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

The 3-methylcytidine (mC) modification is ubiquitous in eukaryotic tRNA, widely found at C in the anticodon loop of tRNA, tRNA, and some tRNA species, as well as in the variable loop (V-loop) of certain tRNA species. In the yeast , formation of mC requires Trm140 for six tRNA substrates, including three tRNA species and three tRNA species, whereas in , two Trm140 homologs are used, one for tRNA and one for tRNA 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 Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G-U-tA of the anticodon loop of tRNA substrates, and this sequence is an identity element because it can be used to direct mC modification of tRNA However, Trm140 recognition of tRNA substrates is different, since their anticodons do not share G-U and do not have any nucleotides in common. Rather, specificity of Trm140 for tRNA is achieved by seryl-tRNA synthetase and the distinctive tRNA V-loop, as well as by tA and iA We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates mC modification of tRNA and tRNA 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.