Nucleic Acids Research Advance Access published online on October 27, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl765
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the 
owska2owski
kowska3ska*
Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University Mi
dzychodzka 5, 60-371 Pozna, Poland
1 Department of Biotechnology and Food Microbiology, August Cieszkowski University of Agriculture Wojska Polskiego 48, 60-627 Pozna, Poland
2 Center for Computational Genomics, The Huck Institutes of the Life Sciences, The Pennsylvania State University, State College PA 16802, USA
3 Department of Cell Biology, Institute of Experimental Biology, Adam Mickiewicz University Umultowska 89, 61-614 Pozna, Poland
*To whom correspondence should be addressed. Tel: +61 829 27 28; Fax: + 61 829 27 30; Email: zofszwey{at}amu.edu.pl
Received September 12, 2006. Revised September 28, 2006. Accepted September 28, 2006.
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 . Using transcripts of intron-less and intron-containing human 
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 human and identification of human gene encoding tRNA methylase responsible for this reaction.
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