumorigenesis. Contact inhibition is also a vital factor in controlling tissue growth and homeostasis. When cells come into contact with neighboring cells, normal proliferating cells typically undergo contact inhibition and stop proliferating in order to avoid overgrowth[START_REF] Mcclatchey | Contact inhibition (of proliferation) redux[END_REF] Components of mammalian tight junction, or adherens junction (AJ) complexes promote mammalian Hippo signaling. For example, loss of Crb3, PALS1 PATJ leads to YAP/TAZ activation in mammalian cells along with polarity defects[START_REF] Varelas | The Crumbs Complex Couples Cell Density Sensing to Hippo-Dependent Control of the TGF--SMAD Pathway[END_REF] Several cell junction proteins such as AMOT (angiomotin) family proteins, LIN7C -catenin, PATJ or MPDZ or E cadherin ZO1, ZO2, NPHP4, and LKB1 too have been identified to interact with core components of the Hippo pathway[START_REF] Chan | Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation[END_REF] (Chan, Lim, Chong, et al., 2011)[START_REF] Dai | Phosphorylation of angiomotin by Lats1/2 kinases inhibits F-actin binding, cell migration, and angiogenesis[END_REF][START_REF] Hirate | The role of angiomotin phosphorylation in the Hippo pathway during preimplantation mouse development[END_REF][START_REF] Michaloglou | The Tyrosine Phosphatase PTPN14 Is a Negative Regulator of YAP Activity[END_REF][START_REF] Zhao | Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein[END_REF]. -Catenin, an adaptor protein between the membrane and actin cytoskeleton for adherens junction (AJ) formation, is another known ABCP regulator that influences Hippo pathway activation. -catenin links the actin cytoskeleton to other adherenes (to external structures). When the Hippo pathway is ON, -catenin binds with YAP and sequesters it at the adherens junction, thereby preventing it from moving into the nucleus (F. X. Yu & Guan, 2013) (W.[START_REF] Wang | PTPN14 is required for the density-dependent control of YAP1[END_REF]. Interestingly members of the Angiomotin (AMOT) family of tight junction proteins regulate YAP/TAZ activity either by binding and activating the Hippo core kinase LATS2[START_REF] Paramasivam | Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor[END_REF], hence promoting YAP/TAZ phosphorylation, or by directly interacting with YAP/TAZ independently of Hippo core kinases[START_REF] Chan | Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation[END_REF] (Chan, Lim, Chong, et al., 2011) (B. Zhao et al., 2011) (W.

  inhibit the activity of RhoA, ROCK or myosin type II result in cytoplasmic translocation and inactivation of YAP/TAZ.([START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF] [START_REF] Wada | Hippo pathway regulation by cell morphology and stress fibers[END_REF]) (B. Zhao et al., 2012).

Figure 28

 28 Figure 28 Mechanotransduction: Mechanical cues regulate YAP/TAZ activities through Rho GTPase and the Hippo pathway. Cell-cell contact, cell stretching, cell tension, extracellular matrix (ECM) stiffness, and changes in cell geometry modulate activities of Rho GTPases, which in turn lead to actin cytoskeleton remodeling. The actin cytoskeleton can control nuclear-cytoplasmic shuttling and transcriptional activities of YAP/TAZ through both LATS1/2-dependent and LATS1/2-independent mechanisms.

Figure 29

 29 Figure 29 Localization of YAP/TAZ at lower vs higher cell density:

Figure 30

 30 Figure 30 Crosstalk: Crosstalk between the Hippo pathway and Wnt and Notch signaling in the intestine: When the Hippo pathway is activated, -catenin in the cytoplasm. When the Hippo pathway is inactive, nuclear YAP1-catenin-induced activation and upregulation of YAP1. Activated YAP1 transcriptionally upregulates expression of Notch receptors, leading to increased activation of Notch signaling and translocation of the NICD to the nucleus. Notch is considered to be a mediator of the Hippo pathway. Adapted from (A. W.[START_REF] Hong | The Hippo pathway in intestinal regeneration and disease[END_REF]

Figure 31

 31 Figure 31 Apical versus basal mechanotransduction via Yki or YAP in different animals:

Figure 32

 32 Figure 32The eight described isoforms of YAP:

Figure 33

 33 Figure 33 Schematic diagram of YAP1 and 2, TAZ, and Yki: Red dot denotes phosphorylation sites. They include Proline-rich region TEAD-binding domain, Sd (Drosophila homolog of mammalian TEADs)-binding domain, WW: WW domain, C-C: coiled-coil region, TAD: transactivation domain, PDZ BD: PDZ-binding domain. Below is the short description of these domains.

