RNAs in Condensate Control: Beyond Equilibrium

Review: Beyond Equilibrium: Roles of RNAs in Condensate Control

This review, authored by Fernando Muzzopappa and Fabian Erdel, explores the pivotal roles of RNAs in regulating nuclear subcompartments, particularly focusing on how active RNA production and processing drive these subcompartments out of thermodynamic equilibrium, thereby modulating their properties.

Introduction to Membraneless Subcompartments

• Cellular Organization: Membraneless subcompartments, also known as biomolecular condensates, are crucial for the spatial and temporal coordination of biological activities within the cell.

• Formation Mechanism: Many of these subcompartments form via phase separation, a process driven by the polymeric and multivalent nature of biomolecules.

• Nuclear Examples: In the cell nucleus, these include:

  • Nucleoli: Sites of rRNA production.

  • Transcriptional condensates: Where mRNAs or noncoding RNAs (ncRNAs) are produced.

  • Nuclear speckles and paraspeckles: Involved in RNA processing and retention.

  • Heterochromatin structures: Package comparatively silent DNA regions.

• RNA's Emerging Role: While RNAs have long been recognized for their role in nuclear architecture, their specific regulatory functions within biomolecular condensates have been elucidated more recently.

Biophysical Perspective of Condensate Formation

• Demixing Process: The formation of biomolecular condensates is a demixing process, incurring an entropic cost that must be offset by favorable intermolecular interactions.

• Molecular Roles: Molecules involved in condensate formation can act in two main ways:

  • 'Clients': Associate with a segment of a chromosome through mere chromatin binding.

  • 'Scaffolds': Establish multivalent interactions with each other (in addition to chromatin binding) through (liquid-liquid/viscoelastic) phase separation.

• Conceptual Roles of RNAs: RNAs can play roles in both scenarios:

  • (i) Binding Platforms: Chromatin-associated RNA molecules can act as binding platforms, recruiting molecules (e.g., RNA-binding proteins, RBPs) to specific genomic loci.

  • (ii) Multivalent Scaffolds: RNAs can bind to molecules within a condensate formed by phase separation, acting as scaffolds, clients, or surfactants to modulate multivalent intermolecular interactions.

• Dynamic Complexity: The active production and export of RNAs from a condensate add further layers of complexity to their regulatory functions.

RNAs as Platforms for Site-Specific Assembly

• Genomic Locus Association: Many nuclear subcompartments are tied to specific genomic loci, owing to the slow movement of chromosomes and the stable binding of proteins to chromatin.

• Nascent Transcripts as Targeting Agents: Chromatin-associated RNAs, particularly nascent transcripts, are critical for targeting proteins to chromatin, thereby mediating subcompartment assembly.

• Prominent Examples:

  • Nucleolus: Assembles around ribosomal DNA repeats encoding rRNAs. Nascent RNAs are essential for nucleolar assembly.

  • Transcriptional Condensates: Form near genes transcribed by RNA Polymerase II, proposed to assemble based on nascent RNAs.

  • Heterochromatin Foci: Linked to an RNA component; nascent major satellite transcripts regulate their properties. While RNA depletion keeps these foci compact, they lose Heterochromatin Protein $1$ and alter dynamic properties.

  • X Chromosome Inactivation: Involves multiple RNA components, including the ncRNA Xist.

• Mechanisms of Regulation: RNAs regulate the stability and composition of nuclear subcompartments through:

  • Recruitment of client molecules that bind chromatin-associated RNAs.

  • Site-specific nucleation of phase-separated condensates (e.g., in X inactivation).

RNAs as Multivalent Scaffolds of Biomolecular Condensates

• Heterotypic Interactions: Intracellular condensates primarily form through heterotypic multivalent interactions among various scaffold molecules, including nucleic acids.

• Biophysical Characteristics of RNAs as Scaffolds:

  • (i) Length and Flexibility: RNAs are relatively long and flexible (cf. Figure 2a and b). This allows them to establish multiple interactions with neighboring molecules. A positive correlation exists between length and phase separation propensity for both RNAs and DNA/chromatin arrays.

  • (ii) High Multivalency: Due to a high density of negative charges, RNAs serve as binding platforms for positively charged partners. Nucleotides also engage in $\pi-\pi$ stacking interactions with aromatic amino acids.

  • (iii) Interaction Affinities: Typical affinities of RNA–protein interactions are similar to DNA–protein interactions (cf. Figure 2c), allowing for sufficiently strong heterotypic interactions with RBPs.

  • (iv) Mobility: Compared to chromosomes, RNAs are quite mobile (cf. Figure 2d), compatible with the dynamic nature of many condensates.

• Role of RNA-Binding Proteins (RBPs):

  • RBPs are enriched in disordered regions (cf. Figure 2e) and have an increased intrinsic disorder compared to other proteins.

  • They can promote RNA-RNA interactions upon RBP–RNA binding, enhance molecular connectivity, and establish spatial subcompartments.

• Conclusion: Both RNAs and RBPs are indicated to form multivalent interaction networks and serve as scaffolds for biomolecular condensates.

RNAs Organize Transcriptionally Active Condensates

Many nuclear substructures not only contain but also actively produce RNAs.

Nucleolus

• Function: Site of rRNA transcription by RNA Polymerase I and ribosome subunit biogenesis.

• Liquid-like Features: Exhibits rounded shape, interfacial tension, and fusion behavior.

