Fig. 4: A37 modifications promote m3C32 incorporation in mt-tRNAThr and mt-tRNASer(UCN).
a Schematic views of synthetic anticodon stem loops (ASL) of mt-tRNAThr (upper) and mt-tRNASer(UCN) (lower) containing the indicated modified nucleotides. Orange U indicates a uridine substituted for pseudouridine. b, c In vitro methylation assays were performed using variants of the mt-tRNAThr ASL (b) or mt-tRNASer(UCN) (ASL) (c), 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 = 636.5; p < 0.0001 (b) and F = 350.8, p < 0.0001 (c)) and significance calculated using Tukey’s multiple comparisons test. RNA was separated by denaturing PAGE, stained with ethidium bromide (EtBr) and labelled RNAs (3H-Me) detected by autoradiography. d Protein extracted from cells transfected with non-target siRNAs (NT) or two different siRNAs against OSGEPL1 or TRIT1 (KD1, KD2) was analysed by western blotting. Representative image of three independent experiments. Asterisk indicates a non-specific cross-reaction of the TRIT1 antibody. e Primer extension was performed on small RNA extracted from siRNA-treated cells as in (d) using [32P]-labelled probes hybridising to mt-tRNAThr (left panel) and mt-tRNASer(UCN) (right panel). Products were separated by denaturing PAGE alongside [32P]-labelled DNA oligonucleotides of the indicated lengths. Signal intensities of the extension products were quantified and the percentage of read-through (Rt) of m3C32 is given as a percentage of the total extension signal. Data from n = 3 independent experiments are shown as mean ± standard deviation. Statistical analysis was done using one-way ANOVA (F = 175.9, p < 0.001 for OSGELP1; F = 38.58, p < 0.001 for TRIT1) and significance calculated using Tukey’s multiple comparisons test. f Fluorescence anisotropy measurements were taken to determine the affinity of recombinant His14-MBP-METTL8 for fluorescein-labelled mt-tRNASer(UCN) ASLs containing different modified nucleotides depicted in (a lower panel). Data from n = 3 independent experiments is show as mean ± standard deviation and dissociation constants (Kds) are given. g In vitro methylation assays were performed as in (b, c) using recombinant His14-MBP-METTL8, [3H]-SAM and the ASLs shown in (a left panel). p-values for data in b, c, e are given in source data; *p < 0.05, ***p < 0.001, ****p < 0.0001. Source data and p values are provided as a Source Data file.
