NADP and NAD Enhance FTO-mediated m6A Demethylation

Fat mass and obesity-associated protein (FTO) is well known to associate with obesity and to remove N6-methyladenosine (m6A) methylation. However, little is known about the regulatory mechanisms of FTO in response to the metabolic environments. Here, we show that NADP and NAD directly binds FTO, increases FTO-mediated m6A oxidation, and enhances adipogenesis. To identify the candidate metabolite(s) directly binding and potentially regulating FTO, we applied metabolite affinity responsive target fluorescence quenching (MARTFQ) to screen a set of obesity related metabolites. The MARTFQ assay is based on the biophysical principle of ligand-induced intrinsic fluorescence quenching of target proteins. Using MARTFQ, we identified NADPH showing the most remarkable ability to bind FTO. Solo NADP(H) or NAD(H) showed more significant ability than ascorbic acid (Vc) to facilitate FTO to demethylate RNA m6A in vitro. And increase of NADP decreased RNA m6A in multi cell lines through enhancing FTO activity. Anti-m6A methylated RNA immunoprecipitation sequencing indicated that NADP-regulated m6A methylated genes showed the significant enrichment in “Transcriptional regulation of white adipocyte differentiation” and “Chromatin modifying enzymes”. Further analyses demonstrated that NADP or high glucose treatment enhanced adipogenesis at least partly through increasing FTO activity. These results shed light on the regulatory mechanism of obesity-associated m6A demethylase FTO in response to metabolic environment.