# Beyond equilibrium: roles of RNAs in condensate control

Fernando Muzzopappa, Fabian Erdel

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Fernando Muzzopappa, Fabian Erdel. Beyond equilibrium: roles of RNAs in condensate control. Current Opinion in Genetics and Development, 2025, 91, pp.102304. 10.1016/j.gde.2024.102304. hal-05385680

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Review

# Beyond equilibrium: roles of RNAs in condensate control

Fernando Muzzopappa and Fabian Erdel

<figure><img src="image_3.png" /></figure>

Membraneless subcompartments organize various activities in the cell nucleus. Some of them are formed through phase separation that is driven by the polymeric and multivalent nature of biomolecules. Here, we discuss the role of RNAs in regulating nuclear subcompartments. On the one hand, chromatin-associated RNA molecules may act as binding platforms that recruit molecules to specific genomic loci. On the other hand, RNA molecules may act as multivalent scaffolds that stabilize biomolecular condensates. The active production and processing of RNAs inside of nuclear subcompartments drives them out of thermodynamic equilibrium and thereby modulates their properties. Accordingly, RNA content and transcriptional activity appear as key determinants of the biophysical and functional nature of nuclear substructures.

**Address**

MCD, Center for Integrative Biology (CBI), University of Toulouse, CNRS, Toulouse, France

**Corresponding authors:**

Muzzopappa, Fernando (fernando.muzzopappa@cnrs.fr),

Erdel, Fabian (fabian.erdel@cnrs.fr)

<figure><img src="image_4.png" /></figure>

@FerMuzzo (Muzzopappa, Fernando)

**Current Opinion in Genetics & Development** 2025, 91:102304

This review comes from a themed issue on **Genome Architecture and Expression**

Edited by Anders Sejr Hansen and Marcelo Nollmann

For complete overview of the section, please refer to the article collection, "Genome Architecture and Expression (2025)"

Available online 14 January 2025

https://doi.org/10.1016/j.gde.2024.102304

0959-437X/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## RNAs as regulators of nuclear organization

The compartmentalization of cellular components enables the spatial and temporal coordination of biological activities. Membraneless subcompartments, or biomolecular condensates, play an important role in this respect and regulate critical cellular processes [1]. Examples in the cell nucleus include nucleoli where rRNAs are produced, transcriptional condensates where mRNAs or noncoding RNAs (ncRNAs) are produced, nuclear speckles and paraspeckles where RNAs are processed

and retained, and heterochromatin structures where comparatively silent DNA regions are packaged [2]. Many of these subcompartments contain RNAs, which have long been known to regulate nuclear architecture [3,4] and whose regulatory roles in the context of biomolecular condensates have emerged more recently.

From a biophysical perspective, the formation of biomolecular condensates represents a demixing process that is associated with an entropic cost, which has to be compensated by favorable intermolecular interactions. Depending on the characteristics of these interactions, demixing can occur via mere chromatin binding, where the respective molecules act as ‘clients’ that associate with a segment of a chromosome or via (liquid-liquid viscoelastic) phase separation, where the molecules act as ‘scaffolds’ that establish multivalent interactions with each other in addition to chromatin binding [5,6]. Conceptually, RNAs may play a role in both scenarios (Figure 1): (i) They can associate with chromatin and provide a binding platform for other molecules, (ii) they can bind to molecules in a condensate that is formed by phase separation, for example, to RNA-binding proteins (RBPs), acting as scaffolds, clients, or surfactants that modulate multivalent intermolecular interactions. Notably, these different roles are not mutually exclusive. The active production of RNAs in a condensate as well as their export from a condensate add an additional layer of complexity to the regulatory roles they can play.

## RNAs as platforms for the site-specific assembly of nuclear subcompartments

Many nuclear subcompartments are associated with specific genomic loci. As chromosomes occupy distinct territories and move only very slowly over larger distances, binding of proteins to chromatin can explain the assembly of positionally stable subcompartments [8]. Chromatin-associated RNAs and particularly nascent transcripts often play roles in targeting proteins to chromatin, thereby mediating the assembly of subcompartments. A prominent example is the nucleolus, which forms around the ribosomal DNA repeats that encode rRNAs. Recent studies have shown that nascent RNAs are key for nucleolar assembly [9–11]. Another example is transcriptional condensates that form in proximity to genes transcribed by RNA Polymerase II [12,13]. These structures have been proposed to assemble based on nascent RNAs [14]. Heterochromatin foci have also been linked to an RNA component, and it has been shown that nascent major satellite transcripts
<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

