The RNA-binding protein PCBP2 is a regulator of microRNAs partition between cell and extracellular vesicles.

While it is accepted that Extracellular Vesicles (EVs)-mediated transfer of microRNAs contributes to intercellular communication, the knowledge about molecular mechanisms controlling the selective and dynamic partition of miRNAs between intracellular and EV compartments is still largely limited. As yet, the interactions between specific RNA-binding proteins (RBPs) and short RNA sequences, have been proved causal for the loading of multiple miRNAs in EVs. Conversely, although in silico and mutagenesis analysis demonstrated the presence/function of specific sequence determinants causal to miRNAs intracellular retention, the interacting protein/s remained unknown. Here, the RBP Poly-C-binding protein 2 (PCBP2, also known as hnRNPE2 or aCP2), was identified as a direct interactor of the CELL-motif: RNA immunoprecipitation (RIP) after UV cross-linking, coupled to RNA pull down, demonstrated that this protein directly binds to miRNAs embedding this sequence and mutagenesis of the motif proved the specificity of its binding. Functionally, PCBP2 knock-down allows the EV-loading of specific intracellular microRNAs. Furthermore, a second requirement for PCBP2 specific binding was identified in SYNCRIP, a previously characterized miRNA EV-loader. SYNCRIP and PCBP2 may contemporarily bind to miRNAs endowed of both hEXO and CELL motifs, as demonstrated by RIP and EMSA assays. Mechanistically, SYNCRIP knock-down appears to limit PCBP2 recruitment. Overall, this body of evidence i) extends PCBP2 known pleiotropic functions to the role of intracellular determinant of miRNAs retention acting as a dominant inhibitor of SYNCRIP function and ii) highlights that multiple proteins/miRNA interactions govern miRNA compartmentalization. Overall design: To investigate PCBP2 functional role in miRNA partition bewteen cell and extrecellular vesicles, we established a 3A cell line in which PCBP2 has been knocked down by shRNA. Then we performed small RNA squencing of shPCBP2 and shCTR cells and their extracellular vesicles.