論文の公開元へStudies on Circadian Clock RNA Methylation and Micturition Rhythm
概要
The circadian clock is composed of clock genes interlocked in transcriptional/post-translational feedback loops (TTFLs) regulating their own expression. The discovery of the basic molecular mechanism of TTFLs at 1984 won the 2017 Nobel Prize in Physiology of Medicine. In the decades since this discovery, most research on TTFLs has focused primally on two spatially and temporally separated processes: “transcription” and “(post)translation”. Yet, how these two processes are connected via RNA level regulation has been poorly understood. Our laboratory found that RNA methylation is necessary for the proper function of the circadian clock, and that when RNA methylation is inhibited, the period length of the circadian clock becomes longer. However, the molecular mechanism underlying this phenomenon is not fully elucidated.
At the physiological level, the oscillations of the core clock genes generated by TTFLs are output as changes in locomotor activity, body temperature, metabolisms, etc. in a cycle of about 24 hours. Historically, recording methods of locomotor activity rhythm and body temperature rhythm have led to great advances in the study of circadian physiology. In addition, the development of the metabolic cage made it possible to measure energy consumption rhythm. However, methods for recording renal excretion and reabsorption rhythms are not well established and new methods with high accuracy are awaiting development.
In Chapter 1, the relationship between the circadian clock and RNA methylation was investigated at the molecular level. Particularly, I focused on studying N6-methyladenosine (m6A), known as the most prevalent internal base modification of mRNA for mammals. I found that the transcripts of a key circadian clock component, Casein kinase 1 delta (Csnk1d), possess an m6A modification in the 3′-untranslated region (3′-UTR). Reduced expression of the m6A writer enzyme Mettl3 led to the upregulation of Csnk1d protein expression, suggesting that the m6A modification negatively regulates Csnk1d translation. To test the importance of m6A of Csnk1d in vivo, I generated mutant mice with a deletion in m6A locus of Csnk1d. The result of locomotor activity measurement demonstrates that the m6A locus is required for the proper oscillation of the circadian clock. Furthermore, I show that two Csnk1d alternative splicing isoforms, Csnk1d1 and Csnk1d2 which have not been characterized so far, exhibit mutually antagonistic functions in the regulation of the circadian clock.
In Chapter 2, I invest my effort to the development of a new system for measuring the circadian rhythm of renal excretion and reabsorption. More specifically, I revised a previously established method, automated Voided Stain on Paper (aVSOP). This improved method allows simultaneous measurement of animal behavior and micturition rhythms, which could not be done so far. Using this system, I monitored simultaneously the locomotor rhythm and micturition rhythm under an experimental jet-lag condition, where the circadian clock function is temporarily impaired.
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