Roles of RNAs in regulating nuclear substructures. (a) Chromatin-associated RNAs may provide binding sites for client molecules, for example, RBPs that are recruited to chromatin [7]. (b) RNAs may establish multivalent interactions with RBPs and act as scaffolds of biomolecular condensates, which may be associated with chromatin or not.

can regulate their properties [15]. In particular, heterochromatin foci remain compact upon RNA depletion but lose heterochromatin protein 1 and change their dynamic properties [16,17]. Inactivation of the X chromosome in females also involves multiple RNA components, including the ncRNA Xist [18]. These examples show that RNAs regulate the stability and composition of nuclear subcompartments. Mechanistically, this may occur through the recruitment of client molecules that merely bind chromatin-associated RNA molecules or through site-specific nucleation of phase-separated condensates (Figure 1). Both mechanisms have, for example, been discussed in the context of X inactivation [19,20].

## RNAs as multivalent scaffolds of biomolecular condensates

After several studies have focused on the role of individual proteins in phase separation, recent advances in the field suggest that most intracellular condensates are formed through heterotypic multivalent interactions among different types of scaffold molecules, including nucleic acids [11]. Based on their general biophysical characteristics, RNAs have several properties that make them good candidate scaffolds [21]. (i) RNAs are relatively long (Figure 2a,b) and flexible [22], which should allow them to establish multiple interactions with neighboring molecules. The positive correlation between length and phase separation propensity has recently been documented for both RNAs [23,24] and DNA/chromatin arrays [25]. (ii) RNA molecules are highly multivalent due to their high density of negative charges, which can serve as binding platforms for positively charged partners, and because nucleotides are free to engage in π-π stacking interactions with aromatic amino acids [26]. (iii) Typical affinities of RNA-protein interactions are similar to those of DNA-protein

interactions (Figure 2c), which should allow RNAs to establish sufficiently strong heterotypic interactions with RBPs. (iv) Compared to chromosomes, which represent the other major type of nucleic acid scaffold in the nucleus, RNAs are quite mobile (Figure 2d), making them compatible with the dynamic nature of many condensates. RBPs, which associate with RNAs in condensates, are also good candidate scaffolds. RBPs are enriched in disordered regions [27] and have a (moderately) increased intrinsic disorder compared to all proteins (Figure 2e). RBPs can promote RNA-RNA interactions upon RBP-RNA binding [28,29], enhance the molecular connectivity [30,31], and set up spatial (sub)compartments [32]. Accordingly, there are several indications that RNAs and RBPs have the ability to form multivalent interaction networks and act as scaffolds of biomolecular condensates.

## RNAs organize transcriptionally active condensates

Several nuclear substructures do not only contain RNAs but actively produce them. A prominent example is the nucleolus, where the transcription of rRNA by RNA Polymerase I and the biogenesis of ribosome subunits take place [39]. These micron-sized structures exhibit several liquid-like features, such as rounded shape, interfacial tension, and fusion behavior upon contact [40]. They are organized in a multilayered structure, where the steps of ribosome biogenesis are spatially coordinated. The fibrillar centers (FC), surrounded by the dense fibrillar components (DFC), are embedded in the largest nucleolar component, the granular component (GC). Ribosome production starts at the FC-DFC interface where pre-rRNAs are transcribed. Pre-rRNA transcripts subsequently diffuse to the DFC for postprocessing, a multi-step reaction that includes RNA cleavage and post-transcriptional modifications. Finally, ribosome subunits
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

Biophysical properties of RNAs and RBPs make them good candidate scaffolds. (a) Radii of gyration of chromatin in human cells [33] and in solution (considered as worm-like chain), of the AlphaFold structures of the entire human proteome (UP000005640), and of RNAs (miRNA, unspliced mRNA, rRNA, and IncRNA) from the human genome (hg38), using a published scaling equation [34]. The median size of each type of RNA is indicated. (b) Size of the BRD4 protein (structure from AlphaFold) compared to the schematic structure of its corresponding unspliced mRNA. (c) Characteristic dissociation constants for dsDNA-protein and ssRNA-protein interactions [35]. (d) Diffusion coefficients of chromatin [36], GFP mono- and multimers [37], and mRNA [38] in living cells. (e) Average PONDR disorder scores of proteins in the human proteome (UP000005640). RBPs were identified using a database [7].

