Distinct substrate specificities of the human tRNA methyltransferases TRMT10A and TRMT10B

Human TRMT10A and TRMT10B exhibit distinct in vitro tRNA substrate specificities. (A) m¹G₉ activity of hTRMT10A on human G₉-containing tRNAs. Labeled tRNA were created by in vitro transcription in the presence of [α³²P]-GTP, resulting in uniform labeling at every G nucleotide. Labeled tRNA were incubated with fivefold dilutions of the enzyme (1.5 mg/mL to 2.4 µg/mL, with the highest concentration indicated by the number shown in the wedge representing each titration) or no enzyme (−) for 1 h. Quenched reactions were digested to single nucleotides using P1 nuclease and analyzed by TLC to resolve product (p*m¹G) from remaining unreacted substrate (p*G). (B) m¹G₉ activity of hTRMT10B on human G₉-containing tRNAs. Reactions were performed identically to A, except that serial dilutions contained 0.3 mg/mL to 0.5 µg/mL enzymes, with the highest concentration indicated by the number shown in the wedge representing each titration as above. (C) m¹G₉ activity of ScTrm10 on human G₉-containing tRNAs. Reactions were performed identically to A, except that serial dilutions contained 2 mg/mL to 3 µg/mL enzyme, with highest concentration indicated by the number shown in the wedge representing each titration as above. (D) m¹A₉ activity of hTRMT10A and hTRMT10B, and T. kodakarensis Trm10 positive m¹A₉ control (Krishnamohan et al. 2019), on human tRNA^Asp. Uniformly A-labeled in vitro transcript of tRNA^Asp was prepared as described in B above (in transcription reactions containing [α³²P]-ATP), and tested with the same assay. Digestion with nuclease P1 generates p*m¹A from methylated product RNA and p*A from unreacted substrate, which are also resolved by TLC.