Danielle A Adekunle

Arnaud Hubstenberger
email: arnaud.hubstenberger@univ-cotedazur.fr

The multiscale and multiphase organization of the transcriptome

Keywords: post-transcriptional regulation, ribonucleoproteins, RNA condensates, RNA phase separation and transition, stress granules, transcriptomics Subjects: Biophysics, Epigenetics, Gene Expression & Regulation, Omics, RNA

Gene expression must be co-ordinated to cellular activity. From transcription to decay, the expression of millions of RNA molecules is highly synchronized. RNAs are covered by proteins that regulate every aspect of their cellular life: expression, storage, translational status, localization, and decay. Many RNAs and their associated regulatory proteins can coassemble to condense into liquid droplets, viscoelastic hydrogels, freeze into disorganized glass-like aggregates, or harden into quasi-crystalline solids. Phase separations provide a framework for transcriptome organization where the single functional unit is no longer a transcript but instead an RNA regulon. Here, we will analyze the interaction networks that underlie RNA super-assemblies, assess the complex multiscale, multiphase architecture of the transcriptome, and explore how the biophysical state of an RNA molecule can define its fate. Phase separations are emerging as critical routes for the epitranscriptomic control of gene expression.

Introduction.

There are hundreds of thousand mRNA molecules [START_REF] Marinov | From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing[END_REF] and millions of total RNA molecules in a single mammalian cell [START_REF] Palazzo | Non-coding RNA: what is functional and what is junk?[END_REF]. Transcriptome organization must, therefore, be highly co-ordinated from transcription to decay. In the nucleus, RNAs are co-transcriptionally bound by regulatory RNA binding proteins (RBPs) that regulate RNA fate. Ribonucleoproteins (RNPs), RNAs complexed with RBPs, are the functional units upon which the mechanisms organizing the transcriptome act. These RNPs can coassemble into super-assemblies called RNA condensates (Figure 1) [START_REF] Banani | Biomolecular condensates: organizers of cellular biochemistry[END_REF]4], that demix from the cytosol or nucleoplasm to provide a means to orchestrate the transcriptome that would not be possible by simple Brownian diffusion. . elegans oocyte are toxic to the cell but reversible. (E) RNPs can stably polymerize into crystalline fibres. Right panel, mutations in the prion-like domain of FUS can induce irreversible fibrous aggregation that are pathological. In (B) and (C), small molecules can freely diffuse into and out of these porous condensates. Image (A-D) are adapted from Hubstenberger et al. [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF], Image (E) from Patel et al. [START_REF] Patel | A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation[END_REF]. also explore how phase separations co-ordinate the organization and fate of large RNA regulons.

The physical nature of RNA phase transitions and the emergent material properties of RNA condensates.

Transcripts are compartmentalized within condensates whose material properties control RNA exchange, dynamics, and biochemical microenvironment. Upon coassembly, RNPs can condense into liquid droplets, semi-liquid hydrogels, solid glass state, or crystal-like solids (Figure 1). In a seminal study, live-imaging demonstrated the liquid nature of micrometersized germ granules [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF]. These droplets dock, fuse, mix, and relax toward a spherical shape under surface tension (Figure 2A). While some droplets are liquid others are viscoelastic (Figure 2A,B) [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. Some RNP gels do not coalesce and can further solidify, limiting intracondensate, or cytosolic exchange [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Kroschwald | Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules[END_REF][START_REF] Lin | Formation and maturation of phase-separated liquid droplets by RNA-binding proteins[END_REF][START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF][START_REF] Patel | A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation[END_REF]. All of these low-density super-assemblies are porous and permeable structures, largely comprised of water, that allow the passive diffusion of small molecules [START_REF] Wei | Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles[END_REF]. The scaffolding components of liquid droplets are disordered [START_REF] Wei | Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles[END_REF][START_REF] Brady | Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation[END_REF]; but some can polymerize into geometrical shapes [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF], or crystalline fibers (Figure 1) [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Lin | Formation and maturation of phase-separated liquid droplets by RNA-binding proteins[END_REF][START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF][START_REF] Patel | A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation[END_REF][START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF][START_REF] Mateju | An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function[END_REF][START_REF] Schwartz | RNA seeds higher-order assembly of FUS protein[END_REF][START_REF] Sun | Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS[END_REF]. Although crystalline aggregates are often associated with degenerative diseases, diffractive assemblies have also been found in myo-granules [START_REF] Vogler | TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle[END_REF]. We have only begun to unravel the influence of these material properties on transcriptome organization. (A) Liquid droplets are typified by their ability to dock, fuse, and relax into spheres under surface tension [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. Molecules can freely diffuse within liquid condensates and exchange at the interface of the condensate and the cytoplasm or nucleoplasm. (B) Solid gel condensates are deformed under mechanical stress, but characteristically recover their initial shape in an elastic response [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF].

Four types of interactions may trigger RNP phase transition to organize the transcriptome.

Characterizing the molecular mechanisms that drive condensate coassembly is critical to elucidating how condensates organize the transcriptome. As demonstrated in simplified reconstituted systems in vitro and overexpression and ectopic expression studies in cellulo, protein-protein, RNA-protein, and RNA-RNA interactions are individually sufficient to drive RNP phase separations (Figure 3). What we have learned from these reconstituted systems is that RNA can either drive condensation, as scaffolding components, or can be recruited into super-assemblies by RBPs.

Model 1. Protein-protein interactions through folded domains.

As demonstrated in synthetic systems, when proteins coassemble into branched networks through stereospecific interactions, the super-assemblies can lead to supramolecular condensates that can reach microns in size [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF]. The more interactions a protein engages in, the lower the concentration required for the protein's superassembly to phase separate (Figure 3). Phase separation depends on two main parameters: the concentration and the multivalency of the scaffolding components. Condensates nucleated by protein-protein interactions can assemble with RNAs through RNA binding domains (RBDs).

Model 2. Protein-protein interactions through intrinsically disordered regions (IDRs).

RBPs are highly enriched in IDRs [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Castello | Insights into RNA biology from an atlas of mammalian mRNA-binding proteins[END_REF]: typically low complexity sequences that do not fold in solution and consequently do not participate to stable stereospecific interactions. To promote self-assembly, some IDRs amino acids may work as 'stickers' promoting weak, promiscuous and multivalent IDR-IDR interactions, while the rest of the disordered sequences function as spacers within the interaction network (Figure 3). Various classes of IDRs, including RGG, G/S-F/Y-G/S and polyQ repeats are soluble at low concentration and self-assemble into viscoelastic droplets that phase separate above a saturation concentration [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF][START_REF] Patel | A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation[END_REF][START_REF] Burke | Residue-by-Residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II[END_REF][START_REF] Conicella | ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain[END_REF][START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF][START_REF] Murakami | ALS/FTD mutationinduced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function[END_REF][START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF][START_REF] Tsang | Phosphoregulated FMRP phase separation models activity-dependent translation through bidirectional control of mRNA granule formation[END_REF][START_REF] Zhang | RNA controls PolyQ protein phase transitions[END_REF]. Highly transient interactions between poorly folded IDR domains promote the liquidity of the system, whose material properties are influenced by IDR composition [START_REF] Quiroz | Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers[END_REF][START_REF] Wang | A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins[END_REF]. Strikingly, artificial droplets mimic the fluidity of endogenous ones and are also sufficient to create ∼5 nm polymer meshworks that exclude molecules larger than 50 kDa. This is similar to what has been seen in germ granules and nucleoli [START_REF] Wei | Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles[END_REF]. Many IDRs carry prion-like sequences, through which disorganized, weak, and transient interactions can develop into strong, irreversible cross-β-strands with crystallin-like order as observed by X-ray diffraction [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Lin | Formation and maturation of phase-separated liquid droplets by RNA-binding proteins[END_REF][START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF][START_REF] Patel | A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation[END_REF][START_REF] Schwartz | RNA seeds higher-order assembly of FUS protein[END_REF][START_REF] Conicella | ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain[END_REF][START_REF] Murakami | ALS/FTD mutationinduced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function[END_REF][START_REF] Zhang | RNA controls PolyQ protein phase transitions[END_REF][START_REF] Decker | Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae[END_REF][START_REF] Han | Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies[END_REF][START_REF] Protter | Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly[END_REF][START_REF] Xiang | The LC domain of hnRNPA2 adopts similar conformations in hydrogel polymers, liquid-like droplets, and nuclei[END_REF]. Therefore, the material properties of IDR-rich RBP granules range from liquid to solid.

