RNA-driven phase transitions in biomolecular condensates.

RNAs and RNA-binding proteins can undergo spontaneous or active condensation into phase-separated liquid-like droplets. These condensates are cellular hubs for various physiological processes, and their dysregulation leads to diseases. Although RNAs are core components of many cellular condensates, the underlying molecular determinants for the formation, regulation, and function of ribonucleoprotein condensates have largely been studied from a protein-centric perspective. Here, we highlight recent developments in ribonucleoprotein condensate biology with a particular emphasis on RNA-driven phase transitions. We also present emerging future directions that might shed light on the role of RNA condensates in spatiotemporal regulation of cellular processes and inspire bioengineering of RNA-based therapeutics.

Dissecting the biophysics and biology of intrinsically disordered proteins.

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.

Direct computations of viscoelastic moduli of biomolecular condensates.

Biomolecular condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model that leverages information regarding intra- and inter-chain contacts, which we extract from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations of phase separation. The key ingredient of the generalized Rouse model is a graph Laplacian that we compute from equilibrium MMC simulations. We compute two flavors of graph Laplacians, one based on a single-chain graph that accounts only for intra-chain contacts, and the other referred to as a collective graph that accounts for inter-chain interactions. Calculations based on the single-chain graph systematically overestimate the storage and loss moduli, whereas calculations based on the collective graph reproduce the measured moduli with greater fidelity. However, in the long time, low-frequency domain, a mixture of the two graphs proves to be most accurate. In line with the theory of Rouse and contrary to recent assertions, we find that a continuous distribution of relaxation times exists in condensates. The single crossover frequency between dominantly elastic vs dominantly viscous behaviors does not imply a single relaxation time. Instead, it is influenced by the totality of the relaxation modes. Hence, our analysis affirms that viscoelastic fluid-like condensates are best described as generalized Maxwell fluids. Finally, we show that the complex shear moduli can be used to solve an inverse problem to obtain the relaxation time spectra that underlie the dynamics within condensates. This is of practical importance given advancements in passive and active microrheology measurements of condensate viscoelasticity.

Phase transition of RNA-protein complexes into ordered hollow condensates.

Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Temperature-dependent reentrant phase transition of RNA-polycation mixtures.

Liquid-liquid phase separation (LLPS) of multivalent biopolymers is a ubiquitous process in biological systems and is of importance in bio-mimetic soft matter design. The phase behavior of biomolecules, such as proteins and nucleic acids, is typically encoded by the primary chain sequence and regulated by solvent properties. One of the most important physical modulators of LLPS is temperature. Solutions of proteins and/or nucleic acids have been shown to undergo liquid-liquid phase separation either upon cooling (with an upper critical solution temperature, UCST) or upon heating (with a lower critical solution temperature, LCST). However, many theoretical frameworks suggest the possibility of more complex temperature-dependent phase behaviors, such as an hourglass or a closed-loop phase diagram with concurrent UCST and LCST transitions. Here, we report that RNA-polyamine mixtures undergo a reentrant phase separation with temperature. Specifically, at low temperatures, RNA-polyamine mixtures form a homogenous phase. Increasing the temperature leads to the formation of RNA-polyamine condensates. A further increase in temperature leads to the dissolution of condensates, rendering a reentrant homogenous phase. This dual-response phase separation of RNA is not unique to polyamines but also observed with short cationic peptides. The immiscibility gap is controlled by the charge of the polycation, salt concentration, and mixture composition. Based on the existing theories of complex coacervation, our results point to a complex interplay between desolvation entropy, ion-pairing, and electrostatic interactions in dictating the closed-loop phase behavior of RNA-polycation mixtures.

Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates.

Living cells organize their internal space into dynamic condensates through liquid-liquid phase separation of multivalent proteins in association with cellular nucleic acids. Here, we study how variations in nucleic acid (NA)-to-protein stoichiometry modulate the condensed phase organization and fluid dynamics in a model system of multicomponent heterotypic condensates. Employing a multiparametric approach comprised of video particle tracking microscopy and optical tweezer-induced droplet fusion, we show that the interfacial tension, but not viscosity, of protein-NA condensates is controlled by the NA/protein ratio across the two-phase regime. In parallel, we utilize fluorescence correlation spectroscopy to quantify protein and NA diffusion in the condensed phase. Fluorescence correlation spectroscopy measurements reveal that the diffusion of the component protein and NA within the condensate core is governed by the viscosity, and hence, also remains insensitive to the changes in NA-to-protein stoichiometry. Collectively, our results provide insights into the regulation of multicomponent heterotypic liquid condensates, reflecting how the bulk mixture composition affects their core versus surface organization and dynamical properties.

Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes.

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide-RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets.

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

Two-Dimensional Crowding Uncovers a Hidden Conformation of α-Synuclein.

