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 FTOs 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 structural insights into how FTO selectively catalyzes m6Am versus m6A demethylation on mRNA, suggests why FTO is sensitive to different 5' cap modifications, and furthers our understanding of how FTO activity is regulated by diverse mechanisms to help control the epitranscriptome.