Profiling dynamic RNA-protein interactions using small molecule-induced RNA editing

RNA binding proteins (RBPs) play an important role in biology and characterizing dynamic RNA-protein interactions in their native context is essential for understanding RBP function. Here, we develop targets of RNA-binding proteins identified by editing induced through dimerization (TRIBE-ID), a facile strategy for identifying and quantifying state-specific RNA-protein interactions based upon rapamycin-mediated chemically induced dimerization and RNA editing. We perform TRIBE-ID with G3BP1, an abundant RBP and core component of stress granules, to study transcriptome-wide G3BP1-RNA interactions during normal conditions and upon oxidative stress-induced liquid-liquid phase separation (LLPS). We quantify editing kinetics in order to infer interaction persistence and show that stress granule formation strengthens preexisting G3BP1-RNA interactions and induces new RNA-protein binding events. Further, we demonstrate that G3BP1 stabilizes its RNA clients in a dose-dependent manner, suggesting that stress granules function as RNA storage depots. Finally, we apply our method to characterize small molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile RNA-protein binding events with temporal control and illuminates the role of LLPS in organizing G3BP1-RNA interactions in the cell.