Published on June 1, 2011 in RNA volume 17.
PubMed ID: 21518805
Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m(3)C) 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 m(3)C 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 m(3)C formation, indicating that subcellular localization of ABP140 to actin filaments is not involved in tRNA modification. m(3)C formation in tRNA(Thr1) could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m(3)C did not form in tRNA(Ser1) in vitro, indicating the absence of a factor(s) required for tRNA(Ser1) m(3)C formation. Thus, ABP140 has been designated TRM140 according to the preferred nomenclature. In addition, we observed a specific reduction of m(3)C formation in HeLa cells by siRNA-mediated knock down of the human ortholog of TRM140.