Figure 5.

5′UTR m⁶A methylation destabilizes CTNNB1 mRNA

(A) m⁶A RIP-qPCR analysis of CTNNB1 mRNA in Con or M3^Mut/− HeLa cells using fragmented RNA. (B) Schematic of the CDS reporter and F-Luc reporter, which contains the CTNNB1 5′UTR before CTNNB1 CDS or reporter genes. (C) The CDS reporter and F-Luc reporter were transfected into Con and M3^Mut/− HeLa cells, respectively. Expression levels of reporter mRNA were detected by quantitative real-time PCR: CTNNB1 mRNA for the CDS reporter, normalized to endogenous CTNNB1 and GAPDH mRNA levels, and F-LUC mRNA for the F-Luc reporter, normalized to R-LUC mRNA. (D) The CDS reporter was transfected into Con or M3^Mut/− HeLa cells for 48 h. Expression levels of exogenous β-catenin (FLAG) were detected by western blot. Band intensities of β-catenin were analyzed using ImageJ and are listed at the bottom of the target bands. (E) The F-Luc reporter was transfected into Con or M3^Mut/− HeLa cells for 48 h. A dual-luciferase assay was performed to measure F-Luc production, which was normalized to R-Luc levels. (F) The CDS reporter was transfected into Con or M3^Mut/− HeLa cells for 48 h and then treated with Act-D for the indicated times. The expression levels of CTNNB1 mRNA were detected by quantitative real-time PCR. (G) The F-Luc reporter was transfected into Con or M3^Mut/− HeLa cells for 48 h and then treated with Act-D for the indicated times. Expression levels of F-LUC mRNA were detected by quantitative real-time PCR. (H) Schematic of m⁶A sites within CTNNB1 mRNA. Mutations of m⁶A sites in the CTNNB1 5′UTR are marked in red. (I) WT and mutated CDS reporters were transfected into HeLa cells. Expression levels of exogenous β-catenin (FLAG) were detected by western blot. Band intensities of FLAG-β-catenin were analyzed using ImageJ and are listed at the bottom of the target bands. (J) WT and mutated F-Luc reporters were transfected into HeLa cells. A dual-luciferase assay was performed to measure F-Luc production, which was normalized to R-Luc levels. (K) Schematic of m⁶A insertions within CTNNB1 mRNA. Insertions of continued m⁶A motifs in the CTNNB1 5′UTR are marked in orange. (L) WT and CDS reporters with inserted m⁶A motifs were transfected into HeLa cells. Expression levels of exogenous β-catenin (FLAG) were detected by western blot. Band intensities of FLAG-β-catenin were analyzed using ImageJ and are listed at the bottom of the target bands. (M) WT and F-Luc reporters with inserted GGAC motifs were transfected into HeLa cells. A dual-luciferase assay was performed to measure F-Luc production, which was normalized to R-Luc levels. (N) YTHDF2-RIP analysis of CTNNB1 mRNA in Con and M3^Mut/− HeLa cells. (O) CDS reporters were transfected into Con and M3^Mut/− HeLa cells. YTHDF2 RIP analysis of reporter mRNA was performed. (P) F-Luc reporters were transfected into Con and M3^Mut/− HeLa cells. YTHDF2-RIP analysis of reporter mRNA was performed. (Q) Expression levels of β-catenin in HeLa cells silencing YTHDF2 were detected by western blot. (R) Expression levels of CTNNB1 mRNA in HeLa cells silencing YTHDF2 were detected by quantitative real-time PCR, (S) Con or M3^Mut/−- HeLa cells were transfected with si-NC or si-YTHDF2 and then treated with CHX for the indicated time. Expression of CTNNB1 mRNA was detected by quantitative real-time PCR. (T) Mechanism of m⁶A regulating the stability of CTNNB1 mRNA. Data are presented as mean ± SD from three independent experiments. Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.