RNA demethylase FTO uses conserved aromatic residues to recognize the mRNA 5' cap and promote efficient m6Am demethylation.

The RNA demethylase FTO acts as a methyl 'eraser' to remove either internal N6-methyladenosine (m6A) or 5' end N6-2'-O-dimethyladenosine (m6Am) modifications on mRNA. FTO has an intrinsic preference and significantly faster demethylation rates in vitro for m6Am modifications located at the 5' mRNA cap structure, but the structural basis for FTO's ability to discriminate m6A versus m6Am modifications has remained unknown. Here we utilize molecular dynamics simulations of FTO-RNA cap complexes to identify conserved aromatic residues on the surface of FTO involved in 5' cap recognition. Subsequent mutagenesis and enzymology experiments validate the specificity of these residues in engaging the 5' cap structure to promote m6Am demethylation. We also identify a nonpolar surface on FTO that interacts with the 2'-O-methyl group of m6Am to impact demethylation kinetics. This work provides the first structure-level insights into how FTO selectively catalyzes m6Am versus m6A demethylation on mRNA and advances our understanding of how FTO activity is regulated by diverse mechanisms to help control the epitranscriptome.