Model 3. RNA-protein interactions.

RNAs can play an important scaffolding role in condensate assembly. RNAs recruit and link RBPs and, accordingly increase local RBP concentration leading to phase separation (Figure 3). In reconstituted systems, it has been observed that increased RNA repeats that recruit RBPs lower the concentration at which RNA-protein complexes demix [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF]. In vitro, IDRs can condense on their own without RNA but at very high concentrations [START_REF] Kato | Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels[END_REF][START_REF] Sun | Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS[END_REF]. To condense at physiological concentrations RBP-RNA interactions are required [START_REF] Lin | Formation and maturation of phase-separated liquid droplets by RNA-binding proteins[END_REF][START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF][START_REF] Schwartz | RNA seeds higher-order assembly of FUS protein[END_REF][START_REF] Zhang | RNA controls PolyQ protein phase transitions[END_REF][START_REF] Hondele | DEAD-box ATPases are global regulators of phase-separated organelles[END_REF][START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]. RBPs must be in excess of RNAs for multiple RBPs to associate with the same RNA molecule and maintain the multivalency required for phase separation. When RNAs are in excess and proteins are limiting, condensate formation is restricted [START_REF] Schwartz | RNA seeds higher-order assembly of FUS protein[END_REF][START_REF] Hondele | DEAD-box ATPases are global regulators of phase-separated organelles[END_REF][START_REF] Maharana | RNA buffers the phase separation behavior of prion-like RNA binding proteins[END_REF]. RNAs also contribute to the material properties of condensates; short and long RNAs limit and increase the viscosity of RNP droplets, respectively [START_REF] Wei | Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles[END_REF][START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF][START_REF] Zhang | RNA controls PolyQ protein phase transitions[END_REF].

Model 4. RNA-RNA interactions.

Even in the absence of protein, RNA-RNA interactions are sufficient to drive phase separation, including at physiological concentrations (Figure 3) [START_REF] Aumiller | RNA-based coacervates as a model for membraneless organelles: formation, properties, and interfacial liposome assembly[END_REF][START_REF] Jain | RNA phase transitions in repeat expansion disorders[END_REF][START_REF] Langdon | mRNA structure determines specificity of a polyQ-driven phase separation[END_REF][START_REF] Van Treeck | RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome[END_REF]. RNAs tend to separate into homotypic clusters. These clusters can either dock without merging or coalesce without mixing, meaning smaller droplets can become embedded in larger ones. One phase can also surround the other as a surfactant [START_REF] Tauber | Modulation of RNA condensation by the DEAD-box protein eIF4A[END_REF]. RNA secondary structures modulated by RBPs can add increased specificity to RNA-RNA interactions [START_REF] Langdon | mRNA structure determines specificity of a polyQ-driven phase separation[END_REF].

The four aforementioned interaction models combine in vivo to drive the coassembly of endogenous condensates. Even in the simplest phase separation model where a single component can condense on its own, many factors control nucleation and modulate saturation concentration above which scaffolding components demix. For many endogenous condensates, multiple components can independently cause demixing, meaning there are many alternative and redundant pathways that can trigger condensate formation. One must distinguish the contribution of the nucleators that seed phase separation, the scaffolding components that drive condensate growth, and the 'hitchhikers' that are passive clients.

The diversity and complex composition of endogenous condensates.

Analyzing condensate composition is central to understanding how condensates organize the transcriptome. Various condensate purifications approaches have been utilized, including differential centrifugation coupled to immunoprecipitation [START_REF] Jain | ATPase-modulated stress granules contain a diverse proteome and substructure[END_REF]7,[START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF][START_REF] Namkoong | Systematic characterization of stressinduced RNA granulation[END_REF][START_REF] Wang | Context-dependent deposition and regulation of mRNAs in P-bodies[END_REF], and a recently developed technology, fluorescence-activated particle sorting (FAPS) [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF]. These approaches revealed that condensates typically consist of numerous protein species; more than 50 proteins coassemble in neuronal granules [7] and hundreds coassemble in nucleoli [START_REF] Andersen | Nucleolar proteome dynamics[END_REF], Pbodies [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF], and SGs [START_REF] Jain | ATPase-modulated stress granules contain a diverse proteome and substructure[END_REF][START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF][START_REF] Youn | High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies[END_REF]. Additionally, they demonstrated that IDRs such as RGG, polyQ, or F/Y-G/S repeats, are extremely enriched in condensates, suggesting that these domains dictate biophysical condensate properties. At the proteomic level, there are many proteinprotein interactions that create dense stereospecific networks, as revealed by studies employing proximity labelling of proteins in these networks [START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF][START_REF] Youn | High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies[END_REF]. The culmination of these studies has made clear that stereospecific protein-protein and IDR-IDR interactions drive coassembly.

Condensate transcriptomes are even more complex than proteomes. P-bodies, SGs, neuronal granules, and germ granules contain hundreds to thousands of mRNA species [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF][START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF][START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Heraud-Farlow | Staufen2 regulates neuronal target RNAs[END_REF]. Although no method exists currently to simultaneously purify distinct cellular condensate subtypes and directly characterize their transcriptome, ATLAS-Seq, a novel fractionation method coupled to RNA-sequencing identified hundreds of clusters of RNAs that co-segregate with their regulatory proteins [START_REF] Adekunle | Transcriptome-wide organization of subcellular microenvironments revealed by ATLAS-Seq[END_REF], supporting a model where RNAs coassemble into supramolecular assemblies that have not yet been described. Small noncoding RNA (ncRNA) content has not been characterized on a transcriptome-wide scale, but miRNAs are stably anchored and numerous proteins from the Argonaute family collect in condensates [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Liu | MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies[END_REF][START_REF] Pillai | Inhibition of translational initiation by Let-7 MicroRNA in human cells[END_REF][START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF][START_REF] Sheu-Gruttadauria | Phase transitions in the assembly and function of human miRISC[END_REF]. Although there are various condensate types, condensates can be classified into two main classes: translation factories and those accumulating translationally repressed RNAs. In fungi, translationally repressed cytosolic mRNAs accrue in SGs, P-bodies, and Whi3 condensates [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF][START_REF] Langdon | mRNA structure determines specificity of a polyQ-driven phase separation[END_REF][START_REF] Wang | Context-dependent deposition and regulation of mRNAs in P-bodies[END_REF], while actively translated glycolytic and translation factors encoding mRNAs condense into translation factories [START_REF] Lui | Granules harboring translationally active mRNAs provide a platform for P-body formation following stress[END_REF][START_REF] Pizzinga | Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth[END_REF]. In metazoans, repressed mRNAs accumulate in Pbodies [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF], SGs [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF], germ granules [START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF], neuronal granules [START_REF] Heraud-Farlow | Staufen2 regulates neuronal target RNAs[END_REF][START_REF] De Graeve | Neuronal RNP granules: from physiological to pathological assemblies[END_REF][START_REF] Formicola | Neuronal ribonucleoprotein granules: dynamic sensors of localized signals[END_REF], myo-granules [START_REF] Vogler | TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle[END_REF], while translated mRNAs can coassemble into translation factories [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF].

RNAs are nucleating factors and condensate organizers.

