Fig. 6: Requirement of m3C32 in mt-tRNAThr/Ser(UCN) for tRNA stability and structure.
a Total RNAs from WT, KO1 and KO2 cells were separated by denaturing PAGE, and mt-tRNAThr/Ser(UCN) and the U6 snRNA were detected using northern blotting. Representative images of three independent experiments (left). Hybridisation signals detected by northern blotting were quantified and the normalised signal intensity from n = 3 independent experiments is shown as mean ± standard deviation (right). Statistical analysis was performed using one-way ANOVA (F = 0.568, ns for mt-tRNAThr; F = 0.5221, ns for mt-tRNASer(UCN)) and significance calculated using Tukey’s multiple comparisons test. b, c Total RNAs from WT, KO1 and KO2 cells (b) or synthetic mt-tRNAThr ASL and mt-tRNASer(UCN) ASL with the modifications indicated (c) were separated by native PAGE, and mt-tRNAThr/Ser(UCN) and the U6 snRNA were detected using northern blotting (b) or sybr gold staining (c). Representative images of three independent experiments. d 1H NMR spectra of unmodified mt-tRNASer(UCN) ASL (black, bottom), its i6A37 modified analogue (green, middle), and the analogous ASL containing both i6A37 and m3C32 modified nucleotides (blue, top) were recorded at 600 MHz. Conditions: 200 µM RNA, 10 mM Na-phosphate buffer pH 7.0, 100 mM NaCl, H2O/D2O 9/1, 10 °C. e CD spectra of unmodified mt-tRNASer(UCN) ASL (black), its ms2i6A37 modified analogue (red), and the analogous ASL containing both ms2i6A37 and m3C32 modified nucleotides (purple) were recorded at 5 µM in 10 mM Na-phosphate buffer pH 7.0, 1 mM MgCl2. NMR samples were prepared once, and CD spectra were recorded at least twice for each sample. p values for data in a are given in source data; ns not significant. Source data and p values are provided as a Source Data file.
