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Sample records for factor messenger rna

  1. Nuclear Export of Messenger RNA

    Science.gov (United States)

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  2. Systemic delivery of factor IX messenger RNA for protein replacement therapy

    Science.gov (United States)

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

    2017-01-01

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

  3. Transforming growth factor-beta1 induces transforming growth factor-beta1 and transforming growth factor-beta receptor messenger RNAs and reduces complement C1qB messenger RNA in rat brain microglia.

    Science.gov (United States)

    Morgan, T E; Rozovsky, I; Sarkar, D K; Young-Chan, C S; Nichols, N R; Laping, N J; Finch, C E

    2000-01-01

    Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth

  4. A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota.

    Directory of Open Access Journals (Sweden)

    Moshe Lapidot

    2015-10-01

    Full Text Available Tomato yellow leaf curl virus (TYLCV is a devastating disease of tomato (Solanum lycopersicum that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo, and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses.

  5. Detection of colony-stimulating factor messenger RNA in single T cells by in situ hybridization

    DEFF Research Database (Denmark)

    Williamson, D J; Owens, T; Pearse, M

    1989-01-01

    RNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity...

  6. Messenger RNA surveillance: neutralizing natural nonsense

    DEFF Research Database (Denmark)

    Weischelfeldt, Joachim Lütken; Lykke-Andersen, Jens; Porse, Bo

    2005-01-01

    Messenger RNA transcripts that contain premature stop codons are degraded by a process termed nonsense-mediated mRNA decay (NMD). Although previously thought of as a pathway that rids the cell of non-functional mRNAs arising from mutations and processing errors, new research suggests a more general...

  7. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs....

  8. ACTH Action on Messenger RNA Stability Mechanisms

    Science.gov (United States)

    Desroches-Castan, Agnès; Feige, Jean-Jacques; Cherradi, Nadia

    2017-01-01

    The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3′-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.

  9. THE LIFETIME OF BACTERIAL MESSENGER RNA

    Energy Technology Data Exchange (ETDEWEB)

    Moses, V.; Calvin, M.

    1963-12-01

    Puromycin, an inhibitor of protein synthesis, appears to act as an inhibitor at additional sites during the induction of {beta}-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 seconds of induction was observed, but puromycin seems to prevent the accumulation of messenger RNA during the period between 20 seconds and the first appearance of enzyme activity after 3 minutes. When cells from a stationary culture are placed in fresh medium containing inducer for {beta}-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, starts within 30 seconds. By contrast, {beta}-galactosidase synthesis is greatly delayed compared with induction during exponential growth. Two other inducible enzymes show similar lags, but malic dehydrogenase, which requires no external inducer, shows no lag. The lags are not due to catabolite repression phenomena. They cannot be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag is also demonstrated by an i{sup -} mutant constitutive for {beta}-galactosidase synthesis. An inhibitor of RNA synthesis, 6-azauracil, preferentially inhibits {beta}-galactosidase synthesis compared with growth in both inducible and constitutive strains. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for normally constitutive proteins. The implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.

  10. Estrogen Regulation of Messenger RNA Stability

    Science.gov (United States)

    1990-08-17

    ribonuclease inhibitor, inhibits activity of RNase A-type enzymes. RNP-CS- ribonucleoprotein consensus sequence (K/R)G(F/Y)(G/A)FVX(F/Y) rRNA - ribosomal...CJ r i ɡ a 5S ^ S i C9 3 3 *» • - M 19 > • h- O C9 ^ h- 5 C9 > l - « < • - U f t - o CJ k u a u Q. < 2 C9 S C9 3 3 "ai t- 41 (9...mRNA molecules will need to be examined. Which of these factors degrade mRNAs? Which factors degrade other types of RNA molecules such as rRNA and

  11. Chemical and structural effects of base modifications in messenger RNA

    Science.gov (United States)

    Harcourt, Emily M.; Kietrys, Anna M.; Kool, Eric T.

    2017-01-01

    A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification -- the epitranscriptome -- is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

  12. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  13. Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

    1994-12-31

    Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

  14. Pseudo-messenger RNA: phantoms of the transcriptome.

    Directory of Open Access Journals (Sweden)

    Martin C Frith

    2006-04-01

    Full Text Available The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo-messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo-messenger RNAs (approximately half of which are transposon-associated among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level.

  15. Expression and alteration of insulin-like growth factor Ⅱ-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zhi-Zhen Dong; Deng-Fu Yao; Deng-Bing Yao; Xin-Hua Wu; Wei Wu; Li-Wei Qiu; Dao-Rong Jiang; Jian-Hua Zhu; Xian-Yong Meng

    2005-01-01

    AIM: To investigate the clinical values of serum free insulin-like growth factor Ⅱ (IGF-Ⅱ) levels and IGF-Ⅱ mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood for diagnosis of HCC and monitoring of extrahepatic metastasis.METHODS: Total RNAs were extracted from HCC tissues or peripheral blood mononuclear cells from patients with HCC, liver diseases devoid of cancer, non-hepatic tumors,and healthy controls, respectively. IGF-Ⅱ cDNAs were synthesized through random primers and reversetranscriptase, amplified by polymerase chain reaction (PCR), and confirmed by DNA sequencing analysis. Serum free IGF-Ⅱ levels in patients with different liver diseases were analyzed by an enzyme-linked immunosorbent assay.RESULTS: The amplified fragments of IGF-Ⅱ mRNA by RT-PCR were identical to originally designed ones with a size of 170 bp and confirmed by sequencing analysis.The dilution experiments revealed that the lowest sensitivity of our system was 2 ng/L of total RNA. The positive frequencies of IGF-Ⅱ mRNA were 100% in HCC tissues,53.3% in para-cancerous tissues, and 0% in non-cancerous tissues, respectively. The serum free IGF-Ⅱ levels were significantly higher in HCC than those in chronic hepatitis or liver cirrhosis. The positive frequency of circulating IGF-Ⅱ mRNA was 34.2% in HCC, no amplified fragment was found in other liver diseases, extrahepatic tumors,and normal controls, respectively. The circulating IGF-Ⅱ mRNA correlated with the stage of HCC, and its positive rate was 100% in HCC with extrahepatic metastasis and 35.5% in HCC with AFP-negative. No significant correlation was found between tumor sizes and circulating IGF-Ⅱ mRNA fragment.CONCLUSION: The abnormal expressions of free IGF-Ⅱ and IGF-Ⅱ mRNA are useful tumor markers for HCC diagnosis, differentiation of extrahepatic metastasis and monitoring postoperative recurrence.

  16. Differential intragraft cytokine messenger RNA profiles during rejection and repair of clinical heart transplants. A longitudinal study

    NARCIS (Netherlands)

    de Groot-Kruseman, Hester A; Mol, Wendy M; Niesters, Hubert G M; Maat, Alex P W; van Gelder, Teun; Balk, Aggie H M M; Weimar, Willem; Baan, Carla C

    2003-01-01

    After clinical heart transplantation, ischemia, acute rejection, and repair mechanisms can trigger the up-regulation of cytokines. To investigate the cytokine profile early after transplantation, we monitored messenger RNA (mRNA) expression levels of tumor necrosis factor-alpha (TNF-alpha), monocyte

  17. Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

    DEFF Research Database (Denmark)

    Barends, Sharief; Zehl, Martin; Bialek, Sylwia

    2010-01-01

    , elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated......The transfer-messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces...... coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A...

