pas le cas alors veuillez m'en excusez, mon étourderie est a blâmer.

First and foremost, I want to thank all the members of my jury, Satoko Yoshizawa, Stéphane Thore, Bruno Sargueil, Jean Cavarelli and Eric Westhof, who agreed to judge my thesis.

Tout d'abord je souhaite remercier les membres de mon jury, Satoko Yoshizawa, Stéphane Thore, Bruno Sargueil, Jean Cavarelli et Eric Westhof, qui ont accepté de juger mes travaux de thèse.

Mes sincères remerciements sont naturellement adressés à Benoît Masquida, mon directeur de thèse, qui a su en trois années me transmettre son intérêt pour la recherche, son expérience du microcosme de la science et bien sur ses connaissances en cristallographie et analyse des structures tridimensionnelles d'ARN. Mes meilleurs souvenirs seront sans aucun doute nos expéditions aux synchrotrons. Merci beaucoup ! Je tiens à remercier Eric Westhof pour m'avoir accueilli au sein de son équipe et nous avoir permis de poursuivre le projet du Lariat-Capping ribozyme mais aussi pour m'avoir permis de participer à deux éditions du RNA meeting.

Je remercie également Ivan Tarassov et Nina Entelis qui nous ont très chaleureusement accueilli et très vite intégré à la vie de leur laboratoire au sein duquel j'ai rafraîchi mes connaissances sur la mitochondrie et je me suis initiée au russe.

Merci à Hubert Becker, pour ses conseils avisés avant et pendant ma thèse mais aussi pour les pauses café ! Merci à toute son équipe pour les discussions enrichissantes que nous avons partagés et tous les p'tits coups de pouces logistiques.

Merci à Catherine Florentz qui à su me guider au moment de choisir ma thèse et m'a motivée pour prendre part à la vie de l'école doctorale des sciences de la vie et de la santé. Cette expérience a été très enrichissante. Merci à notre collaborateur, Henrik Nielsen, qui travaille depuis le début sur le projet du Lariat-Capping ribozyme et qui a produit les données de mutagénèse évoquées dans ce manuscrit. Merci aussi à Steinar Johansen qui, le premier, a observé le Lariat-Capping ribozyme.

Merci à Laurence Drouard et Michel Labouesse pour m'avoir offert la possibilité de participer à la mission de diffusion de l'information scientifique et technique « OpenLAB » deux années consécutives. Merci à Joern

Merci à Pascale Legault qui a encadré mon stage de Master 2 à l'université de Montréal lors duquel je me suis initiée aux techniques de production et de purification d'ARN à grande échelle et ai appris les rudiments de la RMN.

A l'IBMC je souhaite remercier toutes les personnes que j'ai côtoyées et avec qui j'ai travaillées pendant ces trois années. Merci pour vos conseils, vos coups de main, votre soutien. Merci à Laure et Delphine pour leur aide technique.

A l'IPCB je souhaite remercier toutes les personnes du 4 ème étage pour les moments de détente et de convivialité que vous avez organisé. Merci aussi pour votre aide lors du déménagement. Je n'ai passé que quelques mois avec vous mais j'en garderai un très bon souvenir.

Un grand merci à mes collègues doctorants avec qui j'ai partagé les hauts et les bas des manips, les congrès, les pique-niques, la rédaction… Je souhaite remercier mes amis, Anne, Benoît, Cécile, Delphine, Emilie, Franzi, Gilles, Ludovic, Marie, Morgan, Vincent vous avez tous, chacun à votre manière contribués au bon déroulement de cette thèse.

Enfin, vous arrivez en dernier, mais nous n'êtes pas les moins important, merci à tous les membres de ma famille. Plus particulièrement à mes parents qui m'ont soutenus tout au long de ses dix années d'études supérieures et m'ont permis de partir à Leeds et à Montréal ! Merci à toi Anne-Laure ma petite soeur pour tous les moments de complicité que nous partageons. Merci à toi Alexandre qui contribue quotidiennement à mon épanouissement. 1. Introduction.......................................................................................................................................................... 121 2. Article 2 .................................................................................................................................................................. 122 3. Additional results ............................................................................................................................................... 162 4. Discussion .............................................................................................................................................................. 164 B. THE . Introduction.......................................................................................................................................................... 168 2. Article 3 .................................................................................................................................................................. 170 3. Discussion .............................................................................................................................................................. 185 IV

INTRODUCTION

I. INTRODUCTION

Ribonucleic acid is a large family of biological molecules essential to most processes of life. RNA is with DNA and protein one prop on the central dogma of molecular biology. The continuously increasing number of data on genomics, transcriptomics and proteomics indicates that if most eukaryotic genomes are nearly 100% transcribed only about 2 to 10% is translated into protein [START_REF] Jackson | The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells[END_REF].

Hence, most of the RNA in cells is non-coding RNA. This ncRNA has been considered as junk for a long time but now we know that by various mechanisms it can subtly tune gene expression.

In 1989 the Nobel Price in Chemistry was awarded to Thomas R Cech and Sydney Altman for their discovery of the catalytic properties of RNA [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF][START_REF] Kruger | Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena[END_REF]. These catalytic RNA are called ribozymes (ribonucleic acid enzymes) and among all non-coding RNA, ribozymes are the ones I have been interested in for my thesis work.

Naturally occurring ribozymes are divided into two sub-groups. The small ribozymes group includes the hammerhead, hairpin, Varkud satellite, hepatitis delta virus and glmS ribozymes. The second group of ribozymes encompasses group I and II introns, ribonuclease P, the ribosome and the Lariat-Capping ribozyme (LCrz).

All, but the VS ribozyme and LCrz crystal structure have been solved in the last ten years. Nicolai Suslov from the team of Jo Piccirilli, presented the VS ribozyme 3D structure for the first time in November 2012 . In this manuscript I will describe the 2.5 Å crystal structure of the DiLCrz ribozyme, which we have solved in our laboratory during the last couple of years.

To obtain the 3D structure of DiLCrz, our method of choice has been crystallography. We confirmed the unexpected 3D structure of DiLCrz with smallangle x-ray scattering experiments.

We have also been interested in the role in folding of RNA structural motifs through the study of a motif we have identified in the bacterial RNase P crystal structure [START_REF] Reiter | Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA[END_REF], the pk-turn [START_REF] Meyer | A structural module in RNase P expands the variety of RNA kinks[END_REF]. RNA 3D structures are mainly organized around A-form helices interspersed with single-stranded junctions.

These single-stranded junctions orient the Watson-Crick helices by forming complex motifs or modules defined by a consensus sequence and a set of WC and non-WC base pairs (Leontis & Westhof, 2002) and/or architectural features. Therefore, RNA motifs can be predicted from the secondary structure (Jossinet et al, 2010). However, the RNase P and the LCrz crystal structures show that depending on intramolecular tertiary or intermolecular interactions, RNA modules can adopt unexpected motifs.

A. RIBOZYMES IN THE RNA WORLD

The RNA world as suggested by [START_REF] Gilbert | Origin of life: The RNA world[END_REF][START_REF] Gilbert | Origin of life: The RNA world[END_REF] has been mostly supported by the first observation of catalytic RNAs [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF][START_REF] Kruger | Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena[END_REF] and assumed that RNA could have been the first macromolecule of life as it is at the same time a support for the genetic information and a catalyst. In vitro experiments even demonstrated that it is possible to transcribe an RNA template with an RNA polymerase ribozyme [START_REF] Wochner | Ribozyme-catalyzed transcription of an active ribozyme[END_REF].The RNA world hypothesis can be seen as a solution to the chicken and egg problem of what between DNA or protein was first [START_REF] Cech | The RNA worlds in context[END_REF][START_REF] Lehman | RNA in evolution[END_REF].

But how did DNA and protein arise from the RNA world? This question has not been answered yet [START_REF] Dworkin | The roads to and from the RNA world[END_REF] and if the hypothesis of the RNA world in the sense of RNA being the first macromolecule is based on an intellectual hypothesis, it contains some limitations. Primitive conditions on earth might have been favorable for nucleotides to appear [START_REF] Powner | Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions[END_REF] but not ideal for RNA macromolecules to thrive [START_REF] Kawamura | Drawbacks of the ancient RNA-based life-like system under primitive earth conditions[END_REF]. It seems that macromolecules (DNA, RNA and protein) more likely arise from a prebiotic soup in which RNA and peptides would have coexisted. This leads to the alternative hypothesis that RNA catalysts, protein enzymes and modern RNPs are offspring of ancestral RNPs [START_REF] Cech | Crawling out of the RNA world[END_REF].

One good example supporting the "RNP world" is RNase P that performs 5' maturation of pre-tRNA. RNase P are present in the three kingdoms of life and have structurally related RNA subunits, but different protein entities indicating that at least the RNAs should have been present in LUCA [START_REF] Evans | RNase P: interface of the RNA and protein worlds[END_REF]. Bacterial RNase P are ribozymes as their RNA entity is capable of cleaving the 5' leader sequence of pre-tRNA accurately without any protein help in vitro [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF].

However in vivo, bacterial RNA P is associated to proteins, which stabilize the RNA entity into an active conformation. With time, some new proteins appear in RNase P of eukaryotes and Archaea sharing more of the catalytic properties with its RNA component [START_REF] Evans | RNase P: interface of the RNA and protein worlds[END_REF]) [START_REF] Walker | Ribonuclease P: the evolution of an ancient RNA enzyme[END_REF]. Interestingly protein only

RNase P have been found in organelles of some eukaryotes [START_REF] Gutmann | PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis[END_REF]) [START_REF] Holzmann | RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme[END_REF] but their protein components seem not to be evolutionary related to the protein counterparts of RNP RNase P. From an "RNP world" and depending on selective pressure and macromolecules specificity, evolution would have then chosen different roads to catalyse the same reaction.

Nevertheless, if RNA might not be "THE" first macromolecule of life it has a major and indispensible role in modern biology. Indeed RNA is the obligatory intermediate between DNA and proteins but it is not, from far, its only mission in cells [START_REF] Kapranov | Genome-wide transcription and the implications for genomic organization[END_REF]. With the example of RNase P we already understand that RNAs combined with proteins in RNP fulfill diverse processes. These processes range from pre-RNA maturation [START_REF] Green | Pre-mRNA splicing[END_REF], protein synthesis [START_REF] Ban | The complete atomic structure of the large ribosomal subunit at 2.4 A resolution[END_REF]) [START_REF] Nissen | The structural basis of ribosome activity in peptide bond synthesis[END_REF] to telomerase activity [START_REF] Miller | Telomerase recognizes its template by using an adjacent RNA motif[END_REF] [START_REF] Qiao | Triple-helix structure in telomerase RNA contributes to catalysis[END_REF] and recognition of the signal sequence of secreted proteins [START_REF] Walter | Protein translocation across the endoplasmic reticulum[END_REF]. ncRNAs can by various ways finely regulate gene expression (Barrett et al, 2012) [START_REF] Moreira | Unscrambling genetic information at the RNA level[END_REF] for example in the case of siRNA and microRNA that by base pairing to mRNAs regulate their life span and availability for translation [START_REF] Joshua-Tor | Ancestral roles of small RNAs: an Ago-centric perspective[END_REF]. Introns interrupting pre-tRNA, pre-rRNA and pre-mRNA are of different kinds and regulate gene expression via splicing. A more detailed description of introns is made in I.A.

NcRNA also encompass ribozymes. Ribozymes are ribonucleic acid enzymes that, for naturally occurring ones, can be indexed in two sub-groups. The small ribozymes group includes the hammerhead, hairpin, Varkud satellite, hepatitis delta virus and glmS ribozymes. The second group of ribozymes encompass group I, II and III introns, ribonuclease P, the ribosome and the lariat-capping ribozyme.

Lariat-Capping ribozymes

Lariat-capping ribozymes are a new family of ribozymes on which I focused during my thesis. LCrz, initially named GIR1 for Group-I-like ribozyme 1, have been renamed as neither their 3D structure nor their branching reaction supports their original name.

a)

The lariat-capping ribozymes in twin-ribozyme introns LCrz has been observed for the first time in the myxomycete Dydimium iridis [START_REF] Johansen | An intron in the nuclear ribosomal DNA of Didymium iridis codes for a group I ribozyme and a novel ribozyme that cooperate in self-splicing[END_REF] where it is inserted in the apical loop of the P2 stem of DiGIR2 a canonical group IE intron. DiLCrz is followed by a homing endonuclease gene (HEG) which itself contains a spliceosomal intron, I51 (Figure 1). The 1.4 kb twin-ribozyme intron is inserted at position 956 (E. coli SSU rDNA numbering system) in the SSU of the rDNA. Therefore the twin-ribozyme intron is named Dir.S956-1 [START_REF] Johansen | A new nomenclature of group I introns in ribosomal DNA[END_REF]. Later, LCrz has been found in 29 species of the amoeboflagellate genus Naegleria [START_REF] Einvik | Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing[END_REF] where the 1.2 kb twin-ribozyme intron (or twintron) is inserted at position 516. In Nae.S516 [START_REF] Johansen | A new nomenclature of group I introns in ribosomal DNA[END_REF], NaGIR2 belongs to the group IC1 introns, LCrz is inserted in the apical loop of the P6 stem of GIR2 and HEG do not contain a spliceosomal intron (Table 1).

b) Processing pathways of twin-ribozyme introns

Processing pathways of the twin-ribozyme introns are tightly connected to the host life cycle (Figure 3). The first and predominant pathway occurs when conditions of growth are optimal for the amoeba or flagellate at the haploid stages (Figure 4).

This pathway is the self-splicing pathway during which the GIR2 intron is responsible for the twin-ribozyme excision and exon ligation, thus initiating the 18S pre-rRNA maturation. Once the intron is spliced out, LCrz can catalyse its branching reaction releasing the downstream HE pre-mRNA capped by a lariat of three nucleotides (Nielsen et al, 2005;[START_REF] Pyle | Capping by branching: a new ribozyme makes tiny lariats[END_REF]. The 5' three nucleotides lariat substitutes for the conventional m 7 G cap of mRNA. The 5' capped HE pre-mRNA is then cleaved at IPS before being polyadenylated at the 3' end. Last step is the removal of I51 spliceosomal intron of the mRNA (Figure 3) (Vader et al, 1999). No IPS3 is detected for Nae.S516 [START_REF] Decatur | Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme[END_REF]. This pathway benefits to the host as well as to the intron in the sense that mature 18S rRNA and mature HE mRNA are produced.

The second processing pathway leads to the formation of a circular twinribozyme intron in which LCrz is inactive. This circularization pathway is solely mediated by GIR2 and is a general feature of group I introns [START_REF] Nielsen | The ability to form full-length intron RNA circles is a general property of nuclear group I introns[END_REF].

Reopening of the circle seems to be required for LCrz branching reaction to occur.

This pathway is clearly detrimental to the host because exons are not ligated, but favourable to the intron as the FLC has been suggested to promote group I intron mobility [START_REF] Birgisdottir | Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA[END_REF]. FLC are especially abundant after heat shock (unpublished data) indicating that FLC is biologically significant (Figure 3).

The last pathway occurs under starvation conditions that lead to D. iridis encystement. In this pathway, LCrz only is active, leading to the release of a 7.5 kb RNA product cap with the three nucleotides lariat and un-ligated exons (Vader et al, 2002) (Figure 3). The biological relevance of this pathway has not been clarified yet, but it could be a way for the host to down-regulate the level of rRNA expression during starvation while having a stock of HE mRNA ready to be translated upon improvement of growing conditions.

Looking at these three pathways, it is clear that within the twin-intron ribozyme, GIR2 and LCrz actions have to be coordinated. Indeed, LCrz is fully transcribed before GIR2 but should not be active before the latter. Therefore LCrz should adopt an alternative inactive conformation during splicing and FLC pathways to prevent early cleavage of the intron that corresponds to the starvation-induced pathway. How do LCrz and GIR2 coordinate their action? We have no clear evidence yet for RNA-RNA or protein-RNA interactions that could be responsible of this regulation. Spores will be disseminated by the wind and will germinate into new haploid cells (5).

Depending on environmental conditions these new haploid cells will either grow as flagellates, amoeba or cysts (6).

c) Lariat-capping ribozymes' secondary structures

LCrz secondary structure has been determined by comparative sequence analysis, chemical and enzymatic probing and site directed mutagenesis (Einvik et al, 1998b). The secondary structure of LCrz is closely related to the one of group I introns (Figure 5). Thus, helical stems of LCrz are designated as for group I introns.

The secondary structure of the core of LCrz is organized in three domains: ( 1 The critical role of flanking regions of the LCrz for its branching activity has been demonstrated by deletion analysis of the 5' and 3' ends [START_REF] Einvik | Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme[END_REF][START_REF] Jabri | Kinetic and secondary structure analysis of Naegleria andersoni GIR1, a group I ribozyme whose putative biological function is site-specific hydrolysis[END_REF]. One important property of DP2 is that this stem can dissociate to allow the 3' strand to adopt an alternative hairpin fold composed of nucleotides from the 5' end of the downstream HE pre-mRNA. This alternative fold is named HEG P1 and has been observed for the first time in the 5' UTR of the HE pre-mRNA (Vader et al, 1999). HEG P1 is also observed in the pre-catalytic form of DiLCrz [START_REF] Nielsen | A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA[END_REF] or in the post-clivage state of DiLCrz [START_REF] Birgisdottir | Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA[END_REF] were it is involved in the release of the HE pre-mRNA. DP2 and HEG P1 are mutually exclusive structures and present only in the active and inactive states respectively (Figure 6).

The mechanism by which the on and off switch of LCrz is mediated remains to be elucidated.

Group I introns

Group I introns are ribozymes mainly found in the nucleolar rDNA and in mRNA, tRNA and rRNA from organelles of lower eukaryotes. Less than 4% of the identified group I introns are located in prokaryotes and none have been seen so far in Archaea [START_REF] Haugen | The natural history of group I introns[END_REF]. The size of group I ribozyme ranges from ~250 to ~500 nucleotides. Group I introns catalyse a two steps self-splicing event allowing the ligation of the 5' and the 3' exons and the release of the intron [START_REF] Herschlag | Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site[END_REF]. The spliced-out intron can further form a long full length circle (Vicens & Cech, 2009) or truncated circles [START_REF] Tanner | Activity and thermostability of the small self-splicing group I intron in the pre-tRNA(lle) of the purple bacterium Azoarcus[END_REF][START_REF] Zaug | Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction[END_REF]. However group I introns can also undertake the so-called "circularization pathway" that leads to the formation of a full length circle intron but unligated exons [START_REF] Nielsen | The ability to form full-length intron RNA circles is a general property of nuclear group I introns[END_REF][START_REF] Zaug | Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction[END_REF]) (Figure 8). [START_REF] Cech | Crawling out of the RNA world[END_REF]. The circularization pathway leading to unligated exons and formation of a full lenth circularized intron result from a hydrolytic cleavage at the 3' SS ( 6) followed by a transesterification reaction initiated by G (7).

Some group I introns contain ORF encoding homing endonuclease (HE). One role of the HE is cognate intron insertion at DNA level by homing in an intron-less allele at a homologous site via the double strand break repair pathway [START_REF] Belfort | Mechanisms of intron mobility[END_REF][START_REF] Dujon | Group I introns as mobile genetic elements: facts and mechanistic speculations--a review[END_REF]. Evolution of group I introns and their associated HE are correlated as suggested by Goddart and Burt [START_REF] Goddard | Recurrent invasion and extinction of a selfish gene[END_REF]) (Figure 9).

Yet, HEs have two ways to escape the Goddart-Burt cycle. The first one is HEG mobility alone [START_REF] Haugen | The natural history of group I introns[END_REF] and the second one is to acquire new function such as maturase activity [START_REF] Chatterjee | Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease[END_REF]. Indeed, HEs having maturase activity are helpful for the folding of its cognate group I intron into its active conformation, therefore improving self-splicing [START_REF] Belfort | Two for the price of one: a bifunctional intron-encoded DNA endonuclease-RNA maturase[END_REF]. A way for group I introns to escape the Goddart-Burt cycle is to promote their own mobility at RNA level by reverse splicing in homologous and heterologous sites [START_REF] Roman | Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron[END_REF][START_REF] Roman | Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA[END_REF][START_REF] Roman | Integration of the Tetrahymena group I intron into bacterial rRNA by reverse splicing in vivo[END_REF][START_REF] Woodson | Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition[END_REF] (Figure 10). Interestingly, FLC could also allow intron reverse splicing [START_REF] Nielsen | The ability to form full-length intron RNA circles is a general property of nuclear group I introns[END_REF]. Due to group I intron mobility, twintrons are observed when a group I intron is inserted into a pre-existing group I intron (Einvik et al, 1998a;[START_REF] Hafez | The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins[END_REF].

Likewise, HEs can acquire maturase activity not to be lost in the course of evolution, the second group I intron in a twintron could evolve to gain new functions e.g.

branching. As mentioned above, some group I introns require the action of a maturase to fold in their active conformation [START_REF] Russell | RNA misfolding and the action of chaperones[END_REF]. This dependence of the ribozyme toward proteins for folding can be ordered in three categories: [START_REF] Beckert | Synthesis of RNA by in vitro transcription[END_REF] self-splicing allribozyme introns e.g. Tetrahymena, (2) host-factor-dependent optional introns e.g.

S1389 in

Diderma rDNA [START_REF] Wikmark | The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes[END_REF], ( 3) host-factor-dependent obligatory introns e.g. ND5-717 intron in hexacoral mitochondrial genomes [START_REF] Emblem | Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a longterm evolving group I intron[END_REF]. Host-dependent introns often show extended peripheral loop regions [START_REF] Wikmark | The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes[END_REF]. Additionally, the level of dependence of the group I intron toward proteins seems to be correlated with its GC content (Vicens et al, 2008). If the group I intron in need of a protein co-factor does not encode a maturase it will hijack proteins from the host organism (Lambowitz & Caprara, 1999).

Noteworthy, group I intron sequence can be split into stretches of nucleotides located at different position in the genome with a preserved splicing activity.

