2018 Fiscal Year Annual Research Report
Generation and characterization of animal models deficient in RNA methylation.
| Project/Area Number |
17H06257
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| Allocation Type | Single-year Grants |
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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, 准教授 (50711818)
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| Project Period (FY) |
2017-06-30 – 2021-03-31
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| Keywords | circadian / RNA methylation / ck1d |
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| Outline of Annual Research Achievements |
In 2018, we have published two PNAS papers (Fustin JM et al.; Narasimamurthy R et al.) reporting the regulation of Casein kinase 1 delta (Ck1d) expression by m6A mRNA methylation. CRISPR-Cas9-mediated deletion of the methylated locus of Ck1d 3'-UTR in vivo was sufficient to cause an increase in Ck1d expression and a change in the circadian rhythms of the mouse. Interestingly, these investigations lead to the unexpected discovery of two alternatively spliced isoforms of Ck1d, which we decided to focus on, and discovered that they have antagonistic function in the regulation of the circadian clock. While Ck1d1, the canonical isoform, functions as previoulsy reported by phosphorylation its canonical target PER2, leading to its degradation and the acceleration of the circadian clock, Ck1d2 is a new isoform that also phosphorylates PER2 but on a different residue that instead stabilizes PER2 and lead to the lengthening of the circadian period. We performed these investigations in collaboration with Prof David Virshup in DukeNUS in Singapore.
We have generated Ck1d2 knock-out animals, as well as Ck1d1 and Ck1d2 knock-in animals are are currently purifying the genetic background before characterization of the phenotypes.
We have succeeded in generating Mettl3 Ko adult animals using an alternative strategy outlined in the project proposal. We are now using a tamoxifen-induced CRE-recombinase expressed ubiquitously. Animals from 12-weeks old are provided with tamoxifen-containing food. We are currently investigating the phenotype of these animals.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Since tissue-specific Mettl3 knock-out mice were embryonic lethal, we chose a different alternative based on the induction of whole-body Mettl3 knock-out using tamoxifen-induced CRE recombinase. Animals have been obtained successfully and are currently being characterized.
We have identified candidate RNA-binding proteins that regulate Ck1d expression in a m6A-dependent manner and are investigating their involvement in the regulation of the circadian clock. Moreover, we have initiated a collaboration with the laboratory of Prof Katahira in Kyoto University to identify m6A-dependent secondary structures in Ck1d mRNA.
The colonies of Ck1d2 knock-out animals, as well as Ck1d1 and Ck1d2 knock-in animals, are currently being amplified to allow further investigations.
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| Strategy for Future Research Activity |
1: Investigate the regulation of Ck1d by candidate RNA-binding proteins.
2: Investigate m6A-dependent Ck1d mRNA secondary structure.
3: Investigate Mettl3 KO mice circadian phenotype.
4: Generate cell lines expressing only CK1D1 or CK1D2 (using Ck1d KO mouse embryonic fibroblasts)
5: Characterize the circadian phenotype of Ck1d2 knock-out, Ck1d1 and Ck1d2 knock-in animals.
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Research Products
(17 results)