  18. Postage for the messenger: Designating routes for Nuclear mRNA Export

    Science.gov (United States)

    Natalizio, Barbara J.; Wente, Susan R.

    2013-01-01

    Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

  19. Isolation and Translation of Hordein Messenger RNA from Wild Type and Mutant Endosperms in Barley

    DEFF Research Database (Denmark)

    Brandt, Anders Bøving; Ingwersen, J.

    1978-01-01

    Nucleotide sequences of four cDNA clones coding for the carboxy-terminal portion of at least two different B1 hordein polypeptides are presented. The open reading frame in the nucleotide sequence of the the largest clone (pc hor2-4, 720 nucleotides) translates into the 181 carboxy-terminal amino...... was found to be a major constituent of the total messenger RNA population of the endosperm cell. Polyadenylated hordein messenger RNA sedimented at 11S in sucrose gradients and electrophoretic analysis reveals the presence of at least three RNA species with apparent molecular weights of 0.45, 0.36 and 0.......30 megadaltons. The 11S messenger RNA was translated in vitro into hordein precursor polypeptides which are 2–4 kilodaltons larger than the native hordein polypeptides. The endosperm cell of mutant No. 1508 contained twice as much RNA as the wild type endosperm cell but the same amount of polyadenylated 11S RNA...

  20. Messenger RNA patterns in rat liver nuclei before and after treat-ment with growth hormone.

    Science.gov (United States)

    Drews, J; Brawerman, G

    1967-06-09

    Like cortisol, growth hormone enhances RNA synthesis in rat liver nuclei. However, DNA-RNA hybridization experiments show that the application of growth hormone does not stimulate the formation of new species of messenger RNA. The latter phenomenon was observed after treatment with cortisol.

  1. A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

    Science.gov (United States)

    Carson, Sue; Miller, Heather

    2012-01-01

    Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

  2. MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity.

    Science.gov (United States)

    Deng, Youping; Ai, Junmei; Guan, Xin; Wang, Zhaohui; Yan, Bin; Zhang, Daqin; Liu, Chang; Wilbanks, Mitch S; Escalon, Barbara Lynn; Meyers, Sharon A; Yang, Mary Qu; Perkins, Edward J

    2014-01-01

    RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.

  3. Kinetic models for nucleocytoplasmic transport of messenger RNA.

    Science.gov (United States)

    Schröder, H C; Müller, W E; Agutter, P S

    1995-05-21

    Much is known about the mechanism by which mRNAs cross the nuclear envelope (the translocation stage of nucleocytoplasmic transport), but far less is known about the preceding (intranuclear migration/release) and succeeding (cytoplasmic binding) stages. Therefore, existing information suffices for articulating detailed kinetic models of translocation, but not models for the overall mRNA transport process. In this paper, we show that simple kinetic models of translocation can (i) accommodate data about nucleocytoplasmic distributions of endogenous transcripts; (ii) predict the overall effects on these distributions of effectors such as insulin and epidermal growth factor; (iii) throw some light on the mechanism(s) of action of the HIV-1 protein Rev and produce experimentally testable predictions about this mechanism; and (iv) account for the action of influenza virus NS1 protein. However, the simplest forms of translocation models apparently fail to account for some properties of viral regulators such as HIV Rev and adenovirus E1B-E4 complex. To elucidate these topics, less narrowly focused models of mRNA transport are required, describing intranuclear binding/release as well as translocation. On the basis of our examination of translocation models, we suggest some criteria that the requisite broadly based models must satisfy.

  4. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation

    Science.gov (United States)

    Weston, Andrew; Sommerville, John

    2006-01-01

    The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2α, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression. PMID:16769775

  5. Comment on "Length-dependent translation of messenger RNA by ribosomes"

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In recent paper [Phys. Rev. E {\\bf 83}, 042903 (2011)], a simple model for the translation of messenger RNA by ribosomes is provided, and the expression of translational ratio of protein is given. In this comments, varied methods to get this ratio are addressed. Depending on a different method, we find that, roughly speaking, this translational ratio decays exponentially with mRNA length in prokaryotic cell, and reciprocally with mRNA length in eukaryotic cells.

  6. Proteins encoded near the adenovirus late messenger RNA leader segments

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

  7. Translational regulation of MOS messenger RNA in pig oocytes.

    Science.gov (United States)

    Dai, Yanfeng; Newman, Barbara; Moor, Robert

    2005-11-01

    The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3'-untranslated region (3'-UTR). The MOS 3'-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3'-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3'-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3'-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

  8. Messenger RNA exchange between scions and rootstocks in grafted grapevines

    Science.gov (United States)

    We demonstrated the existence of genome-scale mRNA exchange in grafted grapevines, a woody fruit species with significant economic importance. By using diagnostic SNPs derived from high throughput genome sequencing, we identified more than three thousand genes transporting mRNAs across graft junctio...

  9. The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation

    OpenAIRE

    2010-01-01

    Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and trans...

  10. Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia

    OpenAIRE

    Glausier, Jill R; Lewis, David A.

    2011-01-01

    Levels of messenger RNA (mRNA) for the α1 subunit of the GABAA receptor, which is present in 60% of cortical GABAA receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABAA α1 subunit mRNA expression...

  11. TRANSLATION START SITE MULTIPLICITY OF THE CCAAT ENHANCER-BINDING PROTEIN-ALPHA MESSENGER-RNA IS DICTATED BY A SMALL 5' OPEN READING FRAME

    NARCIS (Netherlands)

    CALKHOVEN, CF; BOUWMAN, PRJ; SNIPPE, L; AB, G

    1994-01-01

    The CCAAT/enhancer binding proteins (C/EBP) alpha and beta of the bZIP family of transcription factors each occur as multiple forms due to translation initiation at different in-frame AUG codons from the same messenger RNA. The C/EBP alpha mRNAs of chicken, rat and Xenopus all contain a small 5' ope

  12. Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors

    OpenAIRE

    Gruber, Andreas R; Martin, Georges; Keller, Walter; Zavolan, Mihaela

    2013-01-01

    Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and polyadenylation. A core 3′-end processing complex carries out the cleavage and polyadenylation reactions, but many proteins have been implicated in the selection of polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3′-end...

