Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of ω-NG-monomethylarginine in calf thymus histone H2A

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 HSL7construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hs17 effectively catalyses the transfer of methyl groups from S-adenosyl- [methyl-H-3]-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-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 Hs17. Surprisingly, FLAG-Hs17 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-Hs17 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 Hs17-dependent methylation of endogenous yeast histones. The physiological substrates of the Hs17 enzyme remain to be identified.