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes.

Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding.

As for many intrinsically disordered proteins, order-disorder transitions in the N-terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm-N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single-molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm-N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway-specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm-N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm-N order-disorder transition enabled a complex interplay of phosphorylation and partner-binding steps to modulate its folding landscape.

Molecular mechanism of the chaperone function of mini-α-crystallin, a 19-residue peptide of human α-crystallin.

α-Crystallin is the archetypical chaperone of the small heat-shock protein family, all members of which contain the so-called "α-crystallin domain" (ACD). This domain and the N- and C-terminal extensions are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human αA-crystallin called mini-αA-crystallin (MAC) shows chaperone properties similar to those of the parent protein. Subsequent studies have confirmed the function of this peptide, but no studies have addressed the mechanistic basis for the chaperone function of MAC. Using human γD-crystallin (HGD), a key substrate protein for parent α-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding but also binds weakly and reversibly to HGD (Kd ≈ 200-700 µM) even when HGD is in the native state. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent α-crystallin. Using nuclear magnetic resonance spectroscopy, we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state is involved in facilitating its binding to both the domain interface and core regions during the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides that affect their chaperone activity and can potentially be manipulated for translational studies.

Shedding light on protein folding landscapes by single-molecule fluorescence.

Single-molecule (SM) fluorescence methods have been increasingly instrumental in our current understanding of a number of key aspects of protein folding and aggregation landscapes over the past decade. With the advantage of a model free approach and the power of probing multiple subpopulations and stochastic dynamics directly in a heterogeneous structural ensemble, SM methods have emerged as a principle technique for studying complex systems such as intrinsically disordered proteins (IDPs), globular proteins in the unfolded basin and during folding, and early steps of protein aggregation in amyloidogenesis. This review highlights the application of these methods in investigating the free energy landscapes, folding properties and dynamics of individual protein molecules and their complexes, with an emphasis on inherently flexible systems such as IDPs.

Cataract-associated mutant E107A of human gammaD-crystallin shows increased attraction to alpha-crystallin and enhanced light scattering.

Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ-γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD--namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid-liquid phase separation behavior of E107A-α-crystallin mixtures compared to HGD-α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A-α mixtures differ much more in protein density than those that occur in HGD-α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD-α mixtures, the demixing of phases occurs primarily by protein type while in E107A-α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins--i.e., heterologous γ-α crystallin interactions--primarily due to the change in surface electrostatic potential in the mutant protein.

Direct computations of viscoelastic moduli of biomolecular condensates.

In vitro facsimiles of biomolecular condensates are formed by different types of intrinsically disordered proteins including prion-like low complexity domains (PLCDs). PLCD condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model and information regarding intra- and inter-chain contacts that is extracted from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations. The key ingredient of the generalized Rouse model is the Zimm matrix that we compute from equilibrium MMC simulations. We compute two flavors of Zimm matrices, one referred to as the single-chain model that accounts only for intra-chain contacts, and the other referred to as a collective model, that accounts for inter-chain interactions. The single-chain model systematically overestimates the storage and loss moduli, whereas the collective model reproduces the measured moduli with greater fidelity. However, in the long time, low-frequency domain, a mixture of the two models proves to be most accurate. In line with the theory of Rouse, we find that a continuous distribution of relaxation times exists in condensates. The single crossover frequency between dominantly elastic versus dominantly viscous behaviors is influenced by the totality of the relaxation modes. Hence, our analysis suggests that viscoelastic fluid-like condensates are best described as generalized Maxwell fluids. Finally, we show that the complex shear moduli can be used to solve an inverse problem to obtain distributions of relaxation times that underlie the dynamics within condensates.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart directional motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced enhanced motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

Biomolecular Condensates Can Enhance Pathological RNA Clustering.

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

Determinants of viscoelasticity and flow activation energy in biomolecular condensates.

The form and function of biomolecular condensates are intimately linked to their material properties. Here, we integrate microrheology with molecular simulations to dissect the physical determinants of condensate fluid phase dynamics. By quantifying the timescales and energetics of network relaxation in a series of heterotypic viscoelastic condensates, we uncover distinctive roles of sticker motifs, binding energy, and chain length in dictating condensate dynamical properties. We find that the mechanical relaxation times of condensate-spanning networks are determined by both intermolecular interactions and chain length. We demonstrate, however, that the energy barrier for network reconfiguration, termed flow activation energy, is independent of chain length and only varies with the strengths of intermolecular interactions. Biomolecular diffusion in the dense phase depends on a complex interplay between viscoelasticity and flow activation energy. Our results illuminate distinctive roles of chain length and sequence-specific multivalent interactions underlying the complex material and transport properties of biomolecular condensates.

Heterotypic interactions can drive selective co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex.

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF (mSWI/SNF) complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.