RNAs have emerged as condensate nucleators and structuring factors. The lncRNA NEAT-1 is perhaps the most well-characterized illustration of this. NEAT-1 bridges together RBPs [START_REF] Mao | Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs[END_REF][START_REF] Shevtsov | Nucleation of nuclear bodies by RNA[END_REF] to promote Paraspeckle phase separation. NEAT-1 defines Paraspeckle architecture by creating condensate polarity through the positioning of its 5ʹ and 3ʹ ends at the nucleoplasmic interface and the interring of its core within Paraspeckles [START_REF] Lin | Structural analyses of NEAT1 lncRNAs suggest long-range RNA interactions that may contribute to paraspeckle architecture[END_REF][START_REF] Souquere | Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies[END_REF]. This organizes RBPs along a radial axis around NEAT-1 lncRNAs. The structural role of RNAs in recruiting RBPs to trigger condensation is also evident in histone locus bodies, CBs, nuclear speckles, nuclear stress bodies [START_REF] Shevtsov | Nucleation of nuclear bodies by RNA[END_REF], and nucleoli [START_REF] Berry | RNA transcription modulates phase transition-driven nuclear body assembly[END_REF][START_REF] Falahati | Nucleation by rRNA dictates the precision of nucleolus assembly[END_REF][START_REF] Mitrea | Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA[END_REF].

However, no master scaffolding RNA components have been identified in cytosolic condensates. While a collective of RNAs scaffold P-bodies and SGs [START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF]71], the most abundant RNAs within P-bodies or SGs comprise less than 1% of their transcriptome [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF][START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF], suggesting a more decentralized organization for cytosolic condensates compared with nuclear condensates. Still, RNAs determine condensate structure and composition in the cytosol. This was demonstrated by clustering RNA targets of Pumilio protein by tethering the RBD of Pumilio to an artificial scaffold. RNA clustering was sufficient to trigger P-body-like assembly formation, despite the fact that Pumilio is a non-essential P-body protein [START_REF] Navarro | RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates[END_REF]. Similarly, SG assembly is triggered by RNA release from polysomes which leads to the recruitment of RBPs that assemble SGs [START_REF] Kedersha | Evidence that ternary complex (eIF2-GTP-tRNAiMet)-deficient preinitiation complexes are core constituents of mammalian stress granules[END_REF][74][START_REF] Kedersha | G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits[END_REF]. Unfolded mRNAs trigger a conformational switch and the condensation of the master organizer of SG assembly, G3BP [START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF]. G3BP dimers directly interact with mRNAs, and by recruiting numerous RBPs, acts as a central node within the interaction network whose high degree of multivalency relies on RNAs to phase separate [START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]. Like RNA-protein and protein-protein interactions, RNA-RNA interactions contribute to SG assembly through sense-antisense RNA hybrids [START_REF] Van Treeck | RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome[END_REF]. In protein-free extracts RNA precipitation partially recapitulates the SG transcriptome, suggesting that RNA-RNA interactions are important for SG formation [START_REF] Van Treeck | RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome[END_REF]. The most elegant demonstration to date of mRNAs as condensate organizers is Whi3 RBP condensates in fungi. The secondary structure of mRNAs, modulated by Whi3, control intermolecular RNA-RNA pairing, to dictate whether two mRNA molecules will co-segregate in the same droplet, or separate into distinct droplets [START_REF] Langdon | mRNA structure determines specificity of a polyQ-driven phase separation[END_REF]. Taken together, RNAs are critical to condensate formation: they nucleate RBP-RBP interactions and regulate RNA-RNA liquid-liquid demixing specificity.

Redundant, alternative protein scaffolds create robust super-assemblies.

Multiple components can independently cause demixing, meaning various alternative and redundant pathways can trigger condensate formation. Tethering any of the abundant proteins of CBs, including those non-essential to endogenous CB assembly, to a repetitive DNA array is sufficient to nucleate de novo condensates [START_REF] Kaiser | De novo formation of a subnuclear body[END_REF]. Similarly, the artificial clustering of Pumilio, a non-essential component of P-bodies, described above, demonstrated that an RBD is sufficient to trigger P-body-like assembly formation [START_REF] Navarro | RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates[END_REF]. SGs are perhaps the most striking example of robust and redundant assembly. Even in the absence of stress, overexpression of any of the following components is sufficient to induce SG assembly: G3BP [START_REF] Tourrière | The RasGAPassociated endoribonuclease G3BP assembles stress granules[END_REF], the prion-like domain of TIA-1 [START_REF] Gilks | Stress granule assembly is mediated by prion-like aggregation of TIA-1[END_REF], CPEB1 [START_REF] Wilczynska | The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules[END_REF], Caprin-1 [START_REF] Solomon | Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs[END_REF], DYRK3 [START_REF] Wippich | Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling[END_REF], hnRNPA2, and FUS [START_REF] Molliex | Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization[END_REF]. All of these examples, fit a phase separation model where concentration and multivalency are critical determinants of condensation [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF][START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF].

Condensate coassemblies require two types of protein-protein interactions: folded-folded and IDR-IDR. Several proteins that participate to folded-folded interactions are required for CB, SG, and PB formation [START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF][START_REF] Youn | High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies[END_REF][START_REF] Kedersha | G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF][START_REF] Kaiser | De novo formation of a subnuclear body[END_REF][START_REF] Ayache | P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes[END_REF]. In addition, prion-like IDR-IDR polyQ/N polymers work synergistically with folded domains to assemble P-bodies [START_REF] Decker | Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae[END_REF][START_REF] Protter | Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly[END_REF][START_REF] Hubstenberger | Modifiers of solid RNP granules control normal RNP dynamics and mRNA activity in early development[END_REF]. Folded-folded interactions bring specificity and stability to the system while IDR-IDR interactions may provide droplet fluidity [START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF][START_REF] Vijayakumar | The prion-like domain of Drosophila Imp promotes axonal transport of RNP granules in vivo[END_REF]. Essential protein components in certain environmental contexts were found to be dispensable in others [START_REF] Kedersha | G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits[END_REF][START_REF] Ayache | P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes[END_REF]. This signifies that alternative assembly mechanisms may underlie the plasticity of RNA condensates and clarifies the lack of a unique master scaffolding protein. Master organizers appear to be interchangeable central nodes with the highest degree of multivalent interactions within the condensate network [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF][START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF].

RNA partitioning in RNA condensates: a sorting mechanism.

RNA sorting to condensates is central to understanding how phase separations organize the transcriptome. Granules can be distinguished by their RNA composition [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF][START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Courel | GC content shapes mRNA storage and decay in human cells[END_REF]. Although all mRNAs condense in SGs to some degree, the condensed fraction of mRNAs varies from <1% to >95% [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF]. Any mRNA can transiently dock on condensates, including translated ones, but only repressed mRNAs can be stably anchored, leading to stronger enrichment [START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF][START_REF] Moon | Multicolour single-molecule tracking of mRNA interactions with RNP granules[END_REF]. Similarly, mRNAs condense to various degree in P-bodies [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF], and single-molecule live imaging confirmed that the fraction and dynamics of transcripts that localize to SGs and PBs is different for each mRNA [START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF][START_REF] Wilbertz | Single-molecule imaging of mRNA localization and regulation during the integrated stress response[END_REF]. The wide variations in RNA inclusion suggest that RNA sorting to condensates is fine-tuned. Whether bound in 3ʹUTRs or 5ʹUTRs Argonaute promotes translation repression, but only 3ʹUTR bound RNAs are enriched in P-bodies: translational repression alone is not enough to stabilize RNAs in P-bodies [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF]. From global CLIP analyses, we know that RNA-protein interactions are predictors of RNA enrichment in P-bodies [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF] and live single mRNA imaging demonstrated that these interactions anchor mRNAs and lncRNAs to condensates [START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF][START_REF] Wilbertz | Single-molecule imaging of mRNA localization and regulation during the integrated stress response[END_REF]. From these studies, we can conclude that low specificity and lowaffinity binding allows transient RNA docking, while high specificity and high-affinity docking promotes RNA anchoring. Overall, RNA condensation and solubilization within cytosolic condensates, whose diversity reaches far beyond P-bodies and SGs to include a wide array of cell-type-specific condensates, as well as an increasing list of nuclear bodies, provides a powerful mechanism to sort and organize RNA transcriptome-wide (Figure 4). Novel sequencing approaches such as ATLAS-Seq, suggest that hundreds of RNA supramolecular assemblies remain to be elucidated [START_REF] Adekunle | Transcriptome-wide organization of subcellular microenvironments revealed by ATLAS-Seq[END_REF].

