Evolving specificity of tRNA 3-methyl-cytidine-32 (m³C32) modification: a subset of tRNAs^Ser requires N⁶-isopentenylation of A37

Loss of i⁶A37 from tRNAs^Ser causes their loss of m³C32. (A,B) IGV display tracks for tRNA^SerUGA (A) and tRNA^ThrUGU (B); colors as in Figure 1; note the absence of C32 misincorporations from the tit1Δ cells of A but not B. (C) Bar plot of C32 misincorporations in the mutants indicated. Brackets with asterisks indicate the three Tit1 substrates, all of which have A at position 36 and differ from the four other m³C32-containing tRNAs which contain U at position 36. Error bars indicate standard deviations of four replicates each for WT and tit1Δ, and two replicates each for tit1Δ+tit1-T12A and tit1Δ+tit1⁺. (D) Mass chromatograms of m³C and m⁵C in WT and tit1Δ cells; numbers indicate abundances. (E) Pie chart showing the A37 modification status of tRNAs that carry m³C32; compiled from data in the tRNA modification database (UK, unknown) (Jühling et al. 2009).