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.