are assembled in the GC [41]. The proteins fibrillarin and nucleophosmin, which are enriched in the DFC and GC, respectively, can form immiscible phase-separated droplets *in vitro* and recapitulate basic features of nucleolar organization [42]. Differences in the interfacial tension of the condensates and the RNA-binding domain of both proteins are required for immiscibility. In addition, both proteins interact with rRNA [10,43], which leads to the formation of condensates at lower concentrations [44]. Inhibition of RNA polymerase I induces the disassembly of the canonical nucleolar structure [45], highlighting the key role of rRNA in nucleolar organization and suggesting that rRNA molecules act as multivalent scaffolds. As discussed in the next section, the active production and processing of rRNA drives the nucleolus out of equilibrium and explains features that are not captured by simple equilibrium phase separation models.

Other transcriptionally active subcompartments coincide with the sites where RNA Polymerase II produces mRNAs or ncRNAs. Transcription by RNA Polymerase II involves binding of transcription factors (TFs) to promoters and enhancers, which contact each other. Several TFs can undergo demixing *in vitro* [13,46,47], and both overexpressed [48] and endogenous [49] TFs can form condensates in living cells upon chromatin binding. Recent work has suggested that TFs can undergo surface condensation and cover stretches of chromatin with multiple TF-binding sites [47]. According to this model, TFs will preferentially condense at broad enhancer regions, for example, superenhancers (SEs), along with the coactivator proteins BRD4 and MED1 [13]. Live-cell super-resolution microscopy has documented the presence of transcription-associated nuclear substructures containing these factors [12], and, in particular, a population of comparably large and stable
substructures that represent bona fide condensates at SEs. MED1 accumulation at SEs and concomitant gene activation were shown to depend on TFs such as OCT4 [46]. From these and other studies, a model has emerged in which the cooperative binding of TFs and coactivators to SEs promotes the formation of dynamic condensates that recruit RNA Polymerase II and the required factors to ensure transcriptional activity [50]. Coactivator condensates can be enriched in different types of RNAs, including enhancer RNAs [14], nascent mRNAs [12,13], or long ncRNAs [51]. Transcription-dependent condensates are sensitive to the inhibition of RNA Polymerase II [12], suggesting that the respective RNAs may scaffold them. It should be noted that most transcriptional condensates are quite small and contain only relatively few molecules [12,52,53], posing the question of how well their behavior can be described with concepts from continuum thermodynamics [54].

Two peculiar examples of condensates linked to tran-
scription by RNA Polymerase II are histone-locus bodies
and Cajal bodies. Both structures have been described as
liquid-like condensates that are seeded by transcription
of histone genes and small nuclear RNAs, respectively,
highlighting the role of (nascent) RNAs in condensate
formation [55,56].

Transcription drives biomolecular
condensates out of equilibrium

Living organisms are driven away from equilibrium by
energy-dependent processes. As opposed to the equili-
brium situation, where all concentrations are constant
over time, active processes such as transcription can
change the concentrations or the chemical nature of
molecules over time (Figure 3a). For timescales that are
much shorter than the reaction rate of the active process,
the system can be considered to reside in a quasi-equi-
librium state, and equilibrium phase separation models
can be used to describe it. An example is the formation
and dissolution of P-granules in C. elegans, which has
been considered at local equilibrium [57]. In general, to
comprehensively understand the dynamics of active
condensates, it is instructive to go beyond equilibrium
theories and examine their nonequilibrium behavior
across multiple time scales. We will subsequently touch
upon some nonequilibrium phenomena in the context of
phase separation and highlight their potential implica-
tions for transcriptionally active condensates in the cell
nucleus.

Condensate number

At phase equilibrium, the presence of multiple con-
densates represents a metastable state, and the system
naturally progresses toward a state with a single con-
densate through droplet fusion and Ostwald ripening
[58]. This process can be modulated by the cellular

environment, for example, the motion and fusion of
droplets can be hindered in the presence of obstacles
such as chromosomes [40]. Active modification of phase-
separating components by chemical reactions can induce
the arrest of Ostwald ripening [59,60]. This occurs be-
cause the influx of newly formed molecules competes
with the net flow of molecules from smaller droplets to
larger ones [61]. It has been suggested that this dynamic
arrest of droplet coarsening could describe the stabili-
zation of stress granules [62]. For the nucleolus, it has
recently been suggested that the size of the FC is con-
trolled by active transcription of rRNA and that nascent
rRNAs produced at the interface of the FC and DFC are
stretched to accommodate RBPs, generating a lateral
pressure that suppresses FC growth [63].

