Insight
is our reward
Search

Publications in RNA-binding Protein FUS by NOMIS researchers

Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales

NOMIS Researcher(s)

Published in

July 20, 2023

Non-membrane-bound biomolecular condensates have been proposed to represent an important mode of subcellular organization in diverse biological settings. However, the fundamental principles governing the spatial organization and dynamics of condensates at the atomistic level remain unclear. The Saccharomyces cerevisiae Lge1 protein is required for histone H2B ubiquitination and its N-terminal intrinsically disordered fragment (Lge11-80) undergoes robust phase separation. This study connects single-and multi-chain all-atom molecular dynamics simulations of Lge11-80 with the in vitro behavior of Lge11-80 condensates. Analysis of modeled protein-protein interactions elucidates the key determinants of Lge11-80 condensate formation and links configurational entropy, valency, and compactness of proteins inside the condensates. A newly derived analytical formalism, related to colloid fractal cluster formation, describes condensate architecture across length scales as a function of protein valency and compactness. In particular, the formalism provides an atomisti-cally resolved model of Lge11-80 condensates on the scale of hundreds of nanometers starting from individual protein conformers captured in simulations. The simulation-derived fractal dimensions of condensates of Lge11-80 and its mutants agree with their in vitro morphologies. The presented framework enables a multiscale description of biomolecular condensates and embeds their study in a wider context of colloid self-organization. © Polyansky, Gallego et al.

Research field(s)
Natural Sciences

DOI
10.7554/eLife.80038

Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

NOMIS Researcher(s)

Published in

July 12, 2022

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Research field(s)
Natural Sciences, Physics & Astronomy, Chemical Physics

DOI
10.1073/pnas.2202222119

Co-condensation of proteins with single- and double-stranded DNA

NOMIS Researcher(s)

Published in

March 8, 2022

Biomolecular condensates provide distinct compartments that can localize and organize biochemistry inside cells. Recent evidence suggests that condensate formation is prevalent in the cell nucleus. To understand how different components of the nucleus interact during condensate formation is an important challenge. In particular, the physics of co-condensation of proteins together with nucleic acids remains elusive. Here we use optical tweezers to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) forms liquid-like assemblies in vitro, by co-condensing together with individual DNA molecules. Through progressive forceinduced peeling of dsDNA, buffer exchange, and force measurements, we show that FUS adsorbing in a single layer on DNA effectively generates a sticky FUS-DNA polymer that can collapse to form a liquid-like FUS-DNA co-condensate. Condensation occurs at constant DNA tension for double-stranded DNA, which is a signature of phase separation. We suggest that co-condensation mediated by protein monolayer adsorption on nucleic acids is an important mechanism for intracellular compartmentalization.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1073/pnas.2107871119

Sequence-dependent surface condensation of a pioneer transcription factor on DNA

NOMIS Researcher(s)

Published in

March 1, 2022

Biomolecular condensates are dense assemblies of proteins that form distinct biochemical compartments without being surrounded by a membrane. Some, such as P granules and stress granules, behave as droplets and contain many millions of molecules. Others, such as transcriptional condensates that form on the surface of DNA, are small and contain thousands of molecules. The physics behind the formation of small condensates on DNA surfaces is still under discussion. Here we investigate the nature of transcription factor condensates using the pioneer transcription factor Krüppel-like factor 4 (Klf4). We show that Klf4 can phase separate on its own at high concentrations, but at low concentrations, Klf4 only forms condensates on DNA. Using optical tweezers, we demonstrate that these Klf4 condensates form on DNA as a type of surface condensation. This surface condensation involves a switch-like transition from a thin adsorbed layer to a thick condensed layer, which shows hallmarks of a prewetting transition. The localization of condensates on DNA correlates with sequence, suggesting that the condensate formation of Klf4 on DNA is a sequence-dependent form of surface condensation. Prewetting together with sequence specificity can explain the size and position control of surface condensates. We speculate that a prewetting transition of pioneer transcription factors on DNA underlies the formation and positioning of transcriptional condensates and provides robustness to transcriptional regulation.

Research field(s)
Natural Sciences, Physics & Astronomy, Fluids & Plasmas

DOI
10.1038/s41567-021-01462-2

Glycine-Rich Peptides from FUS Have an Intrinsic Ability to Self-Assemble into Fibers and Networked Fibrils

NOMIS Researcher(s)

Published in

November 2, 2021

Glycine-rich regions feature prominently in intrinsically disordered regions (IDRs) of proteins that drive phase separation and the regulated formation of membraneless biomolecular condensates. Interestingly, the Gly-rich IDRs seldom feature poly-Gly tracts. The protein fused in sarcoma (FUS) is an exception. This protein includes two 10-residue poly-Gly tracts within the prion-like domain (PLD) and at the interface between the PLD and the RNA binding domain. Poly-Gly tracts are known to be highly insoluble, being potent drivers of self-assembly into solid-like fibrils. Given that the internal concentrations of FUS and FUS-like molecules cross the high micromolar and even millimolar range within condensates, we reasoned that the intrinsic insolubility of poly-Gly tracts might be germane to emergent fluid-to-solid transitions within condensates. To assess this possibility, we characterized the concentration-dependent self-assembly for three non-overlapping 25-residue Gly-rich peptides derived from FUS. Two of the three peptides feature 10-residue poly-Gly tracts. These peptides form either long fibrils based on twisted ribbon-like structures or self-supporting gels based on physical cross-links of fibrils. Conversely, the peptide with similar Gly contents but lacking a poly-Gly tract does not form fibrils or gels. Instead, it remains soluble across a wide range of concentrations. Our findings highlight the ability of poly-Gly tracts within IDRs that drive phase separation to undergo self-assembly. We propose that these tracts are likely to contribute to nucleation of fibrillar solids within dense condensates formed by FUS.

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1021/acs.biochem.1c00501

Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing

NOMIS Researcher(s)

Published in

March 1, 2021

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid−liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/s41580-020-00326-6

  • NOMIS Foundation 2024
  • 2024 NOMIS Foundation