The multiscale and multiphase organization of the transcriptome

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The multiscale and multiphase organization of the

transcriptome

Danielle Adekunle, Arnaud Hubstenberger

To cite this version:

Danielle Adekunle, Arnaud Hubstenberger. The multiscale and multiphase organization of the tran-scriptome. Emerging Topics in Life Sciences, 2020, 4 (3), pp.265-280. �10.1042/ETLS20190187�. �hal-03085571�

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The multiscale and multiphase

organization of the transcriptome

Danielle A. Adekunle

1,2

_{; Arnaud Hubstenberger}

3

1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA

02139, U.S.A.

2. Department of Molecular Genetics and Microbiology, UF Genetics Institute, Center

for Neurogenetics, University of Florida, Gainesville, FL, U.S.A

3. Université Côte D'Azur, CNRS, Inserm, iBV, Nice, France

Correspondence: Arnaud Hubstenberger (

Arnaud.HUBSTENBERGER@univ-cotedazur.fr

)

Abstract. 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.

Keywords: post-transcriptional regulation, ribonucleoproteins, RNA condensates, RNA phase

separation and transition, stress granules, transcriptomics

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Introduction.

There are hundreds of thousand mRNA molecules [1] and millions of total RNA molecules in a

single mammalian cell [2]. 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

) [3,4], that demix from the cytosol or nucleoplasm to provide a means to orchestrate

the transcriptome that would not be possible by simple Brownian diffusion.

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].

Phase-separated RNA condensates can contain thousands of transcripts and hundreds of

proteins, providing a higher scale of cellular compartmentalization [5–13]. Nucleoli, Cajal

bodies (CBs), nuclear histones bodies, P-bodies, stress granules (SGs), translation factories,

and cell-type-specific condensates such as myo-granules, neuro-granules, and germ granules

are all well-characterized RNA super-assemblies [3,4]. These granules coassemble, at least in

part, through phase separation, a distinct mode of RNA spatial organization [3,4]. The

architecture of RNA condensates and their regulation by phase transitions provides a

mechanism for synchronizing RNA expression to cellular activity, development, and the

environment, allowing groups of RNAs to function co-ordinately.

This review will examine the various models of molecular interactions that drive RNP phase

separation with a focus on the multiscale and multiphase nature of RNA condensates. It will

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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

micrometer-sized germ granules [14]. 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

) [15]. Some RNP gels do not coalesce and can further solidify, limiting

intracondensate, or cytosolic exchange [16–20]. All of these low-density super-assemblies are

porous and permeable structures, largely comprised of water, that allow the passive diffusion

of small molecules [21]. The scaffolding components of liquid droplets are disordered [21,22];

but some can polymerize into geometrical shapes [15], or crystalline fibers (

Figure 1

) [16,18–

20,23–26]. Although crystalline aggregates are often associated with degenerative diseases,

diffractive assemblies have also been found in myo-granules [27]. We have only begun to

unravel the influence of these material properties on transcriptome organization.

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

(A) Liquid droplets are typified by their ability to dock, fuse, and relax into spheres under surface tension [14,15]. 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 [15].

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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.

Figure 3. Mechanisms of phase separation.

(A) Phase separations are concentration dependent. 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.

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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 [28]. 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 [16,29]: 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 [16,19,20,30–36].

Highly transient interactions between poorly folded IDR domains promote the liquidity of the

system, whose material properties are influenced by IDR composition [37,38]. 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 [21]. 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 [16,18–

20,25,31,33,36,39–42]. 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 [28]. In vitro, IDRs can

condense on their own without RNA but at very high concentrations [16,26]. To condense at

physiological concentrations RBP–RNA interactions are required [18,19,25,36,43,44]. 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 [25,43,45]. RNAs also contribute to

the material properties of condensates; short and long RNAs limit and increase the viscosity

of RNP droplets, respectively [21,32,36].

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

) [46–49]. 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 [50]. RNA secondary structures modulated by RBPs can add

increased specificity to RNA–RNA interactions [48].

