The dynamics of FTO binding and demethylation from the m<sup>6</sup>A motifs

<p><i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A) is considered as a reversible RNA modification occurring more frequently on the GAC than AAC context <i>in vivo</i>, which regulates post-transcriptional gene expression in mammalian cells. m<sup>6</sup>A ‘writers’ METTL3 and METTL14 demonstrate a strong preference for binding AC-containing motifs in living cells. However, this evidence is currently lacking for m<sup>6</sup>A erasers, leaving the dynamics of the internal m<sup>6</sup>A 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 m<sup>6</sup>A ‘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 m<sup>6</sup>A motif-containing RNA sites. FTO overexpression specifically removed m<sup>6</sup>A 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 m<sup>6</sup>A dynamics and the FTO plasticity in biological functions and diseases.</p>