The complex control of RNA organization through phase separation has been evolutionarily selected.

Cells must adapt RNA expression to environmental variations. Changes in temperature, ionic strength, pH, or Redox state may reorganize the transcriptome by passively triggering phase transitions, where RBPs switch from diffuse to condensed states [START_REF] Burke | Residue-by-Residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II[END_REF][START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF][START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF][START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF][START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF][START_REF] Kato | Redox state controls phase separation of the yeast Ataxin-2 protein via reversible oxidation of its methionine-rich low-complexity domain[END_REF][START_REF] Franzmann | Phase separation of a yeast prion protein promotes cellular fitness[END_REF][START_REF] Kroschwald | Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery[END_REF][START_REF] Iserman | Condensation of Ded1p promotes a translational switch from housekeeping to stress protein production[END_REF]. Illustrating the diversity of the responses, PAB1 condensation is triggered by heat [START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF], whereas DDX4 is triggered by cold [START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF]; while FUS and DDX4/LAF-1 condense at high and low salt concentrations, respectively [START_REF] Burke | Residue-by-Residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II[END_REF][START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF][START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF]. Similarly, the pH dependent protonation of G3BP's IDR induces a conformational switch for this SG master protein, promoting its ability to interact and phase separate with unfolded RNAs that are released from polysomes upon stress, which in turn may be critical to limit RNA entanglement [START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF]. To promote cellular fitness, IDRs seem to have been evolutionarily selected to control the temperature or pH levels at which proteins phase separate [START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF][START_REF] Franzmann | Phase separation of a yeast prion protein promotes cellular fitness[END_REF][START_REF] Kroschwald | Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery[END_REF][START_REF] Iserman | Condensation of Ded1p promotes a translational switch from housekeeping to stress protein production[END_REF]. As an example, the stress-induced condensation which inactivates Ded1p protein translation initiation triggers a translational switch from housekeeping to stress protein production. The translation of mRNAs carrying complex 5ʹUTRs is inhibited while the translation of shorter and less structured 5ʹUTRs is promoted [START_REF] Iserman | Condensation of Ded1p promotes a translational switch from housekeeping to stress protein production[END_REF]. Taken together, phase separations have a transcriptome-wide but selective impact on RNA expression.

In addition to these passive regulations, and the active regulations by helicases or chaperones, many protein post-translational modifications (PTM) control phase separations depending on cellular activity, environment, or developmental stage [START_REF] Snead | The control centers of biomolecular phase separation: how membrane surfaces, PTMs, and active processes regulate condensation[END_REF]. Similarly, the number and distribution of direct RNA m6A methylations regulate and influence the composition of the phase-separated transcriptome [START_REF] Ries | M6a enhances the phase separation potential of mRNA[END_REF]. These PTMs and RNA modifications can either work as switches that trigger major transcriptome reorganization from a well-mixed state to a condensed state, or fine-tune condensate dynamics and composition.

The dynamics, composition, and organization of condensates are stress specific [START_REF] Kedersha | G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits[END_REF][START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF][START_REF] Aulas | Stress-specific differences in assembly and composition of stress granules and related foci[END_REF][START_REF] Buchan | Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae[END_REF][100][101]. Twenty percent of SG proteome diversity is stress-dependent [START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF]. The transcriptome of P-bodies and SGs is similarly organized in a stress-specific manner; stress associated RNA motifs were found to be enriched in these granules [START_REF] Khong | The stress granule transcriptome reveals principles of mRNA accumulation in stress granules[END_REF][START_REF] Wang | Context-dependent deposition and regulation of mRNAs in P-bodies[END_REF]. Taken together, the simultaneous condensation or dissolution of RNA droplets in response to the environment provides a mechanism for the synchronization of large pools of RNAs that share the same fate (Figure 4).

Architecture of condensates: channelling RNA through subcompartments.

Despite their liquid nature at the macroscopic level [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF], RNA condensates are not homogenously mixed. Nucleoli and germline P-bodies are subcompartmented by liquid-liquid phase separations (LLPS) [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]102,103]. The biogenesis of ribosomes in nucleoli involves condensate organization around newly synthesized rRNAs in an assembly-line fashion from the core to periphery subcompartments [104]. However, some components may not coassemble through phase separations: 4 out of 6 nucleoli proteins mechanistically analyzed fit a phase separation model, the other proteins were recruited in an active manner [105]. In SGs, super-resolution imaging showed that multiple solid cores with different composition reversibly cluster together sharing the same liquid shell, further illustrating the subcompartmentalization of condensates [START_REF] Jain | ATPase-modulated stress granules contain a diverse proteome and substructure[END_REF][106][107][108][109].

The remodelling of germ granules during development is well-characterized. During early meiosis, liquid germ droplets wet the nuclear pore (Figure 5) [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF]. Electron microscopy analysis has revealed droplet asymmetry along the axis of RNA efflux [110]. Droplets organize around PGL-1/3 proteins that are sufficient to condense [START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]111]. On the nuclear surface, GLH proteins promote germ droplet docking on nuclear pores through hydrophobic FGF repeats [112]. At the cytosolic interface, the FBF-2 translation repressor loads onto exiting mRNAs [113]. These polarized germ condensates further dock with two other condensates, Z granules and Mutator foci [114]. As transcription is arrested and oogenesis progresses, highly fluid germ droplets are released from the pore to the cytosol. They are subsequently engulfed by semi-liquid germline P-bodies, and without mixing become subcompartments of larger condensates (Figure 5) [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. Upon fertilization, condensates dissolve. This allows maternal mRNPs to be sequestered and 'frozen' within droplet gels during oogenesis arrest and quickly mobilized upon embryonic program activation [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. At each mitotic cycle, germ droplets condense posteriorly and somatic P-bodies condense anteriorly to be asymmetrically inherited [START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF][START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]115]. At this developmental stage, RNAs cluster MEG-3 gel cores around liquid PGL-3 droplets [START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF]116,117]. The ability of multiple proteins: PGL-1/3 [START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]111], MEG-3 [116-118], Ddx4/ LAF-1 [START_REF] Elbaum-Garfinkle | The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics[END_REF][START_REF] Nott | Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles[END_REF] to phase separate can mechanistically explain LLPS subcompartmentalization. Compartmentalized granules have also been observed in Drosophila germlines [119][120][121]. From transcription to RNA decay, phase separations channel RNP remodelling through an organized path. The functional unit is no longer an isolated RNA, but RNA regulons: RNAs that are co-segregated through phase separation to be co-regulated (Figure 4). RNAs can be released from droplets into the cytosol, while others are retained in a condensed state, as exemplified by maternal mRNAs during early embryonic development whose release from germ granules correlatess with the different temporal waves of translation activation [START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF]148]. Granule material properties can fine-tune RNA exchange. For example, highly viscous droplets can sequester mRNAs, whereas fluid ones can promote dynamic exchange [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF], which are sites of RNA export. These liquid droplets are subsequently released to the cytosol where they can be engulfed into semi-liquid P-bodies upon oogenesis arrest [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. Fertilization triggers the dissolution of maternal RNPs that were solidified in highly viscous germline Pbodies during the arrest. Embryogenesis promotes asymmetric distribution of maternal RNPs. At the anterior pole, RNPs phase separate into somatic P-bodies [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]115]. At the embryo's posterior pole, RNPs condense into germ granules that contain a liquid core and gel shell [START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF][START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]116]. Scale bars 2 μm.

