Published on June 1, 2011 in RNA volume 17.
PubMed ID: 21518804
The 3-methylcytidine (m(3)C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Delta strains lacks m(3)C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m(3)C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m(3)C and since quantitative analysis shows explicitly that tRNA(Thr(IGU)) from an abp140-Delta strain lacks m(3)C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m(3)C methyltransferase activity, which is specific for tRNA(Thr(IGU)) and not tRNA(Phe) and occurs specifically at C(3)(2). We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m(3)C has a role in translation, since trm140-Delta trm1-Delta strains (also lacking m(2),(2)G(2)(6)) are sensitive to low concentrations of cycloheximide.