UCLA

Protein arginine methylation is an important posttranslational modification in eukaryotes, shown to be involved in the regulation of transcription, the splicing machinery, signaling, and DNA repair. Mammalian protein arginine methyltransferases (PRMT) include a family of nine enzymes that transfer methyl groups onto the omega nitrogen atoms of the guanidino groups of arginine residues, producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA) and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While the other PRMTs have been extensively studied, the roles and activities of two members of this family, PRMT7 and PRMT9, had been less well investigated. Both PRMT7 and PRMT9 are distinguished from other family members with having two methyltransferase-like domains and having acidic residues in an otherwise well-conserved “double-E” substrate-binding motif. My work confirms PRMT7 as the only type III enzyme in the group, with an unusual low temperature optimum for activity and preference for basic residues in an RXR sequence for methylation. I found that mutations of the acidic residues in the double-E motif result in a loss of the specific RXR substrate recognition motif and the appearance of a RG specificity motif typical of many of the other PRMTs. The physiological substrate(s) of PRMT7 remain to be determined, although I found that histone H2B is an effective in vitro substrate. PRMT9, on the other hand, had no reported activity, until I was able to show in a pulldown experiment using HeLa cells that it was associated with two RNA splicing factors. I was able to determine by amino acid analysis that PRMT9 is very specific for methylating the RNA splicing factor SF3B2. This PRMT9-dependent modification reaction produces both MMA and SDMA and thus makes PRMT9 the second example of a type II enzyme in mammals. I found that the position of the methylated arginine residue in SF3B2 is important for PRMT9 recognition, and that the acidic residues in the substrate-binding motif also play an important role in substrate recognition. In addition, mutagenesis studies in the active site cavity of the PRMT7 and PRMT9 enzymes uncovered conserved residues in the substrate binding double-E loop that are important for substrate recognition and residues in the conserved THW motif that are responsible for conferring the methylation activity type. Lastly, I examined the orthologous PRMT7 and PRMT9 enzymes in the nematode Caenorhabditis elegans. I found that the C. elegans PRMT-7 has a distinct substrate preference from the mammalian ortholog, while C. elegans PRMT-9 appears to be biochemically indistinguishable from its human ortholog.