Unraveling the mechanisms behind the packaging of RNA into exosomes

Exosomes are small extracellular vesicles of endosomal origin of about 100nm of diameter. They are used by the cells to transfer information and can contain a wide range of biomolecules. Among many others, exosomes carry coding and non-coding RNAs. The aim of this study is to unravel the
mechanisms behind the exosomal encapsulation of these biomolecules. For that purpose, we have approached this question from three different points of view. First, we studied the differences between cellular and exosomal RNA in order to determine if the class, sequence and function of the RNA molecules determine their encapsulation. Then, we assessed if two RNA binding proteins (Ago2 and GW182), often localized in the site of exosome formation, regulate the loading and export of microRNAs. Finally, we investigated how the chemotherapeutic drug Epirubicin initiates a response in the cell that leads to the exosomal over export of miR-503. In the first part of this study, we have characterized the RNA content of cellular and exosomal RNA on human endothelial cells in depth. Unlike many other profiling studies already published, we have used healthy primary cells to show the basal composition of RNA sub-classes and to find the motifs significantly more present in cells or in exosomes basally. Regarding coding genes, we have shown that mRNA genes can be classified into four different groups depending on the relative export of their regions. Moreover, we proved that RNA duos derived from the same locus and microRNA-mRNA target pairs are found at almost the same ratio in both cells and exosomes. Finally, we have also proven that, even though microRNAs are by far the most studied class of RNAs in exosomes, they represent a very small amount of the total RNA content of these vesicles thus setting the basis to promote a switch in exosomal RNA research towards longer RNAs. In the second part of this thesis, we have been able to conclude that neither AGO2 nor GW182 are involved in general processes of microRNA export based on motif recognition. Finally, in the third and last part, we have identified a new mechanism regulating microRNA export. Under basal conditions, both ANXA2 and hnRNPA2B1 bind to miR-503 in a stable partnership. Epirubicin mediates the export of miR-503 by inducing the separation of hnRNPA2B1 from the complex. In a parallel mechanism, the chemotherapeutic drug induces a reduction in the expression of hnRNPA2B1 that acts as a backup of the main complex-destabilization leading to miR-503 exosomal export.