Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA(CAA)Leu

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(CAA)Leu. Using transcripts of intron-less and intron-containing human tRNA(CAA)Leu 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 pre-tRNA(CAA)Leu and identification of human gene encoding tRNA methylase responsible for this reaction.

[1]  P. Fiedorow,et al.  Intergenic sequences of clustered tRNA genes: new type of genetic marker for phylogenetic studies, with application to the taxonomy of liverworts , 1998, Plant Molecular Biology.

[2]  J. Abelson,et al.  The yeast tRNATyr gene intron is essential for correct modification of its tRNA product , 1983, Nature.

[3]  T. A. Brown,et al.  A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. , 1990, Nucleic acids research.

[4]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Z. Szweykowska-Kulinska,et al.  Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre‐tRNA(Tyr). , 1992, The EMBO journal.

[6]  Marcin Feder,et al.  Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases. , 2004, Nucleic acids research.

[7]  J. Wrzesinski,et al.  A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases. , 1998, Acta biochimica Polonica.

[8]  B. Rost,et al.  Finding nuclear localization signals , 2000, EMBO reports.

[9]  C. Green,et al.  The discovery of new intron-containing human tRNA genes using the polymerase chain reaction. , 1990, The Journal of biological chemistry.

[10]  J. Abelson,et al.  Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo , 1986, Molecular and cellular biology.

[11]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[12]  O. Uhlenbeck,et al.  Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes. , 1990, Biochimica et biophysica acta.

[13]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.

[14]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[15]  Y. Motorin,et al.  Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme. , 1999, RNA.

[16]  H. Beier,et al.  All human tRNATyr genes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon. , 1988, Nucleic acids research.

[17]  M. Paddy,et al.  NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae. , 1998, Gene.

[18]  F. Watt,et al.  The RNA Methyltransferase Misu (NSun2) Mediates Myc-Induced Proliferation and Is Upregulated in Tumors , 2006, Current Biology.

[19]  H. Morris,et al.  Isolation and sequence analysis of two major leucine transfer ribonucleic acids (anticodon Mm-A-A) from a rat tumor, Morris hepatoma 5123D. , 1980, Biochemistry.

[20]  K. Zerfass,et al.  Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context. , 1992, Nucleic acids research.

[21]  Christiane Branlant,et al.  The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite-multisubstrate RNA:Psi-synthase also acting on tRNAs. , 2003, RNA.

[22]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[23]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[24]  M. Matsubara,et al.  Stable tRNA precursors in HeLa cells. , 1984, Nucleic acids research.

[25]  Xiaoyu Zhang,et al.  Methylation of tRNAAsp by the DNA Methyltransferase Homolog Dnmt2 , 2006, Science.

[26]  J. Krajewski,et al.  Mutations of Arabidopsis thaliana pre-tRNA(Tyr) affecting pseudouridylation of U35. , 1995, Biochimica et biophysica acta.