Impact of G-tract RNAs and the DHX36 helicase on stress granule composition and formation

ABSTRACT Stress granules are RNA-protein condensates that form in response to an increase in untranslating mRNPs. 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 promote stress granule formation. Here, we provide three observations arguing that G-tracts capable of forming rG4s do not promote mRNAs partitioning into 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 DHX36 expression, which is thought to unwind G-quadruplexes, the partitioning of endogenous mRNAs was independent of their predicted rG4-forming potential. Third, while mRNAs in stress granules initially appeared to have a higher probability of forming rG4s than bulk mRNAs, this effect disappeared when rG4 motif abundance was standardized by mRNA length. However, we observe that in a G3BP1/2 double knockout cell line, 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. Key Points - G-tract RNAs with quadruplex forming potential in an mRNA do not affect its partitioning into stress granules - mRNA partitioning to stress granules is dependent on mRNA length rather than rG4-forming potential - DHX36, a DEAH-box helicase that unwinds RNA G-quadruplexes, limits stress granule formation Competing Interest Statement T. R. C. is a scientific advisor for Eikon Therapeutics, LincSwitch Therapeutics, Storm Therapeutics, and SomaLogic. R. P. is a co-founder and consultant for Illumen Therapeutics.