Intracellular fluid-like ribonucleoprotein granules (RNP granules) are primarily formed by liquid-liquid phase separation of low-complexity intrinsically disordered protein domains (LCDDs). The arginine-rich (R-rich) LCCDs are ubiquitous in eukaryotic RNA binding proteome, act as multi-valent RNA/protein binding modules for homotypic and heterotypic phase transition, and implicated in c9orf72-related amyotrophic lateral sclerosis (ALS) disease etiology. What is the mechanism by which the phase transition of R-rich LCDDs are regulated? Recently, we showed that RNA can modulate LCDD phase separation behavior by controlling both droplet assembly and dissolution. In presence of RNA, R-rich LCDDs undergo associative liquid condensation into protein/RNA-rich droplets at low RNA-to-LCDD ratios, but form soluble protein-RNA complexes at high RNA-to-LCDD ratios. Using designed and naturally occurring R-rich LCDD sequences as well as the ALS-associated protein Fused in Sarcoma (FUS), here we investigate the molecular driving forces that control this non-monotonous phase transition. Combining quantitative biophysical experiments and polymer physics-based theories, we show that RNA controls (a) mesoscale droplet dynamics, (b) droplet viscoelasticity, and (c) the condensate microenvironment via charge regulated electrostatics and short-range cation-p interactions. Importantly, if the homotypic RNA-RNA interactions are substantial, further complexities are observed in the phase behavior of the system, including the formation of distinct condensed phases (droplets) displaying orthogonal physical and functional properties. Together, our experiment and free-energy surface modeling suggest a remarkable degree of plasticity in the RNP granule phase behavior, manifested by the ability of RNA to control homotypic and heterotypic interactions across different length scales.

RNA controls a switch-like liquid phase transition of low-complexity protein domains

2019 Fall National Meeting

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