  13. Gingival Toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health.

    Science.gov (United States)

    Kennedy, R; Lappin, D F; Dixon, P M; Bennett, D; Riggio, M P

    2017-05-01

    Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. Observational study. Gingival tissue samples were taken post-mortem from 13 horses with no clinical signs of oral disease and 20 horses with periodontitis. mRNA levels of TLR2, TLR4 and TLR9 and cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, IL-12, IL-17 and interferon-γ (IFN-γ) were determined using quantitative real-time PCR. The statistical significance of results was assessed using appropriate t tests. mRNA levels of all TLRs and cytokines were upregulated in equine periodontitis. Significant increases in mRNA levels of TLR2, TLR9, IL-4, IL-10, IL-12 (P≤0.05) and IFN-γ (P≤0.01) were observed for both unweighted and age-weighted analyses of diseased gingival tissue samples compared with healthy gingival samples. In comparisons of samples of periodontitis lesions with healthy gingival control samples from the same horse, significant increases in mRNA levels of TLR4, TLR9, IL-10, IFN-γ (P≤0.05), TLR2, IL-1β and IL-12p35 (P≤0.01) were observed. This study has provided an initial insight into the involvement of the immune system in equine periodontitis. Increased mRNA levels of TLR2, TLR4 and TLR9 indicate substantial microbial challenge in diseased gingival tissue. A mixed Th1/Th2/Th17 cytokine response is produced in equine periodontitis. Further studies are required to more fully characterise the role of the innate immune system in this disease. © 2016

  14. Experiment K-7-22: Growth Hormone Regulation Synthesis and Secretion in Microgravity. Part 2; Hypothalamic Growth Hormone-Releasing Factor, Somatostatin Immunoreactivity, and Messenger RNA Levels in Microgravity

    Science.gov (United States)

    Sawchenko, P. E.; Arias, C.; Krasnov, I.; Grindeland, R. E.; Vale, W.

    1994-01-01

    Immunohistochemical analyses of hypothalamic hormones carried out on tissue from rats flown on an earlier flight (Cosmos 1887) suggested preferential effects on hypophysiotropic principles involved in the regulation of growth hormone secretion and synthesis. We found that staining in the median eminence for peptides that provide both stimulatory (growth hormone-releasing factor, or GRF) and inhibitory (somatostatin, SS) influences on growth hormone secretion were depressed in flight animals relative to synchronous controls, while staining for other neuroendocrine peptides, cortocotropin-releasing factor and arginine vasopressin, were similar in these two groups. While this suggests some selective impact of weightlessness on the two principal central nervous system regulators of growth hormone dynamics, the fact that both GRF- and SS-immunoreactivity (IR) appeared affected in the same direction is somewhat problematic, and makes tentative any intimation that effects on CNS control mechanisms may be etiologically significant contributors to the sequelae of reduced growth hormone secretion seen in prolonged space flight. To provide an additional, and more penetrating, analysis we attempted in hypothalamic material harvested from animals flown on Cosmos 2044 to complement immunohistochemical analyses of GRF and SS staining with quantitative, in situ assessments of messenger RNAs encoding the precursors for both these hormones.

  15. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    Science.gov (United States)

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  16. Messenger RNA fluctuations and regulatory RNAs shape the dynamics of a negative feedback loop

    Science.gov (United States)

    Rodríguez Martínez, María; Soriano, Jordi; Tlusty, Tsvi; Pilpel, Yitzhak; Furman, Itay

    2010-03-01

    Single-cell experiments of simple regulatory networks can markedly differ from cell population experiments. Such differences arise from stochastic events in individual cells that are averaged out in cell populations. For instance, while individual cells may show sustained oscillations in the concentrations of some proteins, such oscillations may appear damped in the population average. In this paper we investigate the role of RNA stochastic fluctuations as a leading force to produce a sustained excitatory behavior at the single-cell level. As opposed to some previous models, we build a fully stochastic model of a negative feedback loop that explicitly takes into account the RNA stochastic dynamics. We find that messenger RNA random fluctuations can be amplified during translation and produce sustained pulses of protein expression. Motivated by the recent appreciation of the importance of noncoding regulatory RNAs in post-transcription regulation, we also consider the possibility that a regulatory RNA transcript could bind to the messenger RNA and repress translation. Our findings show that the regulatory transcript helps reducing gene expression variability both at the single-cell level and at the cell population level.

  17. Messenger RNA Fluctuations and Regulatory RNAs Shape the Dynamics of Negative Feedback Loop

    CERN Document Server

    Martínez, María Rodríguez; Tlusty, Tsvi; Pilpel, Yitzhak; Furman, Itay; 10.1103/PhysRevE.81.031924

    2010-01-01

    Single cell experiments of simple regulatory networks can markedly differ from cell population experiments. Such differences arise from stochastic events in individual cells that are averaged out in cell populations. For instance, while individual cells may show sustained oscillations in the concentrations of some proteins, such oscillations may appear damped in the population average. In this paper we investigate the role of RNA stochastic fluctuations as a leading force to produce a sustained excitatory behavior at the single cell level. Opposed to some previous models, we build a fully stochastic model of a negative feedback loop that explicitly takes into account the RNA stochastic dynamics. We find that messenger RNA random fluctuations can be amplified during translation and produce sustained pulses of protein expression. Motivated by the recent appreciation of the importance of non--coding regulatory RNAs in post--transcription regulation, we also consider the possibility that a regulatory RNA transcri...

  18. Chicken granulosa cells show differential expression of epidermal growth factor (EGF) and luteinizing hormone (LH) receptor messenger RNA and differential responsiveness to EGF and LH dependent upon location of granulosa cells to the germinal disc.

    Science.gov (United States)

    Yao, H H; Bahr, J M

    2001-06-01

    Granulosa cells in the chicken follicle exhibit different phenotypes according to their location relative to the germinal disc (GD). Granulosa cells proximal to the GD (referred to as proximal granulosa cells) are more proliferative, whereas granulosa cells distal to the GD (referred to as distal granulosa cells) are more differentiated. We have shown that epidermal growth factor (EGF) derived from the GD stimulated proliferation of granulosa cells proximal to the GD, whereas extraovarian LH promoted differentiation. We tested the hypothesis that phenotypic differences of granulosa cells are the result of differential responsiveness of granulosa cells to EGF and LH. We found that both granulosa and theca layers of chicken preovulatory follicles expressed mRNA for EGF receptor (EGFr) by polymerase chain reaction (PCR) analysis. However, only the granulosa layer showed differential expression of EGFr and LH receptor (LHr) mRNA. Competitive reverse transcription-PCR revealed that proximal granulosa cells expressed more EGFr mRNA but less LHr mRNA than distal granulosa cells. In addition, proximal granulosa cells proliferated more in response to EGF than their distal counterparts. We further demonstrated that EGF decreased LHr mRNA expression by granulosa cells in a dose-dependent manner, whereas EGF and LH had no effect on EGFr mRNA expression except at one dose of LH (15 ng/ml) that stimulated EGFr mRNA expression. Our findings suggest that EGF derived from the GD influences the phenotypes of granulosa cells. Granulosa cells proximal to the GD exhibit a proliferative phenotype possibly because they are exposed to and are more responsive to GD-derived EGF. Furthermore, GD-derived EGF decreases LHr mRNA expression by proximal granulosa cells and therefore results in less differentiated granulosa cell phenotype. In contrast, granulosa cells distal to the GD are not under the influence of EGF and exhibit a more differentiated phenotype.

  19. Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.