  (Georg Halder, Dupont, & Piccolo, 2012) [START_REF] Morgan | What do mechanotransduction, Hippo, Wnt, and disease[END_REF] and apical and basal signaling[START_REF] Elbediwy | YAP and TAZ in epithelial stem cells: A sensor for cell polarity, mechanical forces and tissue damage[END_REF]. It is not surprising to that altercation or perturbation of such a complex pathway eventually leads to cell proliferation, inhibition of apoptosis, misregulation of cellular differentiation which is are critical aspects of cancer development (Figure34).

Figure 34

 34 Figure 34 Cellular function of YAP/TAZ:

Figure 35 :

 35 Figure 35 : Implications of the Hippo pathway in various cancers and non-cancer diseases:

  a mutation in TBX5 of T-Box family of transcription factors is implicated in Holt-Oram syndrome. It is an autosomal dominant disorder that causes the malformation of upper limbs and cardiac abnormalities. It has been demonstrated

Figure 36

 36 Figure 36 Schematic demonstration of the small-molecule modulators of YAP: These small-molecule modulators of YAP can be classified into three categories: (1) the pink area represents those regulating the upstream molecules of YAP. (2) the green area represents those modulating the phosphorylation of YAP and blocking YAP nuclear translocation, such as Dobutamine. (3) The blue area represents those inhibiting YAP interaction with TEAD1 by directly targeting YAP, such as Verteporfin, VGLL4-mimicking peptide. 1.12Conclusion: Hippo pathway integrates the signals from a verity of growth pathways, dysregulation of Hippo pathway components could lead to cancer and other diseases via vast range of alterations that eventually yield in same molecular result, expression of nuclear localized YAP/TAZ thus targeting the constitutive YAP/TAZ activation could be of therapeutic benefit as already shown in cancer model studies. On the other hand, pharmacological inhibition of Hippo kinases MST1/2 and/or LATS1/2 seems to offer potential therapeutic benefit in an organ-cell specific setting, especially in patients recovering from myocardial injury as transient YAP activation in cardiomyocytes could expand the cardiomyocyte cell pool during

  Diwas Srivastava, Marion de Toledo, Laurent Manchon, Jamal Tazi & François Juge Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, Montpellier, France The mechanisms that contribute to developmental stability are barely known 1 . Alternative splicing (AS) of mRNA precursors expands transcriptomic and proteomic diversity and its extent correlates with organism complexity (REFs). Nevertheless its contribution to developmental robustness has not been documented. Here we show that AS of yorkie (yki) is required for developmental stability in Drosophila. Yki encodes the effector of the Hippo pathway that has a central role in controlling organ growth and regeneration. We identify the splicing factor B52 as necessary for inclusion of yki alternative exon 3 that encodes one of the two WW domains of Yki protein. Compared to the canonical Yki2 isoform containing two WW domains, Yki1 isoform with a single WW domain, has reduced transcriptional and growth-promoting activities, decreased binding to specific partners, and lacks the ability to bridge two proteins containing PPxY motifs. Yet, Yki1 and Yki2 interact similarly with transcription factors and can thus compete in vivo. Flies in which yki AS been abrogated to express only Yki2 isoform show increased fluctuating asymmetry in the wings giving evidence of increased developmental noise. Our results provide the first experimental support that AS participates in developmental robustness and identify yki AS as a new level of control of Hippo pathway. Remarkably, alternative inclusion of the second WW domain is a conserved feature between Yki and its human homolog YAP, indicating that modulation of YAP AS could be a novel strategy to lower YAP activity that is frequently upregulated in cancer cells.

