title: 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 di-methylation 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.
authors.affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark;
authors.forename: Kathrine B
authors.initials: KB
authors.lastname: Sylvestersen
authors.affiliation: §Disease Systems Biology, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Heiko
authors.initials: H
authors.lastname: Horn
authors.affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark;
authors.forename: Stephanie
authors.initials: S
authors.lastname: Jungmichel
authors.affiliation: §Disease Systems Biology, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Lars J
authors.initials: LJ
authors.lastname: Jensen
authors.affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark; michael.lund.nielsen@cpr.ku.dk.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Nielsen
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D045744:Cell Line, Tumor; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008745:Methylation; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D010455:Peptides; D016601:RNA-Binding Proteins; D003609:Dactinomycin; D001120:Arginine