    Science.gov (United States)

    Fox, A; Gittos, M; Harbison, S A; Fleming, R; Wivell, R

    2014-05-01

    Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling.

  20. Cadmium-induced accumulation of metallothionein messenger RNA in rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohi, S.; Cardenosa, G.; Pine, R.; Huang, P.C.

    1981-03-10

    Multiple injections of nontoxic levels of cadmium to a rat result in much higher level of metallothionein (MT) production in the liver than does the single injection. In order to understand the underlying mechanisms we have quantitated and compared the metallothionein-specific messenger RNA contents in the livers following the two induction regimens. Cell-free translation assays coupled with specific immunoprecipitation of MT revealed that MT-mRNA activity in livers of animals multiply injected with Cd is 7- to 10-fold higher than that in livers 4 h after a single Cd-induction. By oligo(dT)-cellulose chromatography, sucrose density gradient centrifugation, and methylmercuric hydroxide-agarose gel electrophoresis this mRNA has been enriched approximately 100-fold from the total RNA. The size of the mRNA is about 400 nucleotides. Hybridization assays with a complementary DNA probe synthesized against the enriched MT-mRNA showed a 4-fold difference in the level of MT-mRNA between the two induction regimens in agreement with the results obtained by the cell-free translation assays. The possible mechanisms for these observations in consideration of the short lived nature of MT-mRNA are discussed.

  1. Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction.

    Directory of Open Access Journals (Sweden)

    Yeon J Lee

    2009-05-01

    Full Text Available Regulation of messenger RNA (mRNA stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A polymerase II-generated poly(A tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A tail-stimulated mRNA turnover in mammalian cells.

  2. Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

    OpenAIRE

    Rincón, A. ; Aguado, C. ; Desviat, L. R. ; Sánchez-Alcudia, R. ; Ugarte, M. ; Pérez, B. 

    2007-01-01

    We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger R...

  3. Circulating thyroid stimulating hormone receptor messenger RNA and differentiated thyroid cancer: A diagnostic meta-analysis

    Science.gov (United States)

    Kong, Chao-Yue; Li, Zhan-Ming; Wang, Li-Shun

    2017-01-01

    Thyroid stimulating hormone receptor messenger RNA (TSHR-mRNA) is over-expressed in thyroid cancer patients, which indicates that TSHR-mRNA is a potential biomarker of thyroid cancer. However, system evaluation for TSHR-mRNA as a diagnostic biomarker of thyroid cancer is deficient. The performance of TSHR-mRNA for thyroid cancer diagnosis was evaluated in this study. Three common international databases as well as a Chinese database were applied for literature researching. Quality assessment of the included literatures was conducted by the QUADAS-2 tool. Totally, 1027 patients from nine studies eligible for the meta-analysis were included in this study. Global sensitivity and specificity for the positivity of TSHR-mRNA in the thyroid cancer diagnosis is 72% and 82%. The value of AUC for this test performance was 0.84. Our meta-analysis suggests that TSHR-mRNA might be a potential biomarker to complete present diagnostic methods for early and precision diagnosis of thyroid cancer. Notably, this findings need validation thorough large-scale clinical studies. PMID:28036261

  4. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  5. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  6. Comment on ``Length-dependent translation of messenger RNA by ribosomes''

    Science.gov (United States)

    Zhang, Yunxin

    2012-02-01

    In a recent paper by Valleriani [Phys. Rev. EPLEEE81539-375510.1103/PhysRevE.83.042903 83, 042903 (2011)], a simple model for the translation of messenger RNA (mRNA) is presented. Using this model, the protein translational ratio r, defined as the ratio of protein translation rate ωtl from mRNA to protein degradation rate ωp, is obtained. The key point in obtaining the translational ratio r is to get the protein translation rate ωtl. In Valleriani 's paper, ωtl is obtained as the mean value of the measured translation rate, which is the ratio of the synthesized protein number to the mRNA lifetime. However, in experiments, different methods might be used to obtain the value of ωtl. Therefore, to apply Valleriani 's model to more general experiments, in this Comment three methods to obtain the translation rate ωtl, and consequently the translational ratio r, are presented. Based on one of the methods which might be employed in most of the experiments, we find that the translational ratio r decays exponentially with mRNA length in prokaryotic cells, and decays reciprocally with mRNA length in eukaryotic cells. This result is slight different from that which was obtained in Valleriani 's paper.

  7. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  8. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

    Directory of Open Access Journals (Sweden)

    Jozef Julian Bujarski

    2013-03-01

    Full Text Available RNA recombination is one of the driving forces of genetic variability in (+-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (i How various factors modulate the ability of viral replicase to switch templates, (ii What is the intracellular location of RNA-RNA template switchings, (iii Mechanisms and factors responsible for non-replicative RNA recombination, (iv Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  9. Exercise does not influence myostatin and follistatin messenger RNA expression in young women.

    Science.gov (United States)

    Jensky, Nicole E; Sims, Jennifer K; Dieli-Conwright, Christina M; Sattler, Fred R; Rice, Judd C; Schroeder, E Todd

    2010-02-01

    We evaluated changes in myostatin, follistatin, and MyoD messenger RNA (mRNA) gene expression using eccentric exercise (EE) and concentric exercise (CE) as probes to better understand the mechanisms of muscle hypertrophy in young women. Twelve women performed single-leg maximal eccentric (n = 6, 25 +/- 1 years, 59 +/- 7 kg) or concentric (n = 6, 24 +/- 1 years, 65 +/- 7 kg) isokinetic knee extension exercise for 7 sessions. Muscle biopsies were taken from the vastus lateralis at baseline, 8 hours after the first exercise session, and 8 hours after the seventh exercise session. In the EE group, there were no changes in myostatin and follistatin (p > or = 0.17); however, MyoD expression increased after 1 exercise bout (p = 0.02). In the CE group, there were no changes in myostatin, follistatin, or MyoD mRNA gene expression (p > or = 0.07). Differences between the EE and CE groups were not significant (p > or = 0.05). These data suggest that a single bout or multiple bouts of maximal EE or CE may not significantly alter myostatin or follistatin mRNA gene expression in young women. However, MyoD mRNA expression seems to increase only after EE.

  10. Characterization of striatal neurons expressing high levels of glutamic acid decarboxylase messenger RNA.

    Science.gov (United States)

    Chesselet, M F; Robbins, E

    1989-07-17

    Two types of labelled cells are detected in sections of rat and mouse striata processed for in situ hybridization histochemistry with 35S-radiolabelled RNA probes complementary to the messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD), the synthesis enzyme for gamma-aminobutyric acid (GABA): numerous lightly, and fewer very densely labelled neurons. In order to determine whether the densely labelled cells correspond to the striatal somatostatinergic neurons with which they share morphological characteristics, the presence of GAD mRNA was examined in brain sections processed successively for dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, a marker of striatal somatostatinergic neurons, and in situ hybridization histochemistry. In addition, the distribution of GABAergic interneurons was analyzed with regard to striatal compartments (striosomes) indicated by patches of dense opiate binding sites. The results show that NADPH diaphorase activity and GAD mRNA do not co-exist in striatal neurons. Furthermore, in contrast to the somatostatinergic neurons which are almost exclusively located in the extrastriosomal matrix, densely labelled GAD cells were present both in the striosomes and the matrix, further suggesting that GABAergic and somatostatinergic neurons form two distinct interneuronal systems in the striatum of rats and mice.

