The mammalian circadian system consists of a central oscillator in the suprachiasmatic nucleus of the hypothalamus, which coordinates peripheral clocks in organs throughout the body. Although circadian clocks control the rhythmic expression of a large number of genes involved in metabolism and other aspects of circadian physiology, the consequences of genetic disruption of circadiancontrolled pathways remain poorly defined. Here we report that the targeted disruption of Nocturnin (Ccrn4l) in mice, a gene that encodes a circadian deadenylase, confers resistance to dietinduced obesity. Mice lacking Nocturnin remain lean on high-fat diets, with lower body weight and reduced visceral fat. However, unlike lean lipodystrophic mouse models, these mice do not have fatty livers and do not exhibit increased activity or reduced food intake. Gene expression data suggest that Nocturnin knockout mice have deficits in lipid metabolism or uptake, in addition to changes in glucose and insulin sensitivity. Our data support a pivotal role for Nocturnin downstream of the circadian clockwork in the posttranscriptional regulation of genes necessary for nutrient uptake, metabolism, and storage.C ircadian clocks are present in most tissues of the body, where they control the expression of 5-10% of the tissue-specific mRNAs through both transcriptional and posttranscriptional regulation (1, 2). The widespread importance of circadian clock regulation is evident in that generalized disruption of normal clock function results in tumor formation, sleep disorders, and metabolic problems (reviewed in refs. 3 and 4). For example, mutations in the central clock genes Clock or Bmal1 result in metabolic changes found in obesity and the metabolic syndrome (5-8), and numerous genes involved in fatty acid, cholesterol, and glucose metabolism in liver are regulated in circadian or diurnal patterns (9-15), indicating that the clock plays a broad role in regulating metabolism. Nonetheless, the large number of genes, metabolic pathways, and cell/tissue types that are under general circadian control impose a major challenge in understanding the molecular details. Further advances in this area require refined understanding of the specific circadian output pathways by which the clocks regulate physiology.For cycling mRNAs to closely reflect daily rhythmic transcriptional drive, their half-lives must be relatively short. There are several examples of rhythmic posttranscriptional regulation in which the mRNA half-life or adenylation state changes over the course of the day (16-19), but very little is known about the mechanisms responsible. A likely contributor is Nocturnin (Ccrn4l, Noc), which has been implicated in the posttranscriptional regulation of mRNA stability and/or translatability by the circadian clock (20). Noc is expressed rhythmically in many tissues, with particularly high-amplitude rhythms in liver where mRNA levels are increased 100-fold in early night (21). Noc is at a pivotal position to play a role in shaping the rhythmic pattern of gene ...

Poly(A) tails are 39 modifications of eukaryotic mRNAs that are important in the control of translation and mRNA stability. We identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. Approximately 80% of these are primarily the result of nuclear adenylation coupled with rhythmic transcription. However, unique decay kinetics distinguish these mRNAs from other mRNAs that are transcribed rhythmically but do not exhibit poly(A) tail rhythms. The remaining 20% are uncoupled from transcription and exhibit poly(A) tail rhythms even though the steady-state mRNA levels are not rhythmic. These are under the control of rhythmic cytoplasmic polyadenylation, regulated at least in some cases by cytoplasmic polyadenylation element-binding proteins (CPEBs). Importantly, we found that the rhythmicity in poly(A) tail length is closely correlated with rhythmic protein expression, with a several-hour delay between the time of longest tail and the time of highest protein level. Our study demonstrates that the circadian clock regulates the dynamic polyadenylation status of mRNAs, which can result in rhythmic protein expression independent of the steady-state levels of the message.

