THE ROLE OF ARGININE METHYLATION IN MIRNA BIOGENESIS INVESTIGATED BY MS-PROTEOMICS

MicroRNAs (miRNAs) are short, non-coding RNA molecules that fine tune gene expression at the post-transcriptional level. The efficient miRNA processing is fundamental to maintain their correct levels in the cell. However, although microRNA biogenesis is a tightly controlled pathway, alterations in this process occur in different pathological conditions, including cancer. Hence, dissecting the molecular mechanisms underlying miRNA biogenesis is crucial to understand how aberrant miRNA patterns are generated. The processing step operated by the Large Drosha Complex (LDC), which is crucial and rate limiting for the production of mature miRNAs, occurs in the nucleus and it consists in the cleavage of the primary miRNAs (pri-miRNAs) into the precursor miRNAs (pre-miRNAs). In our previous MS-based analysis of the cellular methylome, we showed that protein methylation frequently occurs on the majority of the LDC subunits [1], suggesting for the first time the possibility that this post translational modification (PTM) could contribute to the regulation of miRNA biogenesis. To investigate the role of protein methylation in the regulation of the LDC, we thoroughly characterized the methylated sites on the subunits of the complex, thus generating the first comprehensive and high-confident methyl-proteome of the LDC. By modulating the expression of PRMT1, the predominant Protein Arginine Methyltransferase in mammalian cells, we analyzed how methylations change at specific sites of distinct LDC subunits, thus identifying novel PRMT1 substrates. The alteration of the LDC-methylation state upon PRMT1 depletion correlated with an impairment of the pri- to- pre-miRNA processing step, which resulted in the massive deregulation of mature miRNA expression. Although the detailed molecular mechanism remains to be fully deciphered, the results described in this thesis uncover a key role of arginine methylation in the regulation of the LDC activity and, consequently, in miRNA biogenesis.