CD47 interactions with exportin-1 limit the targeting of m⁷G-modified RNAs to extracellular vesicles

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47⁺ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5'-7-methylguanosine (m⁷G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys⁵²⁸ by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m⁷G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m⁷G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m⁷G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m⁷G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.