Substrate Profiling of PRMT1 Reveals Amino Acid Sequences That Extend Beyond the “RGG” Paradigm

Abstract

Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.

Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF

“…Indeed, it was shown previously that SF2/ASF and its close paralog SRp30c are methylated by the human homolog of Hmt1p, PRMT1 (11,21). PRMT1 is a type I methyltransferase that catalyzes the formation of monomethyl and asymmetric dimethyl Arg on proteins (59,60) preferentially at RGG/GRG and related motifs (28,31).…”

Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry

“…Despite the development of these approaches, standard LC-MS workflows for arginine and lysine methylated peptides still suffer from a number of disadvantages. Arginine and lysine methylation lead to missed trypsin cleavage sites and arginine methylation predominantly occurs in conserved glycine/arginine rich sequences called GAR or RGG motifs [22,23]. Methylated peptides therefore frequently contain internal hydrophilic residues, which makes them difficult to capture on hydrophobic stationary phases employed in reversed-phase LC-MS.…”

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…However, the discrepancy in dissociation rate constants is puzzling. Wooderchak et al (2008) also found a distributive behavior of PRMT1 with a peptide containing a single arginine residue, and a distributive mechanism has been described for PRTM6 as well (Lakowski and Frankel , 2008 ). In contrast, Osborne et al (2007) reported PRMT1 to be partially processive in the methylation of a single arginine residue, i.e., monomethyl arginine and dimethyl arginine were found in comparable quantities.…”

“…One caveat is that the oligomeric state of the enzymes might be relevant but was not explicitly considered in any study of PRMT processivity. Whereas K ö lbel et al (2009) found a distributive behavior of PRMT1 at a low concentration, at which the enzyme probably exists as a dimer (Feng et al , 2011 ), others (Osborne et al , 2007 ;Wooderchak et al , 2008 ) used concentrations favoring higher oligomers (Feng et al , 2011 ) but came to conflicting conclusions regarding the processivity. Thus, the oligomeric state of the enzyme is not the most likely explanation for the discrepancies.…”

“…Glycine was found to be important in the + 1 position in an early study using an unidentified methyltransferase activity purified from tissue (Rawal et al , 1995 ). More recent experiments examining a single arginine embedded in an RGG consensus peptide showed that although glycine was among the preferred residues in positions + 1 and + 2, several other amino acids also functioned well for methylation by PRMT1 (Wooderchak et al , 2008 ). Interestingly, although alanine and serine were not tolerated in the + 1 position in this peptide and in a protein that was examined in parallel, both amino acids are found in this position in the C-terminal domain of PABPN1 (Smith et al , 1999 ).…”