  TRiP.HMS00026), UAS-IR Hpo (TRiP.HMS00006), UAS-IR-Tgi (TRiP.HMS00981) and were previously validated in the literature.UAS-Yki1 and UAS-Yki1 S168A transgenes were constructed from pUAS-Yki-V5-His and pUAS-Yki S168A -V5-His clones in pUAS-attB vector, kindly provided by K. Irvine (these clones contain Yki2 isoform cDNA). A XhoI site located between UAS sequences and start codon, flanked by two EcoRI sites, was deleted by EcoRI digestion and re-ligation of these vectors. This generated UAS-Yki2-V5-His and UAS-Yki2 S168A -V5-His. The SfiI-XhoI fragment containing the C-terminal part of Yki2 was replaced by the corresponding SfiI-XhoI amplified from a yki1 cDNA obtained by RT-PCR (third instar larvae). This generated UAS-Yki1-V5-His and UAS-Yki1 S168A -V5-His transgenes. The four constructs UAS-Yki2-V5-His, UAS-Yki2 S168A -V5-His, UAS-Yki1-V5-His and UAS-Yki1 S168A -V5-His were inserted in attP2 site. Injections were performed by Bestgene Inc.yki gene editingTo edit endogenous yki locus by CRISPR/Cas9-mediated Homologous Recombination, a repair construct corresponding to yki gene without introns 2 and 3, and containing a piggyback insertion in intron 1, was created by cloning of multiple PCR fragments amplified with Q5-Taq polymerase (Biolabs). yki gene fragments were amplified from yw flies.PiggyBac transposon containing the reporter gene 3xP3-DsRed (expresses DsRed in the eye), was amplified from pHD-3xFLAG-ScarlessDsRed (DGRC #1367) and cloned into a DraI site present in yki intron 1, thus creating TTAA sequences at both sides of the transposon that are necessary for its excision. We used two guide RNAs targeting exon 2 and exon 4 of yki, cloned into pCFD4 (addgene #49411). The plasmid containing the repair contruct was coinjected with the plasmid encoding the two guides into nos-Cas9 embryos. Injection and screening for positive DsRED flies in the progeny, were performed by Bestgene Inc. Two DsRED-positive lines, validated by PCR, were selected for excision of PiggyBac transposon using a source of transposase (Bloomington #8285). For each line a single excision event was selected to establish a stock. Two independent yki 2only alleles were obtained, called ED1 and ID3. The entire yki locus was sequenced in these lines. We detected polymorphism in introns and silent polymorphism in exons. These variations are present between the yw line used to create the yki 2only construct and the nos-Cas9 line in which the injections were made. Both yki 2only lines display enhanced FA.Molecular biologycDNAs corresponding to MAD, GAF, Sd, Hth, 14.3.3, Hpo and Wbp2 were amplified from third instar larvae RNA by RT-PCR, using a forward primer stating at ATG and reverse primer located just upstream (or sometimes including) the stop codon. Forward primer contains CACC sequence upstream of ATG to orient cloning in pENTR/D-Topo (Invitrogen). All cDNAs were entirely sequenced. Other cDNAs were Cabut (DmCD00765693, DNASU), Tgi (DmCD00765105, DNASU), Mor (Addgene #71048), Ex

  Yki1 isoform at the expense of Yki2:Over the years the Hippo pathway has emerged as a significant player in tissue growth control, stem cell function, regeneration and tumor suppression. During organogenesis, optimum levels of Hippo signaling are crucial for maintaining tissue homeostasis. Overexpression of Hippo signalling results in the formation of smaller organs due to induction of apoptosis[START_REF] Harvey | The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis[END_REF] [START_REF] Pantalacci | The salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila[END_REF] [START_REF] Udan | Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway[END_REF] (S.[START_REF] Wu | hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts[END_REF].With Yki being the central effector of the Hippo pathway in Drosophila where all upstream signals and kinase cascade converge on Yki to regulate the activity of pathway, finding that yki AS is altered by depletion of splicing factor B52 was a major reveal for us. Several studies have shown that the regulation of the transcriptional co-activator Yki is central to the regulation of the Hippo pathway.Indeed we confirmed this AS alteration by western, and RT-PCR B52 depleted S2 cells and Drosophila Wing disc samples. We observed that depletion of B52 induces skipping of yki alternative exon 3 and increases expression of Yki1 at the expense of Yki2 isoform. Thus, Yki protein exists as two isoforms containing one or two WW domains thereafter called Yki1 and Yki2 respectively.

