Extended Data Fig. 3: Validation of modifications inferred from RT-derived signals using mutant V. cholerae strains.
From: Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications
a, Heatmap of misincorporation frequency at position 8 in V. cholerae tRNAs. Most of the misincorporation signals, except for tRNA-Ser1 and tRNA-Gln1A, are eliminated in the ΔthiI strain, consistent with the idea that misincorporation results from the associated modification, s4U. The data for tRNA-Ile2 is not shown (black) because of insufficient read depth (<100 reads). b, Heatmap of misincorporation frequency at position 32 in V. cholerae tRNAs. The signals in tRNAs that are expected to have s2C (tRNA-Arg2A, tRNA-Arg2C, tRNA-Arg3, tRNA-Ser3A, tRNA-Ser3B, and tRNA-Arg4) are eliminated in the ΔttcA strain, whereas the signal in tRNA-Tyr remains due to C to Ψ RNA editing (see Fig 5). The data for tRNA-Ile2 is not shown (black) because of insufficient read depth. c, Heatmap of misincorporation frequency at position 37 in V. cholerae tRNAs. The signals in tRNAs that are expected to have ms2io6A (tRNA-Leu5, tRNA-Phe1, tRNA-Phe2, tRNA-Leu4, tRNA-Trp, tRNA-Cys1, tRNA-Cys2, tRNA-Ser1, and tRNA-Tyr) are eliminated in the ΔmiaA strain, whereas the signals in tRNA species that are predicted to have m1G at position 37 remain. d, Heatmap of misincorporation frequency at position 22 in V. cholerae tRNAs. The signal in tRNA-Tyr was absent in the ΔtrmK strain, suggesting that this signal is derived from m1A. The data for tRNA-Ile2 is not shown (black) because of insufficient read depth. In all panels, representative data from three replicates with similar results for WT and one replicate for knockout strains is shown.
