S. cerevisiae Trm140 has two recognition modes for 3-methylcytidine modification of the anticodon loop of tRNA substrates

The 3-methylcytidine (m(3)C) modification is ubiquitous in eukaryotic tRNA, widely found at C(32) in the anticodon loop of tRNA(Thr), tRNA(Ser), and some tRNA(Arg) species, as well as in the variable loop (V-loop) of certain tRNA(Ser) species. In the yeast Saccharomyces cerevisiae, formation of m(3)C(32) requires Trm140 for six tRNA substrates, including three tRNA(Thr) species and three tRNA(Ser) species, whereas in Schizosaccharomyces pombe, two Trm140 homologs are used, one for tRNA(Thr) and one for tRNA(Ser). The occurrence of a single Trm140 homolog is conserved broadly among Ascomycota, whereas multiple Trm140-related homologs are found in metazoans and other fungi. We investigate here how S. cerevisiae Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G(35)-U(36)-t(6)A(37) of the anticodon loop of tRNA(Thr) substrates, and this sequence is an identity element because it can be used to direct m(3)C modification of tRNA(Phe). However, Trm140 recognition of tRNA(Ser) substrates is different, since their anticodons do not share G(35)–U(36) and do not have any nucleotides in common. Rather, specificity of Trm140 for tRNA(Ser) is achieved by seryl-tRNA synthetase and the distinctive tRNA(Ser) V-loop, as well as by t(6)A(37) and i(6)A(37). We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates m(3)C modification of tRNA(Ser(CGA)) and tRNA(Ser(UGA)) in vitro. In addition, our results show that Trm140 binding is a significant driving force for tRNA modification and suggest separate contributions from each recognition element for the modification.