2014 Fiscal Year Annual Research Report
細胞内メチレーションリズムと時計遺伝子mRNA制御のクロストークの分子基盤
Publicly Offered Research
| Project Area | Crosstalk of transcriptional control and energy pathways by hub metabolites |
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| Project/Area Number |
26116713
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| Research Institution | Kyoto University |
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Principal Investigator |
FUSTIN JM 京都大学, 薬学研究科(研究院), 講師 (50711818)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | Circadian / RNA methylation |
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| Outline of Annual Research Achievements |
In our previous investigations we have demonstrated that RNA methylation regulates the circadian clock. We have used pharmacological and siRNA-mediated inhibition of internal N6-adenosine (m6A) and cap N7-guanosine methylation of mRNA, and showed that without these methylations the period of the circadian clock became longer compared to control. This relationship between RNA methylation and the circadian clock was confirmed in vitro and in vivo. Moreover, we also showed m6A sites within clock genes transcripts, suggesting m6A directly regulated clock gene expression.
In our current investigations we have made good progress in characterising further these mechanisms. Not only we have optimised our protocol for m6A-RNA immunoprecipitation followed by RNA-sequencing of liver mRNA, sampled every four hours during a complete day in order to measure daily variations in m6A methylation. With our collaborators, Prof Manabe Ichiro from the Department of Cardiovascular Medicine at The University of Tokyo Graduate School of Medicine and Prof. Morioka Suimye Masaki from the Department of Bioinformatics at the Tokyo Medical and Dental University, we have recently succeeded in obtaining our first comprehensive circadian survey of m6A RNA methylation and are currently analysing the data.
On the other hand, we have obtained the first chimera mice for the conditional knock-out of the m6A methylase Mettl3, and are currently breeding total KO mice for Fto and Alkbh5, the two m6A demethylases identified to date.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
As explained above our investigation are progressing rather smoothly.
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| Strategy for Future Research Activity |
Starting from now we are seeking to confirm our circadian m6A quantification of the liver transcriptome, and will continue with further analysis of candidate transcripts that may be regulated in a circadian manner via m6A RNA methylation.
We will also systematically identify the mechanism linking m6A RNA methylation with the control of the circadian clock by identifying which core clock transcripts are the most methylated, and what is the role of m6A RNA methylation in the regulation of their expression.
We will analyse the circadian and metabolic phenotypes of Mettl3, Alkbh5 and Fto knock-out mice. Mettl3 total KO mice are dying in utero, which means it will be necessary to use conditional Mettl3 KO mice. Upon germline transmission of our conditional Mettl3 KO allele we will then breed these mice with mice expressing CRE recombinase either specifically in the liver or in the suprachiasmatic nucleus, then analyse the phenotype of tissue-specific Mettl3 KO mice thereby obtained, mainly in circadian locomotor activity behaviour and liver physiology and metabolism. In parallel, colonies of Alkbh5 and Fto knock-out mice, as well as Fto/Alkbh5 double knock-out mice will be amplified and characterised.
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[Journal Article] Isoform- specific monoclonal antibodies against 3β- hydroxysteroid dehydrogenase/ isomerase family provide markers for subclassification of human primary aldosteronism.2014
Author(s)
Doi M, Satoh F, Maekawa T, Nakamura Y, Fustin JM, Tainaka M, Hotta Y, Takahashi Y, Morimoto R, Takase K, Ito S, Sasano H, Okamura H.
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Journal Title
J Clin Endocrinol Metab.
Volume: 99(2)
Pages: 257-62
DOI
Peer Reviewed
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