Interplay between the RBP SYNCRIP and RNA methylation in determining EV miRNA-cargo and functional role in cell-to-cell communication

The RNA-binding protein SYNCRIP is a central mediator of the loading into Extracellular Vesicles (EVs) of specific miRNAs that bear the hEXO consensus core sequence (DDDVWS; D=not C, V=not U, W=A/U, S=C/G). This work identifies a previously uncharacterized dependence of SYNCRIP miRNA binding activity on m6A modification. This has been uncovered in an in vitro model of hepatocellular carcinoma (HCC). Our previous findings reported that SYNCRIP and METTL3 control the composition of the EV cargo. Specifically, METTL3 methylates target miRNAs thus allowing the binding of a second RNA-binding protein (RBP) working as miRNA EV-loader, A2B1. The present work focuses on: i) the impact of SYNCRIP in the specific miRNA loading into HCC-derived small EVs (sEVs); ii) the impact of SYNCRIP on the sEVs ability to induce EMT in non-tumorigenic liver cells; iii) the impact of m6A epitranscriptomic modification on miRNA loading into HCC-derived sEVs and in turn the ability to trigger EMT on recipient cells; iv) the interplay between SYNCRIP and m6A modification in determining SYNCRIP binding to microRNAs. Specifically, we demonstrate that METTL3-dependent methylation allows the recognition of a subset of miRNAs by SYNCRIP, thus promoting their selective export into sEVs. Beyond unraveling this molecular interplay, we also investigated its biological impact on tumor cell ability to influence epithelial target cells. Functionally, sEVs released by HCC cells silenced for SYNCRIP showed an impaired ability to induce EMT in healthy hepatocytes with respect to control HCC-derived sEVs; this reveals the direct EV involvement in the tumor microenvironment and in the establishment of pro-tumorigenic niches. More interestingly, sEVs released by METTL3-silenced HCC cells are unable to induce the expression of mesenchymal markers in target cells, while the epithelial ones remain mostly unaffected. This indicates that epitranscriptomic modifications profoundly impact miRNA EV-loading and, in turn, EV function. The translational relevance of this study resides in the identification of the molecular events and players to be selectively targeted for therapeutic purposes to interfere with EV-mediated tumor progression and metastatization.