Post-transcriptional regulationThe control of gene expression is a complex process. Even after mRNA is transcribed from DNA, mRNAs can undergo many processing and regulatory steps that influence their expression (Keene, 2010). This type of regulation, at the post-transcriptional level, is beneficial, because it allows cells to alter protein levels rapidly without requiring transcript synthesis or processing. mRNA processing begins immediately following transcription with splicing and, in some cases, results in multiple isoforms of the transcript. Upon translocation into the cytoplasm, some mRNAs directly undergo deadenylation and degradation, whereas others can be stored in cytoplasmic compartments, such as processing bodies or stress granules, until they are degraded or re-polyadenylated. Other transcripts interact with the translational machinery and are translated into protein immediately after localization to the cytoplasm. Other mRNAs interact with localization machinery to Summary Circadian rhythms exist in most living organisms. The general molecular mechanisms that are used to generate 24-hour rhythms are conserved among organisms, although the details vary. These core clocks consist of multiple regulatory feedback loops, and must be coordinated and orchestrated appropriately for the fine-tuning of the 24-hour period. Many levels of regulation are important for the proper functioning of the circadian clock, including transcriptional, post-transcriptional and post-translational mechanisms. In recent years, new information about post-transcriptional regulation in the circadian system has been discovered. Such regulation has been shown to alter the phase and amplitude of rhythmic mRNA and protein expression in many organisms. Therefore, this Commentary will provide an overview of current knowledge of post-transcriptional regulation of the clock genes and clock-controlled genes in dinoflagellates, plants, fungi and animals. This article will also highlight how circadian gene expression is modulated by posttranscriptional mechanisms and how this is crucial for robust circadian rhythmicity.

Nocturnin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCs exposed to the PPAR-γ (peroxisome proliferator-activated receptor-γ) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that Noc −/− mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet. Consistent with its role in influencing BMSCs allocation, Noc −/− mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPAR-γ and markedly enhance PPAR-γ transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPAR-γ. Importantly, NOC-mediated nuclear translocation of PPAR-γ is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-γ. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-γ nuclear translocation may be independent of ligand-mediated PPAR-γ activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPAR-γ activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrient-responsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition.

Summary Background Efficient metabolic function in mammals depends on the circadian clock, which drives temporal regulation of metabolic processes. Nocturnin is a clock-regulated deadenylase that controls its target mRNA expression post-transcriptionally through poly(A) tail removal. Mice lacking Nocturnin (Noc−/− mice) are resistant to diet-induced obesity and hepatic steatosis, yet are not hyperactive or hypophagic. Results Here we show that Nocturnin is expressed rhythmically in the small intestine, is induced by olive oil gavage and that the Noc−/− mice have reduced chylomicron transit into the plasma following the ingestion of dietary lipids. Genes involved in triglyceride synthesis, storage and chylomicron formation have altered expression and large cytoplasmic lipid droplets accumulate in the apical domains of the Noc−/− enterocytes. The physiological significance of this deficit in absorption is clear since maintenance of Noc−/− mice on diets that challenge the chylomicron synthesis pathway result in significant reductions in body weight, while diets that bypass this pathway do not. Conclusions Therefore we propose that Nocturnin plays an important role in the trafficking of dietary lipid in the intestinal enterocyes by optimizing efficient absorption of lipids.

The mammalian molecular clock is composed of feedback loops to keep circadian 24-h rhythms. Although much focus has been on transcriptional regulation, it is clear that posttranscriptional controls also play important roles in molecular circadian clocks. In this study, we found that mouse LARK (mLARK), an RNA binding protein, activates the posttranscriptional expression of the mouse Period1 (mPer1) mRNA. A strong circadian cycling of the mLARK protein is observed in the suprachiasmatic nuclei with a phase similar to that of mPER1, although the level of the Lark transcripts are not rhythmic. We demonstrate that LARK causes increased mPER1 protein levels, most likely through translational regulation and that the LARK1 protein binds directly to a cis element in the 3 UTR of the mPer1 mRNA. Alterations of mLark expression in cycling cells caused significant changes in circadian period, with mLark knockdown by siRNA resulting in a shorter circadian period, and the overexpression of mLARK1 resulting in a lengthened period. These data indicate that mLARKs are novel posttranscriptional regulators of mammalian circadian clocks.circadian rhythms ͉ posttranscriptional regulation ͉ RNA binding protein ͉ 3Јuntranslated region ͉ suprachiasmatic nucleus

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3′-untranslated region (3′-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3′-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the 3'-UTR region:, which retains the endogenous 3'-UTR and, where the endogenous 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of 3'-UTR, we analyzed circadian rhythms of mice. Interestingly, mice displayed more than threefold stronger amplitude in bioluminescence rhythms than mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Analysis of the 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in mice, suggesting rhythmic overexpression of PER2 enhances expression of and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of 3'-UTR, miR-24, and PER2 in expression and core clock function.