Fig. 1.
FTO demethylates both internal m6A and cap m6Am in vitro and in vivo. (A and B) UPLC-MS/MS quantification of internal m6A/A and cap m6Am/A ratios in mRNA treated with FTO protein in vitro. Here 400 ng of purified mRNA from HeLa cells were treated with 1 μM of FTO (A) or 0.08 μM of FTO (B) under standard demethylation conditions in 50 μL of reaction mixture for 1 h at 37 °C. (C and D) UPLC-MS/MS quantification of internal m6A/A and cap m6Am/A ratios in mRNA isolated from HeLa (C) and HEK293T (D) cells with or without FTO knockdown. Error bars indicate the mean ± SEM (n = 6, three biological replicates × two technical replicates), determined using an unpaired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.