Global profiling identifies a stress-responsive tyrosine site on EDC3 regulating biomolecular condensate formation.

RNA granules are cytoplasmic condensates that organize biochemical and signaling complexes in response to cellular stress. Functional proteomic investigations under RNA-granule-inducing conditions are needed to identify protein sites involved in coupling stress response with ribonucleoprotein regulation. Here, we apply chemical proteomics using sulfonyl-triazole (SuTEx) probes to capture cellular responses to oxidative and nutrient stress. The stress-responsive tyrosine and lysine sites detected mapped to known proteins involved in processing body (PB) and stress granule (SG) pathways, including LSM14A, FUS, and Enhancer of mRNA-decapping protein 3 (EDC3). Notably, disruption of EDC3 tyrosine 475 (Y475) resulted in hypo-phosphorylation at S161 and S131 and altered protein-protein interactions (PPIs) with decapping complex components (DDX6, DCP1A/B) and 14-3-3 proteins. This resulting mutant form of EDC3 was capable of rescuing the PB-deficient phenotype of EDC3 knockout cells. Taken together, our findings identify Y475 as an arsenic-responsive site that regulates RNA granule formation by coupling EDC3 post-translational modification and PPI states.