Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest

Abstract

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding dimethylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase. Molecular & Cellular Proteomics

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

“…In line with previous reports showing that ERα methylation is a dynamic process, requiring PRMT1 for methylation and JMJD6 arginine demethylase to remove the methylated moiety (56,57), global proteomic analysis also demonstrated that arginine methylation sites in human cells are regulated dynamically by an unknown Arg demethylase during transcriptional arrest (58). It is possible that the Arg demethylation process is a general event regulating cellular Arg methylation level.…”

Histone arginine methylation in cocaine action in the nucleus accumbens

“…The lack of study of this posttranslational modification to date may be due to the scarcity of PRMT substrates, to the intrinsic stability histone R methylation (which is seen as incompatible with a role in regulating cellular pathways), and to the lack of tools for detecting R methylation. Together with recent investigations (33)(34)(35), the present work dispels these earlier impressions, and raises histone R methylation changes in response to repeated cocaine to the rank of other more well-known histone posttranslational modifications implicated in the epigenetic basis of addiction.…”

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

“…Despite the prospective issues associated with sequence database search-derived methylpeptide identifications, the use of orthogonal methylpeptide validation in large scale methylation site discovery studies remains sporadic. Although several such studies have employed heavy-methyl SILAC (6,7,14,16) or other closely related methylpeptide-specific labeling techniques (12,13) to validate methylation sites, others have chosen to bypass orthogonal validation and to instead predominantly rely on the target-decoy approach to provide estimates for high stringency methylpeptide filtering criteria (4,5,8,9,15) or to inform manual data curation (see Table I) (10,11). This irregular use of orthogonal methylpeptide validation is a reflection of the fact that in-depth studies into methylpeptide FDRs have yet to be performed.…”

“…Demonstrations of significantly enhanced methylation site discovery have, for example, been reported from samples generated via pan-specific antibody (8,13) and methyl-lysine binding domain-based (19) pulldowns of methylpeptides, analyzed on Orbitrap Elite and Q Exactive or Orbitrap Velos instruments, respectively, and from samples enriched for arginine-methylated peptides using hydrophilic interaction liquid chromatography (HILIC), analyzed on a Q Exactive instrument (6). Together these contemporary instrument platforms and analytical workflows have enabled thousands of novel methylation sites to be identified from hundreds of proteins in the human proteome (4,6,7,12,13,15), whilst large scale LC-MS/MS characterizations of methylation in other organisms (5, 8 -11, 14, 16) have reinforced the notion that these modifications are widespread and sometimes conserved in eukaryotes (summarized in Table I). …”