A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

Abstract

The 3-methylcytidine (m³C) 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-Δ strains lacks m³C. 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 m³C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m³C and since quantitative analysis shows explicitly that tRNA^Thr(IGU) from an abp140-Δ strain lacks m³C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m³C methyltransferase activity, which is specific for tRNA^Thr(IGU) and not tRNA^Phe and occurs specifically at C₃₂. 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 m³C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m^2,2G₂₆) are sensitive to low concentrations of cycloheximide.