Examples of composite group I introns capable of trans-splicing are all found in the cox1 gene of mitochondria [START_REF] Burger | Group I-intron trans-splicing and mRNA editing in the mitochondria of placozoan animals[END_REF][START_REF] Nadimi | Group I intron-mediated transsplicing in mitochondria of Gigaspora rosea and a robust phylogenetic affiliation of arbuscular mycorrhizal fungi with Mortierellales[END_REF]. Interestingly this ability of group I intron can be used as a molecular biology tool to perform splicing of genes in bacteria (work presented at the RNA meeting 2013 by Ulrich Müller).

a)

Group I introns' secondary structure Group I introns vary considerably in length, sequence and catalytic efficiency nevertheless they all retain a consensus secondary structure composed of helical domains, numbered P1 through P10, and single stranded junction and/or non-WC base-pairs connecting these helical domains [START_REF] Cech | Representation of the secondary and tertiary structure of group I introns[END_REF][START_REF] Davies | Making ends meet: a model for RNA splicing in fungal mitochondria[END_REF][START_REF] Michel | Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure[END_REF]. Domains P1 and P2 compose the substrate domain in which P1 is formed by the 5' end of the ribozyme containing the IGS and the 3' end of the 5' exon. At the top of P1 there is a highly conserved G.U wobble base pair [START_REF] Michel | Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis[END_REF]. The P3-P9 domain is the catalytic domain. It encloses P3, P7, P8 and P9 and is interrupted by the stabilization domain P4-P6 [START_REF] Kim | Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena[END_REF][START_REF] Murphy | An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme[END_REF]. The G-binding pocket accommodating exoG and ωG afterwards (detailed catalytic mechanism is described in I.B.1.b) sits at the top of P7 [START_REF] Michel | Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis[END_REF]) (Figure 11). P10 is a helix formed after the first step of splicing by the 5' end of P1 and the 5' end of the 3' exon.

Figure 11:

Secondary structure diagram of group I introns as proposed by Cech [START_REF] Cech | Representation of the secondary and tertiary structure of group I introns[END_REF]. Only conserved nucleotides [START_REF] Michel | Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis[END_REF] are shown in letters. Stems are colored in the same color as the one of their similar stem in LCrz (see Figure 5). Arrows indicated insertion sites for additional peripheral domains.

Group I introns can contain additional domains budding from terminal or internal loops of helical domains as shown in Figure 11. Additional domain and covariation analysis led to the classification of group I introns into 13 subgroups [START_REF] Michel | Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis[END_REF]. Peripheral domains enhance the stability of the folded ribozyme and/or help the folding of the ribozyme in an active conformation [START_REF] Doudna | Miniribozymes, small derivatives of the sunY intron, are catalytically active[END_REF]van der Horst et al, 1991).

b)

Group I introns' three-dimensional structure Crystal structures of three different group I introns from three diverse origins in three distinct chemical states are available to appreciate group I introns 3D structure.

The first one is the group IC1 intron located in rRNA of Tetrahymena trapped in a pre-catalytic state in which P1 is not present due to the construct design [START_REF] Guo | Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site[END_REF]) (Figure 12A). To be able to crystallize this construct the Cech lab performed in vitro selection for more thermostable ribozymes than the wild-type one [START_REF] Guo | Evolution of Tetrahymena ribozyme mutants with increased structural stability[END_REF]. The second crystal structure is the one of the bacteriophage Twort group IA2 intron [START_REF] Landthaler | Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes[END_REF]. In this crystal structure, P1 is formed by addition in trans of a four nucleotides product analogue, the G.U wobble base pair at the top of P1 is formed and ωG is in the G-binding pocket of P7 [START_REF] Golden | Crystal structure of a phage Twort group I ribozymeproduct complex[END_REF] (Figure 12B). The last group I intron that has been crystallized is the one of the tRNA Ile from Azoarcus [START_REF] Adams | Crystal structure of a selfsplicing group I intron with both exons[END_REF]. It belongs to the subgroup IC3 and it has been engineered to mimic a transition state just after the first step of self-splicing but right before the second step occurs. Therefore this structure is of particular interest for our work as P10 and P1 are formed and ωG lies into its binding pocket (Figure 12C).

These three features allow a fairly straightforward comparison with LCrz crystal structure (see results section, manuscript submitted). All features of tertiary structures of the three group I introns have been described and compared in a book chapter [START_REF] Golden | Group I Introns : Biochemical and Christallographic Characterization of the Active Site Structure[END_REF] and I will only describe some details used for further comparison with LCrz (Figure 13). The G-binding pocket has been identified biochemically using mutant of the group I intron from Tetrahymena and analogues of guanosine (Michel et al, 1989a).

In the three crystal structures and as predicted ωG is forming a base triple interaction with the second and highly conserved G.C base pair of P7. On the top of ωG stacks a single bulged adenosine forming an additional base triple with the first base pair of P7. The third (A-U) base pair of P7 forms as well a base triple interaction with the adenosine of the J6/7 junction. Thus the adenosine of J6/7 tethers the stabilization domain to the catalytic domain but it also shares an essential hydrogen bond between its 2'-hydroxyl group and the 2'hydroxyl and N3 of ωG (Bass & Cech, 1986;[START_REF] Ortoleva-Donnelly | The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme[END_REF]. The G-binding pocket is thus composed of three consecutives base triples (Figure 14). wobble base pair of P1 [START_REF] Strobel | Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site[END_REF]) (Figure 15A). J3/4, J6/6a and J6/7 are tethering P4-P6 to P3-P9 domain (Figure 15B). J2/3 and J8/7 are single stranded junctions important for the recognition of the 5'SS as their nucleotides interact with the minor groove of P1 hence facilitating packing of P1-P2 against P3-P9 domain [START_REF] Pyle | RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme[END_REF][START_REF] Szewczak | A minor groove RNA triple helix within the catalytic core of a group I intron[END_REF]) (Figure 16).

c) Group I introns and the lariat-capping ribozymes

Group I introns and LCrz are tightly related since their sequence and secondary structure are very similar (Figure 5). Hence, the first name given to LCrz was Group-I-like-intron ribozyme 1. As mentioned in I.A.1.a, LCrz are parts of twintrons located in the SSU rDNA of three species of eukaryotes (Einvik et al, 1998a). Very interestingly, one group I twintron has been described recently in the mtDNA at position mS917 in gene encoding the mitochondrial small ribosomal subunit of several species of fungi [START_REF] Hafez | The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins[END_REF]. However LCrz are not catalyzing splicing, but branching in order to cap the downstream HE pre-mRNA with a lariat of three nucleotides (Nielsen et al, 2005). Moreover, comparison of the threedimensional structures of group I introns and DiLCrz shows some important differences (see Results section). Consequently, it is reasonable to suggest that LCrz come from a group I intron that has evolved to acquire new functions [START_REF] Beckert | Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes[END_REF].

Group II introns

Group II introns are large ribozymes, ~300 to ~1000 nucleotides, observed in about 25% of bacteria [START_REF] Dai | Compilation and analysis of group II intron insertions in bacterial genomes: evidence for retroelement behavior[END_REF] as well as in mitochondrial and chloroplast genomes of lower eukaryotes, higher plants (Dai et al, 2003) and archaea [START_REF] Rest | Retroids in archaea: phylogeny and lateral origins[END_REF] and more recently in an annelid worm (Valles et al, 2008). As for group I introns they have to splice out to allow 5' and 3' exons of a given gene to be ligated. Most group II introns enclose an intron encoded protein essential for in vivo self-splicing. Indeed, if in vitro, group II introns are capable of self-splicing it is in salt conditions far from cellular concentration [START_REF] Daniels | Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products[END_REF][START_REF] Jarrell | Group II intron selfsplicing. Alternative reaction conditions yield novel products[END_REF].

The cognate IEP acts as a maturase, which helps the RNA to fold in its active conformation either by stabilizing its active fold or by resolving non-native structures that constitute a kinetic trap [START_REF] Lambowitz | Introns as mobile genetic elements[END_REF][START_REF] Wank | A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA[END_REF]. Group II introns that do not contain ORFs or encode degenerated IEPs rely, like group I introns, on some of the host proteins to act as chaperones (Lambowitz et al, 1999).

Group II introns are not only self-splicing element. They are mobile genetic elements that are capable of insertion in DNA. Once again, for this retrohoming by reverse-splicing event, group II introns rely on their IEPs [START_REF] Meunier | Group II intron stranspose in yeast mitochondria. Structure, function and biogenesis of energy transfer systems[END_REF]. On top of their maturase activity IEPs are reverse transcriptases. After the RNA intron self-splicing the IEP stays bound to it and this RNP complex recognises the DNA to invade. The RNA intron uses its ribozyme activity for reverse splicing and the RT IEP is in charge of transcription of the inserted RNA intron back to DNA (Lambowitz & Zimmerly, 2004).

As a result of retrohoming, one group II intron can be inserted into an already existing one to form the so-called twintron [START_REF] Copertino | Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns[END_REF]Dai & Zimmerly, 2003). Remarkably, depending on the insertion site of the second intron into the first one and to preserve self-splicing capability for both of them they have to adopt a concerted order of reaction [START_REF] Drager | A complex twintron is excised as four individual introns[END_REF][START_REF] Nakamura | Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA research : an international journal for rapid publication of reports on genes and genomes[END_REF].

Interestingly some introns and twintrons do not contain IEP and some IEP have been shown to act in trans on group II introns from unrelated genes (Dai & Zimmerly, 2003;[START_REF] Meng | An intron-encoded protein assists RNA splicing of multiple similar introns of different bacterial genes[END_REF].

Group II introns are not always encoded as one stretch of nucleotides. A functional group II intron, capable of self-splicing, can be reconstituted in trans with two or more separate transcripts [START_REF] Bonen | Trans-splicing of pre-mRNA in plants, animals, and protists[END_REF][START_REF] Glanz | Trans-splicing of organelle introns--a detour to continuous RNAs[END_REF]. This last feature combined with evidence for a similar splicing mechanism designate group II introns as ancestors of the eukaryotic spliceosome [START_REF] Cech | The generality of self-splicing RNA: relationship to nuclear mRNA splicing[END_REF][START_REF] Collins | The question remains: is the spliceosome a ribozyme?[END_REF][START_REF] Sharp | On the origin of RNA splicing and introns[END_REF].

a)

Group II introns' secondary structure Despite low sequence conservation, group II introns adopt a highly conserved secondary structure in which six helical domain (DI to DVI) are distributed around a large central junction [START_REF] Michel | Structure and activities of group II introns[END_REF]. Each helical domain has its own function. DI is the largest domain; it contains sequences for recognition of both exons and most recognition sites for long range tertiary interactions with the other domains [START_REF] Hamill | The receptor for branch-site docking within a group II intron active site[END_REF][START_REF] Su | An obligate intermediate along the slow folding pathway of a group II intron ribozyme[END_REF]van der Veen et al, 1987a). DI, DII, DIII, DV, DVI are implicated in the formation of the catalytic site and the two steps of selfsplicing [START_REF] Costa | Tight binding of the 5' exon to domain I of a group II selfsplicing intron requires completion of the intron active site[END_REF][START_REF] Fedorova | Domains 2 and 3 interact to form critical elements of the group II intron active site[END_REF][START_REF] Qin | The architectural organization and mechanistic function of group II intron structural elements[END_REF]. Nucleotides of the central junction and of DV plus the bulged adenosine of DVI are the most conserved residues of group II introns. DIV is the domain that contains the IEP ORF (Lambowitz & Zimmerly, 2004). Secondary structure of group II introns can be further subdivided into three subgroups namely IIA, IIB and IIC (Michel et al, 1989b;[START_REF] Toor | Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases[END_REF]) (Figure 17A).

b)

Group II introns' three-dimensional structure

In 2008 the Pyle lab has published the first crystal structures of a group IIC intron from the deep-sea bacterium Oceanobacillus iheyensis [START_REF] Toor | Crystal structure of a self-spliced group II intron[END_REF]. This

Group IIC intron has been chosen for crystallization because it is smaller than most studied group IIA and IIB introns. At present, seventeen crystal structures of the same group IIC spliced intron are available each of them displays different features of group II introns, which have been reviewed in detail this year [START_REF] Marcia | Now on display: a gallery of group II intron structures at different stages of catalysis[END_REF].

Succinctly, in the crystal structures, the DI domain wraps around the other domains to scaffold the ribozyme core. The DIV domain, herein replaced by a hairpin, points outward allowing for DIV to accommodate an ORF without impairing the intron folding. DV, which forms the catalytic site, is buried in the ribozyme core.

Unfortunately, in all available structures, the DVI domain containing the bulged adenosine is always missing due to hydrolysis [START_REF] Toor | Tertiary architecture of the Oceanobacillus iheyensis group II intron[END_REF]) (Figure 17B).

c)

Group II introns and the lariat-capping ribozymes

The common feature between group II introns and LCrz is the nature of the branching reaction which leads to the formation of a 2',5' phosphate bond closing an RNA lariat [START_REF] Pyle | Capping by branching: a new ribozyme makes tiny lariats[END_REF]) (Figure 23 This situation is similar to what is observed for small ribozymes (see I.A.5).

Ribonuclease P

RNase P is a large ribonucleoprotein complex observed in the three domains of life [START_REF] Woese | Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya[END_REF] and is composed of one catalytic RNA [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF]) and one or several proteins. The catalytic RNA is homologous in Bacteria, Archaea and Eukarya but the number of protein subunits varies [START_REF] Frank | Ribonuclease P: unity and diversity in a tRNA processing ribozyme[END_REF]. The bacterial RNase P has only one ~14kDa (12 to 15kDa) protein while archaeal RNase P counts four protein subunits and eukaryotic RNase P has from six to twelve protein subunits [START_REF] Hartmann | The enigma of ribonuclease P evolution[END_REF]. Interestingly and as mentioned in the introduction, proteinaceous RNase Ps (PRORP) exist. These PRORP are observed in mitochondria, chloroplasts and nucleus of Arabidopsis thaliana [START_REF] Gutmann | PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis[END_REF] as well as in human mitochondria [START_REF] Holzmann | RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme[END_REF]. However in this manuscript I will only discuss the bacterial RNA-based RNase P from type A [START_REF] Ellis | The RNase P family[END_REF].

The major RNase P role is to process the 5' leader sequence of pre-tRNAs [START_REF] Robertson | Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor[END_REF]. As a ribozyme, the RNA subunit of RNase P bears the catalytic activity [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF][START_REF] Kikovska | Eukaryotic RNase P RNA mediates cleavage in the absence of protein[END_REF][START_REF] Pannucci | RNase P RNAs from some Archaea are catalytically active[END_REF]. However the catalytic RNA dependence to protein subunits for efficient processing of the tRNA is increasing with the number of protein subunits in ribonucleoprotein complex. Noteworthy, RNase P is also responsible for, e.g., 4.5S processing (Peck-Miller & Altman, 1991), tmRNAs maturation [START_REF] Komine | A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli[END_REF] and cleavage of transient structures of E. coli and B. subtilis riboswitches [START_REF] Altman | RNase P cleaves transient structures in some riboswitches[END_REF].

a) RNase P's secondary structure

The secondary structure of the type A RNase P can be subdivided into two sets of nucleotides: (1) the nucleotides forming the highly conserved core of the RNase P RNA common to all five structural classes of RNase P and (2) the nucleotides forming additional domains specific of each structural class. Type A RNase P is present in Bacteria as well as in Archaea pointing to a probable common ancestor.

The core domain has been defined from comparative sequence analysis of RNase P RNA from phylogenetically diverse bacteria [START_REF] Haas | Evolutionary variation in bacterial RNase P RNAs[END_REF]. This minimal core domain has been proven to be active in vitro [START_REF] Siegel | Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA[END_REF]. It contains the five conserved regions where most of the highly conserved residues are observed and the A type helical domain P1, P2, P3, P4, P9, P10 and P11 (Figure 18). The secondary structure of this minimal core is shared by RNase P RNAs of the three domains of life and the conserved regions show high covariation rates. This domain is also designated as C-domain (catalytic domain) as it contains the residues responsible for catalysis. About half of the nucleotides of the RNase P RNA form additional domains that stabilise the core. Some of which are shared between A and B type bacterial RNase P [START_REF] Waugh | The design and catalytic properties of a simplified ribonuclease P RNA[END_REF]. This is the case for the loop of P15 implicated in the recognition of the 3'CCA tail of the pre-tRNA and the helices P7, P8, P9, P10

interacting with the T-loop of the substrate pre-tRNA [START_REF] Harris | Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA[END_REF][START_REF] Knap | Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors[END_REF]. One specific feature of the type A RNase P RNA is the P6 pseudoknot formed by base pairing between residues of the apical loop of P17 with the bulged nucleotides between P5 and P7 (Figure 19). Nucleotides downstream and upstream from the P7 stem define the S-domain (specificity domain) that is responsible for substrate recognition. The 3.8 Å resolution crystal structure of the RNase P holoenenzyme in complex with a tRNA has been solved recently [START_REF] Reiter | Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA[END_REF]. This first lowresolution structure confirms and details the interaction between the holoenzyme and the pre-tRNA. Indeed, nucleotides G19 and C56 from the pre-tRNA D and T loop interact with nucleotides A112 and G147 from the RNase P RNA, respectively. The 3'

ACCA 76 tail of the pre-tRNA is forming three WC base pairs with nucleotides in L15 of the RNase P RNA and one weak interaction is observed between A76 (pre-tRNA) and G253 (RNA from RNase P). The crystal structure reinforces the hypothesis that the protein subunit of RNase P has a role in recognition and positioning of the 5' leader sequence [START_REF] Koutmou | Proteinprecursor tRNA contact leads to sequence-specific recognition of 5' leaders by bacterial ribonuclease P[END_REF][START_REF] Niranjanakumari | Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA[END_REF]

Small ribozymes

Six small ribozyme families are constituted by the hammerhead [START_REF] Prody | Autolytic processing of dimeric plant virus satellite RNA[END_REF], hairpin [START_REF] Buzayan | Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA[END_REF], hepatitis delta virus [START_REF] Sharmeen | Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage[END_REF],

Varkud satellite [START_REF] Saville | A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria[END_REF], glmS [START_REF] Winkler | Control of gene expression by a natural metabolite-responsive ribozyme[END_REF] (Martick & Scott, 2006), HDV [START_REF] Ke | A conformational switch controls hepatitis delta virus ribozyme catalysis[END_REF] and glmS [START_REF] Cochrane | Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme[END_REF] ribozymes represented in cartoon mode. U1A spliceosomal protein used for crystallization in shown in grey.

a)

Biological roles of small ribozymes HMH and hairpin ribozymes have been observed for the first time in the sense and antisense strands of the satellite RNA of the tobacco ring spot virus and in RNA transcripts of avocado sunblotch viroid. HMH and hairpin are responsible for the separation into monomeric segments of the concatamer product resulting from the rolling-circle replication undergone by the satellite RNA [START_REF] Buzayan | Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA[END_REF][START_REF] Hutchins | Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid[END_REF][START_REF] Prody | Autolytic processing of dimeric plant virus satellite RNA[END_REF]. Likewise HDV and VS ribozymes have been found in the satellite RNA virus of human pathogen hepatitis B [START_REF] Sharmeen | Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage[END_REF] and mitochondrial genome of Neurospora crassa, respectively [START_REF] Saville | A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria[END_REF]. Both, HDV and VS ribozymes, cleave the concatameric RNA transcript in which they are inserted [START_REF] Kennell | The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid[END_REF]Perrotta & Been, 1991). But small-ribozymes are not restricted to satellite RNA. Indeed, the glmS ribozyme is located in the 5'UTR of the mRNA encoding the essential enzyme glucosamine-6phosphate (GlcN6P) synthase in some Gram-positive bacteria. glmS is not only a ribozyme but also a riboswitch that needs its co-factor, GlcN6P, to accomplish efficient self-cleavage [START_REF] Winkler | Control of gene expression by a natural metabolite-responsive ribozyme[END_REF]. Moreover, the increasing amount of genomic data and well-suited bioinformatic tools, show that small-ribozymes are ubiquitous in bacteria and eukaryotes [START_REF] Bourdeau | The distribution of RNA motifs in natural sequences[END_REF][START_REF] Ferbeyre | Schistosome satellite DNA encodes active hammerhead ribozymes[END_REF][START_REF] Martick | A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA[END_REF][START_REF] Webb | Widespread occurrence of selfcleaving ribozymes[END_REF].

b)

Small ribozymes and the lariat-capping ribozymes One common feature between hammerhead ribozyme and LCrz is the crucial importance of distal tertiary contacts having major effect on the chemical reaction catalysed by each ribozyme. In the case of the hammerhead ribozyme, a minimal version has been firstly proven active in vitro and crystallized (Pley et al, 1994;Uhlenbeck, 1987). However, twelve years later the crystal structure of a full-length hammerhead ribozyme revealed a tertiary interaction between the bulge of stem I and the apical loop of stem II (Martick & Scott, 2006) that improves cleavage by a 1000-fold in comparison to the minimal version of the hammerhead ribozyme (Canny et al, 2004). In the case of LCrz the peripheral domain DP2-DP2.1 is important as its conformation determines its on or off conformation (see I.B.1.a and Figure 24). In vitro, the reverse reaction of branching, ligation is observed and can completely mask the branching reaction [START_REF] Nielsen | A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA[END_REF]Nielsen et al, 2005).

LCrz can also catalyse irreversible hydrolysis at IPS (Decatur, 1995;[START_REF] Johansen | An intron in the nuclear ribosomal DNA of Didymium iridis codes for a group I ribozyme and a novel ribozyme that cooperate in self-splicing[END_REF]. Interestingly the length of the 5' and 3' flanking regions influence the ratio between the three LCrz-catalysed reactions [START_REF] Einvik | Flanking sequences with an essential role in hydrolysis of a self-cleaving group I-like ribozyme[END_REF][START_REF] Nielsen | A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA[END_REF]) (Figure 24). becomes the first nucleotide at the 5' end of the intron and the 3' end of the 5' exon is freed [START_REF] Cech | In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence[END_REF]Zaug & Cech, 1982). After a conformational rearrangement of the catalytic site to release exoG and accommodate ωG in the G-binding pocket the second step of self-splicing takes place [START_REF] Been | One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity[END_REF]. ωG is a conserved nucleotide of group I intron at its 3' end. The free 3' hydroxyl terminus of the 5' exon then attacks the scissile phosphate downstream from ωG leading to the ligation of the flanking exons and to the release of the intron with ωG at its 3' end (Figure 26). Metal ions surely play a role in RNA folding but also in catalysis by either triggering a nucleophilic attack, stabilizing a leaving group or organizing critical nucleotides in the catalytic site [START_REF] Ae | Metal ions: supporting actors in the playbook of small ribozymes[END_REF][START_REF] Steitz | A general two-metal-ion mechanism for catalytic RNA[END_REF]. In the case of group I introns, different biomolecular approaches allow for the identification of three magnesium ions in the active site of the Tetrahymena intron Does it mean that there are only two metal ions in the catalytic site of group I introns? It is not possible to rule out either model as, crystal structures give a snapshot of one specific state of the ribozyme when biochemical analysis in solution reflects more the malleability necessary to self-splicing of the ribozyme.

