Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates

Significance The RNA modification N6-methyladenosine (m6A) was the first physiological substrate of FTO to be discovered. Recently, cap N6,2′-O-dimethyladenosine (m6Am), internal m6Am, and N1-methyladenosine were also found to be physiological substrates of FTO. However, the catalytic mechanism through which FTO demethylates its multiple RNA substrates remains largely mysterious. Here we present the first structure of FTO bound to N6-methyldeoxyadenosine–modified ssDNA. We show that N6-methyladenine is the most favorable nucleobase substrate of FTO and that the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding FTO’s catalytic mechanism for the demethylation of multiple RNA substrates and shed light on the mechanism through which FTO is involved in diseases or biological processes. FTO demethylates internal N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate—in vitro and in vivo—that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am. Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.