2014 Fiscal Year Research-status Report
Role of RNA methylation in the regulation of gene expression.
| Project/Area Number |
26870283
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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 | RNA methylation / Circadian clock |
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| Outline of Annual Research Achievements |
In this project we are investigating the link between RNA methylation and the circadian clock. We have shown in our previous investigations (Fustin et al., 2013) that inhibiting RNA methylation at the N6 position of internal adenosine residues (m6A) caused the elongation of the circadian period together with a delay in clock gene transcripts processing (RNA export and RNA half-life). This current project aims at further understanding RNA methylation and how it regulates gene expression as well as how it is regulated by the circadian clock. Many questions remains, such as how m6A RNA methylation regulates RNA splicing, processing and translation. These mechanisms are likely to be transcript specific.Moreover, we are seeking to understand what is the tissue specific physiological function of m6A RNA methylation.
We are especially focussing on two points. Firstly, we are measuring variations in RNA methylation in the liver across circadian time. Since many aspect of RNA processing are under circadian control, we surmise that m6A RNA methylation may also shows circadian changes. We are currently analysing the first dataset of m6A RNA methylation of hepatic mRNA sampled every 4 hours across 24 hours.
Secondly, to understand the physiological function of m6A, we are currently establishing mouse colonies deficient in the RNA m6A methyltransferase Mettl3 and in the m6a demethylases Fto and Alkbh5.
We have made reasonable progress on both side of our investigations.
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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
Measuring m6A RNA methylation in the liver across circadian time has proven a challenging undertaking. Establishing the method for reliable quantification of m6A by m6A RNA immunoprecipitation followed by RNA sequencing has taken the most part of last year. The main difficulty was to obtain sufficient sensitivity in RNA sequencing using very limited amounts of methylated RNA, and to scale up RNA immunoprecipitation in order to obtain enough methylated mRNA for analysis. We have recently succeeded in collecting our first dataset, and we can now analyse variations (if any) of m6A RNA methylation and select candidates for further investigations.
The other side of the project is progressing smoothly, since we are now breeding Alkbh5 and Fto knock-out mice in order to obtain double Alkbh5/Fto knock-out. Double knock-out will be required since preliminary results with Alkbh5 revealed and absence of circadian phenotype. However, Alkbh5 knock-out mice show low fertility, limiting breeding efficiency.
We have also obtained chimera mice for a conditional knock-out allele of Mettl3, that are currently being mated for germline transmission.
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| Strategy for Future Research Activity |
We are planning to pursue our investigations on the link between RNA methylation and the circadian clock by first confirming our results on m6A RNA immunoprecipitation/RNA seq results with liver mRNA sampled across circadian time. We will then further analyse interesting candidates and develop the next steps accordingly. We will further investigate how the identified m6A sites in candidate transcripts regulate their expression via RNA processing and what is the physiological relevance of such m6A sites.
Upon establishement of a stable colony of Mettl3 conditional knock-out mice, we will continue this work by breeding these mice to liver- and/or brain-specific CRE-expressing mice for the creation of tissue-specific Mettl3 knock-out mice, since it is already known the lack of Mettl3 in the whole body is embryonic lethal. These mice will then be analysed for circadian parameters.
The breeding and characterisation of Fto knock-out first allele mice will also continue in parallel. We will first analyse the phenotype of whole-body Fto knock-out mice. Fto knock-out mice are likely to be weak and, like Alkbh5, have low fertility. Therefore, we will also establish liver- and/or brain-specific Fto knock-out mice by breeding our knock-out first allele with ubiquitous flippase expressing mice (CAG-FLP) to generate conditional KO Fto allele. Conditional KO mice will then be bred to CRE-expressing mice to generate tissue-specific Fto KO mice, which will then be characterised.
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| Causes of Carryover |
Unfortunately it was not possible to completely use the funds last year due to small variations in consumable prices because of price reduction or campaign.
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| Expenditure Plan for Carryover Budget |
We are planning to use 1,904円 as a part of this year's grant to purchase equipment and various consumables.
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Research Products
(6 results)
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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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