Fig. 7: Effect of m3C32 on mt-tRNAThr/Ser(UCN) aminoacylation and ribosome-association, and mitochondrial translation.
a The numbers of cells in equally seeded cultures of wild-type HEK293 cells (WT) and those lacking METTL8 (KO1 and KO2) were determined over time. Points represent mean total number of cells of n = 3 independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA (F = 0.3165, not significant for 24 h; F = 5.6, p < 0.05 for 48 h; F = 7.425, p < 0.05 for 72 h) and significance calculated using Dunnett’s multiple comparisons test. b Aminoacylation levels of mt-tRNAThr/Ser(UCN)/Met in WT and METTL8 KO cells were analysed using acid urea electrophoresis comparing deacylated (alk; alkaline treated) and acylated (aci; acidic conditions) mitochondrial RNA samples. Arrows indicate bands corresponding to the acylated and non-acylated mt-tRNAs for WT (left) and METTL8 KO (right) cells. Representative images of three independent experiments. c–e Mitochondrial extracts from WT and METTL8 KO cells (KO1 and KO2) were separated by sucrose density gradient centrifugation and fractions collected. Proteins in each fraction were analysed by western blotting (c). Fractions 1–2 (non-mitoribosome-associated mt-tRNAs) and fractions 11–12 (55 S monosomes) were pooled and RNAs extracted from these pools were analysed by northern blotting (d). Representative images of three independent experiments. Ratios of non-mitoribosome-associated (free) and mitoribosome-associated (bound) mt-tRNAs were determined in n = 3 experiments and are shown as mean ± standard deviation (e). Statistical analysis was performed using one-way ANOVA (F = 35.22; p < 0.001 for mt-tRNAThr and F = 13.54, p < 0.01 for mt-tRNASer(UCN)) and significance calculated using Tukey’s multiple comparisons test. f Nascent mitochondrial proteins in WT, KO1 and KO2 cells were labelled with [35S]-methionine, separated by denaturing PAGE, transferred to a membrane and detected using a phosphorimager. Tubulin, detected by western blotting, served as a loading control. Representative images of eight independent experiments. g Levels of de novo synthesised mitochondrial proteins were quantified in n = 8 experiments as in (f) and are shown as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA (F = 18.91, p < 0.0001 for ND5; F = 16.59, p < 0.0001 for COX1; F = 7.345, p < 0.01 for CYTB; F = 16.08, p < 0.0001 for ND2; F = 9.867, p < 0.001 for ND1; F = 6.991, p < 0.01 for COX2/COX3; F = 10.6, p < 0.001 for ATP6; F = 4.442, p < 0.05 for ND6; F = 22.45, p < 0.0001 for ND3; F = 22.35, p < 0.0001 for ND4L/ATP8) and significance calculated using Tukey’s multiple comparisons test. p values for data in a, e and g are given in source data; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Source data and p values are provided as a Source Data file.
