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Updated: Apr 16, 2026
A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Diazirine photocrosslinking recruits activated FTO demethylase complexes for specific N(6)-methyladenosine
Hyun Seok Jeong1, Gosuke Hayashi2, Akimitsu Okamoto1,2
1†Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Abstract:
N(6)-methyladenosine (m(6)A) is a prevalent modification of RNAs. m(6)A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation. We applied diazirine photocrosslinking to the event of m(6)A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m(6)A on the RNA successfully recruited activated FTO complexes with an m(6)A preference. The process of recognition of m(6)A via FTO using diazirine photocrosslinking was controlled by the α-ketoglutarate (α-KG) cosubstrate and the Fe(II) cofactor, which are involved in m(6)A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m(6)A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m(6)A recognition and provides new insights into the dynamic FTO oxidative demethylation process.
Insights
Researchers used diazirine photocrosslinking to study N(6)-methyladenosine (m(6)A) recognition by the FTO demethylase. This method captured FTO complexes, revealing insights into m(6)A demethylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Epigenetics
Background:
- N(6)-methyladenosine (m(6)A) is a key RNA modification influencing mRNA biological pathways and epigenetic regulation.
- The fat mass and obesity associated (FTO) protein is a demethylase that targets m(6)A modifications.
Purpose of the Study:
- To investigate the mechanism of m(6)A recognition by the FTO demethylase.
- To explore the role of diazirine photocrosslinking in studying FTO-m(6)A interactions.
Main Methods:
- Application of diazirine photocrosslinking to RNA containing m(6)A.
- Recruitment of activated FTO complexes to m(6)A-modified RNA.
- Analysis of FTO binding to single-stranded RNAs (ssRNAs) before m(6)A recognition.
Main Results:
- Diazirine photocrosslinking successfully recruited FTO complexes with a preference for m(6)A.
- The recognition process was influenced by the α-ketoglutarate (α-KG) cosubstrate and Fe(II) cofactor.
- FTO was observed to bind to ssRNAs preceding m(6)A recognition.
Conclusions:
- Diazirine photocrosslinking enhances the capture of FTO complexes involved in specific m(6)A recognition.
- This technique provides novel insights into the dynamic process of FTO-mediated oxidative demethylation of m(6)A.
- Understanding FTO's m(6)A recognition mechanism is crucial for RNA epigenetic regulation studies.
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