The RNA binding protein PCBP2 is a regulator of miRNAs partition between cell and extracellular vesicles

In recent years, growing interest has focused on Extracellular Vesicles (EVs) as regulators of cell-to-cell communication: EVs are able to promote changes in gene expression and behavior of recipient cells in several pathophysiological processes by transferring their specific informational cargo of molecules. EVs cargo does not simply reflect the cell of origin content, but rather is defined by dynamic and selective cell-specific loading mechanisms. In particular, it is well recognized that EVs-mediated transfer of microRNAs contributes to intercellular communication, however, the knowledge about molecular mechanisms regulating selective and dynamic miRNA-loading in EVs is limited to a few specific RNA-binding proteins (RBPs) interacting with specific sequence determinants. Moreover, although several sequence motifs causing intracellular retention have been disclosed, the identification of interacting proteins remains unaddressed. Starting from this body of evidence, we focused on the investigation of molecular players responsible for miRNAs intracellular retention. Here, the RBP Poly-C-binding protein 2 (PCBP2, also known as hnRNPE2 or αCP2) was identified as a direct interactor of an intracellular retention (CELL) motif: RNA immunoprecipitation (RIP) after UV cross-linking, coupled to RNA pull-down followed by proteomic analysis, 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 highlights that multiple proteins/miRNA interactions govern miRNA compartmentalization and, specifically, extends PCBP2’s known pleiotropic functions to that of intracellular determinant of miRNAs retention, as a dominant inhibitor of SYNCRIP function.