U13 in a subset of tRNAs is pseudouridylated by TruD in T. thermophilus. (A) Overview of the PRAISE experimental procedure. Pseudouridine sites in tRNAs are labeled by bisulfite treatment. After the desulfonation step, the adapter oligonucleotide is attached to the 3′ end of tRNAs, which are converted to cDNA by reverse transcription followed by NGS. Adapter- and quality-trimmed reads were analyzed by ShapeMapper2 (Busan and Weeks 2018). The NGS experiments were independently repeated (n = 3 for WT and n = 2 for ΔtruD). (B and C) Normalized mutation signals at each nucleotide position in tRNAAsp-GUC for WT (B) and the ΔtruD strain (C). (D) Mutation rates at U13 in the substrate tRNAs of T. thermophilus TruD for WT (green dots) and ΔtruD (orange dots). (E) Sequences from positions 11 to 15 in substrate tRNAs (tRNAAsp-GUC-1-1, tRNAGln-CUG-1-1, tRNAGln-UUG-1-1, tRNAGlu-CUC-1-1, tRNAGln-UUC-1-1) and nonsubstrate tRNAs (e.g., tRNAAla-CGC-1-1, tRNAPhe-GAA-1-1, tRNAMet-CAT-1-1) of TruD are aligned with the target uridine highlighted in blue and compared to other tRNAs.