Self-splicing catalyzed by group II introns is, as for group I intron, a two steps process but the resulting products are different. The first step, also known as branching reaction, is described in I.B.1.a. The second step of splicing is the nucleophile attack of the 3' splice by the 3'OH terminus of the free 5' exon. This last step allows for the release of the lariat intron and ligated exons [START_REF] Daniels | Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products[END_REF][START_REF] Jarrell | Group II intron selfsplicing. Alternative reaction conditions yield novel products[END_REF]) (Figure 25 A). An alternative to this mechanism is the replacement in the first step of splicing of the adenosine of DVI by a water molecule.

Resulting products are ligated exons and a linear intron [START_REF] Jarrell | Group II intron selfsplicing. Alternative reaction conditions yield novel products[END_REF]van der Veen et al, 1987b) (Figure 25 B).

Recent crystal structures [START_REF] Chan | Crystal structure of a group II intron in the pre-catalytic state[END_REF][START_REF] Marcia | Visualizing group II intron catalysis through the stages of splicing[END_REF] and previous biochemical studies confirm that two magnesium and two potassium ions are implicated in the self-splicing reaction [START_REF] Gordon | Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis[END_REF][START_REF] Marcia | Visualizing group II intron catalysis through the stages of splicing[END_REF][START_REF] Sigel | Metal ion binding sites in a group II intron core[END_REF] (Figure 28). Moreover as group II introns have only one catalytic site it should undergo structural rearrangement to allow the two consecutive S N 2 reactions to happen [START_REF] Chanfreau | An RNA conformational change between the two chemical steps of group II self-splicing[END_REF][START_REF] Marcia | Now on display: a gallery of group II intron structures at different stages of catalysis[END_REF]. The transesterification 2'-O is common to all small-ribozymes however, the nucleotide that acts as a general base or a general acid is specific to each ribozyme.

Likewise the N and N+1 nucleotides are specific to one ribozyme. In every case, the reverse reaction, ligation, can be observed.

Hydrolysis

Hydrolysis is the reaction catalysed by RNase P that allows the release of a 5' phosphate mature tRNA and a 3' hydroxyl 5' leader sequence [START_REF] Altman | Tyrosine tRNA precursor molecule polynucleotide sequence[END_REF]. Remarkably RNase P cleaves its substrates in trans and is capable of multiple turnover. Hydrolysis by RNase P RNA is magnesium dependant and suggests an S N 2 -like mechanism [START_REF] Smith | Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis[END_REF]. Nucleotides from the RNase P RNA involved in catalysis are A50, G51 and U52 all located in the P4 stem. The recent crystal structure of the type A RNase P revealed two metal ions at the active site when the pre-cleavage state of the pre-tRNA is mimicked [START_REF] Reiter | Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA[END_REF]) (Figure 30).

MATERIAL &

METHODS

II. MATERIAL AND METHODS

A. CRYSTALLIZATION OF THE LARIAT-CAPPING RIBOZYME

1.

Construct design a)

DiLCrz with substrate in trans First constructs of the LCrz have been designed with sequences of DiLCrz, which is the most extensively studied LCrz out of the three types known. The DiLCrz 166.22 sequence that includes 166 nucleotides upstream the IPS and 22 nucleotides downstream the IPS has been chosen because it almost exclusively performs the branching reaction (Figure 24) (Nielsen et al. Science, 2005). It harbors the peripheral domain DP2-DP2.1 but the alternative hairpin HEG P1 cannot be formed (Nielsen, RNA, 2009). Moreover this sequence is the shortest containing all nucleotides necessary for the branching reaction to take place. The earliest forms of the DiLCrz designed for crystallization were composite with a core ending in the L9 loop and a substrate, added in trans, encompassing the 3' strands of P9, P10 and DP2. In order to present an active-like catalytic site with impeded activity on both hydrolysis and branching the substrate strand of 29 nucleotides harbors a phosphorothioate modified C 230 nucleotide (Figure 31).

To obtain a well-defined 3' end to overcome the random addition of 3'

nucleotides by T7 run-off transcription, an HDV ribozyme is added downstream DiLCrz sequence (Price et al, 1995b;[START_REF] Schurer | A universal method to produce in vitro transcripts with homogeneous 3' ends[END_REF]. The 85 nucleotides HDV ribozyme is placed to cleave right after the last nucleotide, A 222 , of DiLCrz core (Figure 31).

Lastly, to promote crystallization, the P8 loop of DiLCrz, predicted not to be involved in intramolecular contacts [START_REF] Beckert | Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes[END_REF] One major drawback of the LCrz_cp constructs is that their sequences start with a cytosine and this feature is not compatible with efficient in vitro transcription using the T7 RNA polymerase. Instead, the sequence should start with at least two guanosines (Helm et al, 1999). To overcome this matter and obtain a well-defined 5'

end required for crystallization (Price et al, 1995b) we inserted uptstream from the DiLCrz_cp sequence a minimal HMH (Scott et al, 1995b). However we noticed that minimal HMH co-transcriptional cleavage was very weak probably because of the downstream LCrz_cp RNA that folds rapidly and is highly structured therefore competing with HMH folding. We then designed some extended versions of the HMH ribozymes (Figure 33) retaining the important bulge-loop interaction made between stem I and II (Martick & Scott, 2006). Sequence for the HMH extended versions are derived from naturally occurring HMH ribozymes (Khvorova et al, 2003) and take into account that the 3' strand of stem I has to be the sequence of DiLCrz. The preservation of the essential tertiary interaction is verified in silico using the S2S software [START_REF] Jossinet | Sequence to Structure (S2S): display, manipulate and interconnect RNA data from sequence to structure[END_REF]) (Figure 34).

Introduction

To study the massive amount of RNAs identified by transcriptomics analysis, it is important to identify their boundaries in order to grab the "right" molecules. The history of ribozymes well illustrates this idea. At the beginning, the hammerhead ribozyme (HMH) has been characterized in short versions that prevented determining the full extent of the catalytic properties of the natural versions [START_REF] Fedor | Substrate sequence effects on "hammerhead" RNA catalytic efficiency[END_REF].

Thus, biochemical studies showing that hammerhead ribozymes were orders of magnitude more efficient to fold and cleave under low magnesium concentrations if a tertiary interaction between stem I and the loop from stem II was occurring (Khvorova et al, 2003), came years later as a surprise (Figure 1). This finding was further demonstrated by the crystal structure of the Schistosoma hammerhead ribozyme in which setting the correct tertiary interaction leads to a shape of the catalytic site in which the chemical groups responsible for catalysis are ready for in-line attack (Martick & Scott, 2006). This was not the case in crystal structures of the shorter versions (Pley et al, 1994;Scott et al, 1995a) (Figure 1). Other examples like studies on the hairpin (Rupert & Ferre-D'Amare, 2001) and Varkud satellite [START_REF] Lacroix-Labonte | Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture[END_REF] ribozymes also show that the originally defined molecules were not including all the determinants required for their full efficiencies, and further, the vision of a modular architecture of RNA molecules (Masquida et al, 2010). The loss of structural integrity of important modules thus affects the accuracy of RNA architecture, which in the end perturbs the function.

The potential occurrence of functional modules within the ends of RNA molecules justifies that they should be well defined for most studies. Unfortunately, the most widely used enzyme for in vitro production of RNA, the T7 RNA polymerase (T7RNAP), is prone to incorporate residues not encoded by the template at both the 5' (Helm et al, 1999) and 3' ends [START_REF] Milligan | Synthesis of small RNAs using T7 RNA polymerase[END_REF]. These uncoded residues eventually jeopardize experimental strategies such as crystallographic studies, dye labelling for FRET studies, or in general any technic based on RNA ligation [START_REF] Solomatin | Purification of the substrate strand RNA The modified RNA substrate (5' A A G G A A G * C A U C C G G U A U C C C A A G A C A A U G 3') added in trans to the catalytic core (constructs described in II.A.1.a Figure 31) has been chemically synthesized (Dharmacon -Thermo Scientific) and purified by HPLC in the laboratory. 1. Deprotect the 2'-ACE® protected RNA by dissolving the dry pellet of RNA in 400 μL of the 2'-deprotection buffer (100 mM acetic acid adjusted to pH 3.8 with TEMED -Thermo Scientific) and incubating the sample at 60 °C for 30 min[END_REF]. Moreover, any sequence immediately downstream from the promoter cannot be synthesized since T7RNAP requires a specific G-rich starting sequence (Beckert & Masquida, 2011).

The errors made by T7RNAP can be concentrated in the tails of ribozymes located 5' and 3' of the RNA of interest that will be cleaved off (Price et al, 1995a). This strategy can also be adapted to various systems including the production of RNAs with unfavorable 5' sequences [START_REF] Fechter | Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity[END_REF][START_REF] Mörl | Production of RNAs with homogenous 5' and 3' ends[END_REF], and/or to the use of ribozymes in Doudna, 1996). On the 5' side, a hammerhead ribozyme is usually chosen (Figure 2). Only few nucleotides with any sequence downstream from the cleavage site are required for hybridization to fold the active ribozyme. On the 3' side, the hepatitis delta virus (HV) ribozyme is preferred because it is entirely located 3' from the cleavage site and cleaves after any nucleotide, which leaves total freedom to the user. After cleavage, the central RNA is left with 5' OH and 2',3' cyclic phosphate groups.

trans (Ferré-D'Amaré &
As long as the central RNA adopts a simple structure, this strategy does not need specific adaptation. However, if the structure of the central RNA is complex, it may interfere with proper folding of the flanking ribozymes and prevent efficient processing. While a short incubation at a temperature favoring structural rearrangements (~60°C) usually restores cleavage by the 3' HV ribozyme, it is not the case for the 5' hammerhead. This situation happened when we tried to express a lariat capping GIR1 ribozyme (Nielsen et al, 2005) with well defined ends (Figure 2). Nearly no cleavage was observed with a hammerhead version bearing a rather short stem I (5 bp). We assumed that the used truncated hammerhead had sub-optimal activity because it could not form the critical tertiary interactions taking place between stem I and the loop of stem II. Our purpose was to design 5' hammerhead constructs so that their substrate (stem I) resulted from hybridization to a longer sequence of the central lariat capping GIR1 ribozyme (LCrz) while preserving unpaired nucleotides, which interact with the facing loop from stem II.

This tertiary interaction, thereby preserved in the fusion between the hammerhead and the lariat capping GIR1 ribozymes, accelerates folding and catalysis and allows decreasing the required magnesium concentration (Khvorova et al, 2003).

Design of the fusion hammerhead constructs using the program RNAfold [START_REF] Hofacker | Fast folding and comparison of RNA secondary structures[END_REF] and their comparison to wild-type sequences of full length hammerhead ribozymes was carried out. Selected hammerhead sequences with a stem I integrating the 5' end of the lariat capping GIR1 ribozyme (Nielsen et al, 2005) were assayed for efficient cleavage in the course of in vitro transcription reactions (Figure 4). The direct involvement of the bulge in stem I was investigated by determining the observed kinetic constants of the successful candidates (Figure 5 (Canny et al, 2004)). Cloning of efficient fusion hammerhead ribozymes upstream from the lariat capping GIR1 ribozyme, followed by the HδV ribozyme, was performed in plasmid pUC19, further transcribed in vitro as linearized templates. Northern blot analysis showed that the optimized hammerhead ribozymes were very efficient in the full context since they cleave to completion in the course of transcription, thus validating our design (Figure 7). This method validated on two different sequences of the lariat capping GIR1 ribozyme is a practical addition to the molecular biologist's toolbox that addresses defining accurate ends to highly structured RNAs.

Materials

Prepare all solutions using Millipore or DEPC water. All buffers are filtered using a 0.22 m sterile filter and kept at room temperature unless otherwise specified.

In silico design of fusion hammerhead ribozyme sequences

1. RNAfold [START_REF] Hofacker | Fast folding and comparison of RNA secondary structures[END_REF] web server can be used http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi.

Sequence of the full-length wild type hammerhead ribozyme from Schistosoma:

5'GGAUGUACUACCAGCUGAUGAGUCCCAAAUAGGACGAAACGCCGCAAGGCGUCCU GGUAUCCAAUCC3'
Sequence of the full-length fusion hammerhead 6 ribozyme derived from sTRSV+: 2. QIAquick ® Gel Extraction Kit (Qiagen).

3. pUC19 vector (New England Biolabs) stored at a concentration of 1 mg/mL at -20 °C.

4. In-Fusion ® HD Cloning Kit (Clontech).

5. QIAprep ® Spin Miniprep Kit (Qiagen).

6. DNA plasmids were sequenced at GATC Biotech.

2.4

In vitro transcription of the full length RNA using linearized plasmids as templates 3. Methods

In silico design of fusion hammerhead ribozyme sequences

The aim of this step is to design fusion hammerhead ribozymes presenting a hammerhead catalytic core fused to a substrate stem corresponding to the 5' sequence of the lariat capping GIR1 ribozyme. The ribozyme strand presents both ends rooted in stem I, while loops close stems II and III. Stems II and III and the single-stranded regions form the catalytic core and are directly copied from genuine hammerhead sequences presenting tertiary contacts between stem I and the loop of stem II (sTRSV+, CChMVD+, PLMVD+, (Khvorova et al, 2003)). Stem I should be approximately a dozen nucleotides long with a 3' strand being the 5' end of the central RNA, the lariat-capping GIR1 ribozyme in this case (Nielsen et al, 2005). Consequently the 5' strand of stem I is designed to be partialy complementary to the latter and starts with three G residues for transcriptional efficiency (Figure 2). Moreover, a bulge intervening 6 nucleotides after the cleavage site has to be preserved in stem I. The purpose of this bulge is to avoid formation of a perfect duplex which would destroy the tertiary contacts with the loop of stem II (Figure 1) that are crucial for efficient folding and cleavage of the ribozyme (Figure 3 (Canny et al, 2004)). In the sequences resulting from the fusion between hammerhead ribozymes and the lariat-capping GIR1 ribozyme, the presence of (i) a correct three-way junction forming the catalytic core and of (ii) the critical unpaired nucleotides in stem I was systematically checked using RNAfold [START_REF] Hofacker | Fast folding and comparison of RNA secondary structures[END_REF] (see Note 1).

In vitro determination of fusion hammerhead ribozymes cleavage efficiencies

Successful sequences were transcribed from commercial template DNA oligos. The extent of cleavage was monitored in the course of the transcription reactions (Figure 4).

More detailed kinetic analysis of the most efficient transcripts, HMH13, were performed under single-turnover conditions using fusion hammerhead ribozymes consisting of distinct catalytic and substrate strands (Figure 5).

Co-transcriptional cleavage of fusion hammerhead ribozymes

To produce RNAs corresponding to candidate sequences selected in silico by in vitro transcription (Beckert & Masquida, 2011;[START_REF] Milligan | Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates[END_REF], DNA templates preceded by a Class III T7 promoter (5' TAA TAC GAC TCA CTA TA 3') should be produced (Figure 4).

1. Design full-length template DNA oligos, forward and reverse strand that will be used as dsDNA templates for in vitro transcription of the candidates sequences selected in silico (see note 2). 12. Clean the transcription reaction using the Micro Bio-Spin Columns (see note 6).

13. Fractionate each sample on a 10% denaturing PAGE. Run the gel for two hours at 60 W (see note 7).

14. Vacuum dry the gel onto Whatman paper for four hours at 80°C or alternatively seal it in a plastic film and store it at -80°C.

15. Expose an imaging plate for 10 min in a cassette (see note 8).

16. Read the imaging plate using a Bio-imager.

Kinetic analysis of fusion hammerhead ribozymes with trans substrates

This step can be performed as in (Canny et al, 2004) to check whether the bulge in stem I is functional or to determine an optimal magnesium concentration for cleavage (see

Note 9

). The hammerhead cores are obtained by in vitro transcription of dsDNA templates while substrates are obtained by RNA solid phase chemical synthesis (Figure 5).

1. Design two sets of fusion hammerhead ribozymes and their substrates as separate RNA molecules. From this step the catalytic cores of the fusion hammerhead ribozymes will be handled separately from their substrate domains. 5. Purify transcription reactions by 10% denaturing PAGE.

6. Identify RNA bands corresponding to the fusion hammerhead core by UV shadowing.

7. Cut the band of interest out of the gel and place it into a 1.5 mL tube.

8. Immerse the gel band in about 800 µL of water and place the tube in a shaker over-night at 4°C. 9. Decant the elution water and transfer it to a fresh tube.

10. Recover the fusion hammerhead cores by ethanol precipitation of the elution water.

28. Reveal the radioactivity pattern on the imaging plate using a Bio-imager.

29. Quantify the amount of cleaved substrate using appropriate software.

30. Plot the fraction of substrate cleaved versus time and determine k obs by fitting the data to the equation, F Cl = F max (1-e -kt )+F 0 using the KaleidaGraph software (see note 11).

Cloning of ternary ribozyme constructs

The most efficient fusion hammerhead ribozyme is cloned upstream from the lariat capping GIR1 ribozyme followed by an HV ribozyme ending with a restriction 10. Grow the transformed XL-1 Blue Escherichia coli competent cells on LB/Ampicillin (100 µg/mL) plates overnight at 37 °C.

11. Grow 3 mL of LB/Ampicillin (100 µg/mL) cultures from individual colonies.

12. Extract DNA using a miniprep spin kit.

13. Sequence the extracted DNA using the M13 sequencing primers.

14. Prepare glycerol stocks of the clones containing the desired sequences by mixing 1 mL of saturated bacterial culture to 600 μL of sterilized 50% glycerol solution in 2 mL screw cap tubes. Store the glycerol stocks at -80 °C.

In vitro transcription of the ternary ribozyme RNA using linearized plasmids as templates

The transcription reaction of the plasmid template is performed as for the PCR template except that the plasmid is linearized prior to the reaction and that the mass added is greater since the molarity of the template is important.

1. For in vitro transcription, linearize the plasmid with BamHI restriction endonuclease following the enzyme provider instructions. 4. Incubate at 37 °C for 2 h.

5. Incubate half of each transcription at 60 °C for 1 h.

6. Stop transcription reaction by mixing it to an equal volume of urea blue loading buffer 2x.

Northern blot analysis of the cleavage pattern of the ternary ribozyme RNA

1. Design three DNA probes that will be used to hybridize specifically either to the hammerhead ribozyme or to the central RNA or to the HδV ribozyme (see note 17).

2. Label each DNA probe by mixing: 1 M of DNA oligo, 2.5 L of 32 P-ATP (10 mCi/mL, 3000 Ci/mmol), 1 L of T4 polynucleotide kinase (10 U/L), 5 L buffer A 10x, water up to 50 L.

3. Incubate 30 min at 37 °C then 10 min at 65 °C.

4. Clean the labeling reactions using the Micro Bio-Spin Columns (see note 6).

Fractionate samples from in vitro transcription reactions of the ternary ribozyme

RNA on an 8% denaturing PAGE. Load in four consecutive lanes your 60 °C preincubated or not samples in order to be able to compare the cold to the corresponding body-labeled transcription reactions. Repeat the same operation two times. Each set of four lanes will be used for a different DNA probe. Run for 3 hours at 60 W. (see note 7).

6. Transfer the RNA bands from the gel onto positively charged nylon membrane using a vacuum gel drier for four hours at 80 °C.

7. Wash the membrane in water to remove the dried gel particles (see note 18).

8. Crosslink the RNA onto the membrane using a UV crosslinker.

9. Cut the membrane to separate each set of four lanes. From this step you will be dealing with three membranes in parallel.

10. Insert delicately each membrane into a hybridization bottle (see note19).

11. Add 20 mL of pre-hybridization buffer.

12. Incubate at 60 °C for 30 min in a rolling hybridization oven.

13. Add the total amount of one labeled DNA primer per hybridization bottle.

14. Incubate at 37 °C over night in the rolling hybridization oven.

15. Wash each membrane with 20 mL of SSPE 6x 5 min at 37°C in the rolling hybridization oven. Repeat this step three times.

16. Wash each membrane with 20 mL of SSPE 6x 5 min at 42°C in the rolling hybridization oven.

17. Seal each membrane in a plastic film.

18. Expose an imaging plate to the plastic sealed membrane for three hours in a cassette (see note 8, 10).

19. Reveal the radioactivity pattern on the imaging plate using a Bio-imager.

Notes

1. The sequence of the adapted substrate starts after the cleavage site and preserves several unpaired residues in stem I of the ribozyme core to favor tertiary contacts with the loop of stem II. The best situation is when the UAC sequence forms a bulge. When this not possible, avoid building an extended helix by inserting at least non-complementary residues.

2. Do not forget to insert the T7 promoter sequence upstream of the DNA templates.

3. These primers can be the same for all fusion hammerhead ribozymes candidates if you use as primer 1 the sequence of the T7 promoter and as primer 2 the sequence of the central RNA 4. It is not compulsory to add alkaline phosphatase but it is known to increase in vitro transcription yields.

5. We produce our own T7 RNA polymerase, which reduces cost considerably.

6. It is not mandatory to clean transcription reaction although it is preferable to remove not incorporated radio-labeled nucleotides to avoid contamination of the electrophoresis buffer.

7.

To have better defined bands, gels are pre-run for one hour at 60 W.

8. The radioactivity pattern of the gel can as well be obtained on X-ray autoradiogram.

9. This step is not mandatory and was carried out as a proof of principle. It can be performed if the ribozyme construct is inactive to further check for efficiency of a set of hammerhead constructs carrying different kinds of bulges in stem I.

10. Exposure time of the imaging plate depends on the amount of 32 P-labeled material on the membrane.

11. F cl is the fraction of cleaved substrate, F 0 is the initial amount of substrate cleaved before addition of MgCL2, F max is the maximal amount of substrate cleaved, t is the time in minute and k obs is the observed rate of cleavage.

12. For optimal yield, forward and reverse primers should have close melting temperature.

13. Keep your samples on ice while preparing the PCR reaction.

14. Insert your samples in the thermal cycler when the temperature of the sample holder is at 90°C minimum.

15. To avoid any trouble with next steps of the cloning process, 10% of ammonium acetate 3 M pH 5.2 is added to the recovered DNA and precipitated with 3 volumes of pure ethanol. DNA is then re-suspended in 30 L of water.

