FTO separation-of-function mutations alter m⁶A versus m⁶A_m demethylation selectivity on RNA

The RNA demethylase FTO erases N6-methyladenosine (m⁶A) and cap-associated N6,2'-O-dimethyladenosine (m⁶A_m) modifications. However, the molecular basis of its substrate selectivity and the biological effects of m⁶A versus m⁶A_m demethylation in cells remain poorly understood. Here we report two engineered FTO separation-of-function mutants to selectively demethylate either m⁶A or m⁶A_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 m⁶A demethylation but retained wild-type levels of m⁶A_m demethylation, and that FTO L203A could function as a selective m⁶A_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 m⁶A modifications while exhibiting substantially impaired m⁶A_m demethylation, making it a selective m⁶A demethylase. Together, these complementary FTO variants represent the first set of engineered mutations that shift FTO demethylation selectivity between m⁶A and m⁶A_m substrates. These tools enable selective enzymatic removal of m⁶A or m⁶A_m modifications in vitro for sequencing applications, and may facilitate understanding of FTO-mediated m⁶A versus m⁶A_m demethylation in cellular and disease model systems.