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 actinbinding 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.
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