Human TRMT10B exhibits limited tRNA substrate specificity. (A) In vitro methylation assays were performed with four different A9-containing yeast and human tRNAs. Serial dilutions of hTRMT10B (0.3 mg/mL to 0.5 µg/mL), or no enzyme (-) were incubated with the uniformly labeled tRNA and analyzed after nuclease P1 digestion, as described above. (B) Secondary structure diagrams of tRNAs tested in A, with the A9 residue highlighted in red for each tRNA. (C) Multiple sequence alignment of A9-containing human tRNA genes (Juhling et al. 2009). Alignment was performed using Clustal Omega (Sievers et al. 2011), similar nucleotides are highlighted by shading (using BoxShade), and nucleotides unique to tRNAAsp among all other A9 tRNA are starred and shown in red.
