DHX36 modulates stress granule assembly independent of recruitment of mRNAs with G-quadruplex sequence motifs

Stress granules are RNA-protein condensates that form in response to an increase in untranslating mRNPs (messenger ribonucleoproteins). Stress granules form by the condensation of mRNPs through a combination of protein-protein, protein-RNA, and RNA-RNA interactions. Several reports have suggested that G-rich RNA sequences capable of forming G-quadruplexes (rG4s) promote stress granule formation. Here, we provide three observations arguing that G-tracts do not promote messenger RNA (mRNA) accumulation in stress granules in human osteosarcoma cells. First, we observed no difference in the accumulation in stress granules of reporter mRNAs with and without G-tracts in their 3' UTRs. Second, in U-2 OS cell lines with reduced expression of DHX36, which is thought to unwind G-quadruplexes, the accumulation of endogenous mRNAs was independent of their predicted rG4-forming potential. Third, while mRNAs in stress granules initially appeared to have more rG4 motifs than bulk mRNAs, this effect disappeared when rG4 motif abundance was normalized to mRNA length. However, we observed that in a G3BP1/2 double knockout cell line, which strongly inhibits stress granule formation, reducing DHX36 expression rescued stress granule-like foci formation. This indicates that DHX36 can limit stress granule formation, potentially by unwinding trans-rG4s or limiting other intermolecular RNA-RNA interactions that promote stress granule formation.