Very efficient chemically competent Escherichia coli cells can be prepared if all

the material is maintained ice-cold throughout the whole preparation process.

17. The three primers should have close melting temperature and the melting temperature must be above 42 °C 18. Be delicate not to scratch the membrane.

19. Be careful that side of the membrane on which the RNA is crosslinked is facing the inside of the hybridization bottle. There should not be any membrane overlap in the hybridization bottle. **Annealing temperature for PCR of section 3.3 step 3.

As for constructs described in II.A.1.a, a HDV ribozyme is added downstream of LCrz_cp constructs to obtain a well-defined 3' end. 109 circularly permutated constructs have been designed out of which 17 have been tested for crystallization.

The simplest construct is DiLCrz_cp "wild type" that is the 166.22 sequence plus the 5'UUCG3' tetralopp of DP2 (Figure 35). One variant of this DiLCrz_cp construct is DiLCrz_cp_U1A that harbors a U1A binding site in the loop of P8 (Figure 35). Although the diffraction limit of our first crystals was poor (15 Å), DiLCrz_TLR3 construct (Figure 31) conferred a first success at crystallizing the LC ribozyme. We thus decided to take advantage of the circularly permutated constructs and of the tetraloop/tetraloop-receptor motifs to avoid crystallizing a mix of different RNA in order to simplify our protocols. To do so, we engineered stem-loops containing either a tetraloop or a tetraloop receptor, or both (Figure 36). U-rich sequences between the ribozyme core and the inserted motifs and between motifs themselves have been chosen to influence the flexibility of the engineered stems [START_REF] Jaeger | TectoRNA: modular assembly units for the construction of RNA nano-objects[END_REF].

Engineered stems can be inserted in the loop of DP2 and / or in the loop of P8

(Figure 37). 96 different constructs have been designed, out of which we used four for crystallization trials (Figure 38).

LCrz RNA production and purification

All molecular biology methods have been adapted from [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. All solutions have been filtered on 22 μm filter and sterile glass or plastic material has been used wherever possible.

a) Competent Escherichia coli XL-1 Blue cells preparation

Competent Escherichia coli XL-1 Blue cells are prepared in the laboratory according to the below protocol below [START_REF] Inoue | High efficiency transformation of Escherichia coli with plasmids[END_REF].

1. Inoculate a starter culture of 3 mL of LB/Tetracycline (30 μL/mL) with 50 μL of competent cells from the initial strain.

2. Incubate at 18 °C for 6 hours.

3. Inoculate 250 mL of LB/Tetracycline (30 μL/mL) with the 3 mL of starter culture.

4. Incubate at 18 °C until the OD of the bacterial culture reaches 0.6 at a λ= 600 nm.

5. Pre-cool at 4 °C all materials and solutions that will be used from this step and work in the cold room (4 °C). These are very important precautions to take in order to obtain efficient competent cells. 1. Design forward and reverse primers for the successive PCRs (Appendix II).

For optimal yield, forward and reverse primers have been designed to have identical melting temperature. 12. Grow 3 mL of LB/Ampicillin (100 µg/mL) cultures from individual colonies.

13. Extract DNA using a Miniprep Spin Kit (QIAprep ® Spin Miniprep Kit -Qiagen).

14. The extracted DNA is sequenced using the M13 sequencing primers by GATC Biotech (Jakob-Stadler-Platz 7 -78467 Konstanz -Germany). 11. Grow 3 mL of LB/Ampicillin (100 µg/mL) cultures from individual colonies.

12. Extract DNA using a Miniprep Spin Kit (QIAprep ® Spin Miniprep Kit -Qiagen).

13. The extracted DNA is sequenced using the M13 sequencing primers by GATC Biotech (Jakob-Stadler-Platz 7 -78467 Konstanz -Germany).

14. Prepare glycerol stocks of the clones containing the desired sequences by mixing 1 mL of saturated bacterial culture to 600 μL of sterilized 50% glycerol solution in 2 mL screw cap tubes. Store the glycerol stocks at -80 °C.

d) Plasmid preparation

The amount of plasmid needed, hence the amount of bacterial culture, depends on the size of the in vitro transcription you are planning to perform. Typically, I used 300 µg of plasmid DNA to perform a 6 mL in vitro transcription to obtain micrograms of pure RNA for crystallization.

1. Prepare a starter bacterial culture by inoculating 3 mL of LB/Ampicillin (100 µg/mL) with a tip dipped in the glycerol stock of your plasmid of interest. Grow the 3 mL cultures at 37 °C for 6 hours. Starter bacterial culture is not mandatory however it generally leads to a better bacterial growth hence a better DNA plasmid yield.

2. Inoculate 250 mL of LB/Ampicillin (100µg/mL) with the 3 mL of starter culture.

Grow over-night at 37°C.

3. Centrifuge the bacterial culture at 4000 g for 20 min at 4 °C. Discard the supernatant.

4. Extract plasmid from the bacterial pellet with the PureLink ® HiPure Plasmid Filter Maxiprep Kit (Invitrogen).

5. For in vitro transcription, linearize the extracted plasmid with PstI restriction enzyme following the enzyme provider instructions.

6. Check on an agarose gel that the DNA plasmid is fully digested unless this will decrease the yield of the upcoming transcription. 1. Add 10% of ammonium acetate 3 M pH 5.2 to the 6 mL transcription and precipitated with 3 volumes of pure ethanol.

2. Centrifuge at 20 000 g for 20 min in 50 mL BD Falcon TM conical tubes.

3. Discard the supernatant and resuspend the pellet in 1 mL of water.

4. Add to the resuspended pellet 1 mL of urea blue loading buffer 2x (8 M urea, 0.025% xylene cyanol, 0.025% bromophenol blue). 9. Crush the gel band through a syringe.

To check that transcription of

10. For passive elution of the RNA, add 11 mL of water to the crushed gel in a 50 mL tube placed at 4 °C on a roller mixer (Stuart TM Scientific) over-night.

11. Filter the sample through a 150 mL Analytical Filter Unit (Nalgene ®)

12. Add 10% of ammonium acetate 3 M pH 5.2 to the filtrate and precipitated with 3 volumes of pure ethanol.

13. Centrifuge at 27 500 g for 20 min in 50 mL BD Falcon TM conical tubes.

14. Discard the supernatant and resuspend the RNA pellet in 50 μL of water.

15. Determine the RNA concentration from OD measurements at  = 260 nm (Nanodrop -LabTech).

16. Store the pure RNA at -20 °C.

Over-expression and purification of the U1A protein

The 2. Spread the transformed competent cells on a LB/Ampicillin (100 µg/mL) /Chloramphenicol (34 μg/mL) agar plate. Incubate the plate at 37 °C over-night.

3. Prepare a starter bacterial culture by inoculating 3 mL of LB/Ampicillin (100 µg/mL) /Chloramphenicol (34 μg/mL) medium with a tip dipped in selected colony from the agar plate. Grow the 3 mL cultures at 37 °C over-night.

4. Inoculate 2 L of bacterial culture with 1 mL of the starter bacterial culture.

5. Allow bacteria to grow until the DO at  = 600 nm reaches 0.4.

6. Induce U1A gene over expression by adding IPTG at a final concentration of 0.5 mM to the 2 L of medium. Grow at 37 °C for 4 hours.

7. Centrifuge at 4000 g for 30 min at 4 °C.

8. Resuspend the bacterial pellet corresponding to 1 L of bacterial culture in 25 mL of lysis buffer (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 5 μg/mL Leupeptine, 1% Aprotinine (v/v), 0.1% NP-40).

9. Sonicate the sample 10 times for 30 s.

10. Centrifuge at 10 000 g for 10 min at 4 °C.

11. Collect the supernatant and keep it at 4 °C.

12. Check for the presence of the U1A protein in the supernatant by running a SDS 12% PAGE gel (12% acrylamide/bis-acrylamide (37.5:1), 1.5% Tris, 9.4% Glycine, 0.5% SDS).

13. Add PEI dropwise to the supernatant to reach 0.5% finale concentration of PEI.

During this step shake gently and continuously the sample at 4 °C.

14. Centrifuge at 20 000 g for 5 min at 4 °C.

15. Dialyze the lysate twice in buffer A (100 mM KCl, 25 mM HEPES-KOH pH 7.5, 10 mM DTT, 0.5 mM EDTA, 0.5 mM PMSF)

16. Purify by FPLC (ÄKTA TM -GE Healthcare Life Sciences) using an ion exchange column (Sepharose Fast Flow -GE Healthcare Life Sciences). 17. Inject elution buffer B (500 mM KCl, 25 mM HEPES-KOH pH 7.5, 10 mM DTT, 0.5 mM EDTA, 0.5 mM PMSF) to allow progressive elution of the U1A protein.

18. Perform a second purification step using a gel-filtration column (HiLoad TM Superdex TM 75 pg -GE Healthcare Life Sciences) and buffer A.

19. Dialyze the U1A protein in buffer C (10 mM Phosphate buffer pH 7.5, 50 mM KCl, 1 mM DTT).

Perform a cation exchange chromatography (Bio-Scale Ceramic

Hydroxyapatite CHT5-I -BIORAD).

21. Perform an elution gradient with buffer D (10 mM Phosphate buffer pH 7.5, 50 mM KCl, 1 mM DTT, 500 mM (NH 4 ) 2 SO 4 ) to improve the U1A protein purity.

22. Dialyze the U1A protein in storage buffer (10 mM HEPES-KOH pH 7.5, 1 mM DTT, 0.1 mM EDTA).

23. Concentrate the U1A protein by tangential centrifugation at 5000 g for 10 min

(Amicon MWCO 3kDa -Millipore). Store the U1A protein at -80 °C.

Crystallization a) Sample preparation

In the case of purified LCrz_cp, the RNA has been prepared in the desired volume at the necessary concentration (100 M in most cases) in a solution containing 100 mM KCL and 10 mM MES-KOH pH 6.5. The sample has been heated

during 1 min at 95 °C followed by 4 min on ice. MgCl 2 up to 4 mM was then added, and the solution was placed at 55 °C for 10 min then at 4 °C overnight. The homogenous folding of the ribozyme has been verified by running a native 8% PAGE (8% acrylamide/bis-acrylamide (19:1) in TB 1x).

In the case of LCrz core, the RNA was prepared as the LCrz_cp, except that if necessary, the U1A protein was added at the same final concentration as the ribozyme core and the sample was further incubated at 25 °C for 20 min. RNA substrate strand is always added in excess. The sample is then placed at 4 °C overnight. The formation of the binary or ternary complex is verified by gel shift assay.

b) Sitting drops

Initial crystallization conditions were identified using commercial screens (Natrix HT, Index HT, PEGRx HT, Crystal screen HT, Crystal screen cryo HT from To optimize crystals size and diffraction sparse matrix have been designed to span the phase diagram by varying the concentration of salt, precipitant and RNA but also the pH or the temperature (Table 6 to 13). Sitting drops were made as for the initial screening. Initial screening with commercial kits plus screening using sparse matrix screens represent 15 760 sitting drops. Table 8: 48 crystallization conditions sparse matrix with condition F11 (highlighted in purple) from the Index HT (Hampton research ©) used as a starting point. BIS-TRIS pH 6.5 is fixed at 100 mM. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL drops. 12: 4 crystallization conditions sparse matrix with condition A10 (highlighted in purple) from the PEGRx (Hampton research ©) used as a starting point. pH of the buffer is the only parameter that has been modified in the sparse matrix. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL , 600 nL and 800 nL drops.

d) Microbatches

In addition to hanging drops, microbatch experiments have been set up for most of the successful condition, since different setups can influence crystal quality [START_REF] Chayen | Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques[END_REF]. Droplets were prepared in minitray Nunc plates (60 wells) by mixing 2.5 µl of the LCrz RNA at a concentration of 100 µM with 2.5 µl of crystallization solution at various concentrations. Drops were sealed using either 10 µl paraffin oil or crystal adhesive tape or 0.2% agarose gel. We also set up gelmicrobatchs by mixing 2.5 µl of the LCrz RNA with 2.5 µl of crystallisation solution at various concentrations and 0.2% low-melting agarose gel sealed with paraffin oil [START_REF] Lorber | Crystal growth of proteins, nucleic acids, and viruses in gels[END_REF]. Some crystallization conditions have been supplemented with iridium hexamine or various polyamines. All microbatchs were kept at 20°C.

RNA Crystallization condition Microbatch type Additive DiLCrz_cp_U203C

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% -D11 -Index HT-Hpt Research-100 mM BIS-TRIS pH 6. At a final concentration of 60% or 70% of the crystallization condition, crystals were larger than the ones grown in sitting or hanging drop setups and allow us to collect high resolution diffraction data. One general feature regardless of the crystallization condition of the LCrz RNA is that additives, iridium hexamine or polyamines, allow crystals to grow faster and larger, although their diffraction properties were significantly decreased. Taken together optimization in hanging drops and microbatch drops represent 777 handmade drops. All crystallization conditions that allow RNA crystals to grow, in sitting drops, hanging drops and microbatch drops are summarized in Appendix IV. For each successful crystallization condition the following information are given: the LCrz RNA construct, the setup, the crystallization condition and the size of the drop, the time lapse between the day the drops has been made and the picture of the crystals has been taken. When available additional information is given such as the way crystals have been frozen and the best resolution observed.

e) Post-crystallization treatments

Cryoprotective and dehydration treatments have been experienced, as they have been shown to reduce ice-rings and improve diffraction [START_REF] Abergel | Spectacular improvement of X-ray diffraction through fast desiccation of protein crystals[END_REF][START_REF] Alcorn | Progress in rational methods of cryoprotection in macromolecular crystallography[END_REF]. All treatments have been tested on DiLCrz_cp_U203C RNA crystals grown in microbatchs (F12 -Index HT-Hampton Research-2% v/v PEG 400, 100 mM Imidazole pH 7.0, 24% w/v PEGMME 5000) sealed with crystal adhesive tape.

Crystals have been soaked as follows:

-5 or 10 min in 5 l of crystallization solution and 5 or 10 % (v/v) PEG 400 Crystals were stored in CrystalCap HT filled with ethane and frozen in liquid nitrogen.

Data collection and processing

DiLCrz_cp_U203C yielded data quality crystals in condition 0.2 Sodium chloride, 0.1 M HEPES pH 7.5, 25% w/v PEG 3,350. Diffraction data were collected at SLS on beam-line X06DA (PXIII) equipped with a PILATUS 2M detector.

Oscillation frames (0.1°/frame) were collected on crystals undertaking a 20% attenuated beam flux to decrease radiation damage. Crystals were maintained at 100K under a cryo-flux. To solve the structure of the DiLCrz ribozyme de novo by MAD, 4 data sets were collected (Figure 46) on the same crystal (see Results section). Native datasets were also collected up to 2.45 Å resolution. Data were processed using XDS [START_REF] Kabsch | Xds[END_REF] and heavy atoms sites were determined using SHELX package [START_REF] Sheldrick | A short history of SHELX[END_REF].

C. BIOINFORMATIC ANALYSIS OF THE BACTERIAL TYPE A RNASE P

All details for bioinformatic analysis of the motif kinking RNA helices are given in the material and method section of the article and in the supplementary material (see Results section).

RESULTS

&

DISCUSSION

III. RESULTS AND DISCUSSION

A. CRYSTAL STRUCTURE OF THE LARIAT-CAPPING RIBOZYME

Introduction

Out of 2208 families of ncRNA identified in Rfam [START_REF] Gardner | Rfam: Wikipedia, clans and the "decimal" release[END_REF] only 60

families have at least one of its member for which the 3D structure has been solved.

And a glance at the PBD, www.pdb.org, [START_REF] Berman | The Protein Data Bank[END_REF] reveals that even if the number of RNA 3D structures solved increases at a rate of approximately 75 new structures per year for the last 10 years it is far less than for proteins (Figure 47). Xray crystallography is the method of choice to solve 3D structure as for example, 2040 RNA structures, including ribosomes', listed in the NDB are solved by X-ray crystallography in comparison to only 554 RNA structures of rather small size solved by NMR [START_REF] Berman | The nucleic acid database. A comprehensive relational database of three-dimensional structures of nucleic acids[END_REF]. In the below article, we report the crystal structure of the 192 nucleotides DiLCrz at a resolution of 2.5 Å. Size and resolution are comparable with the three known group I introns 3D structures (Cate et al, 1996a;[START_REF] Golden | Crystal structure of a phage Twort group I ribozymeproduct complex[END_REF][START_REF] Lipchock | A relaxed active site after exon ligation by the group I intron[END_REF]. With this new crystal structure we supplement the collection of 3D structures of the ribozyme family. Moreover, previous work on modeling of the DiLCrz ribozyme (Beckert, 2008) and comparison between DiLCrz and group I intron crystal structure allow us to hypothesize that LCrz ribozyme could result from the evolutionary metamorphosis of a group I intron in the context of a twintron (Decatur et al, 1995).

Article

Additional results

We also collected diffraction data for the DiLCrz_active_U203C construct up to 4 Å and solved the structure by the molecular replacement method using the structure of DiLCrz_cp_U203C. Unit cell parameters change significantly between the cp and the active DiLCrz_U203C constructs crystals (Table 16). Some regions of the DiLCrz_active_U203C fit unambiguously in the density map e.g. the P3 stem or the very important DP2/DP2.1/P10 3WJ (Figure 48). However because some nucleotides are clearly positioned differently in the active LCrz in comparison to the post-cleavage state one (Figure 49) it is impossible to confidently build the model at a resolution of 4 Å and hence to proceed to refinement (Table 16). It is for the moment out of question to determine in which state is the LCrz in the crystal as hydrolysis or branching could have occurred during crystallization. We now try to reproduce this crystal to solve the structure de novo using heavy atoms derivatives.

DiLCrz_cp_U203C DiLCrz_active_U203C

Resolution (Å) 2.5 4 that summarizes SAXS parameters for each DiLCrz.

Space group

Methods

MAD MR

Discussion

As described in the introduction, LCrz is part of a twintron as it is embedded into a canonical group I intron, GIR2 (Decatur et al, 1995). There are other examples of a heterogenous twintrons, in the sense that both introns within the twintron are not identical, found in the gene coding for the mitochondrial small subunit ribosomal RNA of fungi where a group IIA intron is inserted within the P9.1 stem of a group IC2 intron [START_REF] Hafez | The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins[END_REF]. More interesting to us, a homogenous group I twintron is as well observed in the SSU rRNA of two species of the Ophiostomatales taxa. The twintron is composed of an external ORF-less group ID intron and an internal group ID intron followed by the ORF of a HEG inserted in the P1 stem of the external intron [START_REF] Hafez | The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins[END_REF]. The latter observation in addition to the previous sequence alignments, secondary structure comparison with Azoarcus group IC3 intron, mutagenesis and modeling data [START_REF] Beckert | Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes[END_REF] lead us to the assumption that LCrz results from the evolutionary drift of a group I intron that under new selective pressure acquired new functions to escape from a probable loss [START_REF] Goddard | Recurrent invasion and extinction of a selfish gene[END_REF]) (Figure 51). using Azoarcus pre-tRNA splicing ribozyme to demonstrate that biotin-GTP can be used as a cofactor to promote the splicing pathway in a SELEX experiment [START_REF] Wilson | In vitro selection of functional nucleic acids[END_REF]. It would also be interesting to solve the 3D structure of LCrz deprived of its DP2/DP2.1 peripheral domain to check whether it would fold into a group I intron fold.

Because LCrz followed by the ORF of a HEG is inserted either in P2 of DiGIR2 or in the P6 stem of NaGIR2 and AlGIR2, we suggest that LCrz together with the gene of its cognate HE invaded GIR2. This event could have happened at least twice in evolution as the GIR2 recipient group I introns are of different class depending on the eukaryote in which the twintron is observed. Still in agreement with the Goddard and Burt cycle, the HE should acquire new function not to be lost [START_REF] Chatterjee | Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease[END_REF][START_REF] Goddard | Recurrent invasion and extinction of a selfish gene[END_REF]. Though HE in Didymium iridis is still capable of intron homing in vivo [START_REF] Johansen | In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis[END_REF]. With respect to this hypothesis, the question whether GIR2 was an ORF-less intron or has lost its HEG is still open. A possibility is that GIR2 was an ORF-containing group I intron and the sequence between the 5' strand of GIR2 and the HE ORF is encoding for an intron insertion site therefore allowing intron reverse splicing at this specific location. LCrz could have emerged from the sequence drift of a group I intron that has promoted its own mobility at the RNA level [START_REF] Hedberg | Nuclear group I introns in self-splicing and beyond[END_REF].

One prospective function of the HE would be to regulate the on-and offconformational switch of LCrz. Indeed, since LCrz is fully transcribed before GIR2, it should not react before self-splicing of GIR2 has been completed since this would lead to unligated exons of the SSU rRNA (Vader et al, 1999). Additionaly, the mutually exclusive DP2 and HEG P1 stems are observed in the on and off states, respectively (Figure 6) [START_REF] Nielsen | A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA[END_REF]. The HE of the twintron could recognize one or the other conformation and modulate the conformational switch. However we cannot exclude that a completely different enzyme could be recruited to regulated LCrz peripheral conformational switch as e.g. group I introns are capable of hijacking proteins to accomplish a maturase function to help their own splicing [START_REF] Garriga | Protein-dependent splicing of a group I intron in ribonucleoprotein particles and soluble fractions[END_REF]Paukstelis et al, 2008). Nonetheless, we cannot exclude that the HE has acquired new functions unrelated to its catalytic activity or to the regulation of the twintron processing.

Nature is likely not altruistic, therefore co-evolution of LCrz and HE for mutual interest is possible especially as LCrz is always found followed by HEG. NaHEG or NaLCrz insertions are not observed solely in Nae.S516 twintron [START_REF] Haugen | The evolution of homing endonuclease genes and group I introns in nuclear rDNA[END_REF][START_REF] Wikmark | Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron[END_REF]. Additionally, having a look back at the three processing pathways that the twintron GIR2/LCrz can undertake (Figure 3), we observe that during the starvation-induced encystement pathway the production of mature SSU rRNA is down regulated while there is an accumulation of the capped 7.5 kb mRNA of the HE [START_REF] Nielsen | The GIR1 banching ribozyme[END_REF]Vader et al, 2002). The capped mRNA of the HE is

B. THE PK-TURN, A NEW RNA KINKING MOTIF

Introduction

RNA motifs are of two types: (1) RNA motifs that only implicate primary sequences as for example the Shine-Dalgarno motif or (2) those that adopt a 3D structure characterized by a set of specific structural features. We focused on the second ones (reviewed in (Masquida et al, 2010)) and when RNA motifs are later discussed in the manuscript they always imply structural characteristics in the sense of the three-dimensional structure.

