N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO

Abstract

We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.

Figure 1. Oxidative demethylation of m⁶A in nucleic acids by FTO

(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.

Figure 2. FTO regulates m⁶A content in mRNA in a FTO-activity dependent manner

(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.

Figure 3. Indirect immunofluorescence analysis of endogenous FTO shows FTO partially colocalizes with nuclear speckles

(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.