Dynamic proximal interaction networks of stress granules during assembly and disassembly reveal RNA regulation important for stress granule formation.

Stress granules (SGs) are biomolecular condensates that transiently assemble during cellular stress and assist in the cellular stress response. SG formation is thought to be driven by multivalent interactions between proteins and transcripts released from translation machineries. Efforts to elucidate the mechanisms underlying SG formation has revealed the importance of protein-protein, protein-RNA and RNA-RNA interactions, which involve protein and RNA scaffolds. However, we lack in understanding additional mechanisms required for SG formation. Here, we present quantitative analyses of multi-bait SG proximity labeling (i.e., BioID) experiments performed during stress time course paralleled with a genome-wide chemical genetic interaction screen. First, we implemented pulse biotin labeling of SG proteome during stress and recovery, augmented by data-independent acquisition (DIA) method. Experimentally, we used a group of bait proteins that effectively label over 50% of the known SG proteome and performed BioID labeling during two different stress conditions (oxidative and hyperosmotic) that induce SG via distinct mechanisms. Through SAINT analysis, we identify ~1,300 high-confident proximal interactors (preys) in steady state, stress, and recovery periods, and predict that about a third of these preys represent SG proteome in either condition. To measure quantitative changes in the SG protein interaction network during SG assembly and disassembly, we utilized DIA-NN analyses to obtain protein abundance and exploited ANOVA tests to determine statistically significant changes. About 20% of preys show significant quantitative changes during assembly and disassembly of SGs, representing a pool of dynamic associations and dissociations, while ~80% remain the same. Regardless of the type of stressor, we observe dynamic proximal interaction changes between SG baits and the components of the CCR4-NOT deadenylase, global regulator of polyA tail lengths. In parallel to the proteomics screening, we utilized a small molecule that can inhibit SG formation, called lipoamide, to interrogate mechanisms underlying SG formation. We identified that CCR4-NOT structural components, CNOT1, CNOT2 and CNOT3, are required for lipoamide inhibitory action on SGs. Based on the function of CCR4-NOT complex, we propose that lengthening of poly(A) tails by loss of CCR4-NOT complex lowers the threshold for phase separation through increasing valency and overcomes the inhibitory effect of lipoamide. Together, we provide dynamic map of SG proximal interaction networks during SG formation and dissolution and propose that regulation of polyA lengths of mRNAs by CCR4-NOT complex is important for SG formation.