FTO demethyltion of N6-methyladenosine depends on the RRACH motif [CLIP-seq]

The fat mass and obesity-associated (FTO) protein is a well-characterized demethylase that removes N6-methyladenosine (m6A) from animal mRNAs. However, it is unclear yet how the demethylation operates in living cells. In this study, we applied genome-wide approaches to study how FTO finds its demethylation targets in human cells. We overexpressed FTO in human HeLa cells, demonstrating that FTO effectively removes m6A from the RRACH motif enriched in the 3’UTR regions and leaves m6A at other motifs unaffected. RRACH elements are clearly enriched at FTO binding sites; however, m6A has a lower tendency to be removed from the FTO-bound RRACH. Taken together with the experimental validaton results, we propose a model in which FTO effectivly recognizes m6A-containing RRACH motifs in RNAs, leading to a faster demethylation and dissociation kinetic than the association, and consequently undectable FTO-mRNA association. However, when FTO binds to the non-m6A RRACH motif, the binding has a lower dissociation kinetics, yielding the detectable FTO binding signals which would enable FTO to have roles in regulating other mRNA processing events. CLIP-seq analysis were performed with anti-FTO antibody in wide-type Hela cells for two biological replicates, as well as in FTO-overexpression Hela cells with anti-Flag antibody. Human FTO gene (GenBank: NM_001080432.2) encoding full length FTO was amplified by PCR and subcloned into pCMV-Tag.2B-flag vector (Stratagene), so FTO–RNA complex is purified by immunoprecipitation by incubating with DynaBeads protein G conjugated with anti-Flag antibody.