MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.
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