  11. Messenger RNA profiling for forensic body fluid identification: research and applications.

    Science.gov (United States)

    Wang, Zheng; Zhang, Su-hua; Di, Zhou; Zhao, Shu-min; Li, Cheng-tao

    2013-10-01

    Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

  12. Messenger RNA Profiling for Forensic Body Fluid Identifica-tion:Research and Applications

    Institute of Scientific and Technical Information of China (English)

    WANG Zheng; ZHANG Su-hua; ZHOU Di; ZHAO Shu-min; LI Cheng-tao

    2013-01-01

    Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link betw een sample donors and actual criminal acts. How ev-er, the conventional body fluid identification methods are prone to various limitations, such as time con-sumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Re-cently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP ) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profil-ing, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibilitywith current DNA extraction and analysis procedure. In the current review ,we provided an overview of the present know ledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possi-ble practical application to forensic casew ork.

  13. Messenger RNA Turnover Processes in Escherichia coli, Bacillus subtilis, and Emerging Studies in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Kelsi L. Anderson

    2009-01-01

    Full Text Available The regulation of mRNA turnover is a recently appreciated phenomenon by which bacteria modulate gene expression. This review outlines the mechanisms by which three major classes of bacterial trans-acting factors, ribonucleases (RNases, RNA binding proteins, and small noncoding RNAs (sRNA, regulate the transcript stability and protein production of target genes. Because the mechanisms of RNA decay and maturation are best characterized in Escherichia coli, the majority of this review will focus on how these factors modulate mRNA stability in this organism. However, we also address the effects of RNases, RNA binding proteins, sRNAs on mRNA turnover, and gene expression in Bacillus subtilis, which has served as a model for studying RNA processing in gram-positive organisms. We conclude by discussing emerging studies on the role modulating mRNA stability has on gene expression in the important human pathogen Staphylococcus aureus.

  14. Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

    Science.gov (United States)

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne

    2014-06-01

    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

  15. MicroRNA Seed Region Length Impact on Target Messenger RNA Expression and Survival in Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Lila E Mullany

    Full Text Available microRNAs (miRNA repress messenger RNAs post-transcriptionally through binding to the 3' UTR of the mRNA with the miRNA seed region. It has been purported that longer seed regions have a greater efficacy on mRNA repression. We tested this hypothesis by evaluating differential expression of miRNAs involved in regulating the immune response, an important mechanism in colorectal cancer (CRC, by seed length category. We subsequently evaluated differential expression of these miRNAs' targets in colonic tissue and the impact of these miRNAs on CRC survival. We determined sequence complementarity between each miRNA seed region and the 3' UTR of each experimentally verified mRNA target gene. We classified miRNAs into groups based on seed regions matching perfectly to a mRNA UTR with six bases beginning at position two, seven bases beginning at position one, seven bases beginning at position two, or eight bases beginning at position one. We analyzed these groups in terms of miRNA differential expression between carcinoma and normal colorectal mucosa, differential colonic target mRNA expression, and risk of dying from CRC. After correction for multiple comparisons, the proportion of the miRNAs that were associated with differential mRNA expression was 0% for the 6-mer, 13.64% for the 7α-mer group, 12.82% for the 7β-mer group, and 8.70% for the 8-mer group. The proportion of miRNAs associated with survival was 20% for the 6-mer group, 27.27% for the 7α-mer group, 10.23% for the 7β-mer group, and 21.74% for the 8-mer group. We did not see a linear relationship between seed length and miRNA expression dysregulation, mRNA expression, or survival. Our findings do not support the hypothesis the seed region length alone influences mRNA repression.

  16. Expression of estrogen receptor and estrogen receptor messenger RNA in gastric carcinoma tissues

    Institute of Scientific and Technical Information of China (English)

    Xin-Han Zhao; Shan-Zhi Gu; Shan-Xi Liu; Bo-Rong Pan

    2003-01-01

    AIM: To study estrogen receptor (ER) and estrogen receptor messenger RNA (ERmRNA) expression in gastric carcinoma tissues and to investigate their association with the pathologic types of gastric carcinoma.METHODS: The expression of ER and ERmRNA in gastric carcinoma tissues (15 males and 15 females, 42-70 years old) was detected by immunohistochemistry and in situ hybridization, respectively.RESULTS: The positive rate of ER (immunohistochemistry)was 33.3% in males and 46.7% in females. In Borrmann Ⅳ gastric carcinoma ER positive rate was greater than that in other pathologic types, and in poorly differentiated adenocarcinoma and signet ring cell carcinoma the positive rates were greater than those in other histological types of both males and females (P<0.05). The ER was more highly expressed in diffused gastric carcinoma than in non-diffused gastric carcinoma (P<0.05). The ER positive rate was also related to regional lymph nodes metastases (P<0.05), and was significantly higher in females above 55 years old, and higher in males under 55 years old (P<0.05). The ERmRNA (in situ hybridization) positive rate was 73.3% in males and 86.7% in females. The ERmRNA positive rates were almost the same in Borrmann Ⅰ, Ⅱ, Ⅲ and Ⅳ gastric carcinoma (P>0.05). ERmRNA was expressed in all tubular adenocarcinoma, poorly differentiated adenocarcinoma and signet ring cell carcinoma (P<0.05). The ERmRNA positive rate was related to both regional lymph nodes metastases and gastric carcinoma growth patterns, and was higher in both sexes above 55 years old but without statistical significance (P>0.05). The positive rate of ERmRNA expression by in situ hybridization was higher than that of ER expression by immunohistochemistry (P<0.05).CONCLUSION: ERmRNA expression is related to the pathological behaviors of gastric carcinoma, which might help to predict the prognosis and predict the effectiveness of endocrine therapy for gastric carcinoma.

  17. Integrated microRNA and messenger RNA analysis in aortic stenosis.

    Science.gov (United States)

    Coffey, Sean; Williams, Michael J A; Phillips, L Vicky; Galvin, Ivor F; Bunton, Richard W; Jones, Gregory T

    2016-11-23

    Aortic valve stenosis (AS) is a major cause of morbidity and mortality, with no effective medical therapies. Investigation into the underlying biology of AS in humans is limited by difficulties in obtaining healthy valvular tissue for use as a control group. However, micro-ribonucleic acids (miRNAs) are stable in post-mortem tissue. We compared valve specimens from patients undergoing aortic valve replacement for AS to non-diseased cadaveric valves. We found 106 differentially expressed miRNAs (p Integrated miRNA/gene expression analysis validated the microarray results as a whole, while quantitative polymerase chain reaction confirmed downregulation of miR-122-5p, miR-625-5p, miR-30e-5p and upregulation of miR-21-5p and miR-221-3p. Pathway analysis of the integrated miRNA/mRNA network identified pathways predominantly involved in extracellular matrix function. A number of currently available therapies target products of upregulated genes in the integrated miRNA/mRNA network, with these genes being predominantly more peripheral members of the network. The identification of a group of tissue miRNA associated with AS may contribute to the development of new therapeutic approaches to AS. This study highlights the importance of systems biology-based approaches to complex diseases.

