Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms.

An intimate link exists between circadian clocks and metabolism with nearly every metabolic pathway in the mammalian liver under circadian control. Circadian regulation of metabolism is largely driven by rhythmic transcriptional activation of clock-controlled genes. Among these output genes, Nocturnin (Noct) has one of the highest amplitude rhythms at the mRNA level. The Noct gene encodes a protein (NOC) that is highly conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, but highly purified NOC possesses little or no ribonuclease activity. Here, we show that NOC utilizes the dinucleotide NADP(H) as a substrate, removing the 2′ phosphate to generate NAD(H), and is a direct regulator of oxidative stress response through its NADPH 2′ phosphatase activity. Furthermore, we describe two isoforms of NOC in the mouse liver. The cytoplasmic form of NOC is constitutively expressed and associates externally with membranes of other organelles, including the endoplasmic reticulum, via N-terminal glycine myristoylation. In contrast, the mitochondrial form of NOC possesses high-amplitude circadian rhythmicity with peak expression level during the early dark phase. These findings suggest that NOC regulates local intracellular concentrations of NADP(H) in a manner that changes over the course of the day.

No abstract

Summary Fine-tuning of transcriptional responses can be critical for long-term outcomes in response to an environmental challenge. The circadian protein Nocturnin belongs to a family of proteins that include exonucleases, endonucleases, and phosphatases and is most closely related to the CCR4 family of deadenylases that regulate the cellular transcriptome via control of poly(A) tail length of RNA transcripts. In this study, we investigate the role of Nocturnin in regulating the transcriptional response and downstream metabolic adaptations during cold exposure in brown adipose tissue. We find that Nocturnin exhibits dual localization within the cytosol and mitochondria, and loss of Nocturnin causes changes in expression of networks of mRNAs involved in mitochondrial function. Furthermore, Nocturnin −/− animals display significantly elevated levels of tricarboxylic acid cycle intermediates, indicating that they have distinct metabolic adaptations during a prolonged cold exposure. We conclude that cold-induced stimulation of Nocturnin levels can regulate long-term metabolic adaptations to environmental challenges.

The circadian rhythm is an evolutionarily conserved process that drives rhythmic behavior and physiology with a period of approximately 24 hours. Circadian rhythms are generated by the heterodimeric transcription factors CLOCK/BMAL1. CLOCK/BMAL1 binds to E‐boxes across the genome and drives the transcription of many genes, including their own repressors Per and Cry. Transcription activation by CLOCK/BMAL1 and its inhibitory feedback loop generated by PER/CRY result in the rhythmic expression of thousands of clock‐controlled genes. Nocturnin (mNOC) is among those genes regulated by CLOCK/BMAL1. mNOC is highly expressed in the liver, where its expression is at maximum in the evening and minimum in the morning. Compared to WT mice, mNOC KO mice exhibit a resistance to high‐fat diet (HFD) induced obesity. Protein sequence analysis reveals that mNOC contains an EEP domain that is conserved in the CCR4 family, whose members function as deadenylases. Interestingly, mNOC also contains a putative mitochondrial targeting signal (MTS) in between two translation initiation sites. To investigate if mNOC does localize to the mitochondria, we performed immunocytochemistry in HEK293 cells overexpressing mNOC that does or does not code for the MTS; and performed a mitochondrial fractionation followed by proteinase k protection assay. To examine mNOC's function in the mitochondria, we overexpressed mNOC in HEK293 cells and performed RT‐qPCR to examine mitochondrial RNA stability. We found that mNOC is able to localize to the mitochondria, but we did not see changes in mitochondrial RNA levels. Further experiments will help conclude if mNOC is targeting mitochondrial translation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.