Fig. 3: YTHDF3 promotes STAT3 mRNA stability and translation efficiency in a manner dependent on METTL3-mediated m6A modification.
A Overlapping genes identified by Ribo-seq, RIP-seq, and RNA-seq were analyzed to identify YTHDF3 targets. B KEGG pathway analysis of the RIP-seq data revealed enriched pathways associated with the YTHDF3-binding genes. C Integrative Genomics Viewer (IGV) image showing the YTHDF3 binding site in the STAT3 mRNA identified by RIP-seq in SiHa and YTHDF3−/− SiHa cells. D An IGV image of the Ribo-seq data confirms the peak enrichment of STAT3 mRNA, suggesting that YTHDF3 binding may enhance its translation. E Ribo-seq analysis revealed altered translation efficiency upon YTHDF3 knockout in SiHa cells. F qPCR analysis was used to compare the relative mRNA levels of STAT3 and IRF7 in the YTHDF3-knockdown SiHa and CaSki cells with those in their respective negative controls. The means and standard deviations from at least three independent experiments are shown. ***p < 0.001, ****p < 0.0001. G Western blot analysis revealed the protein levels of STAT3 and IRF7 in the YTHDF3-knockdown SiHa and CaSki cells compared with their respective negative controls. H Western blot analysis was used to assess STAT3 and IRF7 expression in the YTHDF3-deficient SiHa cells transfected with the YTHDF3 overexpression plasmid versus those transfected with the wild-type plasmid. I Western blot analysis was used to investigate whether STAT3 expression depends on METTL3-mediated m6A modification by comparing STAT3 protein levels in shMETTL3 SiHa and shNC SiHa cells (sh denotes short hairpin RNA-mediated knockdown). J MeRIP-qPCR results showing the m6A enrichment of STAT3 mRNA. The means and standard deviations from at least two independent experiments are displayed. **p < 0.01. K qPCR assays were performed to determine the effect of YTHDF3 on STAT3 mRNA stability after SiHa cells and YTHDF3−/− SiHa cells were treated with 4 µg/mL actinomycin D, and the cells were harvested at 0, 2, and 4 h. The decay rate for each transcript was calculated via the exponential functions of Half-Life. The means and standard deviations from at least three independent experiments are shown. L Western blot analysis showing the effect of YTHDF3 on the protein stability of STAT3 after SiHa and YTHDF3−/− SiHa cells and shNC CaSki and shYTHDF3 CaSki cells were challenged with 200 µg/mL CHX and harvested at 0, 12, and 24 h. M qPCR analysis comparing STAT3 mRNA levels in SiHa and CaSki cells after transfection with vector, wild-type or mutant Flag-tagged YTHDF3 plasmids. The means and deviations from at least two independent experiments are displayed. The data are shown as the means ± SDs; *p <0.05;**p < 0.01; ***p < 0.001; **** p < 0.0001. N Western blot analysis of STAT3 protein levels in SiHa and CaSki cells transfected with vector, wild-type or mutant Flag-tagged YTHDF3 plasmids. STAT3 expression was normalized with GAPDH. The mean ± SD from three experiments was plotted. ns no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. GAPDH was used as loading control.