  18. Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

    Science.gov (United States)

    Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

    2014-01-01

    Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patients from healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients' plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for

  19. DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

    Science.gov (United States)

    Khandelwal, Garima; Jayaram, B

    2012-05-30

    Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

  20. Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse.

    Science.gov (United States)

    Metlapally, Ravikanth; Park, Han Na; Chakraborty, Ranjay; Wang, Kevin K; Tan, Christopher C; Light, Jacob G; Pardue, Machelle T; Wildsoet, Christine F

    2016-11-01

    MicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied. C57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7). Form-deprived eyes showed myopic shifts in refractive error (-2.02 ± 0.47 D; P RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed. Scleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

  1. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    Science.gov (United States)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  2. Light-induced appearance of polysomal poly(A)-rich messenger RNA during greening of barley plants.

    Science.gov (United States)

    Heinze, H; Herzfeld, F; Kiper, M

    1980-10-01

    Changes in polysomal poly(A)-rich mRNA during greening of etiolated barley plants were studied by the technique of cDNA-mRNa hybridization. Hybridizaiton data of the homologous reactions reveal that in etiolated as well as in greened shoots a complexity of 5 X 10(7) nucleotides or about 33000 different average-sized mRNAs are present. These are organized in different abundancy classes with 94% of the total complexicity present in each of the slowest reacting class representing rare messengers. Heterologous hybridizations indicate that 92% of all polysomal poly(A)-rich mRNAs in etiolated shoots are complementary to those of greened and 82% of 'green' poly(A)-rich mRNAs are complementary to white ones. It is shown that the abundant mRNA clases are essentially responsible for these differences. The prevalent classes making up 15% ('white') and 31% ('green') of the poly(A)-rich mRNA mass but comprising only a complexity of 1.8 X 10(4) and 2.1 X 10(4) nucleotides are identical to 50% with each other. Hybridization of isolated prevalent 'green' cDNA with whole 'white' poly(A)-rich mRNA indicates that the additionally appearing 50% prevalent green messengers must be regarded as green-specific, only present in polysomal poly(A)-rich mRNA after illumination. This conclusion is underlined by the hybridization of the 'green' cDNA with total polysomal RNa of etiolated shoots. Evidently appearance of these prevalent messengers in functional polysomes is not caused by a shift from poly(A)-free mRNA to poly(A)-rich mRNA. The results clearly demonstrate that light induces greening by turning on genes or influencing post-transcriptional processing to produce mature green-specific poly(A)-rich mRNA.

  3. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Directory of Open Access Journals (Sweden)

    Joanna J Moser

    Full Text Available BACKGROUND: GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. METHODOLOGY/PRINCIPAL FINDINGS: RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. CONCLUSIONS/SIGNIFICANCE: The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and

  4. Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing.

    Science.gov (United States)

    Shkreta, Lulzim; Bell, Brendan; Revil, Timothée; Venables, Julian P; Prinos, Panagiotis; Elela, Sherif Abou; Chabot, Benoit

    2013-01-01

    For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5' splice site and the U2AF65/U2AF35 complex to the 3' splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively

  5. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control.

    Science.gov (United States)

    Lai, Ming-Chih; Lee, Yan-Hwa Wu; Tarn, Woan-Yuh

    2008-09-01

    Nuclear export of mRNA is tightly linked to transcription, nuclear mRNA processing, and subsequent maturation in the cytoplasm. Tip-associated protein (TAP) is the major nuclear mRNA export receptor, and it acts coordinately with various factors involved in mRNA expression. We screened for protein factors that associate with TAP and identified several candidates, including RNA helicase DDX3. We demonstrate that DDX3 directly interacts with TAP and that its association with TAP as well as mRNA ribonucleoprotein complexes may occur in the nucleus. Depletion of TAP resulted in nuclear accumulation of DDX3, suggesting that DDX3 is, at least in part, exported along with messenger ribonucleoproteins to the cytoplasm via the TAP-mediated pathway. Moreover, the observation that DDX3 localizes transiently in cytoplasmic stress granules under cell stress conditions suggests a role for DDX3 in translational control. Indeed, DDX3 associates with translation initiation complexes. However, DDX3 is probably not critical for general mRNA translation but may instead promote efficient translation of mRNAs containing a long or structured 5' untranslated region. Given that the DDX3 RNA helicase activity is essential for its involvement in translation, we suggest that DDX3 facilitates translation by resolving secondary structures of the 5'-untranslated region in mRNAs during ribosome scanning.

  6. Identification and localization of insulin-like growth factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla.

    Science.gov (United States)

    Batch, J A; Mercuri, F A; Werther, G A

    1996-03-01

    The role of the insulin-like growth factors (IGFs) in hair follicle biology has recently been recognized, although their actions, sites of production, and modulation by the insulin-like growth factor-binding proteins (IGFBPs) have not to date been defined. IGF-I is essential for normal hair growth and development, and may be important in regulation of the hair growth cycle. In many culture systems, IGF-I actions are modulated by the IGFBPs. Thus, if IGFBPs are produced in the human hair follicle, they may play a role in targeting IGF-I to its receptor or may modulate IGF-I action by interaction with matrix proteins. We have used in situ hybridization to localize messenger RNA for the six IGFBPs in anagen hair follicles. Anti-sense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-micrometer sections of adult facial skin were probed. Messenger RNA for IGFBP-3, -4, and -5 were identified, with predominantly IGFBP-3 and -5 mRNA found in the dermal papilla, and to a lesser extent IGFBP-4 mRNA. IGFBP-4 mRNA was also found at the dermal papilla/epithelial matrix border. Messenger RNAs for both IGFBP-4 and -5 were also demonstrated in the dermal sheath surrounding the hair follicle. Messenger RNAs for IGFBP-1, -2, and -6 were not identified. These studies demonstrate specific localization of IGFBP mRNAs in hair follicles, suggesting that they each play specific roles in the local modulation of IGF action during the hair growth cycle.

  7. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

    Science.gov (United States)

    Wilbert, Melissa L; Huelga, Stephanie C; Kapeli, Katannya; Stark, Thomas J; Liang, Tiffany Y; Chen, Stella X; Yan, Bernice Y; Nathanson, Jason L; Hutt, Kasey R; Lovci, Michael T; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B; Morris, Quaid; Hoon, Shawn; Yeo, Gene W

    2012-10-26

    LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Effects of cold stress on hypothalamic corticotrophin-releasing and thyrotropin-releasing hormone messenger RNA levels in chickens

    Institute of Scientific and Technical Information of China (English)

    WANG Jintao; XU Shiwen

    2007-01-01

    There is increasing evidence that stress can activate the hypothalamic-pituitary-adrenal-axis and hypothalamic-pituitarythyroid-axis, and further affect the synthesis and secretion of corticotrophin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH). To evaluate the effect of cold stress on the hypothalamic CRH and TRH messenger RNA (mRNA) levels in Yisha chickens, male Yisha chickens were subjected to acute (1, 6, 12 h) and chronic (5, 10, 20 d) cold stress (12±1)℃. Hypothalami were collected for assessment of mRNA levels by semi-quantitative RT-PCR. Acute stress resulted in a significant decrease of CRH mRNA levels at 6 and 12 h, and a significant increase of TRH mRNA levels at every stress time point. Chronic cold stress resulted in a significant increase of CRH mRNA levels and a significant decrease of TRH mRNA levels compared with the control group at every stress time point. The results suggest that the two genes differently respond to cold stress at the mRNA levels. And the different degrees of cold stress will produce different effects on the identical gene.

