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PRMT1 regulates microRNA biogenesis through the methylation of the Large Drosha Complex

ABSTRACT: MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). We previously reported that the LDC is highly methylated. To investigate the extent of this post-translational modification, we extensively characterized the LDC methyl-proteome by combining heavy methyl SILAC metabolic labelling of cells with the affinity-enrichment of the complex, followed by high resolution mass spectrometry (MS) analysis. We found that arginine (R) methylation is the predominant modification, occurring on multiple sites of 13 of the 23 subunits of the complex. Interestingly, the depletion- or pharmacological inhibition- of PRMT1, the major protein R-methyltransferase in mammals, alters the LDC methylation state and this is linked to a global decrease of miRNA expression, due to the specific impairment of the pri-to-pre-miRNA processing step. Moreover, PRMT1 activity appears to be required for the binding of some LDC subunits to pri-miRNAs. Overall, our study uncovers a previously uncharacterized role of R-methylation in the regulation of the LDC activity, thus effecting miRNA levels in mammalian cells

ORGANISM(S): Homo sapiens

PROVIDER: GSE136418 | GEO | 2019/10/23

REPOSITORIES: GEO

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Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.

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PRMT1 regulates microRNA biogenesis through the methylation of the Large Drosha Complex

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