FTO separation-of-function mutations alter m ⁶ A versus m ⁶ A _m demethylation selectivity on RNA

The RNA demethylase FTO erases N6-methyladenosine (m ⁶ A) and cap-associated N6,2′-O-dimethyladenosine (m ⁶ A _m ) modifications. However, the molecular basis of its substrate selectivity and the biological effects of m ⁶ A versus m ⁶ A _m demethylation in cells remain poorly understood. Here we report two engineered FTO separation-of-function mutants to selectively demethylate either m ⁶ A or m ⁶ A _m modifications on RNA. While investigating the propensity of FTO active site residues to undergo self-hydroxylation, we found that mutations of FTO residue L203 resulted in impaired m ⁶ A demethylation but retained wild-type levels of m ⁶ A _m demethylation, and that FTO L203A could function as a selective m ⁶ A _m demethylase. Conversely, building on our recent work that identified conserved aromatic residues on FTO involved in mRNA 5′ cap recognition, we found that the FTO H232A/W278A double mutant efficiently demethylates m ⁶ A modifications while exhibiting substantially impaired m ⁶ A _m demethylation, making it a selective m ⁶ A demethylase. Together, these complementary FTO variants represent the first set of engineered mutations that shift FTO demethylation selectivity between m ⁶ A and m ⁶ A _m substrates. These tools enable selective enzymatic removal of m ⁶ A or m ⁶ A _m modifications in vitro for sequencing applications, and may facilitate understanding of FTO-mediated m ⁶ A versus m ⁶ A _m demethylation in cellular and disease model systems.