Condensate morphology and material state

Active processes can regulate condensate shape. In the
case of nucleoli, both rRNA transcription and rRNA
processing play a role, which are necessary for nucleolar
stability [45,64]. A recent study indicated that the nu-
cleolar shape is the result of the spatial distribution of
rRNA transcription sites within the nucleolus [65]. From
these sites, the rRNA flows directionally toward the
nucleoplasm. As the rRNA diffuses through each nu-
cleolar subcompartment, it is sequentially modified by
different enzymes until the binding of ribosomal pro-
teins triggers the formation of ribosomal subunits. On
the one hand, rRNA cleavage and binding of these co-
factors decrease the rRNA valency [11,66], leading to
less favorable partitioning and rRNA ejection from the
nucleolus. On the other hand, newly synthesized rRNA
forms a viscous entangled mesh [65]. A recent high-
throughput screening approach showed that mutants
that accumulate early rRNA intermediates enhance this
gel-like behavior [67]. Consequently, polymer en-
tanglement reduces as rRNA is processed, decreasing
the viscosity and increasing rRNA mobility (Figure 3b).
This model suggests that sequential RNA processing is
kinetically controlled by the spatially modulated vis-
coelastic properties of nucleoli. Consistently, depletion
of ATP, which is required for several processing steps
[68], increases nucleolar viscosity [40].

Condensate dynamics and stability

The production of RNAs in transcriptionally active
condensates has been proposed to promote a phenom-
enon called reentrant phase behavior, which means that
the system undergoes two consecutive phase transitions
to reach a final state that resembles the initial state (see
Figure 3a for a quasi-equilibrium representation).
Briefly, the addition of RNA, at equal or lower con-
centration than that required for charge neutralization, to
a homogenous solution of RNA-binding peptides pro-
motes its phase separation by complex coacervation
driven by electrostatic interactions [69]. Subsequent
addition of higher concentrations of RNA generates an
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>

(caption on next page)
Nonequilibrium phenomena in active condensates. (a) Schematic phase diagram of a binary mixture composed of RNAs and RBPs. For each point in the two-phase regime, condensate numbers and sizes may change due to fusion and ripening, even if the concentrations in both phases have equilibrated (left). If the RNA concentration increases due to active transcription, reentrant phase behavior can be observed (right): Condensates first form and then dissolve (arrows depict the trajectory in the quasi-equilibrium representation). Examples of nonequilibrium models for transcriptionally active subcompartments are shown in the following panels. (b) Model for the RNA-dependent regulation of nucleolar morphology. Ribosomal RNA is transcribed at the FC/DFC boundary where it forms a gel-like mesh (left). It is subsequently shortened and disentangled in the DFC (center) and compacted in the GC (right), leading to a decreased rRNA valency and a decreased viscosity, which results in directional flow of rRNA toward the nucleoplasm. (c) Nonequilibrium reentrant phase separation model for transcriptional condensates. Initial binding of TFs at enhancers and promoters leads to surface condensation and enhanced recruitment of RNA Polymerase II, which transcribes short sequences of RNA (left). As the concentration of RNA increases, complex coacervation with positively charged proteins leads to the formation of condensates (center). The nonstoichiometric recruitment of factors into the dense phase could enhance RNA production. When the concentration of RNA surpasses the critical concentration for the reentrant phase transition, the condensate is dissolved by charge inversion (right), and mRNA molecules diffuse away from their production site.

excess of negative charges that drives the transition to a
homogenous state. The latter transition has been ob-
served *in vitro* and in living cells for the RBPs FUS and
TDP43 [70]. The transcriptional regulation by RNA in
coactivator condensates has recently been modeled as
reentrant phase separation [14]. In this model, the initial
recruitment of TFs, coactivators, and RNA Polymerase
II promotes the transcription of short RNAs from en-
hancers and promoters, which in turn enhances the for-
mation of MED1 condensates. The high density of
these proteins favors a transcriptional burst [14,71,72].
However, the formation of a dense phase might not be
required to promote transcription [73]. Further accu-
mulation of RNA transcripts may lead to charge inver-
sion and the dissolution of the transcriptional
condensate, providing a feedback-loop mechanism that
regulates gene expression (Figure 3c). Consistently, in-
hibition of RNA Polymerase II is associated with the
formation of transcriptional condensates with increased
persistence and size [14]. Recently, live-cell microscopy
studies of *Drosophila* embryos corroborated this model
[74]. As pointed out above, most transcriptional con-
densates are quite small, and it remains to be seen how
well they are described by thermodynamic concepts that
have been developed to describe much larger systems
[54]. Mapping the stoichiometries between RBPs and
RNAs in different subcompartments, for example, in
nucleoli and transcriptional condensates, would be
helpful to improve our understanding of these none-
quilibrium structures.