Although RNA granules have been historically classified into discrete types according to specific markers, recent work suggests RNA granules exist in a continuum of phases that guide RNA exchange, and whose liquid-liquid demixing is tightly modulated. SGs are considered compositionally and functionally distinct from P-bodies. In SGs, repressed mRNAs are loaded with translation initiation complexes and the 40S ribosomal subunit, whereas P-body repressors inhibit the loading of initiation factors [START_REF] Kedersha | Evidence that ternary complex (eIF2-GTP-tRNAiMet)-deficient preinitiation complexes are core constituents of mammalian stress granules[END_REF]122]. However, SGs and P-bodies share numerous interactors [START_REF] Jain | ATPase-modulated stress granules contain a diverse proteome and substructure[END_REF][START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF][START_REF] Youn | High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies[END_REF][START_REF] Ayache | P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes[END_REF], and thus compete for these exchanging factors. Shared factors promote docking between P-bodies and SGs, while competition for these factors and interaction stoichiometry within networks control the degree of mixing between coexisting phases [START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]. Exchange of RNA between SGs and P-bodies is bidirectional, as illustrated by live imaging [START_REF] Moon | Multicolour single-molecule tracking of mRNA interactions with RNP granules[END_REF]. However, as described above for nucleoli, competition between weak multivalent interactions and strong stereotypic interactions can also asymmetrically drive RNA flux from one compartment to another [104]. From nuclear to cytosolic condensates, transcripts can be localized through phase separations to distinct compartments, providing a mechanism to organize the transcriptome spatially and temporally and to control associated RNA biochemical reactions and fate.

Condensation is actively limited, and micro-condensates may be underestimated.

Hyper-aggregation has long been known to be induced by overexpressing or mutating prionlike IDRs. RNAi screens demonstrated that multiple genes function to limit condensation and solidification into crystal-like arrays, suggesting that RNP polymerization is the default state [START_REF] Hubstenberger | Modifiers of solid RNP granules control normal RNP dynamics and mRNA activity in early development[END_REF]. In an energy-depleted context condensates grow and solidify, signifying that the cell actively restricts condensation [START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]102]. Helicases ensure transcriptome liquidity. They comprise 10% of the condensate proteome and their depletion causes RNP solidification [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF][START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. By participating in protein-RNA and IDR-IDR interactions, helicases promote condensate assembly, and by disrupting intermolecular RNA-RNA interactions maintain liquidity [START_REF] Hondele | DEAD-box ATPases are global regulators of phase-separated organelles[END_REF][START_REF] Tauber | Modulation of RNA condensation by the DEAD-box protein eIF4A[END_REF]123]. Similarly, chaperones that prevent the accumulation of misfolded proteins regulate granule dynamics [START_REF] Jain | ATPase-modulated stress granules contain a diverse proteome and substructure[END_REF][START_REF] Kroschwald | Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules[END_REF][START_REF] Mateju | An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function[END_REF][124][125][126]. Super-resolution microscopy uncovered that chaperones limit condensate growth to subresolution sizes and further suggested that many biomolecules are supersaturated, forming condensates whose growth is actively limited [127,128]. Mild stresses induce condensates below imaging resolution [START_REF] Riback | Stresstriggered phase separation is an adaptive, evolutionarily tuned response[END_REF] and proximity labelling revealed pre-assembled sub-microscopic SGs under non-stress conditions that may serve as 'seeds' to promote rapid assembly upon stress [START_REF] Markmiller | Context-dependent and disease-specific diversity in protein interactions within stress granules[END_REF][START_REF] Youn | High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies[END_REF]. Illustrating the diversity of mechanisms that limit growth, endoplasmic reticulum tubules promote condensate fission [129]. A decade of research has revealed that RNP's ability to phase separate is the rule rather than the exception, and that condensation is the default state and must be actively limited.

To co-ordinate condensate dissolution to cellular activity, the cell can control multiple parameters that are critical to phase separation: the concentration, interaction strength, and the multivalency of scaffolding components. Nuclear envelope breakdown upon mitosis dilutes nuclear component concentrations that drop below saturation concentrations, and thus induce nuclear condensates dissolution [130]. Signalling pathways induce PTMs that limit interaction strength between scaffolding components to trigger condensate dissolution [START_REF] Snead | The control centers of biomolecular phase separation: how membrane surfaces, PTMs, and active processes regulate condensation[END_REF]. Similarly, helicases and chaperones can disrupt scaffolding interactions leading to condensate collapse. Cells can also express competing ligands that disrupt the multivalency of the central nodes of condensate networks, leading to the dissolution of condensates [START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]. For example, RNA multivalency is critical to germ granule assembly, and soluble RBP can compete with condensates for mRNAs and thus trigger their dissolution [START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]. Thus, the dissolution of condensate can be finely tuned by cellular activity, providing a mechanism to synchronize the release and cytosolic access of large RNA regulons.

Condensation function: sequestration of RNAs vs. catalyzing RNA processing.

RNA condensates organize transcriptional and post-transcriptional regulations. Translation repression associated with mRNA condensation uncouples protein production from mRNA expression on a transcriptome-wide scale [START_REF] Hubstenberger | P-body purification reveals the condensation of repressed mRNA regulons[END_REF]. Condensation could limit translation through two alternative mechanisms: passively sequestering mRNAs away from the translation machinery or catalyzing mRNA processing events that prevent translation. The two alternative models function in vitro, sequestration through FMRP condensation is sufficient to inhibit translation [START_REF] Tsang | Phosphoregulated FMRP phase separation models activity-dependent translation through bidirectional control of mRNA granule formation[END_REF], while condensates catalyze deadenylation that limits translation [START_REF] Sheu-Gruttadauria | Phase transitions in the assembly and function of human miRISC[END_REF]131]. In a third model, non-functional miRISCs are trapped within P-bodies where they can scan potential mRNA targets that traverse granules more transiently [START_REF] Pitchiaya | Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality[END_REF]. In this model, condensates favour the loading of miRNAs onto mRNAs to promote mRNA repression. Of note, very few copies of some transcripts are expressed per cell [START_REF] Marinov | From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing[END_REF] and storing them in dedicated structures like P-bodies could allow them to be more easily 'found'. Condensates also provide a new solvent environment that can passively melt and remodel RNA structures even in the absence of active helicases or chaperones [132], and thus may limit RNA entanglement [START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF], or promote new interactions such as RNA-RNA interactions that are unstable in the cytosolic environment [START_REF] Tauber | Modulation of RNA condensation by the DEAD-box protein eIF4A[END_REF]. Translation factories could promote the cotranslational assembly of protein complexes or ribosome recycling [START_REF] Lui | Granules harboring translationally active mRNAs provide a platform for P-body formation following stress[END_REF][START_REF] Pizzinga | Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth[END_REF][START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF]. Taken together these recent studies suggest that RNA condensates are more than sorting centers that sequester RNA molecules, but may catalyze transcriptome remodelling through specific catalytic activities.

Buffering RNA expression variation through condensation.

Homeostasis maintenance is critical to cellular survival. To do so, cells need to buffer large variations in RNA expression. In a saturated system, when molecules accumulate above the saturation concentration, molecules in excess are buffered by condensates, keeping cytosolic concentrations constant (Figure 3). The same may apply to the overexpression of numerous RBPs that phase separate through self-interactions [133]. However, for complex heterotypic super-assemblies, the saturation concentration depends on scaffolding partners [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF][START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]104,134]. Condensates can also absorb large variations in interaction stoichiometry [START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF]104,134], providing a potential protective mechanism to the cell. For example, upon stress hyper-reactive mRNAs are released from polysomes, drastically changing the interacting ratio between mRNAs and RBPs, which leads to the coassembly of protective SGs [START_REF] Kedersha | Evidence that ternary complex (eIF2-GTP-tRNAiMet)-deficient preinitiation complexes are core constituents of mammalian stress granules[END_REF][74][START_REF] Kedersha | G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Guillén-Boixet | RNAinduced conformational switching and clustering of G3BP drive stress granule assembly by condensation[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]. This property distinguishes condensates from stereospecific complexes, for which variation in stoichiometry between partners often leads to cellular toxicity as monomers tend to interact non-specifically when they accumulate in excess. RNA exchange and partitioning can also be finely tuned within condensates. Interaction strength, client multivalency, or scaffold stoichiometry are predicted to modulate the partitioning of RNAs and control condensate composition [START_REF] Banani | Compositional control of phaseseparated cellular bodies[END_REF][START_REF] Yang | G3BP1 is a tunable switch that triggers phase separation to assemble stress granules[END_REF][START_REF] Sanders | Competing protein-RNA interaction networks control multiphase intracellular organization[END_REF]104,134]. Most biological systems require energy-dependent feed-back loops, but the thermodynamics of phase transitions provides a robust energy-free buffering system to the cell.