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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 [5,7,8,51,52], and a recently

developed technology, fluorescence-activated particle sorting (FAPS) [9]. 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 [10],

P-bodies [9], and SGs [5,11,12]. 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 protein–

protein interactions that create dense stereospecific networks, as revealed by studies

employing proximity labelling of proteins in these networks [11,12]. 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 [6,8,9,13].

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 [53], 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 [9,54–57].

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

[6,48,52], while actively translated glycolytic and translation factors encoding mRNAs

condense into translation factories [58,59]. In metazoans, repressed mRNAs accumulate in

P-bodies [9], SGs [6], germ granules [8], neuronal granules [13,60,61], myo-granules [27], while

translated mRNAs can coassemble into translation factories [62].

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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 [63,64]

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 [65,66]. 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 [64], and nucleoli [67–69].

However, no master scaffolding RNA components have been identified in cytosolic

condensates. While a collective of RNAs scaffold P-bodies and SGs [70,71], the most abundant

RNAs within P-bodies or SGs comprise less than 1% of their transcriptome [6,9], 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 [72]. Similarly, SG

assembly is triggered by RNA release from polysomes which leads to the recruitment of RBPs

that assemble SGs [73–75]. Unfolded mRNAs trigger a conformational switch and the

condensation of the master organizer of SG assembly, G3BP [76,77]. 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

[76,78]. Like RNA–protein and protein–protein interactions, RNA–RNA interactions contribute

to SG assembly through sense–antisense RNA hybrids [49]. In protein-free extracts RNA

precipitation partially recapitulates the SG transcriptome, suggesting that RNA–RNA

interactions are important for SG formation [49]. 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

[48]. 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 [79]. 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 [72]. 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 [80], the prion-like

domain of TIA-1 [81], CPEB1 [82], Caprin-1 [83], DYRK3 [84], hnRNPA2, and FUS [19]. All of

these examples, fit a phase separation model where concentration and multivalency are

critical determinants of condensation [28,70,76,78].

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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 [11,12,75–79,85]. In addition, prion-like IDR–IDR polyQ/N polymers

work synergistically with folded domains to assemble P-bodies [39,41,86]. Folded–folded

interactions bring specificity and stability to the system while IDR–IDR interactions may

provide droplet fluidity [87,88]. Essential protein components in certain environmental

contexts were found to be dispensable in others [75,85]. 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

[28,70,76,78].

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 [6,9,89]. Although all

mRNAs condense in SGs to some degree, the condensed fraction of mRNAs varies from <1%

to >95% [6]. Any mRNA can transiently dock on condensates, including translated ones, but

only repressed mRNAs can be stably anchored, leading to stronger enrichment [56,90].

Similarly, mRNAs condense to various degree in P-bodies [9], and single-molecule live imaging

confirmed that the fraction and dynamics of transcripts that localize to SGs and PBs is different

for each mRNA [56,91]. 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 [9,56]. From global CLIP analyses,

we know that RNA–protein interactions are predictors of RNA enrichment in P-bodies [9] and

live single mRNA imaging demonstrated that these interactions anchor mRNAs and lncRNAs

to condensates [56,91]. From these studies, we can conclude that low specificity and

low-affinity binding allows transient RNA docking, while high specificity and high-low-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 [53].

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 [30,32,34,77,87,92–95].

Illustrating the diversity of the responses, PAB1 condensation is triggered by heat [87],

whereas DDX4 is triggered by cold [34]; while FUS and DDX4/LAF-1 condense at high and low

salt concentrations, respectively [30,32,34]. Similarly, the pH dependent protonation of

G3BP's IDR induces a conformational switch for this SG master protein, promoting its ability

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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 [77]. To promote cellular

fitness, IDRs seem to have been evolutionarily selected to control the temperature or pH

levels at which proteins phase separate [87,93–95]. 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 [95]. 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 [96]. Similarly, the number and

distribution of direct RNA m6A methylations regulate and influence the composition of the

phase-separated transcriptome [97]. 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 [75,87,98–

101]. Twenty percent of SG proteome diversity is stress-dependent [11]. 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 [6,52]. 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 [14], RNA condensates are not

homogenously mixed. Nucleoli and germline P-bodies are subcompartmented by liquid–liquid

phase separations (LLPS) [15,102,103]. 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 [5,106–109].

