RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation

Abstract

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

Ligand Effects on Phase Separation of Multivalent Macromolecules

“…Quantifying the effects of different types of ligands on the change to cA: Experimental characterizations of full binodals are challenging, and accordingly, these measurements have been performed only for a small number of scaffold molecules (37)(38)(39)(40). In contrast, it is easier to measure changes in saturation concentrations in the absence (29) and the presence of ligands (21,33). To set up expectations regarding how saturation concentrations change, we analyze how the ratio changes as a function of divalent ligand concentration (Fig.…”

“…Although scaffolds are the drivers of condensate formation, the solution conditions and concentration regimes in which formation and dissolution of condensates occurs can be regulated by controlling the concentrations of non-scaffold molecules (17,18) or via post-translational modifications to scaffolds (16,19). An example of regulation by non-scaffold molecules comes from recent work on stress granules showing the different effects of Caprin-1 and USP-10 on the stability and integrity of stress granules (12,20,21). This example highlights the need for additional focus on and understanding of regulation by non-scaffold molecules, as dysregulation of condensate formation and dissolution can lead to disease (8,9).…”

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

“…A recent study on the network of protein-protein contacts formed by SARS-CoV-2 structural proteins shows that N interacts with several human 5 ribonucleoproteins known to be involved in the formation of phase-separated protein-RNA granules 28 . Several of these proteins such as G3BP1/2 are composed of disordered regions with sequence characteristics that are known to contribute to stress granule formation via LLPS 29,30 . Stress granules (SGs) are membraneless organelles that store translationally silent mRNA when the cell is exposed to stress to regulate mRNA metabolism 31 .…”

Phase Separation Can Increase Enzyme Activity by Concentration and Molecular Organization

“…Many condensates appear to form through liquid-liquid phase-separation (LLPS), in which oligomerization mediated by multivalent interactions lowers the solubility of proteins and/or nucleic acids sufficiently to form a second phase 1,2,13 . Where measured, the degree of concentration in this phase ranges from 2-to 150-fold for different constituents [14][15][16][17] . Scaffold-like molecules, those that contribute more strongly to formation of the condensate, are typically the most highly concentrated components 14,18 .…”