Biochemical characterization of the substrate specificity of two unique members of the mammalian protein arginine methyltransferase family, PRMT7 and PRMT9

Abstract

Protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and DNA repair. Mammalian protein arginine methyltransferases include a family of nine sequence‐related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA) and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While PRMT1, 2, 3, 4, 6, and 8 have been characterized as type I enzymes, and PRMT5 as a type II enzyme, the role and activity types of the two final members of this family of enzymes, PRMT7 and PRMT9, had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. Both PRMT7 and PRMT9 are distinct members of the family with two methyltransferase or methyltransferase‐like domains and containing acidic residues in otherwise well‐conserved substrate double E binding motif, features not seen in the other PRMT enzymes. Recent work in our laboratory confirmed PRMT7 as the only type III MMA‐forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an R‐X‐R sequence for methylation. Mutations of the acidic residues in the substrate‐binding motif results in a loss of the specific R‐X‐R activity and the appearance of a G‐R‐G specificity typical of many of the other PRMTs. The physiological substrate of PRMT7 has yet to be confirmed, although histone H2B is an effective in vitro substrate. PRMT9, on the other hand, had no reported activity, until immunoprecipitation from HeLa cells showed it pulled down two splicing factors, SF3B2 and SF3B4, in a complex. Amino acid analysis showed that PRMT9 methylates SF3B2 to produce both MMA and SDMA, thus making it the second type II enzyme in mammals. PRMT9 knockdown results in modulation of alternative splicing events. This enzyme appears to be relatively specific for the SF3B2 protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other PRMTs are not recognized by PRMT9. We found that the position of the methylated arginine residue in SF3B2 is important, and the acidic residues in the substrate‐binding motif also play an important role in substrate recognition. Thus, PRMT7 and PRMT9 represent unique members of the mammalian PRMT family.Support or Funding InformationNIH grants GM026020 to S.G.C., DK062248 to M.T.B. and GM007185, a Ruth L. Kirschstein National Research Service Award to A.H.