WC base pairs, mostly distributed in A-type helices, represent 60 to 70% of the total base pairs of a structured RNA [START_REF] Westhof | Predicting and modeling RNA architecture[END_REF] and RNA motifs arose from the match of single stranded nucleotides linking one helix to the others. RNA motifs are defined by a set of weak interactions including non-WC base pairs folding the phosphodiester backbone of the considered RNA strands in a constrained and characteristic manner [START_REF] Leontis | The building blocks and motifs of RNA architecture[END_REF][START_REF] Leontis | Analysis of RNA motifs[END_REF]. RNA motifs consensus sequence encompass all isosteric non-WC base pairs ordered and directed identically to fold in the same three-dimensional structure [START_REF] Leontis | Analysis of RNA motifs[END_REF]. RNA motifs allow additional nucleotides to be inserted if they do not disturb the overall geometry of their folding [START_REF] Abramovitz | Remarkable morphological variability of a common RNA folding motif: the GNRA tetraloop-receptor interaction[END_REF][START_REF] Klein | The kink-turn: a new RNA secondary structure motif[END_REF]. RNA motifs not only join and orient RNA edging helices; they also mediate RNA-RNA or RNA-protein interactions and can be drug targets [START_REF] Ban | The complete atomic structure of the large ribosomal subunit at 2.4 A resolution[END_REF][START_REF] Wimberly | Structure of the 30S ribosomal subunit[END_REF]. Among the well-known RNA motifs are: the loop E [START_REF] Correll | Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain[END_REF][START_REF] Leontis | Effect of magnesium ion on the structure of the 5S RNA from Escherichia coli. An imino proton magnetic resonance study of the helix I, IV, and V regions of the molecule[END_REF], the sarcin-ricin motif (Leontis et al, 2002), the GNRA tetraloop [START_REF] Abramovitz | Remarkable morphological variability of a common RNA folding motif: the GNRA tetraloop-receptor interaction[END_REF], and its receptor (Cate et al, 1996b), the kinkturn or k-turn (Vidovic et al, 2000), the G-ribo [START_REF] Steinberg | G-ribo: a new structural motif in ribosomal RNA[END_REF], the RA motif [START_REF] Grabow | The right angle (RA) motif: a prevalent ribosomal RNA structural pattern found in group I introns[END_REF].

K-turns are widespread motifs present e.g. in the spliceosomal RNA U4 (Vidovic et al, 2000), in riboswitches [START_REF] Blouin | A loop loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control[END_REF][START_REF] Montange | Structure of the S-adenosylmethionine riboswitch regulatory mRNA element[END_REF], in the human RNAse P (Davila [START_REF] Lopez | Conserved and variable domains of RNase MRP RNA[END_REF] and multiple times in rRNA [START_REF] Klein | The kink-turn: a new RNA secondary structure motif[END_REF]. A database dedicated to k-turns is available online: www.dundee.ac.uk/biocentre/nasg/kturn/ [START_REF] Schroeder | A structural database for k-turn motifs in RNA[END_REF]. The k-turn motif is defined by a 15 nucleotides sequence organized in two helices, the canonical one (C-stem) bridged by a bulge of 3 nucleotides to the non-canonical one (NC-tem) capped by two G.A base pairs (Figure 52) [START_REF] Lilley | The structure and folding of kink turns in RNA[END_REF]. The k-turn motif can be inserted in both directions but the angle between C-and NC-stem is always around 60° and the minor grooves of each helix face each other. L1 and L2 nucleotides are stacked onto C-and NC-stem respectively when L3 is bulged out [START_REF] Lilley | The structure and folding of kink turns in RNA[END_REF]. The two G.A pairs are trans sugar edge (G) to Hoogsteen edge (A) pairs and sequence variation of these base pairs is mostly observed at position 2b.2n [START_REF] Cannone | The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs[END_REF][START_REF] Schroeder | Structure and folding of a rare, natural kink turn in RNA with an A*A pair at the 2b*2n position[END_REF][START_REF] Wimberly | Structure of the 30S ribosomal subunit[END_REF]. Stacking and hydrogen bonds hold tightly the RNA motif into its fold. Three hydrogen bonds are crucial: [START_REF] Beckert | Synthesis of RNA by in vitro transcription[END_REF] H bond between 2'O of L1 and N1 of A1n, (2) H bond between 2'O of L3 and the proS nonbridging oxygen of the phosphate group between L1 and L2 and ( 3) an A-minor interaction between the nucleotide at position -1n and either N1 or N3 of the highly conserved adenosine 2b (Figure 52B). K-turn folding can be induced by metal ions [START_REF] Liu | The role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA[END_REF], protein interaction (Turner et al, 2005) and/or long range tertiary interactions [START_REF] Antonioli | Plasticity of the RNA kink turn structural motif[END_REF][START_REF] Schroeder | RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn[END_REF]. K-turns are at least related to two motifs: reverse k-turn [START_REF] Strobel | RNA kink turns to the left and to the right[END_REF]) and pk-turn. 3) an A-minor interaction between the nucleotide at position -1n and N1 of the highly conserved adenosine 2b. In both panels, the longest strand that contains the three bulged nucleotides is shown in purple and the shortest in green.

The pk-turn that we describe in the following article is not just another k-turn, but a new motif that expands the variety of motif able to sharply kink RNA helices.

The pk-turn motif observed in the P16-P17 domain of the 3D structure of the bacterial RNAse P bound to its tRNA substrate [START_REF] Reiter | Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA[END_REF] does not respect any sequence or base pairs geometry requirements of the k-turn motif [START_REF] Lilley | The structure and folding of kink turns in RNA[END_REF].

Nevertheless the pk-turn motif allows the same kinking and twisting of the edging helices than the k-turn.

Because RNA motifs are important from a structural and functional point of view, it is of great interest to be able to spot them in available 3D structures [START_REF] Apostolico | Finding 3D motifs in ribosomal RNA structures[END_REF]Cruz & Westhof, 2011b;[START_REF] Petrov | WebFR3D--a server for finding, aligning and analyzing recurrent RNA 3D motifs[END_REF][START_REF] Sarver | FR3D: finding local and composite recurrent structural motifs in RNA 3D structures[END_REF]Wadley & Pyle, 2004) but also to be able to model them from sequencing data to help determining the 3D structure of large RNA (Cruz & Westhof, 2011b;Masquida et al, 2010;[START_REF] Westhof | Predicting and modeling RNA architecture[END_REF]. New RNA 3D structure is always the time for new motifs to be observed [START_REF] Holbrook | RNA structure: the long and the short of it[END_REF].

Article

,

Discussion

As suggested in our paper and in collaboration with David Lilley's laboratory the commutability of k-turn and pk-turn has been tested [START_REF] Daldrop | The functional exchangeability of pk-and kturns in RNA structure[END_REF]. As a result, the pk-turn can functionally substitute for the k-turn in the SAM-I riboswitch from Thermoanaerobacter tengcongensis [START_REF] Schroeder | RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn[END_REF]. Vice-versa a kturn inserted in either orientation between P16 and P17 of the RNAse P RNA from

Thermotoga maritima can functionally substitute for the pk-turn [START_REF] Reiter | Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA[END_REF].

However in both experiments the activity of recipient catalytic RNA was decreased when the innate RNA motif was replaced [START_REF] Daldrop | The functional exchangeability of pk-and kturns in RNA structure[END_REF]. If the outcome of the pk-turn as a RNA 3D motif inserted between two canonical helices is the same as a k-turn on the overall 3D structure, the intrinsic structural features of the pk-turn differ from the k-turn. The pk-turn motif is suggested to be more flexible than the k-turn as FRET analysis show that it does not fold in the context of a simple duplex in presence of magnesium ions while k-turns do so [START_REF] Liu | The role of specific 2'-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA[END_REF]. This apparent flexibility is compensated by RNAse P folding when it adopts its active conformation as well as by the SAM-I riboswitch. K-turn and pk-turn are not the only motifs found to link and orient P16 to P17 in RNase P. Indeed, 3WJ and 4WJ are as well observed. Taken together these motifs are examples that there are several alternative RNA motifs that can be naturally selected to answer the same overall three-dimensional constraints depending on contextual factors.

Because the pk-turn motif has never been observed before the one revealed by the three-dimensional structure of bacterial RNAse P, any bio-informatic tool could not have detected it. On the contrary sequences expected to fold in a given motif can fold differently under new contextual factors [START_REF] Antonioli | Plasticity of the RNA kink turn structural motif[END_REF]. A well-described example is the reverse k-turn that has only a G to A mutation in a critical G.A pair [START_REF] Strobel | RNA kink turns to the left and to the right[END_REF]. The unexpected folding of the J5/4 junction in DiLCrz provides another example. Indeed, when this junction as been modeled with respect to sequence alignments, secondary structure comparison with group I introns and mutagenesis data, the junction has been built very close to the group I intron junction which serves in recognition of the GoU base pair at the active site (Einvik et al, 1998). Though because the L5-DL2.1 interaction had not been anticipated, the peripheral domain DP2-DP2.1 has been left out in the model. The crystal structure of DiLCrz however revealed that L5-DL2.1 forms a kissing complex that constrains the J5/4 junction to fold differently and to stand away from the ribozyme core (Figure 53). In conclusion, it is possible to predict in silico in which known 3D motif a sequence could fold and to identify known 3D motifs in new RNA 3D structures. But because nature offers too many subtle variations, there should be many motifs supposed to adopt known architectures that remain hidden in databases or on the opposite, many motifs may be incorrectly predicted (Leontis et al, 2002). This demonstrates that experimental evidences remain critical to avoid being misled for 3D motif analysis and their prediction. Despite the high recurrence of known RNA 3D motifs, many more may be observed with the increasing number of studied functional ncRNA (Cruz & Westhof, 2011a) and of large RNA three-dimensional structures solved [START_REF] Holbrook | RNA structure: the long and the short of it[END_REF].

CONCLUSION &

PERSPECTIVES

IV. CONCLUSION AND PERSPECTIVES

We have solved the 2.5 Å crystal structure of the Lariat-Capping ribozyme that was one of the last ribozyme for which no X-ray structure was available. The structure revealed the crucial role of the DP2-DP2.1 peripheral domain that clamps the ribozyme core together by means of two tertiary interactions, the L5-L2.1 kissing complex and the L9-DP2 GNRA-TR interaction, and the family B DP2-DP2.1-P10

3WJ. The other 3WJ P3-P8-P15 belonging to the C family junction (Lescoute & Westhof, 2006) is part of the double pseudoknot P7-P15-P3 crucial to the structure of the catalytic core. The half S-turn mediated by the GoU trans WC base pair atop P15 constitutes a motif related to the sarcin-ricin motif [START_REF] Correll | The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 A resolution[END_REF]. Even though the 3D structure of a post-clevage state mimic of LCrz has been solved, the three nucleotides lariat C 230 A 231 U 232 is folded and tightly constrained by specific interactions of each nucleotides of the lariat with nucleotides from the ribozyme core.

Importantly most crucial interactions are supported by mutagenesis data [START_REF] Jabri | In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity[END_REF]. The structural context thus well influences the 3D motif present in RNAs. One striking example is the pk-turn that we describe in [START_REF] Meyer | A structural module in RNase P expands the variety of RNA kinks[END_REF], which is structurally related to the k-turn [START_REF] Lilley | The structure and folding of kink turns in RNA[END_REF] and reverse k-turn [START_REF] Antonioli | Plasticity of the RNA kink turn structural motif[END_REF], altough it does not fold in isolation but rather needs its harbouring RNA to force it to adopt the right structure conferring it highly dynamic properties that may be important for the function of the ribozyme where it has been spotted. RNA 3D folding and structure result from the interplay between sequences and contextual constraints.

Lariat-Capping ribozymes catalyze a reaction equivalent to the first step of splicing catalyzed by group II introns however in a structural context related to group I introns (Nielsen et al, 2005). Structural similarities and differences between LCrz and group I introns in addition to its localization in a twintron leads us to assume that LCrz results from the evolutionary drift of a group I intron. The branching reaction that arose from the metamorphosis of a group I intron into LCrz is of great interest as very little is known about the evolutionary routes that gave rise to ribozymes in general and more specifically to RNA lariat formation [START_REF] Carlomagno | Structural principles of RNA catalysis in a 2'-5' lariat-forming ribozyme[END_REF] thought to be involved in essential processes such as splicing by group II introns [START_REF] Lambowitz | Group II introns: mobile ribozymes that invade DNA[END_REF] or by the spliceosome in eukaryotes (van der Feltz et al, 2012).

LCrz 3D structure exemplifies that chemically identical reactions can arise from very different structural context. This work also shows that the structure of RNAs is maintained in vivo by constant exposure to qualitative selection pressure and that variation of those may result in the appearance of specific RNAs with new functions.

Considering ecological niches populated by microorganisms helps thus embracing the unknown wealth that should exist in nature when they travel horizontally from a niche to another in various media including oceans which contain billions of viruses and microbes per liter.

In a near future, practical objectives will be to solve the crystal structures of the active form of the LCrz using heavy atoms derivatives and also the crystal

  Pütz de m'avoir permis de faire un monitorat en Biochimie. Merci aux « Openlabistes » et moniteurs qui ont rendus ces deux expériences formidables. Merci à Vincent Olieric pour les nombreux créneaux sur la ligne PXIII au SLS durant lesquels ils nous a accompagnés et a partagé son savoir. Merci à Pierre Roblin pour son accompagnement et son expertise lors des expériences de SAXS sur la ligne SWING de SOLEIL. Merci à Claude Sauter, Pierre Legrand, Andrew Thompson, Alastair McEwen, Pierre Poussin-Courmontagne et Jean Cavarelli. Tous ont contribué aux résultats présentés dans cette thèse. Merci à Stéphane Bellemin-Laponaz pour la synthèse et le dont d'iridium hexamine ainsi qu'à Marat Yusupov pour l'osmium hexamine qu'il nous a offert.

  PK-TURN, A NEW RNA KINKING MOTIF ................................................................................................. 168 1

Figure 1 :

 1 Figure 1: (A) Schematic view of the twin-ribozyme intron with at its extremities the exons (yellow) encoding for the SSU of the ribosome. The splicing group I intron, GIR2, is shown in green, the branching LCrz in blue, the ORF of the HEG in brown with its spliceosomal intron I-51 in pink. 5'splice site (SS), 3'splice site, branching point (BP) and internal processing site (IPS) are pointed out by arrows. Nucleotides forming the lariat (CAU) and G 229 equivalent of G in group I introns are materialized. (B) The twin-ribozyme intron with the secondary structure of GIR2 shown in green interrupted in P2 by the sequence of LCrz and HEG. The 5' and 3' exons are shown in yellow. LCrz secondary structure (blue) is represented as the one of group I introns. HEG (brown) and I-51 (pink) are shown in dashed lines.

Figure 2 :

 2 Figure 2: Sequence alignment of DiLCrz and NaLCrz from 29 different species.

Figure 3 :

 3 Figure 3: Diagram of the three different pathways followed by the twin-ribozyme intron. 5' and 3' exons of the SSU of the ribosome are shown in yellow, the splicing group I intron GIR2 in green, the branching LCrz in blue, HEG in brown and the spliceosomal intron I-51 in pink.

Figure 4 :

 4 Figure 4: Life cycle of the myxomycete Didymium iridis that alternates between a haploid microscopic stage (top part) and a diploid macroscopic stage (bottom part). (1) Sexual fusion of amoeba (drawn on right side) or flagellates (drawn on the left side) carrying different mating alleles (black or white nucleus). (2) Development of the zygote into a multinucleate plasmodium. (3) Development into sporangia. (4) Production of haploid spores by meiosis. Spores will be disseminated by the wind and will germinate into new haploid cells(5). Depending on environmental conditions these new haploid cells will either grow as flagellates, amoeba or cysts(6).

  ) the stabilization domain P4-P5-P6,(2) the substrate domain P10-P15 and (3) the catalytic domain P3-P7-P8-P9. There are however some differences between LCrz and group I introns. First, LCrz lacks the P1 stem and the J8/7 junction. In LCrz P15 is equivalent to P1 and P2 of group I introns. Secondly, LCrz pocesses one additional domain, the peripheral domain DP2-DP2.1. Thirdly, the pseudoknot formed by base pairing of J8/7 from LCrz with the 5' strand of P2 create P15 and allows the formation of the family C(Lescoute & Westhof, 2006) three way junction between P3, P8 and P15. In addition, the J15/7 and the J9/10 junctions are unique features of LCrz.

Figure 5 :

 5 Figure 5: (A) Secondary structure diagrams of DiLCrz compared to Azoarcus group IC3 intron (B). Tertiary interactions are displayed using the LW nomenclature (Leontis & Westhof, 2001). Stacking interactions in LCrz are indicated by an arrow pointing into a rectangle.

Figure 6 :

 6 Figure 6: Secondary structure diagram of DiLCrz predicted off state (left panel) compared to the active state (right panel). When HEG P1 is formed, P10 and DP2 are disrupted. HEG P1 encompass nucleotides from mHEG 5'UTR and exclude the 5' strand of DP2. Arrows point out IPS and BP.

Figure 7 :

 7 Figure 7: Secondary structure diagram of AlLCrz (A), NaLCrz (B) and DiLCrz (C). Main differences between AlLCrz and NaLCrz in comparison to DiLCrz are highlighted in red: (1) the sequence starts in P2.1, J5/4 is variable, P6 stem in longer, L9 comprise more nts and the peripheral regulatory domain is larger and potentially habor one additional stem. Nucleotides forming the post-cleavage lariat are shown in pink and G 229 in blue.

Figure 8 :

 8 Figure 8: Schematic representation of the group I introns' processing pathways. Figure adapted from[START_REF] Nielsen | The ability to form full-length intron RNA circles is a general property of nuclear group I introns[END_REF]. Group I intron is shown in green and exons in yellow. exoG and G are colored in blue. On the left panel the splicing pathway is displayed. It takes places in two consecutive transesterification reactions (1 initiated by exoG) and (2 initiated by U) and further processing (3) of the group I intron lead either to a longer than the full length circle circle (5) or a truncated circle (4)(Vicens & Cech, 2009). The circularization pathway leading to unligated exons and formation of a full lenth circularized intron result from a hydrolytic cleavage at the 3' SS (6) followed by a transesterification reaction initiated by G(7).

Figure 9 :

 9 Figure 9: Goddard and Burt cycle (blue arrows) for group I introns (green) and their cognate HEG (brown) showing a correlated evolution of both genetic elements. When all intron-less alleles have been invaded by group I introns thanks to homing endonucleases action, both elements tends to be lost. One way to escape intron loss is for the intron to promote its own spreading at RNA level (red arrow on the left hand side). To persist HE can either acquire new functions or promote its own mobility (red arrows on the right hand side).

Figure 10 :

 10 Figure 10: Group I intron mobility (Figure adapted from (Hedberg & Johansen, 2013)). Mobility at the DNA (orange) level promoted by HE (brown) shown on the left panel. It implicates a dsDNA break mediated by the HE binding (1) and cleavage (2) at its recognition site (shown in purple) followed by recombination (3) and repair (4). Mobility at the RNA (yellow) level promoted by the intron (green). Steps for intron self-mobility are: (1) transcricription of an I+ gene, (2) group I intron splicing, (3) spliced-out intron recognize an intron insertion site in an I-RNA, (4) intron insertion by reverse-splicing and (5) reverse transcription. I-is an intron-less allele and I+ is an intron-containing allele.

Figure 12 :Figure 13 :

 1213 Figure 12: Schematic diagram of the three different catalytic states in which three different group I introns have been trapped for crystallization. The ribozyme core is represented in green, exons in yellow, P1 in cyan, P10 in pink and G in blue. Figure adapted from[START_REF] Golden | Group I Introns : Biochemical and Christallographic Characterization of the Active Site Structure[END_REF]

Figure 14 :

 14 Figure 14: Three-dimensional structure of the Azoarcus group IC3 intron G-binding pocket (A). A129.G128-C178 and A127.A131-U176 base triples sandwich the G.G130-C177 base triple therefore forming the G-binding pocket. (B). Detailed view of the base triple holding G in its binding pocket (C). Nucleotides from P7 are in orange, from J6/7 in green and G in blue.

Figure 15 :

 15 Figure 15: (A) Three-dimensional structure of the Azoarcus group IC3 intron showing the implication of J4/5 (beige) at the 5' splice site. Detail of A-minor interactions of A58 and A87 from J4/5 with the G.U wobble base pair of P1 (cyan). (B) Secondary structure diagram of the Azoarcus group I intron with nucleotides tethering P4-P6 domain to P3-P9 domain shown in colors.

Figure 16 :

 16 Figure 16: Three-dimensional structure of the Azoarcus group IC3 intron showing interaction of J2/3 (grey) and J8/7 (orange) with the minor groove of P1 (cyan).

Figure 17 :

 17 Figure 17: Group II introns secondary structure diagram (A) and 3D structure (B). In green is shown the largest domain DI harboring exons binding sites (EBS 1 to 3). DIV (in grey) can contain an ORF (grey dashes) in its apical loop. In the crystal structure DIV I present as a simple stem loop. DVI (purple) containing the reactive and highly conserved adenosine is missing in the crystal structure. Exons are shown in orange.

Figure 18 :

 18 Figure 18: Secondary structure diagram of the consensus structure among RNase P from Bacteria, Archaea and Eukaryotes. The five universally conserved sequence regions are labeled CRI-V. Regions that are colored have homologous structural elements in all three domains of life and regions in black are specific unique element for each phylogenetic group.

Figure 19 :

 19 Figure 19: Secondary structure diagram of the bacterial RNase P (Figure adapted from (Reiter et al, 2010)) where all stem-loops belonging to the catalytic domain are shown in green and all stem-loop belonging to the substrate domain in orange.

