Proteomic tracking extracellular vesicle RNA interactors through orthogonal labelings in recipient immune cells

Extracellular vesicles (EVs) mediate cell-cell communication by transferring RNAs that modulates recipient cell function. However, the RNA-protein interactome in EV-recipient cell communication remains poorly understood due to technical limitations. We developed a novel EV RNA-protein interaction profiling by targeted crosslinking and SILAC (EV-RIP-Tag) strategy to systematically capture EV RNA-protein interactions in recipient cells. This approach enabled the first large-scale profiling of EV RNA-binding proteins (RBPs) in recipient cells. Our dynamic study of tumor-derived EVs in Jurkat T cells provided new insights into the temporal process of EV RNA uptake. Additionally, profiling EV RBPs from wild-type and IDH1-mutant ICC cell lines revealed significant changes in the RBP landscape within CD8+ T cells. These alterations were reversed upon treatment with the IDH1 inhibitor AG120, highlighting a potential mechanism for IDH1 mutation-mediated immune suppression. Our findings offer novel insights into EV-mediated immune modulation and present a powerful tool for studying the molecular mechanisms of cancer-associated immunosuppression.