Fig. 3: In vitro reconstitution of tRNA D16 and D17 modifications.
a Subcellular localization of human DUS1L-Myc. U87 cells were transfected with a plasmid expressing DUS1L-Myc. Confocal microscopic images were obtained after immunostaining of Myc-tagged proteins (green) and DAPI staining (blue). Scale bar, 10 µm. b Purified His-DUS1L resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Protein size markers are indicated. c LC-MS analysis of total RNA nucleosides after incubating the reaction mixture with (+) or without (−) His-DUS1L or NADPH. Total RNA from DUS1L KO or control LNZ308 cells was used as the substrate RNA. The values are the relative levels to the mean D levels in total RNA of control cells, normalized by the uridine peak area of the same samples. Means ± s.e.m. from n = 5 samples each. d LC-MS analysis of RNase T1-digested fragments of tRNATyr(GUA) purified from total RNA after incubating the reaction mixture with or without His-DUS1L or NADPH, using total RNA from DUS1L KO LNZ308 cells as the substrate RNA. XICs of unmodified, single D-modified, and doubly D-modified 3-mer fragments (positions 16–18) with the indicated m/z are shown. e LC-MS analysis of total RNA nucleosides after incubating the reaction mixture in the same way as in (c), with (+) or without (−) NADPH or NADH. Means ± s.e.m. from n = 4 samples each. ****P < 0.0001, *P < 0.05, n.s., not significant by the Brown–Forsythe and Welch ANOVA tests. Exact P-values are included in the Supplementary Data 1.
