Validation of m1A9 methylation product catalyzed by hTRMT10B. (A) Extracted ion chromatograms of the nucleoside (left) and the oligonucleotide (right) analyses of tRNAAsp transcript (with and without hTRMT10B incubation indicated by black and gray arrows, respectively). The nucleoside analysis confirmed that m1A (the only ribonucleoside modification detected) is only observed in the transcript incubated with hTRMT10B. The oligonucleotide analysis revealed the presence of the unique and sequence-specific UU[m1A]G oligonucleotide, confirming m1A at position 9 of tRNAAsp. Mass spectra information is shown in Supplemental Figure 2. (B,C) Methylation activities of hTRMT10B variants with inactivating mutations in the conserved SAM binding residues. Activity assays were performed with either single variant G231R or double variant G231R/G232R purified enzymes (0.3 and 0.2 mg/mL, respectively), with tRNAs uniformly labeled at G nucleotides (B) or A nucleotides (C), as in Figure 2.
