| 研究実績の概要 |
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.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
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.
|
