THE ROLE OF PROTEIN ARGININE METHYLATION IN RBP-RNA INTERACTION MODULATION AND ITS IMPLICATIONS IN CANCER STRESS RESPONSE INVESTIGATED BY MS-PROTEOMICS

Various post-translational modifications (PTMs) have been described to regulate RNA-binding protein (RBP) activity, subcellular localization, and interactions with other proteins or RNAs. Proteome-wide experiments recently carried out in our group have shown that RBPs are the most abundant arginine (R)-methylated proteins. Protein Arginine Methyltransferases (PRMTs) are the enzymes responsible for the deposition of methylation on arginine. Recent evidence has indicated that R-hypomethylation could influence RBP phase-separation and consequent formation of Membrane-Less Organelles (MLOs). In my Ph.D. project, we implemented a quantitative proteomic approach to profile global changes of RBP-RNA interactions upon the modulation of R-methylation, both directly by the use of PRMT inhibitor and indirectly through cisplatin (CDDP)-induced PRMT1 re-localization on chromatin. In particular, by coupling the Orthogonal Organic Phase Separation (OOPS) strategy with mass spectrometry (MS) analysis, we profiled RNA-protein interaction in dependence on R-methylation remodeling. Biochemical and immunofluorescence analysis validated the differential association of a set of RBPs with RNA upon PRMT1 inhibition but also that altered modification is linked to MLOs formation. We then applied our strategy in ovarian cancer in the context of CDDP-induced protein R-methylation rewiring to understand if it may affect RBP-RNA interaction. Preliminary analysis of the significantly regulated RBPs from a new OOPS-MS experiment carried out in these conditions revealed a strong modulation of RBP-RNA interaction and suggested new interesting targets. We are currently analyzing the R-methylation state of the most promising RBPs and in parallel the RNAs which interact differentially with the RBPs in the same conditions.