**Conclusion**

RNAs regulate the global nuclear architecture and are
important components of several nuclear substructures.
Their biophysical properties allow RNAs to play various
roles in recruiting molecules and in scaffolding con-
densates, regulating their spatial location, molecular
composition, and material state. As opposed to proteins
that are imported into the nucleus from the cytoplasm,
RNAs are actively produced in various nuclear sub-
compartments, which drives these structures out of
equilibrium, impacting their number, morphology, and
stability. Thus, the transcriptional activity and the RNA
content of nuclear condensates can serve as fingerprints
to distinguish different classes of nuclear substructures

with distinct biophysical properties. Future challenges
include to distinguish in which cases RNAs act as re-
cruitment platforms, condensate scaffolds or both, to
further elucidate the link between their higher-order
structure and their role in condensates, and to clarify
which phenomena are the cause or consequence of
biological activity.

**Data Availability**

No data were used for the research described in the art-
ticle.

**Declaration of Competing Interest**

The authors declare no conflict of interest.

**Acknowledgements**

We apologize to all colleagues whose work could not be cited due to length limitations. We would like to thank Argyris Papantonis and the anonymous reviewer for the helpful feedback.

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In this work, the authors used single-molecule microscopy experiments to characterize the formation of TF condensates on DNA. They showed that Klf4 can form foci on DNA below its critical concentration of de-mixing in solution. These surface condensates are formed in two steps: At low concentration, Klf4 binds to chromatin (adsorbed state), while at higher concentration, it switches to a condensed state. Contrary to chromatin binding, where an increase in the protein concentration tends to lead to nonspecific binding, surface condensation enables sequence-dependent condensate localization rather independently of bulk protein concentration.

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In this work, the authors use a mathematical model that takes into account the nucleolar architecture and rRNA transcription to predict the size of the FC compartment. Based on their model, they propose that nascent rRNAs, which are stretched toward the DFC compartment, generate a pressure that counteracts the FC interfacial tension and suppresses FC growth. In agreement with this hypothesis, they showed that mild inhibition of RNA Polymerase I promotes the growth of the FC compartment.

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In this work, the authors show that the mobility of rRNA is considerably slower than that of nucleolar proteins. The directed motion of rRNAs radiates from the transcription site and defines the shape of the nucleolus. The flow of rRNA is promoted by the transcription of long nonprocessed rRNAs that form an entangled gel. Subsequent processing of the rRNAs leads to a decrease of their entanglement and vascularity. As a result, rRNA becomes more compact and mobile until it exits the nucleolus. This work links the complex viscoelastic properties of the nucleolus with its biological function.

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71. Schneider N, Wieland F-G, Kong D, Fischer AAM, Hörner M, Timmer J, Ye H, Weber W: *Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.* *Sci Adv* 2021, **7**:eabd3568.

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73. Trojanowski J, Frank L, Rademacher A, Mücke N, Grigaitis P, Rippe **K**: *Transcription activation is enhanced by multivalent interactions independent of phase separation.* *Mol Cell* 2022, **82**:1878-1893 e10.

In this work, the authors used modular synthetic TFs and live-cell microscopy to explore the link between phase separation and transcription activation. Optogenetic recruitment of different activation domains with different phase separation propensities showed that multivalent interactions can stabilize TFs on the promoter and enhance transcription. However, analysis of this activation process at different concentrations allowed the authors to contrast conditions where TFs form condensates
and where TFs merely bind to promoter sequences. Comparing these
sets of conditions, the authors found that multivalent interactions are
linked to the formation of phase-separated condensates, but that this
process is not required to promote transcription.

74. Cho C-Y, O'Farrell PH: Stepwise modifications of transcriptional

* hubs link pioneer factor activity to a burst of transcription. *Nat Commun* 2023, 14:4848.

This work used a combination of live-cell microscopy with molecular
sequestration of different TFs to show that Zelda forms dynamic clusters
in the nucleus of *Drosophila* embryos. These are followed by con-
densate formation of BRD4, with the concomitant dissolution of TF
clusters. In a similar manner, BRD4 condensates nucleate RNA
Polymerase II clusters, which require initiation of the transcription to
form a condensate that is later dissolved by sustained transcription.