Figure 20 :

 20 Figure 20: Three-dimensional structure of the type A bacterial RNase P holoenzyme bound to a substrate tRNA (A). (B) is the same as (A) after a 180° rotation along the y axis. (C) is a zoom in the structure after a rotation of 90° along the y axis of view (A). Details shown are, contacts between the tRNA (T and D loop, 5'CCA3' tail and at the 5' maturation site) and the RNase P. The pk-turn motif between P16 and P17 is shown in grey. As in Figure 19, the Cdomain is colored in green and the S-domain in orange. The tRNA is shown in pink and the protein component of RNase P in blue.

  and twister (work presented by Breaker lab at the RNA meeting 2013). Their sizes vary from 50 to 150 nucleotides and despite their very diverse 3D structures (Figures 21), they all catalyse the same 2'-O transesterification cleavage reaction described in I.B.1.c.

Figure 21 :

 21 Figure 21: Crystal structures of the hairpin(Rupert & Ferre-D'Amare, 2001), hammerhead(Martick & Scott, 2006), HDV[START_REF] Ke | A conformational switch controls hepatitis delta virus ribozyme catalysis[END_REF]) and glmS[START_REF] Cochrane | Structural and chemical basis for glucosamine 6-phosphate binding and activation of the glmS ribozyme[END_REF] ribozymes represented in cartoon mode. U1A spliceosomal protein used for crystallization in shown in grey.

Figure 22 :

 22 Figure 22: Reactions catalyzed by known naturally occurring ribozymes. (A) Transesterification reaction that occurs during the first step of splicing by group I and II introns is chemically identical. Differences are: (1) the nucleophile in group I intron is provided by an exogenous guanosine (exoG) and is induced by the 3'O when the nucleophile in group II intron is embedded in the intron's sequence and is induced by the 2'O and (2) the 3' product of the first step of splicing is a linear group I intron with exoG bound at the 5' end when group II form a lariat closed by a 2'5' phosphate bond. (B) Transesterification reaction catalyzed by all small naturally occurring ribozymes. It involves attack of the 2'O on the adjacent 3'P leading to the release of a 5' product with a 2'3' cyclic phosphate 3'end and a 3' product with a 5'OH 5'end. (C) Hydrolysis catalyzed by RNase P is triggered by the nucleophilic attack of a water molecule onto the scissile phosphate between the 5' leader sequence of the tRNA and the tRNA. The resulting products are 5'P 5'end for the 3' product and à 3'OH 3'end for the 5' product.

Figure 23 :

 23 Figure 23: The branching reaction catalyzed by LCrz is triggered by the nucleophilic attack of the 2'O from U232 onto the close in space 5'P of C230. The resulting 5' product is a 3'OH 3'end (blue) and the resulting 3' product is a lariat of three nucleotides closed by a 2'5' phosphate bond (pink).

Figure 24 :

 24 Figure 24: (A) The three types of reaction that LCrz can undertake (1) is branching, (2) ligation and (3) irreversible hydrolysis. (B) Secondary structure diagram of LCrz harboring the DP2/DP2.1 peripheral domain on which systematic deletion has been performed to test the number of nucleotides required of branching or hydrolysis. Numbers pointing at arrows indicate the distance from the IPS in the number of nts. (C) Schematic diagram showing effect on the three reactions that LCrz can undertake depending on the length of its 3'end.

Figure 25 :

 25 Figure 25: The two pathways that group II introns can undertake for splicing. (A) Step 1: The 2'O of the highly conserved adenosine from DVI domain attacks the 5' splice site. The 5' exon is released with 3'OH 3'end and the intron is circularized. Step (2) the 3'O of the 5' exon attacks the 3' splice site. Exons are ligated and the lariat intron is freed. Both steps are highly reversible. This pathway if reversed allows for group II introns reverse splicing. (B) Hydrolytic pathway in which a water molecule plays the role of the highly conserved adenosine. Resulting intron is linear and no reverse splicing is possible. (Figure adapted from (Pyle, 2008)).

Figure 26 :

 26 Figure 26: Diagram of the two steps of splicing performed by group I introns. The first step of splicing (3) is triggered by an exogenous guanosine (exoG) when the second step (5) of splicing triggered by the uridine involved in the G.U wobble base pair of P1and belonging to the 3'end of the 5'exon. (Figure adapted from[START_REF] Golden | Group I Introns : Biochemical and Christallographic Characterization of the Active Site Structure[END_REF]).

(

  [START_REF] Hougland | Functional identification of catalytic metal ion binding sites within RNA[END_REF][START_REF] Shan | Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction[END_REF][START_REF] Szewczak | Identification of an active site ligand for a group I ribozyme catalytic metal ion[END_REF] (Figure panel 27 A). Crystal structures discussed in I.A.2.c confirmed the presence of only two out of three metal ions at the active site (Golden, 2008; Lipchock & Strobel, 2008; Stahley & Strobel, 2005) (Figure panel 27 B and C).

Figure 27 :

 27 Figure 27: (A) Biochemical model for metal ion interactions in the active site of the Tetrahymena intron. Metal ions at the active site of Azoarcus group I intron and their interactions shown in a schematic view (B) and in the crystal structure (C). Hydrogen bonds are represented with dashed lines.

Figure 28 :

 28 Figure 28: Metal ion cluster at the active site of group II intron from Oceanobacillus iheyensis. The three metal ions are interconnected via residues of the intron. Potassium ion is displayed in violet and magnesium in yellow. Hydrogen bonds are shown as dashed lines.

Figure 29 :

 29 Figure 29: Detailed three-dimensional structure of the hammerhead active site with nucleotide N and N+1 being C17 and C1.1 respectively (pink). A methyl group replaces the 2'O from C17 to prevent cleavage during crystallization. The general base is G12 while the general acid is G8 (blue).

Figure 30 :

 30 Figure 30: Two metal ions (yellow) at the active site of the type A bacterial RNase P holoenzyme bound to a mature tRNA substrate (pink) and a 5' tRNA leader sequence mimic (violet). RNase P catalytic domain is shown in green and hydrogen bonds in black dashed lines.

  , has been engineered in two ways: (1) by adding a binding site for the spliceosomal protein U1A (Ferre-D'Amare & Doudna, 2000; Oubridge et al, 1994) and (2) by adding a tetraloop receptor motifs (TR) capped by means of a tetraloop (TL) (Ferre-D'Amare et al, 1998) (Figure 31).

Figure 31 :

 31 Figure 31: Diagram of the five DiLCrz constructs with the substrate in trans. In vitro transcription starts with the two guanosines at the 5' end of DP2 of the DiLCrz core shown in black. The 29 nucleotides substrate strand is highlighted in orange and the nucleotide C 230 harboring a phosphorothioate group is designated by an asterisk. The three nucleotides forming the lariat are shown in pink and the guanosine G 229 is shown in light blue. Five alternatives engineered P8 loop, U1A binding sequence or TL-TR motif, are shown in red. Spacer sequences between the ribozyme core and TR and between TR and TL are shown in grey. The HDV ribozyme added to avoid heterogeneous 3' end of DiLCrz due to in vitro run off transcription with the T7 RNA polymerase is shown in green.

Figure 32 :

 32 Figure 32: Design of the DiLCrz circular permutation. (A) Secondary structure of the 166.22 wild type sequence of the DiLCrz with the three nucleotides C 230 , A 231 and U 232 forming the post-cleavage lariat in pink and G 229 equivalent to group I ribozyme G in blue. The in vitro transcription start site is at G 64 in DP2. The sequence is annotated as in the twintron. (B) Circular permutation construct of the DiLCrz (DiLCrz_cp) with the in vitro transcription start site at C 230 . The DP2 stem is closed by a 5'UUCG3' tetraloop shown in green. Non-WC base pairs are shown in respect to the Leontis-Westhof nomenclature (Leontis & Westhof, 2001). Empty rectangles represent base stacking interactions.

Figure 33 :

 33 Figure 33: (A) Highly structured LCrz RNA (black) folding in the course of the transcription impairs folding and efficient cleavage of the minimal HMH ribozyme (orange) but not of the extended HMH (pink). The cleavage site for the HMH ribozyme is shown in blue. (B) Crystal structure of the full length version of the HMH ribozyme is shown in pink(Martick & Scott, 2006) with nucleotides implicated in the stem I -stem II tertiary interaction in green. Crystal structure of the minimal HMH is shown in orange(Scott et al, 1995b). For both HMH structures, nucleotides either side of the scissile phosphate bond are shown in blue and nucleotides positioned differently depending on the HMH sequences lengths are shown in pink sticks (full length HMH) or orange sticks (minimal HMH).

Figure 34 :

 34 Figure 34: In silico predicted tertiary interactions (base pairs implicated are shown in green) between the internal bulge of stem I and the apical loop of stem II in extended versions of the HMH ribozyme (pink) from diverse origins bearing the 5' sequence of DiLCrz_cp (black) as the 3'strand of stem I.

2

 2 In vitro determination of fusion hammerhead ribozymes cleavage efficiencies 2.2.1Co-transcriptional cleavage of fusion hammerhead ribozymes 1. DNA oligos are used as template for amplification by PCR creating the dsDNA template required for the in vitro transcriptions of the full-length hammerhead ribozymes from Schistosoma and full-length fusion hammerhead ribozymes HMH6, HMH9, HMH10 and HMH13. Forward (+) and reverse (-) strands for each hammerhead ribozyme: primers used for PCR amplification of the dsDNA template: Primer 1: 5' TAATACGACTCACTATA3' Primer 2 for fusion hammerhead ribozymes: 5'GGGATACCGGATGGAC3' Primer 2 for hammerhead from Schistosoma: 5'GGATTGGATACCAG3' 3. Phusion High-Fidelity DNA Polymerase at a concentration of 2000 U/mL (New England BioLabs inc) stored at -20 °C.

2 .

 2 Purify by 10% denaturing PAGE the complementary DNA oligos that will form the dsDNA templates used for in vitro transcription of the different ribozymes' cores. 3. Mix 20 g of the forward and the reverse pure template DNA oligos and add KCl up to a final concentration of 500 mM. To hybridize the template DNA oligos heat the sample for 1 min at 95°C and allow it to cool down to room temperature for 10 min. 4. Set up 200 μL transcription reactions containing: 40 g of dsDNA template, 40 μL of TMSDT 5x Buffer, 5 mM ATP, 5 mM GTP, 5 mM CTP, 5 mM UTP, 0.8 μL of alkaline phosphatase (1 U/μL) (see note 4), T7 RNA polymerase as indicated by your provider (see note 5), water up to 200 L.

  endonuclease site used for linearization to permit run-off transcription. Since cloning is the best choice for storing matrices in the laboratory's library, it is advised to anticipate the cloning step at the very beginning of the sequence design by adding a 5' sequence either corresponding to the desired restriction enzyme recognition sequence or to the recombination sequence specified in the cloning kit.All lariat capping GIR1 RNA constructs from Didymium iridis (DiGIR1) were transcribed from plasmid DNA templates obtained by cloning of an insert generated by 3 successive PCR amplifications. The 5' PCR primers were designed to contain an EcoRI restriction site, a T7 promoter, the sequence of the co-transcriptional self-cleaving hammerhead ribozyme and the 5' sequence of the DiGIR1 ribozyme. The 3' PCR primers included a BamHI restriction site, a post-transcriptional self-cleaving HδV ribozyme and the 3' sequence of the lariat capping DiGIR1 ribozyme (Figure6).

2 .

 2 Set up 50 μL transcription reactions containing: 2.5 g of linearized plasmid DNA, 10 μL of TMSDT 5x Buffer, 5 mM ATP, 5 mM GTP, 5 mM CTP, 5 mM UTP, 0.2 μL of alkaline phosphatase (see note 4), T7 RNA polymerase as indicated by your provider (see note 5), water up to 50 μL. 3. In parallel set up 50 L transcription reactions containing: 2.5 g of linearized plasmid DNA, 10 L of TMSDT 5x Buffer, 5 mM ATP, 5 mM GTP, 5 mM CTP, 2.5 mM UTP, 25 mCi 32 P-UTP (10 mCi/mL, 3000 Ci/mmol), 0.2 μL of alkaline phosphatase (1 U/μL) (see note 4), T7 RNA polymerase as indicated by your provider (see note 5), water up to 50 L.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Mutually exclusive theoretical secondary structures of the pre-RNA ternary ribozyme transcript. If the pre-transcript presents an unfolded inactive hammerhead and a folded lariat-capping GIR1 ribozymes, the situation is unfavorable (left panel). However, the opposite situation (right panel) favors maximal cleavage during the in vitro transcription reaction. The bulge in stem I potentially forms a more stable structure with the loop closing stem II than if stem I is closed by a couple of additional A-U pairs. An arrow indicates the cleavage site.

Figure 3 :

 3 Figure 3: Secondary structure diagrams of the fusion hammerhead ribozyme candidates (outputs from RNAfold) show a correct catalytic core structure and unpaired nucleotides in stem I. The secondary structure of the hammerhead ribozyme from Schistosoma is shown as a reference model with nucleotides from the bulge in stem I interacting with the loop from stem II circled. Some of the circled unpaired nucleotides in candidates HMH6, 9 and 13 potentially interact with the loop from stem II.

Figure 4 :

 4 Figure 4: Body-labeled RNA pattern from a standard transcription reaction of the fulllength hammerhead ribozymes fused to the 13 nucleotides at the 5' end of the lariatcapping GIR1 ribozyme. This step is meant to check whether ribozymes with the modified sequence, but lacking the main part of the lariat-capping GIR1 ribozyme, cleave in the course of the transcription reaction. The main RNA species numbered 1 to 3 are visible on the gel, (1) uncleaved hammerhead, (2) cleaved hammerhead core, (3) 13-nt cleaved substrate. HMH10, used as a negative control, carries a mutation C15 with respect to the Schistosoma hammerhead ribozyme numbering (see Figure 1B).

Figure 5 :

 5 Figure 5: Example of kinetic analysis of trans fusion hammerhead lariat capping GIR1 ribozymes. The model hammerhead from Schistosoma (left panel) is compared to HMH13 (right panel). A 50-fold excess of the in vitro transcribed hammerhead ribozyme core was added to the 3' 32 P-labeled RNA substrate substrate to achieve single turnover conditions. Two kinds of cores (and substrates in the case of the Schistosoma ribozyme) were used. The first kind preserved the bulge in stem I whereas the second kind forms a duplex with perfect complementarity. Cleavage is observed only when the bulge is present. The fraction of cleaved substrate was plotted versus time. Data were fitted to the equation F cl =F max (1-e -kt )+F 0 to determine k obs values. The Schistosoma ribozyme presents a k obs similar to HMH13, k obsSch =0.013±4.7 . 10 -3 min -1 and k obsHMH13 =0.016±8.3 . 10 - 3 min -1 showing that our designed hammerheads are as efficient as the Schistosoma model ribozyme at 0.1 mM MgCl 2 .

Figure 6 :

 6 Figure 6: Description of the PCR strategy of the DNA template encoding the ternary ribozyme system. A plasmid containing a 5' truncated lariat-capping GIR1 ribozyme (LCrz) was used as a template for the first PCR amplification. Two further PCR steps created the flanking hammerhead and hepatitis  virus (HV) ribozymes, a T7-promotor and restriction sites for cloning (EcoRI, BamHI).

Figure 7 :

 7 Figure 7: Northern blot analysis of a transcription reaction of the ternary ribozyme construct. Two hours transcription Body-labeled (T) and cold (N) reactions were performed and fractionated side by side by PAGE. Northern blot analysis was performed using a set of probes specific for (i) HV, (ii) LCrz, and (iii) the hammerhead ribozymes.

Figure 35 :

 35 Figure 35: Diagram of two DiLCrz circularly permutated constructs: DiLCrz wild type in black with the HMH13 ribozyme upstream (pink) and the HDV ribozyme downstream (green). Unmodified P8 loop in shown in black and the alternative U1A binding sequence in red. The three nucleotides forming the post-cleavage lariat are circled in pink and G 229 in blue. In vitro transcription starts at HMH ribozyme 5' end (black arrow).

Figure 36 :

 36 Figure 36: Engineered stems to be inserted in place of L8 and/or LP2. TL and TR motifs are shown in red and spacer sequences in grey. (A) Stems containing TL and TR motifs. (B) Stems containing TL motifs. These stems will be inserted in combination with a stem containing a TR motif in the same RNA construct. (C) Stems containing TR motifs.

Figure 37 :

 37 Figure 37: Diagram of DiLCrz_cp construct with open L8 and DP2 where the engineered stems presented in Figure 37 are to be inserted. Nucleotides forming the lariat are shown in pink and G 229 in blue.

Figure 38 :

 38 Figure 38: Four DiLCrz_cp harboring engineered stems shown in Figure 37 that have been used for crystallization trials. (A) and (B) harbor a TL motif in place of LP2 and a TR motif in place of L8. (C) and (D) harbor a TR motif in place of LP2 and a TL motif in place of L8. Nucleotides forming the lariat are shown in pink and G 229 in blue.

Figure 39 :Figure 40 :

 3940 Figure 39: Diagram of DiLCrz_cp_P2-1 construct that has one A-U base pair less (in red) in the DP2 stem in comparison the DiLCrz_cp construct. The 5'UUCG3' tetraloop closing DP2 is shown in green, the nucleotides forming the post-cleavage lariat are shown in pink and G 229 in blue.

Figure 41 :

 41 Figure 41: Diagram of two AlLCrz circularly permutated constructs. (A) AlLCrz_cp_long in black with the HMH9 ribozyme upstream (pink) and the HDV ribozyme downstream (green). The AP2.1 stem in closed by a 5'UUCG3' tetraloop. The three nucleotides forming the postcleavage lariat are circled in pink and G 229 in blue. In vitro transcription starts at HMH ribozyme 5' end. (B) AlLCrz_cp_court after cleavage of both, upstream and downstream, ribozymes in the course of the transcription. AlLCrz_cp_court is shorter of 12 nucleotides in AP2.1 in comparison to AlLCrz_cp_long.

6 . 7 . 10 .

 6710 Centrifuge the bacterial culture at 2500 g fro 10 min at 4 °C. Discard the supernatant. Resuspend the pelleted cells in 80 mL of TBjap (10 mM Pipes, 15 mM CaCl 2 , 250 mM KCl pH adjust pH to 6.7, add MnCl 2 to a concentration of 55 mM and DMSO to 1.96%). 8. Keep the suspension on ice for 10 min.9. Centrifuge the bacterial culture at 2500 g for 10 min at 4 °C. Discard the supernatant. Resuspend the pelleted cells in 18.6 mL of TBjap and DMSO to a final concentration of 7%. 11. Keep the suspension on ice for 10 min. 12. Very delicately aliquot the competent cells in Eppendorf tubes and immediately freeze them in liquid nitrogen. 13. Quickly store aliquots of competent cells at -80 °C. b) Cloning of the full length constructs All LCrz RNA constructs have been transcribed from plasmid DNA templates obtained by cloning of an insert generated by 2 or 4 successive PCRs. The 5' PCR primers have been designed to contain an EcoRI restriction site, a T7 promoter, the sequence of the co-transcriptional self-cleaving HMH ribozyme (for the circularly permutated constructs only) and the 5' sequence of LCrz. 3' PCR primers included a BamHI restriction site, a post-transcriptional self-cleaving HDV ribozyme and the 3' sequence of the LCrz (Figure 42 and Appendix I).

Figure 42 :

 42 Figure 42: Scheme of the successive PCRs necessary to clone full length constructs including EcoRI restriction site (blue), the T7 promoter (pink), extended HMH ribozyme (orange), LCrz (black), HDV ribozyme (green) and BamHI restriction site (cyan).

9 .

 9 Insert the purified PCR product into a pUC19 plasmid using In-Fusion ® HD Cloning Kit (Clontech). An alternative protocol has also been used: mix a ½ molar ratio of pUC19 plasmid that has been double digested by EcoRI and BamHI and purified on agarose gel and the insert that has also been double digested by EcoRI and BamHI and purified on agarose gel with 0.5 L of PEG 6000 24% w/v (Hampton Research) and water up to 17.5 L. Incubate the sample at 65 °C for 5 min. Add 0.5 L of T4 DNA ligase (30 U/L-Thermo Scientific) and 2 L of T4 DNA ligase 10x (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP pH 7.8 at 25 °C-Thermo Scientifc). Incubate the sample at 20 °C for 1 hour. 10. Directly transform competent Escherichia coli XL-1 Blue cells (prepared as described in II.B.2.a) with either the In-Fusion ® HD Cloning Kit product or with 10 L of the ligation reaction. 11. Spread the transformed competent Escherichia coli XL-1 Blue cells samples on Petri dish containing LB/Ampicillin (100 µg/mL) and incubate overnight at 37 °C.

15 .

 15 Prepare glycerol stocks of the clones containing the desired sequences by mixing 1 mL of saturated bacterial culture to 600 μL of sterilized 50% glycerol solution in 2 mL screw cap tubes. Store the glycerol stocks at -80 °C. c) Site directed mutagenesis Because the U203C mutation shown to stabilize NaLCrz (Jabri & Cech, 1998) resulting from a PCR artefact in DiLCrz_cp, has been established indispensable for crystallization of DiLCrz (Figure 43) and does not impair branching (Figure 44)

Figure 43 :

 43 Figure 43: (A) Crystal packing contacts between nucleotides neighboring the U203>C mutation in P15 of one DiLCrz_cp (cyan) and nucleotides of P7 of a symmetry-related DiLCrz_cp molecule (light pink). (B) Superposition of the A113-C203 WC trans base paire (cyan) of P15 to a canonical A-U WC trans base pair (orange).

Figure 44 :

 44 Figure 44: Cleavage assay of the U203C mutated DiLCrz_active. [α-32P] UTP body labeled wild type active DiLCrz and mutated U203C active DiLCrz have been assayed for cleavage efficiency. RNA is denatured at 95°C for 1 min and refolded in 1 M KCl, 25 mM MgCl2, 10 mM Acetate pH 5.0 at 45 °C for 5 min. An aliquot is taken right after refolding, t=0 min. To trigger the cleavage reaction, 4 volumes of 1 M KCl, 25 mM MgCl2, 47,5 mM HEPES pH 7.5 is added to the folded RNA. Cleavage reaction takes place at 45 °C for 4 h(Decatur, 1995). Aliquots are taken at t=30 min, t=60 min, t=120 min and t=240 min. The reaction is stopped by addition of EDTA-urea blue loading buffer. Aliquots are loaded on a 8% denaturing PAGE. Three distinct products are observed: full-length DiLCrz is the uncleaved ribozyme (188 nt), cat is the catalytic core (166 nt) and sub is the cleaved substrate(22 nt).

