Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA Leu (CAA).
We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA Leu (CAA). Using transcripts of intron-less and intron-containing human pre-tRNA Leu (CAA) genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 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-tRNALeu 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 m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of humanpre-tRNA Leu (CAA) and identification of human gene encoding tRNA methylase responsible for this reaction.
Brzezicha
Bartosz
B
Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznań, Poland.
Schmidt
Marcin
M
Makalowska
Izabela
I
Jarmolowski
Artur
A
Pienkowska
Joanna
J
Szweykowska-Kulinska
Zofia
Z
D044503:5-Methylcytosine / Q000378:metabolism*; D000926:Anticodon / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism*; D012356:RNA, Transfer, Leu / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000926:Anticodon; D012322:RNA Precursors; D012356:RNA, Transfer, Leu; D029701:Saccharomyces cerevisiae Proteins; C091001:pre-tRNA(Leu); D044503:5-Methylcytosine; D008780:Methyltransferases; C515610:NSUN2 protein, human; D012359:tRNA Methyltransferases; C024845:NCL1 protein, S cerevisiae