  9. Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond.

    Science.gov (United States)

    Vallazza, Britta; Petri, Sebastian; Poleganov, Marco A; Eberle, Florian; Kuhn, Andreas N; Sahin, Ugur

    2015-01-01

    In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

  10. Contrasting responses to interferon β-1b treatment in relapsing-remitting multiple sclerosis: Does baseline interleukin- 12p35 messenger RNA predict the efficacy of treatment?

    NARCIS (Netherlands)

    Boxel van-Dezaire, A.H.H.; Trigt van-Hoff, S.C.J.; Killestein, J.; Schrijver, H.M.; Houwelingen, J.C. van; Polman, C.H.; Nagelkerken, L.

    2000-01-01

    Interferon (IFN)-β treatment is effective in relapsing-remitting multiple sclerosis (RR-MS) via an as yet unidentified mechanism. In the present study, we investigated whether the expression of messenger RNA (mRNA) encoding the interleukin (IL)-12 subunits p40 and p35, IL-12 receptor chains, IL-18,

  11. A study of ribonucleoproteins: The sequence of rabbit 18S ribosomal RNA and the identification of proteins associated with messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Connaughton, J.F. Jr.

    1989-01-01

    This study considers the functional role of ribosomal RNA and messenger ribonucleoproteins in the translational regulation of gene expression. The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. Rabbit 18S RNA was cleaved with either T{sub 1} ribonuclease or RNase H, using a Pst 1 DNA linker to generate a unique set of overlapping fragments spanning the entire molecule. Both intact and fragmented 18S RNA were end-labeled with {sup 32}P and base-specifically cleaved enzymatically and chemically. Nucleotide sequences were determined from long polyacrylamide sequencing gels run in formamide. To assess functional roles of RNA in gene expression, specific mRNA-protein interactions were also examined. Eukaryotic mRNA is associated with specific proteins that may be important in translational regulation and mRNA stability; mRNP complexes were reconstituted in a message-dependent, cell-free rabbit reticulocyte translation system, using unique mRNA species transcribed in vitro with SP6 polymerase. Transcripts of both rabbit and human {beta}-globin cDNA were labeled with {sup 32}P either throughout the molecule ore selectively at the 5{prime} and 3{prime} terminus.

  12. Proopiomelanocortin messenger RNA levels are increased in the anterior pituitary of the sheep fetus after adrenalectomy in late gestation.

    Science.gov (United States)

    McMillen, I C; Antolovich, G C; Mercer, J E; Perry, R A; Silver, M

    1990-09-01

    We have investigated the effect of bilateral adrenalectomy at 116-119 days' gestation on the levels of the messenger (m) RNA for proopiomelanocortin (POMC) in the anterior pituitary of the fetal sheep and in the ovine placentome during late gestation (134-136 days' gestation). After fetal adrenalectomy there was a significant (p less than 0.001) and sustained increase in circulating ACTH concentrations in the adrenalectomised group (1,838 +/- 155 ng/l at 130-136 days) when compared with the intact control group (131 +/- 25 ng/l at 130-136 days). The mean levels of POMCmRNA relative to 18S RNA were also significantly higher (p less than 0.001) in the adrenalectomised fetal sheep pituitaries (2.8 +/- 0.12; n = 4) than in the intact/control fetal sheep pituitaries (1.31 +/- 0.13; n = 4). In contrast to the findings in the anterior pituitary, POMCmRNA was not detected in RNA extracted from the placentomes of either the adrenalectomised or intact fetal sheep. There was also a significant arteriovenous difference in ACTH concentrations in the umbilical circulation in both adrenalectomised and intact fetal sheep at 134-136 days' gestation. This study demonstrates therefore that the fetal adrenals act to suppress POMCmRNA levels in late gestation and also that the increase in circulating ACTH after adrenalectomy originates from the pituitary and not the placentome.

  13. Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Gerdes, Kenn

    2008-01-01

    Prokaryotic toxin-antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro...

  14. Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

    Science.gov (United States)

    Kobayashi, Y; Peterson, B C; Waldbieser, G C

    2015-04-01

    This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P muscle was increased (P growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

    DEFF Research Database (Denmark)

    Reinert, Line; Shi, B; Nandi, S;

    2006-01-01

    MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification e...

  16. Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum.

    Science.gov (United States)

    Willcutts, M D; Morrison-Bogorad, M

    1991-11-19

    The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.

  17. Partial purification and characterization of the messenger RNA for cell fibronectin.

    Science.gov (United States)

    Fagan, J B; Yamada, K M; de Crombrugghe, B; Pastan, I

    1979-08-10

    Fibronectin mRNA has been partially purified by guanidine extraction, oligo-(dT)-cellulose chromatography and sucrose density gradient centrifugation. We obtain a fraction which programs a wheat germ in vitro translation system to synthesize a polypeptide species which co-electrophoreses with fibronectin in SDS-polyacrylamide gels and which is immunoprecipitated with affinity purified fibronectin-specific IgG. Analysis of this RNA fraction by methyl mercury hydroxide-agarose gel electrophoresis reveals the presence of a band accounting for 30 percent to 50 percent of the ethidium bromide-staining material in the fraction. The RNA of this band has an estimated molecular weight of about 3 million daltons and is greatly reduced in the corresponding RNA fraction from RSV transformed CEF. This RNA has been tentatively identified as fibronectin mRNA.

  18. Messenger RNA vaccine based on recombinant MS2 virus-like particles against prostate cancer.

    Science.gov (United States)

    Li, Jinming; Sun, Yanli; Jia, Tingting; Zhang, Rui; Zhang, Kuo; Wang, Lunan

    2014-04-01

    Prostate cancer (PCa) is the most diagnosed cancer in the western male population with high mortality. Recently, alternative approaches based on immunotherapy including mRNA vaccines for PCa have shown therapeutic promise. However, for mRNA vaccine, several disadvantages such as the instability of mRNA, the high cost of gold particles, the limited production scale for mRNA-transfected dendritic cells in vitro, limit their development. Herein, recombinant bacteriophage MS2 virus-like particles (VLPs), which based on the interaction of a 19-nucleotide RNA aptamer and the coat protein of bacteriophage MS2, successfully addressed these questions, in which target mRNA was packaged by MS2 capsid. MS2 VLP-based mRNA vaccines were easily prepared by recombinant protein technology, nontoxic and RNase-resistant. We show the packaged mRNA was translated into protein as early as 12 hr after phagocytosed by macrophages. Moreover, MS2 VLP-based mRNA vaccines induced strong humoral and cellular immune responses, especially antigen-specific cytotoxic T-lymphocyte (CTL) and balanced Th1/Th2 responses without upregulation of CD4(+) regulatory T cells, and protected C57BL/6 mice against PCa completely. As a therapeutic vaccine, MS2 VLP-based mRNA vaccines delayed tumor growth. Our results provide proof of concept on the efficacy and safety of MS2 VLP-based mRNA vaccine, which provides a new delivery approach for mRNA vaccine and implies important clinical value for the prevention and therapy of PCa.