The remodelling of germ granules during development is well-characterized. During early

meiosis, liquid germ droplets wet the nuclear pore (

Figure 5

) [14]. 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 [44,111]. 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

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semi-liquid germline P-bodies, and without mixing become subcompartments of larger

condensates (

Figure 5

) [15]. 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 [15]. At each mitotic cycle, germ droplets

condense posteriorly and somatic P-bodies condense anteriorly to be asymmetrically

inherited [8,14,15,115]. At this developmental stage, RNAs cluster MEG-3 gel cores around

liquid PGL-3 droplets [8,116,117]. The ability of multiple proteins: PGL-1/3 [44,111], MEG-3

[116–118], Ddx4/ LAF-1 [32,34] to phase separate can mechanistically explain LLPS

subcompartmentalization. Compartmentalized granules have also been observed in

Drosophila germlines [119–121]. 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

).

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 P-bodies 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) Some 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 [8,148]. Granule material properties can fine-tune RNA exchange. For example, highly viscous droplets can sequester mRNAs, whereas fluid ones can promote dynamic exchange [15].

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Figure 5. Condensate architecture is remodelled during germline development.

In early oogenesis, germ droplets wet nuclear pores [14], 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 [15]. Fertilization triggers the dissolution of maternal RNPs that were solidified in highly viscous germline P-bodies during the arrest. Embryogenesis promotes asymmetric distribution of maternal RNPs. At the anterior pole, RNPs phase separate into somatic P-bodies [15,115]. At the embryo's posterior pole, RNPs condense into germ granules that contain a liquid core and gel shell [8,14,44,116]. 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 [73,122]. However, SGs and P-bodies share

numerous interactors [5,9,11,12,85], 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 [78]. Exchange of RNA between SGs and P-bodies is bidirectional, as illustrated by live

imaging [90]. 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

prion-like 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

[86]. In an energy-depleted context condensates grow and solidify, signifying that the cell

actively restricts condensation [15,102]. Helicases ensure transcriptome liquidity. They

comprise 10% of the condensate proteome and their depletion causes RNP solidification

[9,15]. By participating in protein–RNA and IDR–IDR interactions, helicases promote

condensate assembly, and by disrupting intermolecular RNA–RNA interactions maintain

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liquidity [43,50,123]. Similarly, chaperones that prevent the accumulation of misfolded

proteins regulate granule dynamics [5,17,24,124–126]. 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 [87]

and proximity labelling revealed pre-assembled sub-microscopic SGs under non-stress

conditions that may serve as ‘seeds’ to promote rapid assembly upon stress [11,12].

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 [96].

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 [78]. For example,

RNA multivalency is critical to germ granule assembly, and soluble RBP can compete with

condensates for mRNAs and thus trigger their dissolution [44]. 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 [9]. 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 [35], while condensates catalyze deadenylation that limits translation [57,131]. In

a third model, non-functional miRISCs are trapped within P-bodies where they can scan

potential mRNA targets that traverse granules more transiently [56]. 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 [1] 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 [77], or promote new interactions such as RNA–RNA interactions that are

unstable in the cytosolic environment [50]. Translation factories could promote the

co-translational assembly of protein complexes or ribosome recycling [58,59,62]. Taken together

these recent studies suggest that RNA condensates are more than sorting centers that

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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

[28,70,76,78,104,134]. Condensates can also absorb large variations in interaction

stoichiometry [70,104,134], 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 [73–78]. 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 [70,76,78,104,134]. 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 [59]. In metazoans, germ granules are asymmetrically inherited

in the germline [14,44,117]. Consistent with the transmission of an epigenetic signal, germ

granule mutants become RNAi defective, express somatic transcripts, and become sterile over

generations [114,136–139]. These condensates accumulate small ncRNAs and their regulatory

proteins are essential for ensuring transgenerational epigenetic inheritance [114,140–146].

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.

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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.

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.

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

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