We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9. This enzyme, the product of theYML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA Arg3 and 5-methylcarbonylmethyl-2-thiouridine in tRNA Glu . In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37°C to paromomycin, a translational inhibitor. These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress.In the last few years, it has become increasingly evident that S-adenosylmethionine (AdoMet)-dependent methyltransferases play a wide range of roles in cell physiology. Unlike other covalent modifications such as phosphorylation that occur mainly on proteins, methylation has been observed in DNA, RNA, proteins, lipids, polysaccharides, and small molecules (15,35,36,38). As a result of such diversity, identifying the methyltransferases and their substrates has been challenging. Although more than 25 methylated nucleotides have been documented for eukaryotic tRNAs alone, only a few of the corresponding enzymes and their genes have been identified (16,47).In 1999, using conserved AdoMet binding motifs, we identified 26 putative methyltransferases (designated F1 to F26) encoded by the genome of the yeast Saccharomyces cerevisiae (40). Biochemically, we have been able to show that two of these species are in fact methyltransferases; F3 (YDR465c) catalyzes the transfer of a methyl group to the delta-nitrogen atom of arginine residues in a novel protein posttranslational reaction (40, 58), while F9 (YER175c) catalyzes the methyl esterification of the small molecule trans-aconitate (9). Recently, F1 (YDL201w) was found to be responsible for formation of 7-methylguanosine at position 46 of tRNA (3).One major approach in identifying novel enzymes has been the comparison of the methylation spectra of strains lacking the putative methyltransferase and those of their isogenic wildtype parents by separation techniques such as sodium dodecyl sulfate (SDS) gel electrophoresis (26, 59). In the yeast S. cerevisiae, one can study biological methylation in vivo by incubating cells with, the major biological methyl donor (26). As a result, all methyl-accepting species such as RNAs, proteins, and small molecules can become radiolabeled and the fate of the methylated species can be followed biochemically. One can then look for differences in the methylation spectra between a mutant strain and its parent.In all domains of life, tRNAs are highly modified posttranscriptionally (46, 47). Methylation reactions account for the majority of these modifications. In the yeast S. cerevisiae, eight tRNA methyltransferases have been identified so far. The TRM1 gene product forms N 2 ,N 2 -dimethylguanosine at position 26 (14). TRM2 encodes a protein that forms 5-methyluridine at position 54 (41). TRM3 encodes a pr...