Further

  investigation could be directed towards finding small molecules that manipulate the alternative splicing of YAP as a way to modulate YAP activity in cancer cells. Thus our research should open new perspectives for modulation of the Hippo pathway in cancer cells by altering YAP alternative splicing.The regulation of the Hippo pathway has evolved into a complex network over the past two decades, with new players added at an unprecedented pace. Our study shall add even more complexity into this regulatory network.induced by chemical causes the Hippo pathway to switch its role from being anti-apoptotic to apoptotic. They showed that upon stress stimulation by caffeine, Yki interacts with the stress-responsive transcription factor p53 in flies with loss of Hippo components such as Wts or Hpo (Figure39a & b). We asked how our heterozygous yki 1only & homozygous yki 2only would respond to caffeine treatment and what will be the effect of overexpression of Yki1 or Yki2 activated and non activated isoforms? Unfortunately, we were unable to reproduce the pro-apoptotic function in our experiments. It could be due to the technical problem with the medium. At this point, we decide to focus on generating the yki 2only flies without any mutation and resume the characterization studies when we obtain it (Figure39 c).

1.3.3 Trans-acting factors regulating Alternative splicing

	SREs recruit trans-acting proteins which can either activate splicing or repress it. The best-
	known splicing factors are the proteins belonging to the SR (serine-arginine rich) and hnRNP
	(heterogeneous nuclear ribonucleoproteins) protein families. Proteins belonging to SR protein
	family are known to enhance splicing. SR proteins have one or two RRM (RNA recognition
	motif) protein domains and an RS (arginine-serine rich) domain. The RRM domain mediates
	SR protein binding with intronic splicing enhancers (ISEs) and Exonic splicing enhancers
	(ESEs) on the RNA, while RS domains promote spliceosome assembly and activate the
	splicing. In addition to the SR protein family, there are other SR-related proteins, that also
	activate splici	(Roland Tacke, Tohyama,
	Ogawa, & Manley, 1998). The mechanism by which splicing activation can happen is by
	splicing regulator proteins that can interact with and stabilize the binding of U1 snRNP or
	other spliceosomal components on pre-mRNA that initiate splicing. As another possibility,
	splicing activators can interact with other splicing factors (like TIA proteins), and indirectly

  knocked down 8 Drosophila SR proteins (SC35, SF2, SRp54, XL6, Rbp1, B52, Rsf1, and Rbp1-like) in S2 cells via RNAi (below 10% in each case)

individually and did RNA-seq analysis to in order to reveal the extent to which SR proteins targets overlap with each other and to what extent SR proteins cooperate with each other to regulate splicing. They only focused on five simplest types of AS which are cassette exons, competing donor or acceptor sites, mutually exclusive exons, and intron retention (Figure

Table 1 : Conservation of Hpo pathway core components between Drosophila and mammals.

