Fig. 5: Methylation substrate recognition by METTL8.
a, b In vitro transcribed mt-tRNAThr (a) or synthesised mt-tRNASer(UCN) ASL containing ms2i6A37 (b) or derivatives containing individual nucleotide substitutions within the anticodon loop were subjected to methylation assays with His14-MBP-METTL8 and [3H]-SAM. Tritium incorporated into the RNA was measured by scintillation counting. Bar plots show mean counts per minute (CPM) of n = 3 independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA (F = 214.6; p < 0.0001 (a) and F = 277.1, p < 0.0001 (b)) and significance calculated using Tukey’s multiple comparisons test. RNA was also separated by denaturing PAGE, stained with ethidium bromide (EtBr) and labelled RNAs (3H-Me) were detected by autoradiography (lower panel). Representative images from three independent experiments. c Schematic view of chimeric tRNA/ASLs used for in vitro methylation assays. Upper panel shows the anticodon loop of mt-tRNAThr (unmodified; green) or also with the anticodon stem (green shading) within mt-tRNAAsn. Lower panel shows a hybrid of the anticodon loop of mt-tRNASer(UCN) with the anticodon stem of mt-tRNAPhe. d, e The chimeric RNAs represented in (c) were used for in vitro methylation assays with recombinant His14-MBP-METTL8 and [3H]-SAM. Tritium incorporated into the RNA was measured by scintillation counting. Bar plots show mean counts per minute (CPM) of n = 3 independent experiments ± standard deviation. Statistical analysis was performed using one-way ANOVA (F = 190.5; p < 0.0001) and significance calculated using Tukey’s multiple comparisons test (d) or using a two-tailed unpaired Student’s t test (ns non-significant) (e). RNA was also separated by denaturing PAGE, stained with ethidium bromide (EtBr) and labelled RNAs (3H-Me) were detected by autoradiography. Representative images from three independent experiments. f CD spectra of unmodified mt-tRNASer(UCN) ASL (black) (top), its ms2i6A37 modified analogue (red) (bottom), and the analogous ASL containing A38C (green) (bottom) and G35A (blue) (top) substitutions were recorded at 5 µM in 10 mM Na-phosphate buffer pH 7.0, 1 mM MgCl2. CD spectra were recorded at least twice for each sample. p values for data in a, b, d, e are given in source data; ***p < 0.001, ****p < 0.0001, ns not significant. Source data and p values are provided as a Source Data file.