  19. [Synthesis of messenger-like RNA in plasma cels producing different clases of immunoglobulins].

    Science.gov (United States)

    Nikitin, A V; Babichev, V A

    1976-01-01

    Electrophoretic analysis of the nuclear poly A RNA cells of the MOPC 21, MOPC 406 and MOPC 41 plasmocytomas demonstrated a similarity in their distribution by the mol wt. Kinetics of the label accumulation in the nuclear poly A RNA of different plasma cells was unitypical. Individual peculiarities of the distribution of the cytoplasmic poly A RNA were expressed for each individual line of the myeloma cells. The majority of the molecules of the heterogenous RNA in the plasma cells were subjected to posttranscription polyadenylation.

  20. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells.

    Science.gov (United States)

    Soundararajan, Sridharan; Chen, Weiwei; Spicer, Eleanor K; Courtenay-Luck, Nigel; Fernandes, Daniel J

    2008-04-01

    We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.

  1. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    Science.gov (United States)

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  2. Fecal bile acid excretion and messenger RNA expression levels of ileal transporters in high risk gallstone patients

    Directory of Open Access Journals (Sweden)

    Miquel Juan

    2009-12-01

    Full Text Available Abstract Background Cholesterol gallstone disease (GS is highly prevalent among Hispanics and American Indians. In GS, the pool of bile acids (BA is decreased, suggesting that BA absorption is impaired. In Caucasian GS patients, mRNA levels for ileal BA transporters are decreased. We aimed to determine fecal BA excretion rates, mRNA levels for ileal BA transporter genes and of regulatory genes of BA synthesis in Hispanic GS patients. Results Excretion of fecal BA was measured in seven GS females and in ten GS-free individuals, all with a body mass index 2O3 (300 mg/day for 10 days, and fecal specimens were collected on the last 3 days. Chromium was measured by a colorimetric method, and BA was quantitated by gas chromatography/mass spectroscopy. Intake of calories, nutrients, fiber and cholesterol were similar in the GS and GS-free subjects. Mean BA excretion levels were 520 ± 80 mg/day for the GS-free group, and 461 ± 105 mg/day for the GS group. Messenger RNA expression levels were determined by RT-PCR on biopsy samples obtained from ileum during diagnostic colonoscopy (14 GS-free controls and 16 GS patients and from liver during surgery performed at 8 and 10 AM (12 GS and 10 GS-free patients operated on for gastrointestinal malignancies, all with a body mass index Conclusion Hispanics with GS have fecal BA excretion rates and mRNA levels of genes for ileal BA transporters that are similar to GS-free subjects. However, mRNA expression levels of Cyp7A1 are increased in GS, indicating that regulation of BA synthesis is abnormal in Hispanics with GS.

  3. In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

    Energy Technology Data Exchange (ETDEWEB)

    Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.; Ivell, R.

    1987-05-29

    Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.

  4. Expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNA in the human CNS: a {sup 33}P in situ hybridization study

    Energy Technology Data Exchange (ETDEWEB)

    Peckys, D.; Landwehrmeyer, G.B. [Department of Neurology, Albert-Ludwigs-University Freiburg, Neurozentrum, Breisacherstrasse 64, D-79106 Freiburg (Germany)

    1999-02-01

    The existence of at least three opioid receptor types, referred to as {mu}, {kappa}, and {delta}, is well established. Complementary DNAs corresponding to the pharmacologically defined {mu}, {kappa}, and {delta} opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using {sup 33}P-labelled RNA probes. In the present study we report the regional and cellular expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the {mu} and {kappa} opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the {delta} opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between {mu} opioid receptor receptor binding and {mu} opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous

  5. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

    Directory of Open Access Journals (Sweden)

    Akira Ito

    Full Text Available Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and citrate synthase (CS, which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1 and aggrecan (ACAN, was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y-box 9 (SOX9, which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and

  6. Regulation of amylase messenger RNA concentration in rat pancreas by food content.

    OpenAIRE

    Giorgi, D; Bernard, J. P.; Lapointe, R.; Dagorn, J C

    1984-01-01

    Regulation of the expression of pancreatic amylase genes was studied by comparing groups of rats fed diets with high (75%), intermediate (20%) and low (11%) carbohydrate content. Animals on the high carbohydrate diet had nine times as much amylase mRNA as those on low carbohydrate diet, and twice as much as the intermediate group, as determined by filter hybridization of equal amounts of total pancreatic RNA to an excess of a cloned rat amylase cDNA probe. Parallel results were obtained when ...

  7. Intestinal adaptation after extensive small bowel resection: differential changes in growth and insulin-like growth factor system messenger ribonucleic acids in jejunum and ileum.

    Science.gov (United States)

    Ziegler, T R; Mantell, M P; Chow, J C; Rombeau, J L; Smith, R J

    1998-07-01

    The distal small bowel exhibits greater adaptive growth than proximal segments after partial small intestine resection. To explore this process, we evaluated adaptive cellularity, intestinal insulin-like growth factor (IGF) system messenger RNA (mRNA) transcripts, and effects of recombinant IGF-I treatment in jejunum and ileum of adult rats. Gastrostomy-fed animals underwent 80% jejuno-ileal resection or intestinal transection and reanastomosis without resection, followed by infusion of human recombinant IGF-I (2.4 mg/kgXday) or vehicle. After 7 days, resected rats demonstrated modest adaptive growth in jejunum and marked cell proliferation in ileum. Resection increased IGF-I mRNA in both jejunum (183%) and ileum (249%) and up-regulated IGFBP-4 mRNA levels in both tissues. IGFBP-3 mRNA fell significantly in ileum after resection. IGF-I infusion modestly increased ileal cellularity after resection, but had no effect in jejunum. IGF-I markedly increased IGFBP-3 mRNA levels in jejunum after both transection and resection. These data confirm that bowel resection induces greater adaptive growth in ileum than jejunum. IGF-I administration modestly increases ileal, but not jejunal, growth after resection. Increased levels of intestinal IGF-I and IGFBP-4 mRNA suggest roles for IGF-I and IGFBP-4 in mediating small bowel adaptation. Higher levels of jejunal IGFBP-3 mRNA may be related to limited jejunal vs. ileal growth after extensive jejuno-ileal resection.

  8. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts.

    Science.gov (United States)

    Wrenzycki, C; Wells, D; Herrmann, D; