G3BP1 is an RNA-binding protein (RBP) that functions as the central scaffold for stress granule (SG) assembly. Under stress, G3BP1 phase separates with mRNA and recruits multiple RBPs to form dynamic, multi-component SGs. Abnormal persistence of SGs is linked to impaired disassembly, suggesting that SGs undergo age-dependent transitions in material properties. Here, we employ in vitro condensates composed of G3BP1 and RNA as SG mimics and quantitatively track their age-dependent dynamics. Using active and passive nanoscale rheology with optical tweezers, we demonstrate that metastable G3BP1 condensates continuously evolve from a terminally viscous to a terminally elastic state, accompanied by nanoscale cage formation. Mechanistically, we show that electrostatic interactions between two oppositely charged intrinsically disordered regions, IDR1 and IDR3, of G3BP1 dictate the timescale of condensate aging. Strikingly, RNA modulates these interactions in a length- and structure-dependent manner, thereby delaying dynamical arrest of G3BP1 condensates. In contrast, CAPRIN1 binding to the NTF2L dimerization domain only mildly accelerates aging, suggesting that RNA-protein interactions, rather than dimerization, dominate the aging dynamics. Overall, our work provides a high-resolution mechanical characterization of SG mimics during aging, revealing how two oppositely charged IDRs, RNA features, and protein partners govern G3BP1 condensate metastability, with direct implications for age-dependent dynamical arrest of SGs in cells in disease contexts.
Singh et al. (2026) studied this question.