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

Abstract

The 3-methylcytidine (m³C) modification is ubiquitous in eukaryotic tRNA, widely found at C₃₂ 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 <i>Saccharomyces cerevisiae</i>, formation of m³C₃₂ requires Trm140 for six tRNA substrates, including three tRNA^Thr species and three tRNA^Ser species, whereas in <i>Schizosaccharomyces pombe</i>, 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 <i>S. cerevisiae</i> Trm140 protein recognizes its six tRNA substrates. We show that Trm140 has two modes of tRNA substrate recognition. Trm140 recognizes G₃₅-U₃₆-t⁶A₃₇ of the anticodon loop of tRNA^Thr substrates, and this sequence is an identity element because it can be used to direct m³C modification of tRNA^Phe However, Trm140 recognition of tRNA^Ser substrates is different, since their anticodons do not share G₃₅-U₃₆ 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 t⁶A₃₇ and i⁶A₃₇ We provide evidence that all of these components are important in vivo and that seryl-tRNA synthetase greatly stimulates m³C 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.