(a) Quantification of the m⁶A/A ratio in mRNA by LC-MS/MS. Significant increase of the m⁶A level was observed in both FTO siRNA-treated HeLa and 293FT cells after 48 h. *, P-value < 0.05, Student’s t-test; means ± s.e.m. for n = 8 experiments. (b) The m⁶A/A ratio of mRNA samples isolated from cells with overexpressed FTO. The overexpression of wild-type FTO decreased the m⁶A content significantly. * P-value < 0.05, Student’s t-test; means ± s.e.m. for n = 8 experiments.

(a) Proposed oxidative demethylation of m⁶A to A in DNA and RNA by FTO in the presence of iron(II) and α-KG. (b, c) As shown by HPLC profiles of digested substrates, 20 mol% of FTO can completely demethylate m⁶A in ssRNA and ssDNA in 3 h at room temperature at neutral pH. (d) About 40% demethylation of m⁶A in a RNA stem loop could be observed under the same reaction conditions.

(a) HeLa cells were co-stained with antibodies against FTO and speckle marker SC35 in the presence or absence of transcription inhibitor actinomycin D (ActD). Yellow dots indicated by white arrows correspond to the colocalized spots of FTO (Red) with SC35 (Green) (N = 150). Upon transcription inhibition, the FTO loci number decreased in the nucleoplasm, but its speckle distribution became more pronounced. (b) Quantitative foci analysis of SC35, Pol II-S2P, and FTO with (+), or without (−) ActD treatment. 30, 37 and 67 cells were randomly selected for foci analysis of SC35, Pol II-S2P, and FTO, respectively. The t-test using two-way ANOVA in Grouped Analyses of Prism5 software was applied for statistical analysis. P-value less than 0.01 was considered a significant difference. The mean numbers of foci per cell for each sample with and without ActD treatment were shown above, and below the corresponding P-value, respectively. The foci number of Pol II-S2P and FTO decreased significantly upon transcription inhibition.