2018 Fiscal Year Research-status Report
Translation control of circadian rhythms
| Project/Area Number |
18K14755
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
MILLIUS ARTHUR 国立研究開発法人理化学研究所, 生命機能科学研究センター, 国際特別研究員 (80624858)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Ribosomal profiling / Circadian rhythms / Period2 / Translational Repression / uORFs / RNA |
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| Outline of Annual Research Achievements |
Defects in circadian rhythms are related to health problems, such as obesity, cancer, and depression, and understanding circadian RNA is important for developing new treatments. Most mammalian proteins have circadian cycles of production and degradation, but de novo transcription is only responsible for a small fraction of this rhythmicity. We used ribosomal profiling to understand RNA translation in liver from circadian-entrained mice that were transferred to constant darkness conditions over a 24-h period. We compared translation levels to absolute protein levels from 20 selected circadian proteins using mass spectrometry. We observed a delay between translation and peak protein levels for several circadian genes and a wide range of translation efficiencies. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs including the core clock gene Per2. Increased uORFs in 5' UTRs (but not uORF length or position) decreased ribosome binding in downstream ORFs and reduced expression of synthetic reporter constructs in vitro and in single cells. Mutation of the Per2 uORF increased luciferase reporter expression in 3T3 cells, and genomic Per2 uORF mutation using CRISPR/Cas9 increased PER2 expression in PER2:Luc MEFs. These results suggest that uORFs generally repress translation and that the uORF in Per2 is important for high-amplitude circadian rhythms.
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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
We are using Crispr/Cas9 genetic editing to mutate the Per2 uORF at the endogenous locus in PER2:LUC reporter MEF cells. In addition, we are mutating wild-type mice using Crispr/Cas9 to determine how mutation of the Per2 uORF affects behavioral rhythms. We discovered four homozygous mutant mice (two males and two females) in the F0 generation and are planning to backcross these mice to wild-type mice and to each other to generate homozygous mice to phenotype. Progress has proceeded more smoothly than initially planned because we developed international collaborations with researchers in Japan and Australia to facilitate the data analysis and mice phenotype parts of the study.
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| Strategy for Future Research Activity |
We plan to phenotype the homozygous Per2 mutant mice for activity rhythms and expression of Per2 in the liver and brain. We plan cross to cross the mice to activity reporter mice, such as Bmal1-Luc mice, to determine the effect of mutant Per2 expression on classic clock machinery and perform RNA sequencing and ribosomal profiling to determine how transcript expression and translation is altered by altered Per2 production. We hypothesize that mutant mice will be less resistant to jet-lag and will conduct artificial jet-lag experimental. We plan to conduct in silico screening to identify candidate RNA-binding proteins (RBPs) that may alter translation, determine which RBPs are circadian in the liver, and use RNA structure sequencing to determine where RBPs might bind RNA.
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| Causes of Carryover |
I transferred my affiliation from RIKEN to iFReC at Osaka University on April 1, 2019 to collaborate more closely with the labs of Daron Standley and Shizuo Akira. Professor Standley is an expert in analyzing next-generation sequencing data and Professor Akira is a world-renown immunologist who has discovered an RNA-degrading enzyme called Regnase 1, which is important for immune cell activation. I am planning to develop RNA structure sequencing in collaboration with both labs as well as use my newly developed ribosomal profiling technique to understand how the Regnase family of proteins alter the translation of RNA transcripts. Therefore, I reserved some of the budget from FY2018 to use for starting these projects in the Akira and Standley labs in FY2019.
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