Related Experiment Videos
Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates
Xiao Zhang1, Lian-Huan Wei1, Yuxin Wang2
1Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Abstract:
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.
Insights
The FTO enzyme demethylates various RNA and DNA molecules, including N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6A). This study reveals FTO
Area of Science:
- Biochemistry and Molecular Biology
- Epigenetics and RNA Modifications
Background:
- FTO (Fat mass and obesity-associated protein) is a demethylase enzyme with known activity on N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6A_m) in RNA, and N1-methyladenosine (m1A) in tRNA.
- Evidence suggests FTO can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U) in DNA, but the precise mechanisms of substrate recognition and catalysis remain unclear.
Purpose of the Study:
- To elucidate the molecular basis of FTO's broad substrate specificity and catalytic mechanisms for diverse RNA and DNA modifications.
- To provide structural insights into FTO's interaction with N6-methyladenosine (m6A) and related methylated nucleobases.
Main Methods:
- In vitro and in vivo demethylation assays to assess FTO activity on various substrates.
- X-ray crystallography to determine the structure of human FTO bound to 6mA-modified single-stranded DNA (ssDNA).
Main Results:
- FTO exhibits extensive demethylation activity on both internal m6A and cap m6A_m in mRNA.
- Crystal structure reveals FTO's interaction with 6mA-modified ssDNA, highlighting N6-methyladenine as the most favorable nucleobase substrate.
- FTO displays similar demethylation activity on internal m6A and m6A_m within the same RNA sequence, indicating substrate specificity is primarily driven by nucleobase interactions within the catalytic pocket.
Conclusions:
- FTO's substrate specificity is largely determined by interactions with the nucleobase, not the ribose ring, explaining its activity on multiple m6A-related substrates.
- RNA sequence and tertiary structure can influence FTO's catalytic efficiency.
- Findings provide a structural foundation for understanding FTO's demethylation mechanisms and designing targeted chemical probes or therapeutics.