Condensate epitranscriptomics control gene expression.

The epitranscriptome consists of RNP structural organizations and PTMs that control gene expression and are transgenerationally inherited. The ability of the PolyQ-rich RBP, Whi3 super-assemblies in yeast to be asymmetrically inherited illustrates that phase separations regulate translation to mediate epigenetic memory [135]. Some translation factories can also be asymmetrically inherited [START_REF] Pizzinga | Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth[END_REF]. In metazoans, germ granules are asymmetrically inherited in the germline [START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]117]. Consistent with the transmission of an epigenetic signal, germ granule mutants become RNAi defective, express somatic transcripts, and become sterile over generations [114,[136][137][138][139]. These condensates accumulate small ncRNAs and their regulatory proteins are essential for ensuring transgenerational epigenetic inheritance [114,[140][141][142][143][144][145][146].

Conclusion.

Phase transitions have emerged as an essential compartmentalization mechanism to provide a higher scale of transcriptome organization. From bacteria to eukaryotes, this mode of organization is conserved and may have provided one of the first organization mechanisms in the RNA world that preceded current forms of life. Great progress has been made in mechanistically dissecting phase separations. It is clear, we are only beginning to uncover their biological function.

Figure 1 .

 1 Figure 1. RNP coassembly can induce phase separation. Left panel, schematic representations of different states of RNP organization. Right panel, confocal image of the, respective, fluorescently labelled RNP superassembly. (A) Soluble RNPs, 'M', and small molecules 'O', can be homogenously mixed within the cytoplasm or nucleoplasm. Right panel, repressed mRNPs homogenously mixed in the cytosol of a C. elegans oocyte (B) Dynamic, multivalent RNP interactions can induce demixing of RNPs into semi-liquid or liquid droplets. Right panel, repressed mRNPs condensed into a viscoelastic droplet within the cytosol of a quiescent C. elegans oocyte. (C) Stronger interactions can promote RNP coassembly into amorphous hydrogels. Right panel, heat shock-induced stress granules in C. elegans oocytes. (D) RNPs can polymerize into regular arrays. Right panel, square sheet granules induced by the loss of function of CGH-1 helicase in C. elegans oocyte are toxic to the cell but reversible. (E) RNPs can stably polymerize into crystalline fibres. Right panel, mutations in the prion-like domain of FUS can induce irreversible fibrous aggregation that are pathological. In (B) and (C), small molecules can freely diffuse into and out of these porous condensates. Image (A-D) are adapted from Hubstenberger et al. [15], Image (E) from Patel et al. [20].

Figure 2 .

 2 Figure 2. Characteristic properties of liquid droplet and solid gel RNP super-assemblies.

Figure 3 .

 3 Figure 3. Mechanisms of phase separation. (A) Phase separations are concentration dependent.

Figure 4 .

 4 Figure 4. The impact of phase separations on transcriptome organization. (A) RNP droplet dissolution can work as a developmental or environmental switch and synchronize the simultaneous release within the cytosol of large RNA regulons. For example, numerous nuclear condensates, as well as cytosolic Pbodies dissolve upon mitosis [130,147]. (B) The condensation of RBPs can alternatively inhibit their interaction with mRNA targets, as observed upon Pab1, Ded1p, or Whi3 condensation upon stress [87,95,135].(C) SomeRNAs can be released from droplets into the cytosol, while others are retained in a condensed state, as exemplified by maternal mRNAs during early embryonic development whose release from germ granules correlatess with the different temporal waves of translation activation[START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF]148]. Granule material properties can fine-tune RNA exchange. For example, highly viscous droplets can sequester mRNAs, whereas fluid ones can promote dynamic exchange[START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF].

Figure 5 .

 5 Figure 5. Condensate architecture is remodelled during germline development. In early oogenesis, germ droplets wet nuclear pores[START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF], which are sites of RNA export. These liquid droplets are subsequently released to the cytosol where they can be engulfed into semi-liquid P-bodies upon oogenesis arrest[START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]. Fertilization triggers the dissolution of maternal RNPs that were solidified in highly viscous germline Pbodies during the arrest. Embryogenesis promotes asymmetric distribution of maternal RNPs. At the anterior pole, RNPs phase separate into somatic P-bodies[START_REF] Hubstenberger | Translation repressors, an RNA helicase, and developmental cues control RNP phase transitions during early development[END_REF]115]. At the embryo's posterior pole, RNPs condense into germ granules that contain a liquid core and gel shell[START_REF] Lee | Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins[END_REF][START_REF] Brangwynne | Germline P granules are liquid droplets that localize by controlled dissolution/condensation[END_REF][START_REF] Saha | Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism[END_REF]116]. Scale bars 2 μm.

Above a specific concentration threshold, RNPs will condense and the size of the condensates will increase according to the amount of RNPs in excess of this saturation concentration. While RNPs accumulate within condensates, cytosolic RNP concentration remains constant. (B) Alternative reconstituted models that induce phase separation in vitro: Model 1. Multivalent interactions of folded protein domains into highly connected networks can induce phase separation. Increased protein multivalency leads to a lowering of the saturation concentration required for condensate formation. Model 2. Unfolded disordered protein regions trigger phase separation through weak and promiscuous but highly multivalent interactions. Model 3. RNA scaffolds can bridge RBPs into clusters, lowering their local concentration and triggering phase separation. Model 4. Multivalent RNA-RNA interactions promote demixing independent of proteins. In Model 1 and Model 2, RNAs can be passively recruited as clients of RBPs.

Funding.

Core funding for this work was provided by CNRS and Inserm, and grants from the ATIP-Avenir program to AH. This work has also been supported by the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-01.

Summary.

• Thousands of transcripts and hundreds of proteins coassemble into RNA condensates. • Phase separations provide a higher scale of transcriptome organization.

• Phase transitions synchronize the transcriptome with cellular activity.

• Diffuse, liquid, semi-liquid, and solid RNP states influence RNA fate.

Competing Interests.

The authors declare that there are no competing interests associated with the manuscript.

Author contributions.

D.A. and A.H. contributed equally to the manuscript.

Abbreviations.

CBs : Cajal bodies IDRs : intrinsically disordered regions LLPS : liquid-liquid phase separations ncRNA : noncoding RNA PTM : post-translational modifications RBDs : RNA binding domains RBPs : RNA binding proteins RNPs : ribonucleoproteins SGs : stress granules


Marie-Hélène Ruz
email: ruz@univ-littoral.fr

Caroline Rufin-Soler

Arnaud Héquette

Rachel Révillon

Anne-Peggy Hellequin

Philippe Deboudt

Vincent Herbert

Olivier Cohen

Florian Lebreton

Lucie Le Goff

François G Schmitt

Denis Marin

Climate Change and Risk Perceptions in Two French Coastal Communities

Keywords: Climate change, coastal hazards, risk perception, adaptation strategies

Understanding public perception of climate change risks in coastal communities is a prerequisite for effective climate adaptation. This study aimed at evaluating the potential impacts of climate change on two coastal communities on the northern coast of France by producing prospective maps of the coastline by 2065 and at assessing the local inhabitants' risk perception and preferred strategies for facing future coastal risks through a survey of 285 households. The solutions inhabitants considered the more appropriate to preserve their living environment while adapting to the potential impacts of climate change were analyzed. A vast majority of the inhabitants is aware of coastal risks in their municipality, but their feeling of exposure is spatially-influenced depending on the distance to the hazard source. Conversely, no relationship was found between preferred strategies for facing future coastal risks and proximity to the shoreline. In the municipality where only soft defense structures are present, more inhabitants opt for the "hold the line" solution, while in the other municipality where a portion of the coast is protected by a seawall, a much larger proportion of the surveyed residents prefer the adaptation option.

INTRODUCTION

Climate change will likely have dramatic impacts on coastal communities that will be increasingly affected by coastal erosion and/or marine flooding during the 21 st century [START_REF] Barnard | Dynamic flood modeling essential to assess the coastal impacts of climate change[END_REF] as a result of sea-level rise and possible changes in wind/wave conditions and storm regimes [START_REF] Cazenave | Sea level rise and its coastal impacts[END_REF][START_REF] Mölter | Review on the projections of future storminess over the North Atlantic European region[END_REF]. Population density is already considerably higher in the coastal zone compared to inland areas and the population at risk of marine flooding may increase by several hundred million people in the next decades due to population growth and sea-level rise [START_REF] Jongman | Global exposure to river and coastal flooding: long term trends and changes[END_REF][START_REF] Neumann | Future coastal population growth and exposure to sea-level rise and coastal flooding -A global assessment[END_REF]. Climate change will therefore result in increasing coastal hazards posing threats to coastal communities that will require appropriate adaptation and/or mitigation strategies.

Public perception of risks can constrain and influence political, social and economic action [START_REF] Leiserowitz | Climate change risk perception and policy preferences: the role of affect, imagery, and values[END_REF], and a better understanding of risk perceptions of coastal residents can be a key factor for implementing effective adaptation strategies to coastal hazards and climate change [START_REF] Button | Coastal vulnerability and climate change in Australia: public risk perceptions and adaptation to climate change in non-metropolitan coastal communities[END_REF][START_REF] Carlton | Climate change and coastal environmental risk perceptions in Florida[END_REF]. Although studies conducted on risk perception of coastal populations generally show that coastal inhabitants who may be potentially affected by natural coastal hazards (e.g., marine flooding) are often aware of local risks [START_REF] Elrick-Barr | Perceptions of risk among households in two Australian coastal communities[END_REF][START_REF] Kellens | An analysis of the public perception of flood risk on the belgian coast[END_REF][START_REF] Michel-Guillou | Representations of coastal risk (erosion and marine flooding) among inhabitants of at-risk municipalities[END_REF], there is limited information on the perception by coastal populations of future coastal hazards that will likely increase with climate change despite a growing interest for these issues during recent years [START_REF] Mills | Perceived and projected flood risk and adaptation in coastal Southeast Queensland, Australia[END_REF][START_REF] Ngo | Drivers of flood and climate change risk perceptions and intention to adapt: an explorative survey in coastal and delta Vietnam[END_REF] This paper presents preliminary results from a research project aimed at (1) assessing the risk perceptions of coastal residents of two coastal communities of northern France threatened by coastal erosion and marine flooding and at (2) evaluating the strategy preferences of the populations to face coastal changes that will likely be caused by climate change in the next 50 years. For both sites, recent coastal evolution raises the problem of vulnerability to coastal erosion and marine flooding that will increase in the next future due to rising sea level and possible higher frequency of extreme events associated with climate change (IPCC, 2013). These two sites were chosen to implement a participative project with local population and stakeholders in order to try to develop solutions of adaptation to climate change. One of the main originality of this project lies in the development of a participatory action research involving scientists from geosciences, oceanography, humanities and social sciences, with local populations to develop coconstruction adaptation strategies to cope with climate change, which will ultimately be shared with coastal managers, state _________________________________________________________________________________________________ stakeholders and decision-makers. This article focuses on the results of a questionnaire survey carried out among the local inhabitants of the two studied municipalities concerning coastal risk perception and preferred strategies for facing future coastal hazards associated with climate change.

Study Area

This study was conducted at two localities of the coast of northern France where some properties are threatened by coastal erosion or marine flooding. At both sites, coastal dunes, which represent the only defense against the sea for some of the households, have experienced erosion and shoreline retreat during the last decades [START_REF] Chaverot | Changes in storminess and shoreline evolution along the northern coast of France during the second half of the 20 th century[END_REF][START_REF] Crapoulet | Variations in the response of the dune coast of Northern France to major storms as a function of available beach sediment volume[END_REF]. The first site is Wissant, a small coastal resort (981 permanent residents) located on the French coast of the Dover Strait (Fig. 1A). An extensive section of the sea front is protected by a seawall promenade that suffered massive damages on several occasions in recent years, and which is now armored with riprap. In the western part of the village, a real estate development is directly threatened by the erosion of the coastal dune (Fig. 1B) that locally resulted in a shoreline retreat of more than 200 m during the second half of the 20 th century. Coastal erosion continues to occur nowadays, causing dramatic shoreline retreat that may exceed 10 m during major storms such as the ones that hit Western Europe during the fall-winter 2013-2014 [START_REF] Crapoulet | Variations in the response of the dune coast of Northern France to major storms as a function of available beach sediment volume[END_REF].

The second site is Oye-Plage, a village of 5260 inhabitants between Calais and Dunkirk (Fig. 1A). A Nature Reserve called "The Platier d'Oye" has the particularity of hosting a housing estate (les Ecardines) where 153 houses constructed in the mid 70's are located below the highest tide level (Fig. 1C). This estate is protected from the sea by an eroding sand dune less than 100 m wide. In the central area of the Platier d'Oye, shoreline retreated by about 50 m since 1949. Behind the coastal dunes, the estate is at risk of marine flooding and State Authorities have implemented a plan for coastal safety (Plan de Prévention des Risques Littoraux). This plan imposes to the property owners to build stairs to access the attic and roof windows to enable their rescue in case of flooding.

METHODS

Several public meetings were organized in 2017-2018 at each survey site for presenting the objectives and the results of the project to the local population. In addition, a door-to-door survey was conducted in the two coastal communities.

Prospective Mapping of Future Coastal Risks and Communication to Population

Historical shoreline changes since the middle of the 20 th century, based on series of orthorectified vertical aerial photographs from 1949 to 2015, were presented during one of the first meetings for identifying the areas that underwent erosion during the last decades. Prospective maps of the coastal zone in 50 years (relative to 2015), based on documented shoreline evolutions and taking into account future sea level rise were then produced. These maps were based on high-resolution airborne LiDAR topographic data (vertical error < 15 cm) obtained in 2016 as baseline topography and on a sea level rise projection of 23 cm by 2065 corresponding to the RCP6.0 scenario (IPCC, 2013). In addition, the maximum water level during a 100 year return period storm event was calculated, based on tide gauge records from nearby tide stations and on wave data recorded at an offshore wave buoy. The highest rates of shoreline change observed between 1949 and 2015 were selected for projecting the shoreline position in 2065. The generated maps not only show the expected shoreline position in 2065, and corresponding land loss, but also the areas along the coast where marine flooding will likely take place during a significant storm event. These prospective maps were presented to the local population during public meetings in order to raise public awareness concerning the nature and location of coastal hazards that may threaten these coastal communities during the next decades and to incite reactions from the inhabitants for seeking solutions to the foreseen problems. These maps could also be consulted online by the inhabitants through the website of the project (https://cosaco.univ-littoral.fr/). During following meetings, possible technical solutions and strategies for facing these future risks were presented and discussed with the inhabitants.

Survey Data Collection

After the presentation to the local populations of the prospective maps and possible solutions for coping with future coastal change, the door-to-door survey was conducted in June and July 2018. Because the surveyed municipalities extend several kilometers inland, our investigations were limited to the _________________________________________________________________________________________________ coastal fringe to restrict our survey to the coastal population. Permanent residents as well as second home owners were surveyed. In Wissant the proportion of vacation homes is very significant (67%) compared to Oye-Plage (10%).

