Fig. 2: CRACI profiles D sites in HepG2 tRNA in a quantitative manner.

a The identified D sites in cytoplasmic tRNAs (ct-tRNAs) from G2 cells, marked in pink color. b The number of D sites identified by CRACI in HepG2 ct-tRNAs, which are located at positions 16, 17, 20, 20a, and 47. c Heatmap displaying D modification stoichiometry at high-confidence D sites in HepG2 ct-tRNAs. A representative tRNA isoform in each tRNA class is shown. The mutation ratio was calculated as the average of two biological replicates. d Comparative analysis of D stoichiometry at positions 16/17, 20, 20a, and 47 in HepG2 ct-tRNAs, with violin plots illustrating the distribution of modification stoichiometry. Each point was calculated as the average of two biological replicates. e CRACI detected eight D20 sites that overlap very well with the 10 previously reported D20 sites installed by DUS2L⁴⁵. The remaining two tRNAs harbor not only D but also acp³U at position 20, resulting in mutation signatures even without treatment. Venn diagrams illustrating the overlap of CRACI-detected D sites with 5-ClUrd-iCLIP targets in human ct-tRNAs at f D16/17 and g D20 sites, installed by ‘writer’ proteins DUS1L and DUS2L, respectively⁴⁷. h Venn diagram showing the overlap of CRACI-detected D47 sites with 5-FUrd-iCLIP targets in human ct-tRNAs, corresponding to the ‘writer’ protein DUS3L¹⁴. i Identification of D sites by CRACI in mitochondrial tRNAs (mt-tRNAs) from human HepG2 cells. j The overlap of CRACI-detected mt-tRNA D sites with a previously published mass spectrometry dataset⁴⁹. k Quantitative observation of mt-tRNA D sites by CRACI, demonstrating the concordance with mass spec data. Two biological replicates are used for CRACI and shown here. The quantitative D ratios for RNA-MS were obtained from a published mass spectrometry dataset⁴⁹.