• Multilayered Structure: Spatially coordinates ribosome biogenesis steps:

  • Fibrillar Centers (FC): Surrounded by Dense Fibrillar Components (DFC).

  • Dense Fibrillar Components (DFC): Embedded in the Granular Component (GC).

  • Granular Component (GC): The largest nucleolar component.

• Process Flow: Ribosome production begins at the FC–DFC interface with pre-rRNA transcription. Pre-rRNA transcripts then diffuse to the DFC for post-processing (cleavage, post-transcriptional modifications). Finally, ribosome subunits assemble in the GC.

• Protein Interactions: Fibrillarin (enriched in DFC) and nucleophosmin (enriche in GC) can form immiscible phase-separated droplets in vitro, mimicking nucleolar organization. Differences in interfacial tension and RNA-binding domains are crucial for immiscibility. Both proteins interact with rRNA, leading to condensate formation at lower concentrations.

• rRNA's Scaffolding Role: Inhibition of RNA Polymerase I disassembles the nucleolus, underscoring rRNA's key role as a multivalent scaffold.

Other RNA Polymerase II-Driven Condensates

• Location: Coincide with sites where RNA Polymerase II produces mRNAs or ncRNAs.

• Mechanism: Involves transcription factor (TF) binding to promoters and enhancers, which physically interact.

  • TFs can undergo demixing in vitro and form condensates in vivo upon chromatin binding.

  • A model suggests TFs preferentially condense at broad enhancer regions (e.g., superenhancers, SEs) with coactivators like BRD4 and MED1.

• Superenhancer (SE) Condensates: Live-cell microscopy reveals transcription-associated nuclear substructures at SEs containing these factors, forming large and stable condensates. MED1 accumulation at SEs and gene activation depend on TFs (e.g., OCT4).

• Model of Transcriptional Condensates: Cooperative binding of TFs and coactivators at SEs promotes dynamic condensate formation, recruiting RNA Polymerase II and other essential f actors for transcription.

• RNA Content: These coactivator condensates can be enriched in enhancer RNAs, nascent mRNAs, or long ncRNAs.

• RNA Scaffolding: Transcription-dependent condensates are sensitive to RNA Polymerase II inhibition, suggesting that these RNAs serve as scaffolds.

• Size Considerations: Most transcriptional condensates are small and contain few molecules, raising questions about the applicability of continuum thermodynamics models.

• Specific Examples:

  • Histone-locus bodies: Seeded by transcription of histone genes.

  • Cajal bodies: Seeded by transcription of small nuclear RNAs.

  • Both highlight the importance of (nascent) RNAs in condensate formation.

Transcription Drives Biomolecular Condensates Out of Equilibrium

Living systems operate away from equilibrium due to energy-dependent processes.

• Equilibrium vs. Non-Equilibrium: In contrast to equilibrium states where concentrations are constant, active processes like transcription change molecular concentrations or chemical nature over time. For short timescales, a quasi-equilibrium state might exist, but a comprehensive understanding requires examining non-equilibrium behavior.

Condensate Number

• Equilibrium Behavior: At phase equilibrium, multiple condensates represent a metastable state, tending towards a single condensate via droplet fusion and Ostwald ripening.

• Cellular Modulation: Obstacles like chromosomes can hinder droplet motion and fusion.

• Active Arrest of Ostwald Ripening: Chemical reactions actively modifying phase-separating components can arrest Ostwald ripening. This occurs when the influx of newly formed molecules balances the net flow from smaller to larger droplets. Examples include stress granules and nucleoli.

  • For the nucleolus, rRNA transcription controls the size of FCs. Nascent rRNAs at the FC/DFC interface are stretched, generating lateral pressure that suppresses FC growth.

Condensate Morphology and Material State

• Active Regulation of Shape: Active processes regulate condensate shape, notably in nucleoli, where rRNA transcription and processing are vital for stability.

• Nucleolar Shape and rRNA Flow: Nucleolar shape is influenced by the spatial distribution of rRNA transcription sites. rRNA flows directionally from these sites towards the nucleoplasm.

• Sequential rRNA Processing: As rRNA diffuses through nucleolar subcompartments, it is sequentially modified by enzymes. Ribosomal protein binding triggers ribosomal subunit formation.

  • Valency Decrease: rRNA cleavage and co-factor binding reduce rRNA valency, leading to less favorable partitioning and rRNA ejection.

  • Viscoelastic Properties: Newly synthesized rRNA forms a viscous entangled mesh (Figure 3b). Polymer entanglement decreases as rRNA is processed, reducing viscosity and increasing rRNA mobility.

  • Kinetic Control: This model suggests that sequential RNA processing is kinetically controlled by the spatially modulated viscoelastic properties of nucleoli.

  • ATP Dependence: Depletion of $\text{ATP}$, required for several processing steps, increases nucleolar viscosity.

Condensate Dynamics and Stability

• Reentrant Phase Behavior: The production of RNAs in transcriptionally active condensates is proposed to promote reentrant phase behavior, where a system undergoes two consecutive phase transitions to return to a state resembling the initial one (cf. Figure 3a for a quasi-equilibrium representation).

• Mechanism:

  • Initial Phase Separation: Adding RNA (at equal or lower concentration than needed for charge neutralization) to a homogenous solution of RNA-binding peptides promotes phase separation through complex coacervation, driven by electrostatic interactions.

  • Redissolution: Subsequent addition of higher RNA concentrations can cause the condensate to redissolve.