RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development

Fate and Complex Pathogenic Effects of Dioxins and Polychlorinated Biphenyls in Obese Subjects before and after Drastic Weight Loss

BACKGROUND: In humans, persistent organic pollutants (POPs) are stored primarily in adipose tissue. Their total body burden and their contribution to obesity-associated diseases remain unclear. OBJECTIVES: We characterized POP total body burden and their redistribution in obese individuals before and after drastic weight loss and compared these values with a variety of molecular, biological, and clinical parameters. METHODS: Seventy-one obese subjects were enrolled and underwent bariatric surgery. Blood and adipose tissue samples were obtained at different times from these individuals as well as from 18 lean women. RESULTS: POP content (17 dioxins/furans and 18 polychlorinated biphenyl congeners) in different adipose tissue territories was similar, allowing us to assess total POP body burden from a single biopsy. Total POP body burden was 2 to 3 times higher in obese than in lean individuals. We also found increased expression of some POP target genes in obese adipose tissue. Drastic weight loss led to increased serum POPs and, within 6-12 months, to a significant 15% decrease in total polychlorinated biphenyl body burden. Importantly, serum POP levels were positively correlated with liver toxicity markers and lipid parameters, independently of age and body mass index. CONCLUSIONS: POP content in adipose tissue and serum correlate with biological markers of obesity-related dysfunctions. Drastic weight loss leads to a redistribution of POPs and to a moderate decrease of their total body burden

Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome

13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs

Nocturnin Expression Is Induced by Fasting in the White Adipose Tissue of Restricted Fed Mice

The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues. Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT) of mice housed in 12∶12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes

HIF2α reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma

<p>Abstract</p> <p>Background</p> <p>HIF2α/EPAS1 is a hypoxia-inducible transcription factor involved in catecholamine homeostasis, vascular remodelling, physiological angiogenesis and adipogenesis. It is overexpressed in many cancerous tissues, but its exact role in tumour progression remains to be clarified.</p> <p>Methods</p> <p>In order to better establish its function in tumourigenesis and tumour angiogenesis, we have stably transfected mouse neuroblastoma N1E-115 cells with the native form of HIF2α or with its dominant negative mutant, HIF2α (1–485) and studied their phenotype <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p><it>In vitro </it>studies reveal that HIF2α induces neuroblastoma cells hypertrophy and decreases their proliferation rate, while its inactivation by the HIF2α (1–485) mutant leads to a reduced cell size, associated with an accelerated proliferation. However, our <it>in vivo </it>experiments show that subcutaneous injection of cells overexpressing HIF2α into syngenic mice, leads to the formation of tumour nodules that grow slower than controls, but that are well structured and highly vascularized. In contrast, HIF2α (1–485)-expressing neuroblastomas grow fast, but are poorly vascularized and quickly tend to extended necrosis.</p> <p>Conclusion</p> <p>Together, our data reveal an unexpected combination between an antiproliferative and a pro-angiogenic function of HIF2α that actually seems to be favourable to the establishment of neuroblastomas <it>in vivo</it>.</p

Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3beta signaling pathway regulates BMAL1 protein stability and activity.GSK3beta phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3beta-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3beta pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.These findings uncover a previously unknown mechanism of circadian clock control. The GSK3beta kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems

Body weight, metabolism and clock genes

Biological rhythms are present in the lives of almost all organisms ranging from plants to more evolved creatures. These oscillations allow the anticipation of many physiological and behavioral mechanisms thus enabling coordination of rhythms in a timely manner, adaption to environmental changes and more efficient organization of the cellular processes responsible for survival of both the individual and the species. Many components of energy homeostasis exhibit circadian rhythms, which are regulated by central (suprachiasmatic nucleus) and peripheral (located in other tissues) circadian clocks. Adipocyte plays an important role in the regulation of energy homeostasis, the signaling of satiety and cellular differentiation and proliferation. Also, the adipocyte circadian clock is probably involved in the control of many of these functions. Thus, circadian clocks are implicated in the control of energy balance, feeding behavior and consequently in the regulation of body weight. In this regard, alterations in clock genes and rhythms can interfere with the complex mechanism of metabolic and hormonal anticipation, contributing to multifactorial diseases such as obesity and diabetes. The aim of this review was to define circadian clocks by describing their functioning and role in the whole body and in adipocyte metabolism, as well as their influence on body weight control and the development of obesity