Fig. 5

METTL1 downregulation suppresses protein synthesis, proliferation and tumour growth in vivo. A Global protein synthesis rate measured by flow cytometry analysis of OP-puromycin (OP-puro) incorporation reflects reduced protein synthesis in PC3 METTL1 KO cells compared to the control (WT). Fluorescence was normalised to cell size (FSC) in WT and METTL1 KO cells. Two biological and three technical replicates and the mean ± SD are shown. B Translation initiation and regulatory factors are displaced from the cap of mRNAs in METTL1 KO cells. Log₂ fold change (FC) binding of the indicated translation initiation and regulatory factors to m⁷G-cap-coated sepharose beads in PC3 METTL1 KO vs. WT cells. Densitometry data were normalised to the input. Mean ± SEM, n = 3. C Anti-TOG RNAs block the 5'TOG-dependent displacement of translation initiation factors in vitro. Log₂ FC of translation initiation factors bound to synthetic biotinylated-5'TOG in PC3 WT cells transfected with 5'TOG + Anti-TOGs (ANT) compared to PC3 WT cells transfected with 5'TOG + scramble RNAs (TOG). Densitometry data were normalised to the input. Mean ± SEM, n = 4. D Displacement of translation initiation factors from mRNA caps is TOG-dependent and can be reversed by expressing anti-TOG RNAs. Log₂ fold change (FC) of m⁷G-cap-bound translation initiation factors in PC3 WT and METTL1 KO cells transfected with biotinylated-5'TOG (TOG) or anti-TOG RNA (ANT) versus cells transfected with scramble RNA oligonucleotides. Densitometry data were normalised to the input. Mean ± SEM, n = 3. Original wester blots are shown in supplementary figure S5 (B, C, D). E Growth curves of PC3 METTL1 KO, WT, and parental cells (PC3). Mean ± SD, n = 3. The dotted line represents the average growth of WT and parental or KO cells. F Reduced cell division rate, as measured by BrdU incorporation. G METTL1 depletion increased apoptosis in PC3 METTL1 KO cells. Flow cytometry analysis of Annexin V staining. Mean ± SD, n = 3. H Reduced spheroid formation capacity in PC3 METTL1 KO cells. Means ± SD, n = 3. The dotted line represents the average values of all WT or KO cells. I Tumour growth in xenografted PC3 METTL1 KO and WT cells in athymic nude mice reflects impaired tumour formation in the absence of METTL1. Mean ± SEM, n = 10. J-L Protein expression (J) and m⁷G methylation levels of tRNAs (K, L) of PC3 METTL1 KO cells ectopically expressing a doxycycline-inducible HA-tagged wild-type (WT) or a catalytic dead mutant (AFPA) version of METTL1. PC3 METTL1 KO cells were infected with an empty vector (eV) as a control. Methylene blue staining was used as the loading control (L, bottom panel). Mean ± SD, n = 3 (L). M, N Proliferation (M) and spheroid formation capacity (N) were dependent on METTL1 catalytic activity. PC3 METTL1 KO cells re-expressing METTL1 (WT) or catalytic dead mutant (AFPA) compared to METTL1 KO cells infected with empty vector (eV). Mean ± SD, n = 6. O, P 5'TOG transfection induces apoptosis and reduces cell proliferation. Percentage of apoptotic (O) and growth rates (P) of PC3 METTL1 KO and WT cells after transfection with synthetic 5'TOGs (TOG) or anti-TOG (ANT) RNAs. Controls (Cont) were transfected with scramble RNAs. Mean ± SD, n = 6 (O), n ≥ 10 (P). Statistical tests: Two-way ANOVA (E, I, L), one-way ANOVA (F, G, H), and one-tailed Student’s t-test (A-D, M-P). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001