DEAD-Box Helicase 3 Modulates the Non-Coding RNA Pool in Ribonucleoprotein Condensates During Stress Granule Formation

Stress granule formation is a type of liquid–liquid phase separation in the cytoplasm, leading to RNA–protein condensates that are associated with various cellular stress responses and implicated in numerous pathologies, including cancer, neurodegeneration, inflammation, and cellular senescence. One of the key components of mammalian stress granules is the DEAD-box RNA helicase DDX3, which unwinds RNA in an ATP-dependent manner. DDX3 is involved in multiple steps of RNA metabolism, facilitating gene transcription, splicing, and nuclear export and regulating cytoplasmic translation. In this study, we investigate the role of the RNA helicase DDX3's enzymatic activity in shaping the RNA content of ribonucleoprotein (RNP) condensates formed during arsenite-induced stress by inhibiting DDX3 activity with RK-33, a small molecule previously shown to be effective in cancer clinical studies. Using the human osteosarcoma U2OS cell line, we purified the RNP granule fraction and performed RNA sequencing to assess changes in the RNA pool. Our results reveal that RK-33 treatment alters the composition of non-coding RNAs within the RNP granule fraction. We observed a DDX3-dependent increase in circular RNA (circRNA) content and alterations in the granule-associated intronic RNAs, suggesting a novel role for DDX3 in regulating the cytoplasmic redistribution of non-coding RNAs. Overall design: U2OS cells were maintained in high-glucose Dulbecco's Modified Eagle Medium (ATCC, Manassas, VA, USA ) supplemented with 10% fetal bovine serum (Mediatech Inc., Manassas, VA, USA) and 1% penicillin/streptomycin (HyClone, GE Healthcare Life Sciences, Pittsburgh, PA, USA) at 37 °C in a humidified atmosphere with 5% CO2, as previously described [21]. Cells were pre-treated with 6 µM RK-33 (MedChemExpress Monmouth Junction, NJ, USA), stock solution 5mM DMSO (MilliporeSigma Burlington, MA, USA D8418) for 1 h. Sodium arsenite stress was induced by treating cells with sodium arsenite, 500 µM NaAsO2 (MilliporeSigma, Burlington, MA, USA S7400), for 60 min. Basal conditions were defined as treatment with 0.12% DMSO for 2 h, corresponding to the duration and timing of RK-33 addition in treated samples.