Study of the molecular machinery controlling miRNA sorting in exosomes: a role for PCBP2

INTRODUCTION AND AIM OF STUDY Exosomes are small, cell-secreted vesicles that carry specific repertoires of proteins and RNAs to recipient cells, and the selective transfer of proteins and RNAs in the exosomal cargo represents an important means of inter-cellular communication. While exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and exosomal RNA cargo selection is deregulated in pathological conditions, mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Our aim is to investigate the role of RNA-binding protein PCBP2 in the molecular machinery that determines the miRNAs loading into the exosome or the intracellular retention. MATERIAL AND METHODS Biotin miRNA pull-down experiment; RNA immunoprecipitation UV-cross linking; stable silencing of PCBP2; collection and characterization of exosomes on basis of their size and markers expression; analysis of PCBP2 silencing on the miRNAs exsport’ (qRT-PCR); co-immunoprecipitation. RESULTS Starting from the analysis of a small RNA sequencing (Santangelo et al., 2016), a common putative CYTO motif is identified by intracellular microRNAs’ sequence analysis. By mass spectrometry analysis, among the protein interactors able to specifically bind to hCYTO enriched miRNAs, we also identified PCBP2. PCBP2 is localized in the intracellular compartment. PCBP2 knock-down impairs sorting of miRNAs in exosomes determining a greater export of some miRNA. DISCUSSION Concerning microRNA partition, while current knowledge points to a key role for the exosome mediated transfer in cell-to cell communication, the processes controlling the selective compartmentalization between cell and EV, in physiology and disease, are still largely uncharacterized. Recent evidence highlighted the loading in exosomes of miRNAs in dependence of different RNA-binding proteins. This study has focused on the characterization of the role of PCBP2 as molecular retention factor capable of binding a CYTO motif and of blocking the sorting of miRNAs. These new insights into the mechanisms of miRNA exosomal sorting process open the way for the possible selective modification of the miRNAs’ retention motif.