Figure 4.

m⁶A methylation on both the 5′UTR and CDS regions of TGFB1 mRNA controls its translation efficiency. (a) Reporters for 5′UTR and CDS regions of TGFB1 mRNA. Potential m⁶A sites were mutated (GGAC to GGCC); (b) wild type (WT) and mutated reporters were transfected in HeLa cells for 48 h. Expression levels of reporter mRNA were measured by qRT-PCR: FLUC mRNA for 5′UTR reporter, normalized to RLUC mRNA levels; TGFB1 mRNA for the CDS reporter, normalized to GAPDH mRNA levels; (c) the WT-5′UTR reporter was co-transfected with TK-Rluc reporter in control and Mettl3^Mut/− HeLa cells for 48 h. Dual-luciferase assay was performed to measure F-Luc production, which was normalized to R-Luc levels; (d) WT-5′UTR or Mutant-5′UTR reporter were co-transfected with TK-Rluc reporter in HeLa cells for 48 h. Dual-luciferase assay was performed to measure F-Luc production, which was normalized to R-Luc levels; (e) WT-CDS reporter was transfected in control and Mettl3^Mut/− HeLa cells for 48 h. Expression levels of exogenous TGFβ1 (His) were measured by Western blot; (f) WT-CDS or Mutant-CDS reporter were transfected in HeLa cells for 48 h. Expression levels of exogenous TGFβ1 (His) were measured by Western blot; (g) Translation efficiency of WT and TGFβ1 mutant is defined as the quotient of reporter protein production divided by mRNA abundance [11]. For 5′UTR, reporter protein production was determined from dual-luciferase assay; for CDS, reporter protein production was analyzed by ImageJ from Western blot. Data are presented as means ± SD from three independent experiments. Student’s t-test, n.s, no significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control.