(A) Schematic for analysis of orthogonal SNORD13-substrate pairs. Exemplary sequencing traces are provided in Supplemental Figure S9. (B) Bar graph of ac4C levels at site corresponding to SSU-1842 in orthogonal SNORD13-substrate pairs. Values represent ac4C-dependent misincorporation rates normalized relative to the WT h45-ITS1 and WT SNORD13 pair, which was set to equal 100%. Values represent n = three biological replicates, analyzed by two-tailed Welch's t-test in comparison to WT (ns = not significant, [*] P < 0.05, [**] P < 0.01, [***] P < 0.001, and [****] P < 0.0001). Sequences of SNORD13 18S-A mutant and h45-ITS1 18S-A mutant are provided in the Supplemental Information. (C) Overview of SNORD13 structure-function relationships probed in this study.
