Identification of human tRNA:m(5)C methyltransferase catalysing intron-dependent m(5)C formation in the first position of the anticodon of the [Formula: see text]

We identified a human orthologue of tRNA:m(5)C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C(34) in the intron-containing yeast [Formula: see text]. Using transcripts of intron-less and intron-containing human [Formula: see text] genes as substrates, we have shown that m(5)C(34) is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m(5)C(34) formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNA(Leu) transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m(5)C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human [Formula: see text] and identification of human gene encoding tRNA methylase responsible for this reaction.