Figure 45 :

 45 Figure 45: Diagram showing the position of the U to C mutation (orange) in P15 stem of (A) DiLCrz_active, (B) AlLCrz and (C) NaLCrz.

  e)Large scale in vitro transcription of the full length RNA using linearized plasmids as templates Large scale in vitro transcription (up to 6 mL) is indispensable to obtain micrograms of pure RNA for crystallization 1. Set up 6 mL transcription reaction containing: 300 g of linearized plasmid DNA, 1.2 mL of TMSDT 5x Buffer (200 mM Tris-HCl pH 8.1, 110 mM MgCl 2 , 5 mM spermidine, 25 mM DTT, 0.05% Triton), 5 mM ATP, 5 mM GTP, 5 mM CTP, 5 mM UTP, 6 μL of alkaline phosphatase (1 U/L in TMSDT 1x -Sigma-Aldrich), T7 RNA polymerase as indicated by your provider (we produce our T7 RNA polymerase in the laboratory), water up to 6 mL.2. Incubate at 37 °C for 4 h.

3 .

 3 Instantly incubate at 60 °C for 1 hour to allow HDV ribozyme cleavage. f) LCrz RNA purification Four different methods have been tried to purify the LCrz RNA: native PAGE, denaturing PAGE, denaturing PAGE combined to HPLC and BioTrap (Schleicher & Schuell). Only the following protocol using denaturing PAGE purification allows us to obtain RNA suitable for crystallization.

  PMRIQYAKTD SDIIAKMK) that is added to the RNA harboring a U1A binding site in the loop of P8 (constructs described in II.A.1.a Figure 31 and II.A.1.b Figure 35) has been produced in the laboratory. We thank Adrian Ferré d'Amaré for giving us the plasmid pU1A_31H36R containing the U1A gene. 1. Transform BL21 Star TM (DE3)pLysS One Shot® (Invitrogen TM ) competent cells with 6 ng of plasmid containing the sequence for U1A over-expression.

Table 9 :

 9 48 crystallization conditions sparse matrix with condition F12 (highlighted in purple) from the Index HT (Hampton research ©) used as a starting point. HEPES pH 7.5 is fixed at 100 mM. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL drops.

Table 10 :

 10 48 crystallization conditions sparse matrix with condition H6 (highlighted in purple) from the Index HT (Hampton research ©) used as a starting point. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL drops.

Table 11 :

 11 29 crystallization conditions sparse matrix with conditions D11, F11, F12 (highlighted in purple) from the Index HT (Hampton research ©) and F1, F3 from the PEGRx HT (Hampton research ©) used as a starting point. pH of the buffer is the only parameter that has been modified in the sparse matrix. This screen has been used to crystallize DiLCrz_cp, DiLCrz_cp_U203C, DiLCrz_active and DiLCrz_active_U203C at 20 °C in 400 nL drops.

- 5

 5 or 10 min in 5 l of crystallization solution and 5 or 10 % (v/v) PEG 600 -5 or 10 min in 5 l of crystallization solution and 5 or 10 % (v/v) MPD -5 or 10 min in 5 l of crystallization solution and 5 or 10 % (v/v) ethylene glycol In order to solve the structure of the DiLCrz_cp using heavy atoms derivatives, crystals have been soaked as follows (Heras & Martin, 2005): 1. 5 min in 5 l of crystallization solution and 0 or 5 or 10 % (v/v) PEG 400 (optional) 2. 5 min in 5 l of crystallization solution and 0 or 5 or 10 % (v/v) PEG 400 and 1 mM osmium hexamine or iridium hexamine or cobalt hexamine 3. Few seconds in 5 l of crystallization solution and 0 or 5 or 10 % (v/v) PEG 400 and 2 mM MgCl 2

Figure 46 :

 46 Figure 46: Fluorescence scan performed on iridium hexamine soaked crystals to determine wavelengths at which data should be collected to solve the structure of DiLCrz_cp_U203C de novo by MAD.

Figure 47 :

 47 Figure 47: Histogram showing the yearly growth of structures solved by X-ray (Data taken from PDB website). RNA structures are shown in blue with the exact number in brackets. In red is the total number of X-ray structures registered in the PBD up to the 29 th of July 2013.

Figure 48 :

 48 Figure 48: 3D structures of P3 stem (A) and DP2/DP2.1/P10 3WJ (B) in the 2Fo-Fc composite omit maps of DiLCrz_active_U203C. Stems are colored as in the article of the crystal structure of DiLCrz.

Figure 49 :

 49 Figure 49: 3D structures of the two first base pairs of P7 stem clearly shifted from the 2Fo-Fc composite omit maps of DiLCrz_active_U203C.

Figure 50 :

 50 Figure 50: SAXS shapes of DiLCrz_cp_U203C (A) and DiLCrz_active_U203C (B). Stem loops of DiLCrz are schematically drawn to orient the SAXS shapes and colored as in the article of the crystal structure of DiLCrz. (C) Superposition of SAXS curves recorded for the two forms of DiLCrz. (D)Table that summarizes SAXS parameters for each DiLCrz.

Figure 51 :

 51 Figure 51: A model for the metamorphosis of a group I intron into LCrz. Figure adapted from (Beckert et al, 2008). (A) Azoarcus group I intron a step just before the second step of splicing undertakes some mutation in J8/7, P2 and P3 that allows for partial alternatives base pairing between the substrate and the catalytic domains. (B) Loss of the 5' exon favors secondary structure's remodeling especially of the long stretch of nucleotides left unpaired in P1. (C) All structural elements are in place to perform the branching reaction. (D) Loss of structural element not necessary to the energetic stabilization of the active conformation of the new LCrz.

Figure 52 :

 52 Figure 52: (A) Consensus sequence of the k-turn motif numbered as defined by David Lilley (Lilley, 2012). Characteristic G.A base pairs at the beginning of the NC-stem are represented according to LW nomenclature (Leontis & Westhof, 2001). (B) 3D structure of the k-turn motif present in the SAM-I riboswitch (Stoddard et al, 2010) labeled as the consensus sequence. Circled number show: (1) H bond between 2'O of L1 and N1 of A1n, (2) H bond between 2'O of L3 and the proS non-bridging oxygen of the phosphate group between L1 and L2 and (3) an A-minor interaction between the nucleotide at position -1n and N1 of the highly conserved adenosine 2b. In both panels, the longest strand that contains the three bulged nucleotides is shown in purple and the shortest in green.

Figure 53 :

 53 Figure 53: Comparison between the modeled J5/4 junction of DiLCrz (A)(Beckert, 2008) and the J5/4 junction observed in the crystal structure (B). J5/4 in the 3D structure comprising the peripheral domain DP2-DP2.1 is shaped to tailor the L5/DL2.1 kissing complex (C) and is made of two A-minor and base stacking interactions.

  structures and SAXS shapes of the two others LCrz from Naegleria sp. and Allovahlkampfia sp. Indeed, if LCrz appeared independently several times in the course of evolution, these structures will help better understand how different structural solutions have been selected to respond to the same problem which is to trigger LCrz catalysis mainly after splicing of the whole intron. The structures will unravel how domains that differ from DiLCrz are organized but also if the L5-L2.1 kissing complex conferring to DiLCrz its unique signet ring shape is preserved or whether elements inherited from the ancestral group I ribozyme inserted within the ribosomal intron have evolved to form different motifs of interaction. Crystallization of NaLCrz and AlLCrz might require more engineering than DiLCrz implying highthroughput probing experiments to refine the secondary structure of their large peripheral domain. In a more distant future experiments working with the full-length twintron should help understanding how splicing by GIR2 and branching by LCrz are coordinated to regulated SSU rRNA production.

   Natrix HT TM F1: 0.05 M Na cacodylate trihydrate pH 6.0, 30% v/v MPD  100 M  400 nL  Sitting drop  20 °C  2 weeks  Natrix HT TM G7: 0.08 M Strontium chloride hexahydrate, 0.02 M Magnesium chloride hexahydrate, 0.04 M Na cacodylate trihydrate pH 7.0, 20% v/v MPD, 0.012 M Spermine tetrahydrochloride  100 M  400 nL  Sitting drop  20 °C  4 weeks  Natrix HT TM G8: 0.08 M NaCl, 0.04 M Na cacodylate trihydrate pH 7.0, 30% v/v MPD, 0.012 M Spermine tetrahydrochloride  100 M  400 nL  Sitting drop  20 °C  2 weeks  Natrix HT TM G10: 0.04 M LiCl, 0.08 M Strontium chloride hexahydrate, 0.02 M Magnesium chloride hexahydrate, 0.04 M Na cacodylate trihydrate pH 7.0, 30% v/v MPD, 0.012 M Spermine tetrahydrochloride  100 M  400 nL  Sitting drop  20 °C  6 weeks  Natrix HT TM H4: 0.08 M KCl, 0.02 M Magnesium chloride hexahydrate, 0.04 M Na cacodylate trihydrate pH 7.0, 50% v/v MPD, 0.012 M Spermine tetrahydrochloride  Index HT TM D7: 0.1 M BIS-TRIS pH 6.5, 25% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  400 nL  Sitting drop  20 °C  4 weeks  Index HT TM D8: 0.1 M HEPES pH 7.5, 25% w/v Polyethylene glycol 3,350  100 M  400 nL  Sitting drop  20 °C  6 months  Index HT TM D10: 0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol monomethyl ether 5,000  100 M  400 nL  Sitting drop  20 °C  6 days  Index HT TM D11: 0.1 M BIS-TRIS pH 6.5, 28% w/v Polyethylene glycol monomethyl ether 2,000  Frozen in liquid ethane  2.8 Å  100 M  400 nL  Sitting drop  20 °C  4 weeks  Index HT TM E1: 0.2 M Calcium chloride dihydrate, 0.1 M BIS-TRIS pH 6.5, 45% v/v (+/-)-2-Methyl-2,4-pentanediol  100 M  400 nL  Sitting drop  20 °C  4 weeks  Index HT TM E4: 0.2 M Ammonium acetate, 0.1 M HEPES pH 7.5, 45% v/v (+/-)-2-Methyl-2,4pentanediol  100 M  400 nL  Sitting drop  20 °C  6 months  Index HT TM E9: 0.05 M Ammonium sulfate, 0.05 M BIS-TRIS pH 6.5, 30% v/v Pentaerythritol ethoxylate (15/4 EO/OH)  100 M  400 nL  Sitting drop  20 °C  4 weeks  Index HT TM F10: 0.2 M Sodium chloride, 0.1 M BIS-TRIS pH 5.5, 25% w/v Polyethylene glycol 3 Index HT TM H6: 0.2 M Sodium formate, 20% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  2.8 Å  100 M  400 nL  Sitting drop  20 °C  9 weeks  PEGRx HT TM B8: 0.1 M BIS-TRIS pH 6.5, 20% w PEGRx HT TM F3: 0.1 M Sodium malonate pH 8.0, 0.1 M Tris pH 8.0, 30% w/v Polyethylene glycol 1,000  Frozen in liquid ethane  5  Nuc-Pro HTS G6: 100 mM Sodium acetate, 50mM ADA pH 6.0, 15% v/v MPD  100 M  400 nL  Sitting drop  20 °C  6 months  Nuc-Pro HTS G9: 100 mM Sodium chloride, 50 mM MES Sodium salt pH 6.0, 23% v/v MPD  100 M  400 nL  Sitting drop  20 °C  6 weeks  0.1 M Sodium malonate pH 8.0, 0.1 M Tris pH 7.0, 30% w/v Polyethylene glycol 1,000  Frozen in liquid nitrogen  3  Index HT TM F12: 0.2 M Sodium chloride, 0.1 M HEPES pH 7.5, 25% w/v Polyethylene glycol 3 Natrix HT TM E11: 0.075 M NaCl, 0.002 M Calcium chloride dihydrate, 0.05 M Na cacodylate trihydrate pH 6.0, 30% w/v 1,6-Hexanediol, 0.0005 M Spermine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.12 M BIS-TRIS pH 6.5, 33.6% w/v Polyethylene glycol monomethyl ether 2,000  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  6 days  0.14 M BIS-TRIS pH 6.5, 39.2% w/v Polyethylene glycol monomethyl ether 2,000  100 M  5 L  Microbatch in gel sealed with paraffin oil  20 °C  5 weeks  0.12 M BIS-TRIS pH 6.5, 33.6% w/v Polyethylene glycol monomethyl ether 2,000, 1%low melting agarose  Frozen in liquid ethane  10 Å  100 M  5 L  Microbatch in gel sealed with paraffin oil  20 °C  5 weeks  0.14 M BIS-TRIS pH 6.5, 39.2% w/v Polyethylene glycol monomethyl ether 2,000, 1%low melting agarose  Frozen in liquid ethane  10 Å  100 M  5 L  Microbatch in gel sealed with paraffin oil  20 °C  5 weeks  0.16 M BIS-TRIS pH 6.5, 44.8% w/v Polyethylene glycol monomethyl ether 2,000, 1%low melting agarose  Frozen in liquid ethane  10 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.24 M Sodium chloride, 0.12 M BIS-TRIS pH 6.5, 30% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.28 M Sodium chloride, 0.14 M BIS-TRIS pH 6.5, 35% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.32 M Sodium chloride, 0.16 M BIS-TRIS pH 6.5, 40% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.36 M Sodium chloride, 0.18 M BIS-TRIS pH 6.5, 45% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.22 M Sodium chloride, 0.11 M BIS-TRIS pH 6.5, 27.5% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  3.6 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.28 M Sodium chloride, 0.14 M HEPES pH 7.5, 35% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  4 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.32 M Sodium chloride, 0.16 M HEPES pH 7.5, 40% w/v Polyethylene glycol 3,350  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.36 M Sodium chloride, 0.18 M HEPES pH 7.5, 45% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  2.7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 weeks  0.120 M NaCl, 0.0032 M Calcium chloride dihydrate, 0.08 M Na cacodylate trihydrate pH 6.0, 48% w/v 1,6-Hexanediol, 0.008 M Spermine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 weeks  0.135 M NaCl, 0.0036 M Calcium chloride dihydrate, 0.09 M Na cacodylate trihydrate pH 6.0, 54% w/v 1,6-Hexanediol, 0.009 M Spermine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 weeks  0.135 M NaCl, 0.0036 M Calcium chloride dihydrate, 0.09 M Na cacodylate trihydrate pH 6.0, 54% w/v 1,6-Hexanediol, 0.009 M Spermine  Frozen in liquid ethane  To many crystals in the loop  100 M  5 L  Microbatch sealed with crystal tape  20 °C  5 weeks  0.16 M BIS-TRIS pH 6.5, 44.8% w/v Polyethylene glycol monomethyl ether 2,000  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3 weeks  0.28 M Sodium chloride, 0.14 M BIS-TRIS pH 6.5, 35% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  7 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3 weeks  0.32 M Sodium chloride, 0.16 M BIS-TRIS pH 6.5, 40% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  5 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3 weeks  0.28 M Sodium chloride, 0.14 M HEPES pH 7.5, 35% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  2.5 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3 weeks  0.32 M Sodium chloride, 0.16 M HEPES pH 7.5, 40% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  3.7 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3 weeks  0.36 M Sodium chloride, 0.18 M HEPES pH 7.5, 45% w/v Polyethylene glycol 3,350  Frozen in liquid ethane  4 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  3 weeks  0.32 M Sodium chloride, 0.16 M BIS-TRIS pH 6.5, 40% w/v Polyethylene glycol 3,350, Spermidine 12 mM  Frozen in liquid nitrogen  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.06 M BIS-TRIS pH 6.5, 16.8% w/v Polyethylene glycol monomethyl ether 2,000, 1 mM Iridium hexamine  Frozen in liquid nitrogen  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.12 M BIS-TRIS pH 6.5, 33.6% w/v Polyethylene glycol monomethyl ether 2,000, 1 mM Iridium hexamine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.14 M BIS-TRIS pH 6.5, 39.2% w/v Polyethylene glycol monomethyl ether 2,000, 1 mM Iridium hexamine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.16 M BIS-TRIS pH 6.5, 44.8% w/v Polyethylene glycol monomethyl ether 2,000, 1 mM Iridium hexamine  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.24 M Sodium chloride, 0.12 M BIS-TRIS pH 6.5, 30% w/v Polyethylene glycol 3,350, 1 mM Iridium hexamine  Frozen in liquid nitrogen  7 Å  100 M  5 L  Microbatch sealed with paraffin oil  20 °C  5 days  0.32 M Sodium chloride, 0.16 M BIS-TRIS pH 6.5, 40% w/v Polyethylene glycol 3,350, 1 mM Iridium hexamine  Frozen in liquid nitrogen  7 Å  100 M  5 L  Microbatch sealed with crystal tape  20 °C  10 days  0.12 M Succinic acid pH 7.0, 0.12 M BICINE pH 8.5, 36% v/v Polyethylene glycol monomethyl ether 550, 1 mM Iridium hexamine  100 M  5 L  Gel Microbatch sealed with paraffin oil  20 °C  4 weeks  0.1 M BIS-TRIS pH 6.5, 28% w/v Polyethylene glycol monomethyl ether 2,000, 1% low melting agarose  Frozen in liquid nitrogen  10 Å  100 M  5 L  Gel Microbatch sealed with paraffin oil  20 °C  4 weeks  0.2 M Sodium chloride, 0.1 M BIS-TRIS pH 6.5, 25% w/v Polyethylene glycol 3,350, 1% low melting agarose  Frozen in liquid nitrogen  10 Å  100 M  5 L  Gel Microbatch sealed with paraffin oil  20 °C  4 weeks  0.2 M Sodium formate, 20% w/v Polyethylene glycol 3,350, 1%low melting agarose DiLCrz_active Picture  100 M  400 nL  Sitting drop  20 °C  12 weeks  Natrix HT TM D5: 0.1 M Potassium chloride, 0.015 M Magnesium chloride hexahydrate, 0.05 M TRIS hydrochloride pH 7.5, 10% v/v Polyethylene glycol monomethyl ether 550  100 M  5 L  Microbatch sealed with crystal tape  20 °C  3  0.2 M Ammonium acetate, 0.1 M BIS-TRIS pH 6.5, 25% w/v Polyethylene glycol 3,350 DiLCrz_TLR3 Picture  72 M  200 nL  Sitting drop  20 °C  5 weeks  Natrix HT TM E11: 0.075 M NaCl, 0.002 M Calcium chloride dihydrate, 0.05 M Na cacodylate trihydrate pH 6.0, 30% w/v 1,6-Hexanediol, 0.0005 M Spermine  72 M  400 nL  Sitting drop  20 °C  3 weeks  Natrix HT TM E11: 0.075 M NaCl, 0.002 M Calcium chloride dihydrate, 0.05 M Na cacodylate trihydrate pH 6.0, 30% w/v 1,6-Hexanediol, 0.0005 M Spermine  Frozen in liquid ethane  25 Å  40 M  400 nL  Sitting drop  20 °C  10 weeks  60 mM Magnesium choride, 190 mM Sodium choride, 100 mM Sodium citrate pH 5.

Table 1 :

 1 Comparisons of the principal features of the twin-ribozyme from D. iridis and Naegleria.

		Dir.S956-1	Nae.S516
	Microorganism	Dydimium iridis	29 out of 70 Naegleria
		(Panama 2 isolate)	strains sequenced
	Insertion site of the twintron	Position 956 in SSU rRNA	Position 516 in SSU rRNA
	Insertion site of LCrz-HEG	P2 stem of DiGIR2	P6 stem of NaGIR2
	GIR2	IE type group I intron	IC1 type group I intron
	Intron in HEG	Spliceosomal intron of 51 nts

and 25). However

	, if the reaction catalyzed by
	both ribozymes is similar from a chemical point of view, all other features such as,
	nucleotides implicated in branching, size of the lariat, global 2D and 3D structures,
	biological role and context etc. are specific to each ribozyme. This implies that in the
	course of evolution different RNAs have been selected to catalyze the same reaction.

  Set up 50 μL transcription reactions containing: 1 g of the cleaned PCR reaction, 10 μL of TMSDT 5x Buffer, 5 mM ATP, 5 mM GTP, 5 mM CTP, 2.5 mM UTP, 25 Ci

	32 P-UTP (10 mCi/mL, 3000 Ci/mmol), 0.2 μL of alkaline phosphatase (1 U/μL)
	(see note 4), T7 RNA polymerase as indicated by your provider (see note 5),
	water up to 50 μL.
	9. Incubate at 37 °C for 2 h.
	10. Remove a 12 μL aliquot of each transcription reaction after 30 min, 1 h and 2 h.
	11. Stop the transcription by mixing the sample to an equal volume of urea-
	containing loading buffer 2x.
	5. Check the efficiency of amplification by mixing 5 μL of the PCR reaction to 1 μL
	glycerol blue loading buffer 6x and load on to a 2% (w/v) agarose gel.
	6. After ethidium bromide staining, observe the DNA migration pattern by UV
	shadowing.

2. Design primers for PCR amplification of the template DNA oligos. (see note 3). 3. Prepare 50 L of PCR reaction containing: 300 ng of each DNA template, 0.5 μL of the Phusion High-Fidelity DNA Polymerase (2000 U/mL), 10 μL of the Phusion HF Buffer 5x, 1.2 M of forward primer, 1.2 M of reverse primer, 400 M dNTPs mix, water up to 50 μL.

4. Run the PCR reaction in a thermal cycler following the set up displayed in Table

1.

7. Clean the PCR reaction using the Micro Bio-Spin Columns.

8.

  M of forward primer, 1.2 M of reverse primer, 400 M dNTP mixture, water up to 50 μL (see note 13).3. Run the PCR reaction in a thermal cycler following the set up displayed in Table1(see note 14).

	4. Mix the PCR reaction to 10 μL glycerol blue loading buffer 6x and load the entire
	PCR reaction onto a 2% (w/v) agarose gel.
	5. After ethidium bromide staining, observe the DNA migration pattern under UV
	shadowing.

1. Design forward and reverse primers for the successive PCRs (see note 12). 2. Prepare 50 L of PCR reaction containing: 50 pg of template plasmid DNA, 0.5 μL of the Phusion High-Fidelity DNA Polymerase (2000 U/mL), 10 μL of the Phusion HF Buffer 5x, 1.2 6. Cut the DNA band of interest out of the gel. 7. Recover the DNA insert in 30 L of water with a gel extraction kit (see note 15).

