The dynamics of FTO binding and demethylation from the m6A motifs

N6-methyladenosine (m6A) is considered as a reversible RNA modification occurring more frequently on the GAC than AAC context in vivo, which regulates post-transcriptional gene expression in mammalian cells. m6A ‘writers’ METTL3 and METTL14 demonstrate a strong preference for binding AC-containing motifs in living cells. However, this evidence is currently lacking for m6A erasers, leaving the dynamics of the internal m6A modification under debate recently. We analysed three recently published FTO CLIP-seq data sets and two generated in this study, one of the two known m6A ‘erasers’. FTO binding peaks from all cell lines contain RRACH motifs. Only those from K562, 3T3-L1and HeLa cells were enriched in AC-containing motifs, while those from HEK293 were not. The exogenously overexpressed FTO effectively binds to m6A motif-containing RNA sites. FTO overexpression specifically removed m6A modification from GGACU and RRACU motifs in a concentration-dependent manner. These findings underline the dynamics of FTO in target selection, which is predicted to contribute to both the m6A dynamics and the FTO plasticity in biological functions and diseases.