RNA demethylase FTO uses conserved aromatic residues to recognize the mRNA 5' cap and promote efficient m⁶A_m...

The RNA demethylase FTO acts as a methyl 'eraser' to remove either internal N ⁶-methyladenosine (m⁶A) or 5' end N ⁶-2'-O-dimethyladenosine (m⁶A_m) modifications on mRNA. FTO has an intrinsic preference and significantly faster demethylation rates in vitro for m⁶A_m modifications located at the 5' mRNA cap structure, but the structural basis for FTO's ability to discriminate m⁶A versus m⁶A_m 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 m⁶A_m demethylation. We also identify a nonpolar surface on FTO that interacts with the 2'-O-methyl group of m⁶A_m to impact demethylation kinetics. This work provides the first structural insights into how FTO selectively catalyzes m⁶A_m 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.