title: Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A.
abstract: The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.
authors.affiliation: The Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA (University of California, Los Angeles), Los Angeles, CA 90095-1569, USA.
authors.forename: Tina Branscombe
authors.initials: TB
authors.lastname: Miranda
authors.forename: Joyce
authors.initials: J
authors.lastname: Sayegh
authors.forename: Adam
authors.initials: A
authors.lastname: Frankel
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Katz
authors.forename: Mark
authors.initials: M
authors.lastname: Miranda
authors.forename: Steven
authors.initials: S
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D015870:Gene Expression; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013950:Thymus Gland; D019323:omega-N-Methylarginine / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D019323:omega-N-Methylarginine; D008780:Methyltransferases; C077013:PRMT2 protein, human; D011484:Protein-Arginine N-Methyltransferases; C120666:HSL7 protein, S cerevisiae; D011494:Protein Kinases