understand the physics of protein and RNA phase separation
Biomolecular condensates (or membraneless organelles), comprised mostly of nucleic acids and proteins, usually form via the process of phase separation. Those macromolecular assemblies play extremely important roles in virtually all aspects of cellular biology: from minimizing cellular noises, regulating transcription, translation, chromatin structure and dynamics, to protein quality control, viral assembly and immune response. Importantly, dysregulation of condensate assembly or dissociation is often correlated with protein and RNA misfolding, and is indicative of the onset of various diseases, including neurodegeneration, viral infections, cardiac disease, and cancer. It is, therefore, imperative to characterize the underlying mechanisms governing the formation and dissolution of biomolecular condensates, as well as the structures and dynamics of their constituent RNAs and proteins.
We are developing & leveraging novel theoretical & computational frameworks to uncover the general principles regulating condensate formation, stability, structures, and properties. Such knowledge will open a new door for the engineering of new cellular functions, assist the development of novel biomaterials, as well as unlock a unique approach for advanced therapeutic interventions for many diseases. In particular:
Determine the structures and dynamics of constituent RNAs and proteins in condensates
Elucidate the relationship between the molecular structures, condensate composition, and its morphology & material properties
Understand the effects of environmental factors on the formation and growing mechanisms of RNA-protein condensates to develop therapeutic strategies for condensate-related diseases
publications
The Folding of Germ Granule mRNAs Controls Intermolecular Base Pairing in Germ Granules and Maintains Normal Fly Development
Siran Tian, Hung T. Nguyen, Ziqing Ye, Silvi Rouskin, D. Thirumalai, and Tatjana Trcek
Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration, creating an elevated potential for intermolecular base pairing. However, the type and abundance of intermolecular base pairing in mRNA clusters is poorly characterized. Using single-molecule super-resolution microscopy, chemical probing for base accessibility, phase separation assays, and simulations, we demonstrated that mRNAs remain well-folded upon localization to germ granules. While most base pairing is intramolecular, mRNAs still display the ability for intermolecular base pairing, facilitating clustering without high sequence complementarity or significant melting of secondary structure. This base pairing among mRNAs is driven by scattered and discontinuous stretches of bases appearing on the surface of folded RNAs, providing multivalency to clustering but exhibits low probability for sustained interactions. Notably, engineered germ granule mRNAs with exposed GC-rich complementary sequences (CSs) presented within stable stem loops induce sustained base pairing in vitro and enhanced intermolecular interactions in vivo. However, the presence of these stem loops alone disrupts fly development, and the addition of GC-rich CSs exacerbates this phenotype. Although germ granule mRNAs contain numerous GC-rich CSs capable of stable intermolecular base pairing, they are primarily embedded by RNA folding. This study emphasizes the role of RNA folding in controlling the type and abundance of intermolecular base pairing, thereby preserving the functional integrity of mRNAs within the germ granules.
Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences
Hiranmay Maity, Hung T. Nguyen, Naoto Hori, and D. Thirumalai
Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of the monomer for (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the free energy (GS) of the ground state, which is an SH, is used to predict the self-association kinetics. As the monovalent salt concentration, CS, increases, ΔGS and GS increase, which decreases the rates of dimer formation. In contrast, ΔGS for shuffled sequences, with the same length and sequence composition as (CAG)31, is larger, which suppresses their propensities to aggregate. Although demonstrated explicitly for (CAG) polymers, the finding of inverse correlation between the free energy gap and RNA aggregation is general.
Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation
Hiranmay Maity, Hung T. Nguyen, Naoto Hori, and D. Thirumalai
Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid–liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA–RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum—which depends on the exact sequence, n, and ionic conditions—is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.
Condensates in RNA repeat sequences are heterogeneously organized and exhibit reptation dynamics
Although it is known that RNA undergoes liquid–liquid phase separation, the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamic properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders. After establishing that the simulations reproduce key experimental findings, we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high-viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid–liquid phase separation in RNA, which is not easily discernible in current experiments.