285 people were interviewed, 162 in Wissant and 123 in Oye-Plage [START_REF] Révillon | L'évaluation des représentations des habitants face aux risques côtiers à Oye-Plage et Wissant[END_REF]. The survey consisted of 99 questions covering the following topics: (1) housing, (2) attachment to the land and coastal activities, (3) coastal risks, (4) knowledge of coastal risk and feeling of exposure, (5) memory of past risks and vision of the future, (6) information about coastal risks, and (7) knowledge about coastal protection techniques and strategies. Four possible scenarios were proposed for future shoreline management strategies: (1) "hold the line" (i.e., protect the shoreline by strengthening the defenses against the sea), (2) adapt, depending on individual and collective stakes, (3) relocation (retreat) of activities and goods, or (4) do nothing.

RESULTS

In both coastal communities, male and female were equally represented. In Wissant, 48.8% of the respondents were permanent residents and 50.6% were second home owners, the remaining 0.6% corresponding to tourists, whereas in Oye-Plage the permanent residents accounted for 89.5% of the respondents, which is close to the proportion of main residences in this municipality. At Wissant, the lower representativeness of the number of second home owners in our survey compared to the overall proportion of secondary homes could be explained by the absence of some second home owners in July.

A large proportion (≈80%) of surveyed residents own their houses and the majority of them own their house for more than 20 years (56 % in Wissant, 53 % in Oye-Plage). The respondents over 55 years old are 48.8 % at Oye-Plage and 59.2 % at Wissant and in both municipalities the retired persons are in large numbers (30%). The socio-professional categories and the level of education (thus the level of income) is however very different between the two investigated municipalities. In Wissant, more than 40% of respondents have high managerial and professional occupations and a high level of education with 46% of the surveyed residents having a high to very high education level (master's or doctoral degree). In Oye-Plage, intermediate and lower services and technical occupations predominate (70%), residents with a high and very high level of education representing only 17% of the sample.

Perception of Coastal Risks

A large majority (about 80%) of surveyed residents is aware of coastal risks in their municipality. However, when they are asked if their house is exposed or not to coastal erosion and/or to marine flooding, the results are very different (Fig. 2). In Oye-Plage, 35% of the residents consider that their house is not threatened by a coastal risk while 64% believe their house is at risk (Fig. 2B). In Wissant, 59% of the residents do not think their home is exposed to a coastal risk and 42% consider that their house is exposed (Fig. 2A). At both sites, the feeling of exposure to coastal risks varies spatially, the majority of the residents who responded that their home is endangered living close to the shoreline. In Wissant, the feeling of exposure to coastal risks is clearly spatially-related, virtually all of the inhabitants of the old village located on a height (mainly permanent residents) are not concerned by coastal risks, while the people living close to the beach (mainly second home owners) are aware that their house can be endangered (Fig. 2A).

When the residents were asked what the major causes of coastal risks are, the first cause identified is climate change, with 28% of the surveyed residents at Oye-Plage and 25% at Wissant believing that climate change is the main reason of coastal erosion. A similar result was obtained for marine flooding, climate change being the first response at Oye-Plage (20%) and at Wissant (23%).

_________________________________________________________________________________________________ Preferred Strategies for Coping with Future Coastal Risks

The results of the survey show that the preferred strategies for facing future coastal risks are by far adaptation (depending on individual and collective stakes) and protection (hold the line). At Oye-Plage, adaptation and protection strategies are equally preferred with 41% and 43% respectively (Fig. 3B), whereas at Wissant, the majority of the surveyed residents prefer adaptation measures (53%) and 25% opt for holding the line (Fig. 3A). The third preferred strategy is relocation at both sites while very few inhabitants (≤ 5%) consider "do nothing" as a scenario for the future. Conversely to what was observed with risk perceptions, the preferred strategies do not appear to be spatially-controlled as some residents living on the seafront at Wissant, for example, opt for the relocation solution while some individuals living far from the sea in the old village are inclined to favor the "hold the line" solution (Fig. 3A).

DISCUSSION

These results show that in both coastal communities, most of the inhabitants are aware that the littoral strip is exposed to coastal risks. At both study sites, no relationship between education level and perception of risk was found, conversely to what was observed in the Netherland by [START_REF] Botzen | Dependence of flood risk perceptions on socioeconomic and objective risk factors[END_REF] who noticed that more highly educated individuals have a lower flood risk perception in their study of flood risk perception. In the present study, the personal perception of coastal risks by inhabitants is strongly influenced by the location of their house, and notably by the distance to the shoreline. These results are consistent with previous studies that showed that coastal risk perception depends, at least partly, on the distance to the hazard, people leaving farther away from a hazard source generally exhibiting lower levels of perceived risks [START_REF] Lechowska | What determines flood risk perception? A review of factors of flood risk perception and relations between its basic elements[END_REF][START_REF] O'neill | The impact of perceived flood exposure on flood-risk perception: the role of distance[END_REF].

Concerning the preferred strategies for facing future coastal risks, the proximity to the shoreline is not a determinant factor. A large proportion of inhabitants surveyed in both communities do not consider relocation as an option for responding to climate change (Fig. 3), this result being in line with a number of previous studies showing that retreat is generally rejected by coastal residents (e.g., [START_REF] Douglas | Coastal flooding, climate change as environmental justice: identifying obstacles and incentives for adaptation in two metropolitan Boston Massachusetts communities[END_REF]. At Oye-Plage, more people (43%) want protection at any cost compared to Wissant (25%). In Wissant, people possibly feel more protected by the presence of a hard coastal defense structure, which is not the case at Oye-Plage where only soft defense structures (wooden groins and sand fences) are present on the upper beach. Furthermore, at Oye-Plage the plan for coastal safety that obliges the property owners to adapt their home in the area at risk of flooding could be an additional reason for preferring the "hold the line" option for a large proportion of the residents.

In both coastal municipalities, the surveyed residents identify climate change as the first cause of coastal risks, showing that a significant proportion of the population is aware of the impacts of climate change. This differs from the findings of [START_REF] Spence | Perceptions of climate change and willingness to save energy related to flood experience[END_REF] who found no evidence that living in areas vulnerable to climate change influence the perception of risks related to climate change. The high level of awareness of climate change risks in the studied communities may be due to the implementation of a plan for coastal safety in both municipalities, but also to the presentation to the local population of prospective maps of future coastal risks with climate change.

CONCLUSIONS

One of the conclusion of this study is that a large proportion of the surveyed population living in two coastal municipalities of northern France clearly identify climate change as a major cause of coastal risks, showing that most residents are aware of _________________________________________________________________________________________________ the potential impacts of climate change. The results presented in this paper also highlight that the feeling of exposure to coastal risks in these coastal communities is spatially-influenced, the distance to the hazard source being a major factor of risk perception. Conversely, no relationship was found between preferred strategies for facing future coastal risks and proximity to the shoreline. Preliminary analyses suggest that the level of education may influence preferred strategies. Nevertheless, further analyses of other socio-economic factors are needed for strengthening these first results.

Figure 1 .

 1 Figure 1. (A) Location of the studied sites : 1: Wissant, 2: Oye-Plage; (B) Photograph of the housing estate threatened by coastal erosion in Wissant (source: Amis de Wissant); (C) Oblique aerial view of the housing estate at Oye-Plage protected from marine flooding by an eroding coastal dune.

Figure 2 .

 2 Figure 2. Perception of coastal risks (coastal erosion and/or flooding) by the coastal population of (A) Wissant and (B) Oye-Plage. The pie charts present the results for the total surveyed population in each municipality, while the aerial photographs show only a portion of the surveyed territory, especially for the lower photograph (B) showing mainly the Ecardines housing estate at Oye-Plage.

Figure 3 .

 3 Figure 3. Preferred strategies at (A) Wissant and (B) Oye-Plage for facing future coastal risks associated with climate change. The pie charts present the results for the total surveyed population in each municipality, while the aerial photographs are insets showing a portion of the surveyed territory.

ACKNOWLEDGMENTS

This work was funded by the Fondation de France through the project COSACO and by the Région Hauts-de-France through the project AD'ACPOP. The authors are grateful to the Mayors of Wissant and Oye-Plage who graciously lent us their communal hall for the public meetings. The inhabitants of Wissant and Oye-Plage are also thanked for their participation to the project.