Figure 5.

YTH N⁶-methyladenosine RNA binding protein 1 (YTHDF1) promotes TNF/NF-kB signalling in CRC through promoting RELA (p65) mRNA translation. (A) Differentially expressed genes between CT26 cells with and without knockout of Ythdf1 were enriched in TNF signalling pathway identifying by Ribo-seq (left). Heatmap of genes on TNF signalling pathway (right). (B) Methylated RNA immunoprecipitation (MeRIP) sequencing on CT26 cells, showing m⁶A modifications on last exon or 3’UTR of RELA (p65) mRNA. (C) Scheme showing the design of primers for MeRIP-qPCR to validate m⁶A modifications on p65 mRNA. Potential m⁶A sites were highlighted in red (upper). MeRIP-qPCR primers are indicated by arrows. MeRIP-qPCR validated m⁶A modification with primers #2 and #3 (lower). (D) RNA immunoprecipitation sequencing with anti-YTHDF1 antibody (YTHDF1-RIP) showing the enrichment of p65 mRNA compared with IgG control (left). YTHDF1-RIP-qPCR with primers specific to murine p65 mRNA (right). (E) Western blot of key effectors of TNF/NF-κB signalling pathway on knockout of Ythdf1. (F) Western blot analysis performed with indicated human CRC cells overexpressing wild-type (WT) or mutant (mut) YTHDF1, or YTHDF1 knockdown with shRNA. Non-targeted shRNA (shNC) were used as control. (G) YTHDF1-RIP-qPCR with primers specific to human p65 mRNA. (H) Expression of p65 and phospho-p65 was determined by western blot in colon tumours from the intestine-specific Ythdf1 knockin mice and wildtype littermates. cKI: conditional knockin (two tailed t-test (C, D, G)).