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

<jats:p>The 3-methylcytidine (m<jats:sup>3</jats:sup>C) modification is widely found in eukaryotic species of tRNA<jats:sup>Ser</jats:sup>, tRNA<jats:sup>Thr</jats:sup>, and tRNA<jats:sup>Arg</jats:sup>; at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA<jats:sup>Ser</jats:sup>. 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 <jats:italic>Saccharomyces cerevisiae</jats:italic>, and find that tRNA<jats:sup>Thr(IGU)</jats:sup> from <jats:italic>abp140</jats:italic>-Δ strains lacks m<jats:sup>3</jats:sup>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 <jats:italic>S</jats:italic>-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that <jats:italic>ABP140</jats:italic> is required for m<jats:sup>3</jats:sup>C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m<jats:sup>3</jats:sup>C and since quantitative analysis shows explicitly that tRNA<jats:sup>Thr(IGU)</jats:sup> from an <jats:italic>abp140</jats:italic>-Δ strain lacks m<jats:sup>3</jats:sup>C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or <jats:italic>Escherichia coli</jats:italic> has m<jats:sup>3</jats:sup>C methyltransferase activity, which is specific for tRNA<jats:sup>Thr(IGU)</jats:sup> and not tRNA<jats:sup>Phe</jats:sup> and occurs specifically at C<jats:sub>32</jats:sub>. 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<jats:sup>3</jats:sup>C has a role in translation, since <jats:italic>trm140-Δ trm1-Δ</jats:italic> strains (also lacking m<jats:sup>2,2</jats:sup>G<jats:sub>26</jats:sub>) are sensitive to low concentrations of cycloheximide.</jats:p>