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

Abstract

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 tRNAThr1 and tRNASer1. 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 tRNAThr1 could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m3C did not form in tRNASer1 in vitro, indicating the absence of a factor(s) required for tRNASer1 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.