O'Connell et al.

Cytosolic mRNA regulation during cellular stress occurs through the formation of membrane-less organelles like P-bodies (PB) and stress granules (SG), yet their compositional and functional dynamics throughout stress are poorly understood. We combine APEX-sequencing and high-content imaging to analyse the human transcriptomes associated with PBs and SGs during oxidative stress and recovery. Our findings reveal P-bodies undergo extensive compositional remodelling during stress, and these changes are retained during recovery. In contrast, SG-associated transcripts closely resemble the cytosol, highlighting distinct differences between the organelles. PB-associated mRNAs during stress show increased AU-rich elements and oncogenic content relative to the cytosol. We find similar SG-PB dynamics extending our analysis to chemotherapy stress. These results uncover critical differences between PB and SG, suggesting a unique role for P-bodies in stress adaptation. Our study provides a valuable resource of cytosolic membrane-less organelle-associated transcripts.