RNA structure directs RNA partitioning and is actively disrupted inside stress granules to enable cellular recovery

RNA structures play important roles in liquid-liquid phase separation. However, how it is regulated during stress response and stress granule formation is still under studied. Here, we performed in vivo RNA structure probing before and after sodium arsenite treatment, and in stress granules. While RNAs generally become more double-stranded upon stress, they maintain their single-strandedness inside stress granules. We showed that RNA single-strandedness enables increased inclusion inside stress granules and that stress granule-enriched RNAs form fewer intra- and intermolecular RNA-RNA interactions. Additionally, several RNA binding proteins including SRSF1 are enriched in differential structure regions. eCLIP analysis revealed that SRSF1 binds to single-stranded regions along RNAs, and increased SRSF1 binding enabled better inclusion of RNAs in stress granules, whereas depletion of SRSF1 decreased stress granule formation under low amounts of stress. We also observed the active unwinding of RNAs inside stress granules by helicases, including DDX3X, and showed that inhibition of DDX3X results in slower dissolution of stress granules during recovery. Our study reveals the existence of multiple mechanisms to maintain RNA single-strandedness inside stress granules and to allow reversibility of stress granule formation, highlighting the importance of regulating RNA structure to enable cellular plasticity and response. Overall design: To study dynamic changes in RNA structures and RNA-RNA interactions during arsenite stress, we performed in vivo click Selective 2-Hydroxyl Acylation and Profiling Experiment (icSHAPE) and proximity ligation sequencing (SPLASH). Specifically, we stressed HeLa cells with sodium arsenite (NaAsO2) for an hour and then allowed the cells to recover for 1 or 2 hours by removing sodium arsenite from the media. At each of these time points (untreated, 1-hour arsenite stress, 1-hour recovery, and 2-hour recovery), we treated the cells with the SHAPE compound, NAI-N3, to map local secondary structures. As both intra and inter-molecular RNA-RNA interactions are possibly involved in the process of biomolecular condensate formation, we performed SPLASH to determine the intra- and inter molecular RNA-RNA interactions in untreated and stressed cells. Additionally, we also performed ribosome profiling (Ribo-seq) to study the translation efficiency of the cellular transcripts during stress and recovery.