BPS2026 – Molecular details of RNA in biomolecular condensates

Fused in sarcoma (FUS) is an intrinsically disordered RNA-binding protein that assembles with RNA and other components to form ribonucleoprotein (RNP) granules in cells. In a test tube, FUS undergoes phase separation to form condensates that recapitulate the unusually dense interiors of membraneless organelles (MLOs). Although work from our group and others has significantly advanced understanding of how protein-protein interactions stabilize condensates, the principles that govern protein-RNA contacts in condensates remain poorly defined. Using NMR spectroscopy, molecular dynamic simulations, and biochemical assays, we show that RNA incorporation significantly remodels condensates formed by FUS disordered domains, lowering density and increasing local and diffusional motion of protein molecules. Whereas charge-charge interactions dominate FUS-RNA binding in dilute solution, within condensates additional contacts including π-π stacking and hydrogen bonding also contribute to binding. Furthermore, in the condensed phase environment we use NMR to observe a striking shift in the structural ensemble of a model G-quadruplex RNA, where a sizable fraction of this otherwise extremely stable structure becomes unfolded. Hence, these structures may be remodeled within condensates. Together, these findings update our view of disordered protein-RNA interaction, protein-RNA co-phase separation, and provide new insights into how RNP granules may function in cells.