Figure 2. Rho-seq detects D on yeast tRNAs.

(A) Distribution of the 228 tRNA D-sites according to their relative position on tRNA.

(B) Upper panel: primer extension assays specific to tRNA^Asp_GUC (containing a putative Dus2-dependent D20 based on Rho-seq) performed on R+ total RNA from WT, single dus (Δdus2), and triple dus (Δdus1–3-4) mutant strains. Bottom panel: primer extension assays specific to tRNA^Arg_UCG (putative Dus1-dependent D16–17 and putative Dus2-dependent D20 based on Rho-seq) performed on R+ total RNA from WT, single dus (Δdus1, Δdus2), and triple dus (Δdus1–3-4, Δdus2–3-4) mutant strains.

(C) D-ratio profiles along the entire 18S rRNA in R+ and R− conditions in WT or Δd4 strains. The m¹acp³y modification (blue) naturally blocks a RT reaction. The m⁷G modification (orange) is sensitive to R labeling and therefore blocks the RTase in R+ conditions independently of the strain. Representative example for SPRRNA.43 is shown.

(D) Distribution of 372 detected D-sites in fission yeast within tRNAs and mRNAs.

(E) Putatively dihydrouridylated mRNAs have up to three distinct D-sites on their sequence. ala1 and fhn1 are the two mRNAs dihydrouridylated at three different positions (orange triangles).

(F) General features of the 125 protein-coding genes whose RNA products are modified.

(G) Effect of the assignment of test and control conditions in the Rho-seq analysis pipeline. Sixteen permutation tests were performed in which the properly committed information was randomly mixed. Error bars represent max and min numbers of detected D-sites upon permutation.

(H) Boxplot representation of WT R+ D-ratio distribution for tRNAs (228 values) and mRNAs (143 values) and corresponding means highlighted by orange dashed lines. Blue dashed lines indicate the D-ratios of R+ 100% D spike-in and 50% D spike-in.