Abstract TP308: FUS Contributes to Neurofunctional circRNAs Packaging Into Small Extracellular Vesicles Under Hypoxia Condition

Abstract

Small extracellular vesicles (sEVs) contain abundant circular RNAs (circRNAs), contributing to a wide range of cellular processes. However, the precise process dictating the circRNAs packaging into sEVs remains unclear. This study revealed an innovative loading mechanism for particular circRNAs into sEVs during hypoxia, facilitated by the Fused in Sarcoma (FUS) protein and stress granules (SGs). We noted that the hypoxic sEVs has a higher abundance of FUS than that in normal sEVs. And the living cell images and immunofluorescence results indicate that, before loading into sEVs, FUS translocated from the nucleus and forms aggregate spots with marker protein CD63 in cytoplasmic SGs under hypoxia condition. Concurrently, by using the FISH staining and 5-EU click chemistry, we observed that FUS facilitates the specific cytoplasmic circRNAs recruit into SGs during hypoxia. Upon relief of hypoxic conditions, with the degradation of stress granules, the cytoplasmic FUS will gradually transport into sEVs with SGs-recruited circRNAs. Knocking out FUS dramatically disrupts the SGs recruitment of circRNAs to stress granules and significantly diminishes circRNA abundance in sEVs, leading to the accumulation of circRNAs in cytoplasm. Remarkably, by employing a newly transcribed RNA sequencing (RICK-seq), we found that the FUS medicated sEVs transport mechanism may be applicable to a class of neurofunctional circRNAs. Additionally, based on the observation of molecular docking and truncated peptides, we demonstrated that the zf_RanBP domain is essential for FUS interacting with specific circRNAs, which further highlights the critical role of FUS in the sEVs transport process of circRNAs during hypoxia stress. In general, our finding uncovered a FUS-medicated novel transport mechanism of specific circRNAs into sEVs during hypoxia, and indicate the potential therapeutic avenues for regulating the cargo of sEVs in hypoxia-related pathologies.