Dynamic Proximal Interactomics and Chemical Genetic Screening Reveal CCR4-NOT Sequestration in Stress Granules as a Mechanism for Transcript Stabilization

Living cells adapt to stress by protecting essential resources, such as stabilizing transcripts. Stress granules, which form during stress through the coalescence of polyadenylated transcripts released from polysomes and RNA-binding proteins, are implicated in post-transcriptional regulation, although their precise roles remain contested. To address this, we generated dynamic proteomic landscapes of stress granules during assembly and disassembly under oxidative and hyperosmotic stress using multi-bait BioID profiling combined with quantitative mass spectrometry. Our analysis reveals dynamic rewiring of stress granule proximal interaction networks, identifying RNA-binding proteins involved in translation repression and mRNA decay, including the CCR4-NOT deadenylase complex, as universal molecular signatures of condensed stress granules. Using the CCR4-NOT deadenylase complex as a model, we demonstrate the function of dynamic components associated with stress granules. Complementary genome-wide chemical genetic screening identified CCR4-NOT components as regulators of stress granule formation. Reduced CCR4-NOT activity, which lengthens poly(A) tails, enhanced stress granule assembly, while shortened poly(A) tails inhibited it. Furthermore, stress-induced sequestration of the CCR4-NOT complex to stress granules coincided with known global poly(A) tail lengthening. These findings suggest that stress granule condensation promotes transcriptome stabilization by regulating CCR4-NOT localization as part of an adaptive stress response. This work underscores the power of integrating quantitative proteomics with chemical genetics to advance our understanding of biomolecular condensates in diverse biological processes.