Publications

• A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop.

  D'Silva S., Haider S.J., Phizicky E.M.

  The 3-methylcytidine (m³C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the c ... >> More

  The 3-methylcytidine (m³C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Δ strains lacks m³C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m³C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m³C and since quantitative analysis shows explicitly that tRNA(Thr(IGU)) from an abp140-Δ strain lacks m³C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m³C methyltransferase activity, which is specific for tRNA(Thr(IGU)) and not tRNA(Phe) and occurs specifically at C₃₂. We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m³C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m²,²G₂₆) are sensitive to low concentrations of cycloheximide. << Less

  RNA 17:1100-1110(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Actin-binding protein ABP140 is a methyltransferase for 3-methylcytidine at position 32 of tRNAs in Saccharomyces cerevisiae.

  Noma A., Yi S., Katoh T., Takai Y., Suzuki T., Suzuki T.

  Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m³C) is found at position 32 of the tRNAs for Thr and Ser. We used a systematic reverse genetic approach combined with mass spectro ... >> More

  Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m³C) is found at position 32 of the tRNAs for Thr and Ser. We used a systematic reverse genetic approach combined with mass spectrometry (ribonucleome analysis), and identified the actin-binding protein ABP140 as the protein responsible for m³C formation in both tRNA(Thr1) and tRNA(Ser1). ABP140 consists of an N-terminal actin-binding sequence and a C-terminal S-adenosylmethionine (Ado-Met) binding motif. Deletion of the actin-binding sequence in ABP140 did not affect m³C formation, indicating that subcellular localization of ABP140 to actin filaments is not involved in tRNA modification. m³C formation in tRNA(Thr1) could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m³C did not form in tRNA(Ser1) in vitro, indicating the absence of a factor(s) required for tRNA(Ser1) m³C formation. Thus, ABP140 has been designated TRM140 according to the preferred nomenclature. In addition, we observed a specific reduction of m³C formation in HeLa cells by siRNA-mediated knock down of the human ortholog of TRM140. << Less

  RNA 17:1111-1119(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans.

  Xu L., Liu X., Sheng N., Oo K.S., Liang J., Chionh Y.H., Xu J., Ye F., Gao Y.G., Dedon P.C., Fu X.Y.

  Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry ... >> More

  Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m³C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of <i>trm141</i> as the second gene responsible for m³C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m³C formation in mammals as well. Here, we report the discovery and characterization of three distinct m³C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m³C in specific tRNAs and that METTL8 only contributes m³C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in <i>METTL2</i> and <i>-6</i> null-mutant cells, of m³C in total tRNA, and primer extension analysis located METTL2-modified m³C at position 32 of tRNA^Thr isoacceptors and tRNA^Arg(CCU) We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. <i>METTL8</i>, however, modified only mRNA, as determined by biochemical and genetic analyses in <i>Mettl8</i> null-mutant mice and two human <i>METTL8</i> mutant cell lines. Our findings provide the first evidence of the existence of m³C modification in mRNA, and the discovery of METTL8 as an mRNA m³C writer enzyme opens the door to future studies of other m³C epitranscriptomic reader and eraser functions. << Less

  J. Biol. Chem. 292:14695-14703(2017) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.