 1

	Drosophila Melanogaster proteins	Human proteins
	Core components
	Hippo (Hpo)	Serine/theonine kinase 4/3 MST1/MST2 or
		STK4/STK3
	Salvador (Sav)	Salvador (SAV1 or WW45)
	Warts (Wts)	Large tumor suppressor Kinase 1/2 (LATS1/2)
	MOB as tumor supressors (Mats)	Mps one binder kinase activator-like 1A/1B
		(MOB1A/MOB1B or collectively MOB1)
	Yorkie (Yki)	Yes-associated protein (YAP), Transcriptional co-
		activator with PDZ binding motif (TAZ) or
		(WWTR1)
	Upstream Modulators
	Fat (Fat)	Angiomotin AMOT
	Expanded (Ex)	Ferm domain-containing protein 6 (FRMD6)
	Merlin (Mer)	Neurofibromin (NF2)
	Kibra (Kib)	Kibra (KBR)
	Crumbs (Crb)	CRB1-CRB3
	Ras association family member (Rassf)	Ras association domain-containing protein 1-6
		(RASSF1-6)
	Scrib	SCRIB
	Mask	MASK1,MASK2
	Wbp2	WBP2
	Dlg	DLG1-DLG4
	Lgl	LGL1, LGL2
	Jub	Ajuba, LIMD1, WTIP
	Downstream mediators
	Scalloped (Sd)	TEA domain family member 1/2/3/4
		(TEAD1,TEAD2,TEAD3,TEAD4)
	Tondu-domain-Containing growth inhibitor	Transcription cofactor vestigial-like protein 4
	(Tgi)	(VGLL4)
	Homothorax (Hth)	Meis1
	Tsh	--------
	Mad

  used taxol or nocodazol to arrest the cells in G2/M cycle and investigated what member of the Hippo pathway are regulated during mitosis. They Cells adapt and overcome to constant internal and external stresses that endanger their integrity. So it is not surprising to find that several stress signals can modulate Yki/YAP/TAZ activity in vivo. In 1996 when (Taylor, Wang, & Erikson, 1996) had determined MST1/2 is activated by a high concentration of arsenite or heat shock, the components of the pathway were still not yet grouped together; thus a little attention was paid. Later in two decades when the Hippo pathway was established, it incited new interests in looking for stress signals that can modulate the kinase cascade of the Hippo Pathway. Further, MST1/2 is shown to be activated by oxidative stress caused by H2O2

	reported that YAP is phosphorylated at T119, S289, and S367 by cell cycle kinase CDK1
	revealing an additional regulator of YAP mediated activity.
	1.8.1.6 Stress signals as upstream regulators:

2 Inhibitors of Hippo signaling as anti-cancer drug candidate:

  put forward a patented compound IBS008738 that activates TAZ and promotes myogenesis in mouse myoblast cells (C2C12 cells). This compound facilitates muscle repair after cardiotoxin-induced muscle injury and prevents steroid induced muscle atrophy in mice. They argue that this compound can be used realistically for old patients who suffer from sarcopenia and confined to bed. Due to such confinement, they lose skeletal muscle in lower limbs and often become disabled. The TAZ activator, IBS008738 can be administered to such elderly patients to maintaining muscle volume until the patient can resume exercise. The short term application can further downside the worries of TAZ activation leading to oncogenesis or inducing indolent tumors.

	1.11.

  used MAJIQ (Modeling Alternative Junction Inclusion Quantification) to identify Local

	Splicing Variations (LSV) in two previously published datasets from Bradley et al.
	(#GSM1552264, # GSM1552267) and Brooks et al. (#GSM627333, #GSM627334 and
	#GSM627343). Each dataset contains two replicates of control cells and B52-depelted
	RNAseq. The reads were subjected to standard quality control (QC) and filters criteria
	according to the following parameters: (1) trimming and cleaning reads that aligned to
	primers and/or adaptors, (2) reads with over 50% of low-quality bases (quality value
	one read, and (3) reads with over 10% unknown bases (N bases). We have used a software
	called Trimmomatic (v0.36) to remove primers and also to remove bad quality reads. After
	filtering, we removed short reads (<36bp), the remaining reads are called "clean reads" and

stored as FASTQ format. Reads were aligned to Drosophila melanogaster reference genome release 6.19 using STAR (Spliced Transcripts Alignment to a Reference). STAR output were stored in BAM files. BAM files were then submitted to MAJIQ analysis pipeline. LSV definitions were generated and quantified by MAJIQ. MAJIQ applies several normalization factors to the raw values before to compute normalized PSI (Percent Selected Index) and

Acknowledgements

Altogether these results indicate that B52 depletion reduces growth at least in part through the Hippo pathway and suggest that it lowers Yki activity by modifying alternative splicing of yki mRNAs. We, therefore, explored the functional differences between the two Yki isoforms.

To compare the activity of Yki isoforms, we created UAS-Yki1 and UAS-Yki2 transgenic flies for GAL4-mediated overexpression, by site-specific integration. Compared to Yki2, overexpression of Yki1 isoform induces weaker overgrowth phenotypes in the posterior domain of wings (Fig. 2a) or in the eyes (Suppl. Fig. 3). We analyzed expression of Yki reporter genes ex-lacZ and diap-lacZ following Yki isoforms overexpression in the posterior domain of wing discs. Expression of both transgenes is moderately increased by Yki1 overexpression as compared to Yki2 overexpression (Fig. 2b,c). We also analyzed expression of a bantam miRNA sensor, which expression is inversely correlated to the level of ban miRNA, a direct target of Yki. Yki1 induces a weaker decrease of bam-sensor expression than Yki2 isoform (Fig. 2c). Together these results show that Yki1 has a reduced activity compared to Yki2 in vivo.

Yki1 and Yki2 isoforms differ respectively by the absence or presence of alternative exon 3, which includes the second WW domain (WW2). Numerous studies have demonstrated that, in the context of Yki2 isoform, WW domains are required for interaction with several partners containing PPxY motifs such as Ex, Hpo, Wts, NcoA6, Wbp2 and Tgi 8- 15 . The absence of one WW domain in Yki1 is supposed to reduce interaction with such partners, but this assumption has not been experimentally demonstrated. Moreover binding of proteins that interact with the N-terminal part of Yki, such as Sd or 14.3.3, may also be affected if the structure of the protein is modified by the absence of the domains encoded by exon 3. It is therefore important to address these interactions in the context of full-length proteins and with a quantitative assay. To this end we developed a dual-luciferase co-Immunoprecipitation (co-IP) method in S2R+ cells inspired by the DULIP method 16 described in mammalian cells. Our assay monitors co-IP between a bait protein fused to Flagtagged-Firefly luciferase and a prey protein fused to HA-tagged-Renilla luciferase (Fig. 3a).

Quantification of luciferases activities ratio after IP with anti-Flag antibody gives a rapid and sensitive readout of the interaction between the bait and the prey. Flag-Firefly-Yki1 and Flag-Firefly-Yki2 were used as baits for interaction with thirteen known Yki partners fused to Renilla luciferase as preys (Fig. 3b). We used Flag-Firefly as a control for non-specific interactions. By this approach, we detected significant interactions between Yki isoforms and all proteins tested, from modest co-IP with GAF, MAD and Cbt, to high and very high interaction for the other proteins. We found that both isoforms Yki1 and Yki2 interact At first, we succeeded to establish Yki1 isoform expressing flies. We determined that the homozygous (yki 1only / yki 1only ) were lethal at the embryonic stage and we even determined by using the null allele of yki, i.e. yki B5 that the flies were lethal due to the mutation that we created. However for an unknown reason we do not detect expression of neither Yki1 protein nor yki1 mRNA in these flies. Therefore this allele is not a true yki 1only allele but a null.

After another attempt we were able to generate Yki2 isoform expressing flies and here to we found out that it contains a small deletion of 4 amino acid residues at the repair site in exon 4, thus we named the allele as yki 2-only and nevertheless we decided to characterize these flies and in the meantime we worked on new strategy to create good yki 2-only flies without. We reasoned that such a small mutation might not change the behavior of this isoform completely. We discovered that homozygous yki 2-only / yki 2-only flies were viable without any visible phenotype. What was surprising for us to find that yki 2-only / yki 2- only flies depicted 50% lethality which we did not expect given the flies had no visible phenotype (Figure 38). Could it be due to the lack of Yki1 which is absolutely required at the early stage of development? Alternatively, could it be an issue of parental fertility which is affected? Or is it merely due to the modification of the Yki2 peptide due to the minor mutation?

Appendix Another argument that we tried to investigate was if the lack of phenotype in adult flies was is simply because in normal developmental conditions the Yki1 isoform has less activity, but in case of stress, it increases to counteract the severity of Yki2's transcriptional output.

Indeed [START_REF] Di Cara | The Hippo pathway promotes cell survival in response to chemical stress[END_REF] showed the loss of Hippo pathway component during stress

GMR>UAS-Yki1

S168A GMR>UAS-Yki2, ,GMR>UAS-Yki2, ey>UAS-Yki2 S168A GMR>UAS-Yki2 S168A did not yield any different phenotype.

Role of B52 in cell competition:

To further explore the link between B52 and cell competition, we generated the B52 mutant clones by mitotic recombination in wing imaginal disc. Interestingly we observed that B52 -/-clones are eliminated over time via apoptosis. This could be due to the phenomenon of cell competition (Appendix-Figure 40 a). This phenomenon was first described by the analysis of a dominant mutation in a Drosophila Minute (M) gene that affects ribosomal biogenesis and therefore slows down translation and protein synthesis.

While homozygous mutations (M/M) of these genes are cell lethal, heterozygous (M/+) adults are viable and fertile, although they suffer a developmental delay.

Interestingly, clones of heterozygous cells (M/+) growing in a wild type wing disc were not underrepresented in the adult wing, as expected, but completely eliminated [START_REF] Morata | Minutes: Mutants of Drosophila autonomously affecting cell division rate[END_REF]. The eliminated M/+ cells are referred to as "looser cells," and surrounding wild type cells are referred to as "winner cells" (Appendix-Figure 40b). The elimination induces the compensatory proliferation of winner cells, and thus the organ retains the correct size. We induced, B52 -/-mutant clones in disc where the surrounding cells have a mutation in the minute gene (M/-) and we observed no elimination suggesting that indeed the elimination was due to cell competition. Thus cell competition is a context-dependent phenomenon since the looser cells survive when surrounded by looser cells and gets eliminated when surround by winner cells (Appendix Figure 40c). Since we observed the B52 -/-clones were eliminated via apoptosis, we wondered if we could rescue these clone by co-overexpression of caspase inhibitor. To realize this, we used MARCM technique [START_REF] Luo | A protocol for mosaic analysis with a repressible cell marker (Marcm) in drosophila[END_REF]. Indeed we observed partial rescue of B52 mutant clones via expression of P35, a caspase inhibitor and Flag-B52 (Appendix Figure 41). According to literature several pathways are involved in cell competition including Hippo pathway [START_REF] Tyler | Genes affecting cell competition in drosophila[END_REF], cell polarity [START_REF] Agrawal | Neoplastic transformation and aberrant cellcell interactions in genetic mosaics of lethal(2)giant larvae (lgl), a tumor suppressor gene of Drosophila[END_REF] [START_REF] Brumby | scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila[END_REF]) [START_REF] Woods | The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions[END_REF], JAK/STAT [START_REF] Rodrigues | Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis[END_REF] Dpp signaling [START_REF] Moreno | Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development[END_REF]. In one of our primary analysis of RNAseq data by (Bradley et al., 2015) [START_REF] Brooks | Regulation of alternative splicing in Drosophila by 56 RNA binding proteins[END_REF] we saw that indeed B52 alters the splicing of the yki, dlg and STAT92E.

First, we checked the overexpression of Yki2 in B52 mutant clones via MARCM technique, the idea was to be able to rescue the B52 mutant clones from getting eliminated because if B52 depletion leads to more production of Yki1 isoform the maybe overexpressing would prevent B52 mutant clones from elimination. However, to this end, we did not see any rescue (Appendix Figure 42).

Appendix Figure 42 : Rescue of B52 mutants by Yki1 and Yki2:

Just by expressing Yki1 or Yki2 the elimination of B52 mutant clones cannot be rescued efficiently.

Abstract:

The Hippo pathway is a conserved pathway involved in tissue growth and tumor suppression.

Studies have demonstrated its significance in the development of human cancers. This cascade controls the activity of the transcription co-activator Yorkie (Yki) in flies and Yes-associated protein (YAP) in mammals. Due to Alternative Splicing (AS), both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains. Since WW domains are essential for interaction with specific partners, the alternative inclusion of this domain in Yki/YAP protein may remodel their interaction network and therefore their activity.