Figure 7.

The Valence of Arginine Residues within the RG-Rich Region Determines the Efficiency of G3BP Phase Separation

(A) Phase separated fraction of 5 μM G3BP1 or G3BP2 as a function of PEG-20K concentration without RNA (mean, SD, fit, n = 16 FOVs).

(B) Phase-separated fraction of G3BP1 and G3BP2 as a function of G3BP concentration with poly(A) RNA and 1% PEG-20K (mean, SD, fit, n = 16 FOVs).

(C) R_H of G3BP2 at pH 7 and 85 mM KCl (solid line) or after increasing the KCl to 400 mM (dashed line), as determined by DLS (mean average of 6 measurements).

(D) DLS measurement of the oligomeric species of G3BP2 at pH 7 and 85 mM KCl or after increasing the KCl to 400 mM (mean average of 6 measurements).

(E) Model depicting an RNA-mediated conformational transition of G3BP into a phase-separation competent state (top panel). Under physiological conditions, G3BP adopts a compact state (left) that is stabilized by intramolecular interactions between the RG-rich region and the acidic region. The compact state inhibits G3BP phase separation. Upon stress, polysomes disassemble, and mRNAs are released in an unfolded protein-free state. Binding of unfolded mRNA to G3BP outcompetes the intramolecular interactions between the RG-rich and the acidic regions. RNA-bound G3BP adopts an expanded conformation in which the RG-rich region becomes exposed to engage in protein-protein and protein-RNA interactions. These new interactions among RG-rich regions stabilize clusters of G3BP1 bound to RNA (bottom panel). RNA-mediated G3BP clusters allow physical crosslinking of RNA molecules to form inhomogeneous protein-RNA condensates.

See also Figure S7.