Fig. 6

Loss of m⁷G tRNA methylation results in distinct translational programmes. A Schematic overview of nascent polypeptide OP-puro-labelling and enrichment followed by LC–MS/MS peptide identification and quantification analysis (upper panel). Lower panel shows common differentially expressed nascent proteins ranked in a volcano plot according to their statistical p-value (-Log₁₀ pV) and their relative abundance ratio (Log₂ FC) between four replicates of PC3 WT and METTL1 KO cells. Coloured dots represent statistically (p-value < 0.05) upregulated (red) and downregulated (blue) proteins in METTL1 KO cells. B Gene Ontology (GO) category enrichment of biological processes in significantly (p-value < 0.05) upregulated (UP, FC > 1) or downregulated (Down, FC < 1) nascent proteins in METTL1 KO cells compared to WT cells. Categories were ranked according to their statistical P-value (-Log₁₀ pV) and fold enrichment of genes found for each category. C No correlation between differentially expressed proteins (protein log₂ FC) and their mRNA (protein log₂ FC) was observed in PC3 WT vs. METTL1 KO cells. Coloured dots represent significant (p-value < 0.05) differentially expressed proteins (blue), mRNAs (yellow) or both (red) for each gene. D Representative polysome profile in WT (grey line) and METTL1 KO PC3 cells (red line) (left panel) shows reduced translation in METTL1 KO cells. The fraction of the abundance of each mRNA in each polysome fraction is shown with respect to the content in all fractions, reflecting increased translation of IRF9 and ISG15 in METTL1 KO cells. Mean ± SEM, n = 3. The right boxplot represents the fold change (FC) of mRNA content in the polysome fraction relative to non-polysome fractions. E Increased IRF9, ISG15 and STAT1 protein expression observed in PC3 METTL1 KO cells is 5'TOG-dependent. Protein expression levels are represented as log₂ fold change for WT (grey bars) transfected with 5'TOG RNA (TOG) versus scramble control RNA (Ct), and METTL1 KO cells (red bars) transfected with anti-TOG RNAs (ANT) versus scramble control RNAs (Ct). Mean ± SEM, n = 3. Original western blots are shown in supplementary figure S7. F, G STAT1-dependence of apoptosis (F) and growth rates (G) of METTL1 KO and WT PC3 cells. KO cells were transfected with siScramble or siSTAT1. Mean ± SD, n = 6 (F, G). Statistical tests: One-tailed Student’s t-test was used (E–G). *p < 0.05, **p < 0.01, ***p < 0.001