8. Insert the purified PCR product into a pUC19 plasmid using a cloning kit. 9. Directly transform XL-1 Blue Escherichia coli competent cells with the recombinant plasmid product (see note 16).

Table 1 :

 1 PCR program

	Step	Temperature (°C)	Time	Nb of cycles
	Initial denaturing	95	3 min	1
	Denaturing	95	30 s
	Annealing	45*/50**	1 min	30
	Elongation	72	1 min
	End	4	10 min	1
	*Annealing temperature for PCR of section 3.2.1 step 4.

Table 2 .

 2 Insert samples in the thermal cycler when the temperature of the sample holder is at 90 °C minimum.

	2. Prepare 50 L of PCR1 reaction containing: 50 pg of template plasmid DNA,
	0.5 μL of Phusion High-Fidelity DNA Polymerase (2000 U/mL -New England
	BioLabs inc), 10 μL of Phusion HF Buffer 5x (New England BioLabs inc), 1.2
	M of forward primer, 1.2 M of reverse primer, 400 M dNTP mixture, water
	up to 50 μL. Samples are kept on ice while preparing the PCR reaction.
	3. Run the PCR reaction in a thermal cycler following the set up displayed in
	Step	Temperature (°C)	Time	Nb of cycles
	Initial denaturing	95	3 min	1
	Denaturing	95	30 s
	Annealing	50	1 min	30
	Elongation	72	1 min
	End	4	10 min	1

Table 2 :

 2 PCR program to produce the full-length constructs inserts

	4. Mix the PCR reaction to 10 μL glycerol blue loading buffer 6x (50% glycerol,
	0.1% xylene cyanol, 0.1% bromophenol blue) and load the entire PCR
	reaction onto a 2% (w/v) agarose gel (2 g of agarose for routine use -Sigma
	Aldrich -dissolved in 100 mL of TBE 1x).
	5. After ethidium bromide staining, observe the DNA migration pattern with UV
	light.
	6. Cut the DNA band of interest out of the gel.
	7. Recover the DNA insert in 30 L of water with the QIAquick ® Gel Extraction Kit
	(Qiagen). To avoid any trouble with next steps of the cloning process, 10% of
	ammonium acetate 3 M pH 5.2 is added to the recovered DNA and
	precipitated with 3 volumes of pure ethanol. DNA is then re-suspended in 30
	L of water.

Table 3 .

 3 Insert samples in the thermal cycler when the temperature of the sample holder is at 90 °C minimum.

	2. Prepare 50 L of PCR reaction containing: 50 ng of template plasmid DNA,
	0.5 μL of the Phusion High-Fidelity DNA Polymerase (2000 U/mL -New
	England BioLabs inc), 10 μL of the Phusion HF Buffer 5x (New England
	Step	Temperature (°C)	Time	Nb of cycles
	Initial denaturing	99	3 min	1
	Denaturing	95	30 s
	Annealing	50	1 min	15
	Elongation	72	1 min 30 s
	End	4	10 min	1

BioLabs inc), 1.2 M of forward primer, 1.2 M of reverse primer, 400 M dNTP mixture, 10% DMSO, water up to 50 μL. Samples are kept on ice while preparing the PCR reaction.

3. Run the PCR reaction in a thermal cycler following the set up displayed in

Table 3 :

 3 PCR program for site directed mutagenesis

	4. Mix the PCR reaction to 10 μL glycerol blue loading buffer 6x (50% glycerol,
	0.1% xylene cyanol, 0.1% bromophenol blue) and load the entire PCR
	reaction onto a 2% (w/v) agarose gel (2 g of agarose for routine use -Sigma
	Aldrich -dissolved in 100 mL of TBE 1x).
	5. After ethidium bromide staining, observe the DNA migration pattern with UV
	light.
	6. Cut the DNA band of interest out of the gel.

10. Spread the transformed competent Escherichia coli XL-1 Blue cells on LB/Ampicillin (100 µg/mL) Petri dish and grow overnight at 37 °C.

and 5. LCrz RNA construct RNA concentration in μM Incubation temperature in °C Drop size in nL Crystallization screen Index HT -Hampton Research Crystallization screen PEGRx HT -Hampton Research Crystallization screen Natrix HT -Hampton Research Crystallization screen Crystal screen HT -Hampton Research Crystallization screen Crystal screen Cryo HT -Hampton Research Crystallization screen JBScreen Nuc-Pro HTS -JenaBioscience

	DiLCrz_cp	100 20	400	x	x	x			x
	DiLCrz_cp_U203C	88	20	400			x	x	x
	DiLCrz_cp_U203C	100 20	400	x	x	x			x
	DiLCrz_cp_U203C	100 20	400			x
	DiLCrz_cp_U203C	200 20	400			x
	DiLCrz_cp_U203C	300 20	400			x
	DiLCrz_cp_U203C	500 20	400			x
	DiLCrz_cp_U203C	100 30	400			x
	DiLCrz_cp_U203C	200 30	400			x
	DiLCrz_cp_U203C	300 30	400			x
	DiLCrz_cp_U203C	500 30	400			x
	DiLCrz_cp_P2-1	100 20	400	x	x	x			x
	DiLCrz_cp_U203C_P2-1	100 20	400	x	x	x			x
	DiLCrz_cp_P2TL9_P8TR17	100 20	400	x	x	x			x
	DiLCrz_cp_P2TL16P8_TR23	100 20	400	x	x	x			x
	DiLCrz_cp_P2TR18P8_TL12	100 20	400	x	x	x			x
	DiLCrz_cp_P2TR22_P8TL13	100 20	400	x	x	x			x
	DiLCrz_active	100 20	400	x	x	x			x
	DiLCrz_U203C_active	100 20	400	x	x	x	x	x	x
	AlLCrz_cp_U252C_long	100 20	400	x	x	x			x
	AlLCrz_cp_court	100 20	400	x	x	x			x
	AlLCrz_cp_U252C_court	100 20	400	x	x	x			x
	NaLCrz_cp_U261C_long	100 20	400	x	x	x			x
	NaLCrz_cp_U261C_court	100 20	400	x	x	x			x

Hampton Research © and Nuc-Pro HTS from Jena Bioscience). Crystallization trays were prepared with the Mosquito ® robot (TTP Labtech) in 96 wells Corning © plates by mixing a volume of LCrz RNA with an equal volume of reservoir solution.

Crystallization plates were kept at different temperatures. All crystallization trials are enlisted in Table

4

Table 4 :

 4 Summary table of the crystallization trials done with the Mosquito® robot in 96 wells sitting drop set-ups. Crosses indicate that this crystallization screen has been tested. Drop size as indicated corresponds to the volume of RNA plus the volume of crystallization solution.

	LCrz ARN	LCrz core RNA

concentration in μM LCrz substrate RNA concentration in μM U1A protein concentration in μM Incubation temperature in °C Drop size in nL Crystallization screen Index HT -Hampton Research Crystallization screen PEGRx HT -Hampton Research Crystallization screen Natrix HT -Hampton Research Crystallization screen JBScreen Nuc-Pro HTS -JenaBioscience

	DiLCrz_TLR1	180 240	20	400	x	x	x	x
	DiLCrz_TLR1	133 200	20	400			x
	DiLCrz_TLR1	180 240	37	400			x
	DiLCrz_TLR1	180 240	20	200			x
	DiLCrz_TLR2	100 150	20	400	x	x	x	x
	DiLCrz_TLR3	72 96	20	400	x	x	x	x
	DiLCrz_TLR3	133 200	20	400			x
	DiLCrz_TLR3	72 96	37	400			x
	DiLCrz_TLR3	72 96	20	200			x
	DiLCrz_TLR3	72 96	20	2000		x	x
	DiLCrz_TLR3	50 75	20	400		x	x
	DiLCrz_TLR4	120 160	20	400	x	x	x	x
	DiLCrz_TLR4	133 200	20	400			x
	DiLCrz_TLR4	120 160	37	400			x
	DiLCrz_TLR4	120 160	20	200			x
	DiLCrz_cp_U1A 100	100 20	400	x	x	x	x
	DiLCrz_cp_U1A 100	100 20	1000		x
	DiLCrz_cp_U1A 50	50 20	1000		x

Table 5 :

 5 Summary table of the crystallization trials done with the Mosquito® robot in 96 wells sitting drop set-ups for LCrz constructs in complex with an RNA substrate or the U1A protein. Crosses indicate that this crystallization screen has been tested. Drop size as indicated corresponds to the volume of RNA plus the volume of crystallization solution.

Table 6 :

 6

		96 crystallization conditions sparse matrix the condition A9 (highlighted in purple)
	from the JBScreen Nuc-Pro HTS (JenaBioscience) used as a starting point. Sodium citrate
	pH 5.6 is fixed at 100 mM. This screen has been used to crystallize DiLCrz_TLR3 at 20 °C,
	30 °C and 37°C in 400 nL drops at concentration of: -[DiLCrz_TLR3] = 40 M, [RNA substrate strand] = 53 M
	-[DiLCrz_TLR3] = 72 M, [RNA substrate strand] = 96 M -[DiLCrz_TLR3] = 140 M, [RNA substrate strand] = 187 M
		1	2	3	4	5	6	7	8	9	10	11	12
	A	Sodium Malonate (mM)
		(w/v) PEG 400 (%)

Table 7 :

 7 48 crystallization conditions sparse matrix with condition F3 (highlighted in purple) from the PEGRx HT (Hampton research ©) used as a starting point. Tris pH 8.0 is fixed at 100 mM. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL drops.

			1	2	3	4	5	6	7	8	9	10	11
	A	NaCl (mM) (w/v) PEG 400 (%)	100 5	200 5	100 15	200 15	100 20	200 20	100 25	200 25	100 35	200 35	100 45	200
	B	NaCl (mM)
		(w/v) PEG 1500 (%)

Table 13 :

 13 4 crystallization conditions sparse matrix with condition C1 (highlighted in purple) from the PEGRx (Hampton research ©) used as a starting point. pH of the buffer is the only parameter that has been modified in the sparse matrix. This screen has been used to crystallize DiLCrz_cp_U203C at 20 °C in 400 nL , 600 nL and 800 nL drops.

			pH 3 30%	pH 4 30%	pH 5 30%	pH 6 30%
		c)	Hanging drops
	Again, to optimize crystal size and diffraction, larger drops in Linbro 24 wells
	RNA		Reservoir solution		[RNA] M	T (°C)
	DiLCrz_TLR3	75 mM Sodium chloride, 2 mM Calcium chloride dihydrate, 50 mM Sodium cacodylate trihydrate 0.5 mM Spermine pH 6.0, 30% w/v 1,6 Hexanediol,	and 48 [LCrz core] = 24 [RNA substrate strand] = 36	4, 20,37
		75 mM Sodium chloride, 2 mM
	DiLCrz_TLR3	Calcium chloride dihydrate, 50 mM Sodium cacodylate trihydrate 0.5 mM Spermine pH 6.0, 30% w/v 1,6 Hexanediol,	[LCrz core] = 48 and 96 [RNA substrate strand] = 64	4, 20, 37
	DiLCrz_TLR3	75 mM Sodium chloride, 2 mM Calcium chloride dihydrate, 50 mM Sodium cacodylate trihydrate 0.5 mM Spermine pH 6.0, 30% w/v 1,6 Hexanediol,	[LCrz core] = 72 and 144 [RNA substrate strand] = 96	4, 20,37
	DiLCrz_TLR3	75 mM Sodium chloride, 2 mM Calcium chloride dihydrate, 50 mM Sodium cacodylate trihydrate 0.5 mM Spermine pH 6.0, 30% w/v 1,6 Hexanediol,	[LCrz core] = 96 and 192 [RNA substrate strand] = 128	4, 20,37
	RNA		Reservoir solution		[RNA] M	T

plates (Hampton Research) have been prepared by mixing 1 L of RNA to 1 L of the reservoir solution (Table

14

). Reservoir volume is 150 L.

Table 14 :

 14 Summary table of the crystallization trials in 2 L hanging drops with a reservoir of 150 L.

Table 15 :

 15 Summary table of the crystallization trials in 5 L microbatch drops. The crystallization conditions are given at concentration corresponding to 100%. Percentages indicated are final concentration of a given crystallization solution in a microbatch drop.

Table 16 :

 16 Table summarizing cell parameters, resolution, refinement statistics and method for 3D structure resolution for DiLCrz_U203C cp and active constructs.

  In collaboration with partners' laboratory upcoming objectives are to develop a strategy exploiting LCrz as a biotechnological tool. With the 3D crystal structure of LCrz now in hands we can, as we did for HMH ribozyme, optimize new versions of LCrz capable of cleaving unrelated substrate. Stability of the thereby mRNA capped by the three nucleotides lariat can be exploited to increase production of protein in prokaryotes and eukaryotes. LCrz can even be imagined as a therapeutic tool if engineered to be an efficient trans acting ribozyme capable of cleaving undesirable viral RNA or defective innate RNA in cells in a multiple turnover manner. One question to be addressed in the native context of this twintron is how transcription, translation and mRNA decay are regulated in eukaryotes when RNA polymerase II is not involved? It would then be possible to imagine a system in which regulation of a transcript level depends on the RNA polymerase I rather than on RNA polymerase II. LCrz could be as well used for gene therapy if inserted upstream the ORF of protein that is not sufficiently expressed because e.g. its corresponding mRNA is degraded before translation since it lacks a cap. Stable capped mRNA can as well be used as RNA sponges e.g. to modulate siRNA level. Encouraging preliminary results show that lariat-capped mRNA can be expressed and tracked in E. coli, yeast and mammalian cells.

	Turner B, Melcher SE, Wilson TJ, Norman DG, Lilley DM (2005) Induced fit of RNA on
	binding the L7Ae protein to the kink-turn motif. RNA 11: 1192-1200
	Moreover mRNA capped by a three nucleotides lariat (native HE mRNA or Uhlenbeck OC (1987) A small catalytic oligoribonucleotide. Nature 328: 596-600
	reporter gene mRNA) can be used to explore all steps of translation from translation Vader A, Johansen S, Nielsen H (2002) The group I-like ribozyme DiGIR1 mediates alternative processing of pre-rRNA transcripts in Didymium iridis. European journal of initiation to mRNA degradation. Indeed complex systems could be investigated biochemistry / FEBS 269: 5804-5812
	exploiting the fact that pre-mRNA of the HE in GIR2/LCrz twintron is a RNA
	polymerase I transcript although it is matured and translated as if it wore a RNA Paukstelis PJ, Chen JH, Chase E, Lambowitz AM, Golden BL (2008) Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. Nature 451: 94-97 Peck-Miller KA, Altman S (1991) Kinetics of the processing of the precursor to 4.5 S Vader A, Nielsen H, Johansen S (1999) In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron. The EMBO journal 18: 1003-1013 Vagin A, Teplyakov A (2010) Molecular replacement with MOLREP. Acta crystallographica Section D, Biological crystallography 66: 22-25 Valles Y, Halanych KM, Boore JL (2008) Group II introns break new boundaries: presence in a bilaterian's genome. PloS one 3: e1488 van der Feltz C, Anthony K, Brilot A, Pomeranz Krummel DA (2012) Architecture of the spliceosome. Biochemistry 51: 3321-3333 van der Horst G, Christian A, Inoue T (1991) Reconstitution of a group I intron self-splicing reaction with an activator RNA. Proceedings of the National Academy of Sciences of the United States of America 88: 184-188 van der Veen R, Arnberg AC, Grivell LA (1987a) Self-splicing of a group II intron in yeast mitochondria: dependence on 5' exon sequences. The EMBO journal 6: 1079-1084 van der Veen R, Kwakman JH, Grivell LA (1987b) Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation. The EMBO journal 6: 3827-3831 Vicens Q, Cech TR (2009) A natural ribozyme with 3',5' RNA ligase activity. Nature chemical biology 5: 97-99 Vicens Q, Paukstelis PJ, Westhof E, Lambowitz AM, Cech TR (2008) Toward predicting self-splicing and protein-facilitated splicing of group I introns. RNA 14: 2013-2029 Vidovic I, Nottrott S, Hartmuth K, Luhrmann R, Ficner R (2000) Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. Molecular cell 6: 1331-polymerase II transcript. Barrett LW, Fletcher S, Wilton SD (2012) Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements. Cellular and molecular life RNA, a naturally occurring substrate for RNase P from Escherichia coli. Journal of 1342 molecular biology 221: 1-5 sciences : CMLS 69: 3613-3634 Perrotta AT, Been MD (1991) A pseudoknot-like structure required for efficient self-Wadley LM, Pyle AM (2004) The identification of novel RNA structural motifs using COMPADRES: an automated approach to structural discovery. Nucleic acids research 32: Bass BL, Cech TR (1986) Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid cleavage of hepatitis delta virus RNA. Nature 350: 434-436 6650-6659
	precursor. Biochemistry 25: 4473-4477

  Sitting drop  20 °C  10 weeks  15 mM Magnesium choride, 130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol 400 Sitting drop  20 °C  6 months  Natrix HT TM A11: 0.01 M Magnesium chloride hexahydrate, 0.05 M Sodium cacodylate trihydrate pH 6.0, 1.0 M Lithium sulfate monohydrate  Frozen in liquid ethane  15 Å  100 M  400 nL  Sitting drop  20 °C  6 months  Natrix HT TM B6: 0.01 M Magnesium acetate tetrahydrate, 0.05 M Sodium cacodylate trihydrate pH 6.5, 1.3 M Lithium sulfate monohydrate  Frozen in liquid ethane  4.3 Å  100 M  400 nL  Sitting drop  20 °C  8 weeks  Natrix HT TM C4: 0.05 M Magnesium sulfate hydrate, 0.05 M HEPES sodium pH 7.0, 1.6 M Lithium sulfate monohydrate Index HT TM E9: 0.05 M Ammonium sulfate, 0.05 M BIS-TRIS pH 6.5, 30% v/v Pentaerythritol ethoxylate (15/4 EO/OH)

	6, 25% v/v
	Polyethylene glycol 400
	 40 M  400 nL
	 Sitting drop  140 M  20 °C  10 weeks  400 nL  Sitting drop
	 20 °C  10 weeks
	 100 M  400 nL
	 Sitting drop  20 °C
	 2 L  48 M  6 months 
	 Hanging drop  20 °C
	 7 weeks

 130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol  72 M  400 nL  Sitting drop  20 °C  10 weeks  130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol  140 M  400 nL  Sitting drop  20 °C  10 weeks  130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol  72 M  400 nL   15 mM Magnesium choride, 130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol 400  72 M  400 nL  Sitting drop  20 °C  10 weeks  15 mM Magnesium choride, 130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 5% v/v Polyethylene glycol 400  72 M  400 nL  Sitting drop  20 °C  10 weeks  30 mM Magnesium choride, 130 mM Sodium choride, 100 mM Sodium citrate pH 5.6, 25% v/v Polyethylene glycol 400  Natrix HT TM E11: 0.075 M NaCl, 0.002 M Calcium chloride dihydrate, 0.05 M Na cacodylate trihydrate pH 6.0, 30% w/v 1,6-Hexanediol, 0.0005 M Spermine  100 M  400 nL 

Results -Article 2

Results -Article 3

Acknowledgement

This work was supported by the Centre National de la Recherche Scientifique and the University of Strasbourg through a Ph.D. grant to MM. We are grateful to Laure Schaeffer for technical assistance.

Indeed alternative conformations of HEG P1 and DP2 are observed exclusively in the inactive and active forms of LCrz respectively (see I.A.1.c and Figure 6). Moreover, the crystal structure of LCrz revealed that the peripheral domain acts as a clamp tightening the ribozyme core (see Results section).

Each small-ribozyme has been extensively studied ( [START_REF] Lilley | Ribozymes and RNA Catalysis: RSCPublishing[END_REF] and (Ferre-D'Amare & Scott, 2010) for review). Therefore it is now possible to use them as powerful tools in molecular biology (Price et al, 1995). During my PhD, I used hammerhead and HDV ribozymes to obtain LCrz to be used for crystallography with well-defined 5' and 3' ends, respectively. This process is detailed in II.A.1.b.

B. CHEMICAL REACTIONS CATALYSED BY RIBOZYMES

The range of natural chemical reactions catalyzed by ribozymes is limited to transesterification reactions and hydrolysis, but depending on the structural environment the resulting products are diverse (Figure 22). One exception is the ribosome in which rRNA catalyses peptidyl transfer.

Abstract

RNA-mediated biological processes usually require precise definition of 5' and 3' ends.

RNA ends obtained by in vitro transcription using T7-RNA-polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists in inserting an RNA with known boundaries in between two ribozymes, usually a 5'-hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.

7.

Structure determination and model refinement

The resulting experimental electron density map of DiLCrz_cp_U203C allowed to build an initial model using Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] which was later refined with Phenix [START_REF] Adams | PHENIX: a comprehensive Pythonbased system for macromolecular structure solution[END_REF] [START_REF] Kruger | Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena[END_REF] .However the model proved to be difficult to refine because at this resolution the molecular replacement solution largely dominates the data. We now turn towards heavy atom derivatives to solve this problem.

Molecular visualizations and structures illustrations were performed using PyMOL (Schrödinger).

B. SMALL-ANGLE X-RAY SCATTERING EXPERIMENTS

LCrz RNA have been produced and purified as for crystallization. All LCrz RNA have been prepared in 25 L at the concentration of 6 mg/mL in a solution containing 100 mM KCL and 10 mM MES-KOH pH 6.5. The sample has been heated

during 1 min at 95 °C followed by 4 min on ice. MgCl 2 up to 4 mM was then added, the solution was placed at 55 °C for 10 min then at 4 °C overnight. The homogenous folding of the ribozyme has been verified by PAGE under native conditions.

All details for SAXS data collection and analysis are given in the supplementary material of the article describing the DiLCrz_cp_U203C crystal structure (see Results section).

protected from degradation at its 5' end by the lariat of three nucleotides and at the 3' end by additional nucleotides belonging to the 3' exon and GIR2. The accumulated mRNA would therefore be ready to be translated when the myxomycete comes out of encystement and a large amount of HE could be produced [START_REF] Nielsen | The GIR1 banching ribozyme[END_REF]. If the HE regulates the on and off switch of LCrz this phenomenon would allow for rapid up-regulation of the mature SSU rRNA production allowing the cell to quickly restore the optimal level of ribosome and therefore of the resulting cellular functions.

APPENDIX