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

Mol Cell Proteomics. 2014 Aug;13(8):2072-88. doi: 10.1074/mcp.O113.032748. Epub 2014 Feb 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arginine / metabolism*
  • Binding Sites / drug effects
  • Cell Line, Tumor
  • Dactinomycin / pharmacology
  • Gene Expression Regulation / drug effects
  • HEK293 Cells
  • Humans
  • Methylation
  • Peptides / chemistry
  • Peptides / isolation & purification*
  • Proteomics / methods*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Transcription, Genetic* / drug effects

Substances

  • Peptides
  • RNA-Binding Proteins
  • Dactinomycin
  • Arginine