Proteomic Tracking Extracellular Vesicle RNA Interactors in Recipient Immune Cells through Orthogonal Labelings.

RNA cargos in extracellular vesicles (EVs) mediate intercellular communication by engaging RNA-binding proteins (RBPs) in recipient cells. Here, we introduce extracellular RNA interactome capture through orthogonal labelings (ERICOL), a targeted cross-linking and quantitative chemical proteomic strategy based on metabolic labeling of RNAs and stable isotope labeling of proteins for systematic profiling of EV RBPs in recipient cells. Time-resolved analysis of tumor-derived EVs in Jurkat T cells revealed dynamic patterns of EV RNA uptake and RBP engagement. Further profiling in primary human CD8+ T cells treated with wild-type or IDH1 mutant intrahepatic cholangiocarcinoma (ICC)-derived EVs uncovered IDH1 mutation-driven alterations in the EV RBP landscape. ERICOL offers a powerful strategy for comprehensive profiling of EV RNA interactome dynamics and provides mechanistic insights into EV-mediated immune modulation.