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

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• The RNA demethylase FTO removes N6-methyladenosine (m⁶A) and cap-associated N6,2 -O-dimethyladenosine (m⁶A m) modifications.
• The molecular mechanisms governing FTO's substrate selectivity and the distinct cellular impacts of m⁶A versus m⁶A m demethylation are not fully understood.

Purpose of the Study:

• To engineer FTO separation-of-function mutants for selective demethylation of m⁶A or m⁶A m RNA modifications.
• To investigate the roles of specific FTO active site residues in substrate selectivity.
• To provide tools for studying the biological significance of distinct RNA demethylation events.

Main Methods:

• Site-directed mutagenesis was used to create FTO variants, including L203A, H232A, and W278A.
• Enzyme activity assays were performed to assess the demethylation capabilities of wild-type and mutant FTO proteins on m⁶A and m⁶A m substrates.
• The selectivity of engineered FTO variants for m⁶A versus m⁶A m was evaluated.

Main Results:

• The FTO L203A mutant selectively demethylates m⁶A m while retaining impaired m⁶A demethylation.
• The FTO H232A/W278A double mutant selectively demethylates m⁶A with substantially impaired m⁶A m demethylation.
• These complementary FTO variants represent the first engineered mutations to shift demethylation selectivity between m⁶A and m⁶A m.

Conclusions:

• Engineered FTO variants provide unprecedented selectivity for m⁶A or m⁶A m RNA demethylation.
• These tools enable selective enzymatic modification removal in vitro for sequencing applications.
• The findings facilitate deeper understanding of FTO-mediated demethylation in cellular and disease models.