Diazirine photocrosslinking recruits activated FTO demethylase complexes for specific N(6)-methyladenosine recognition.
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.
†Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Hyun Seok
HS
Jeong
‡Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Gosuke
G
Hayashi
†Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.|‡Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Akimitsu
A
Okamoto
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000071516:Alpha-Ketoglutarate-Dependent Dioxygenase FTO; D003432:Cross-Linking Reagents / Q000737:chemistry; D003978:Diazomethane / Q000737:chemistry*; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry; D055668:Photochemical Processes; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D003432:Cross-Linking Reagents; D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D011506:Proteins; D011994:Recombinant Proteins; D003978:Diazomethane; D012313:RNA; C010223:N-methyladenosine; D000071516:Alpha-Ketoglutarate-Dependent Dioxygenase FTO; C526492:FTO protein, human; D000241:Adenosine