Discovery of the β-barrel–type RNA methyltransferase responsible for N(6)-methylation of N(6)-threonylcarbamoyladenosine in tRNAs

Methylation is a versatile reaction involved in the synthesis and modification of biologically active molecules, including RNAs. N(6)-methyl-threonylcarbamoyl adenosine (m(6)t(6)A) is a post-transcriptional modification found at position 37 of tRNAs from bacteria, insect, plants, and mammals. Here, we report that in Escherichia coli, yaeB (renamed as trmO) encodes a tRNA methyltransferase responsible for the N(6)-methyl group of m(6)t(6)A in tRNA(Thr) specific for ACY codons. TrmO has a unique single-sheeted β-barrel structure and does not belong to any known classes of methyltransferases. Recombinant TrmO employs S-adenosyl-L-methionine (AdoMet) as a methyl donor to methylate t(6)A to form m(6)t(6)A in tRNA(Thr). Therefore, TrmO/YaeB represents a novel category of AdoMet-dependent methyltransferase (Class VIII). In a ΔtrmO strain, m(6)t(6)A was converted to cyclic t(6)A (ct(6)A), suggesting that t(6)A is a common precursor for both m(6)t(6)A and ct(6)A. Furthermore, N(6)-methylation of t(6)A enhanced the attenuation activity of the thr operon, suggesting that TrmO ensures efficient decoding of ACY. We also identified a human homolog, TRMO, indicating that m(6)t(6)A plays a general role in fine-tuning of decoding in organisms from bacteria to mammals.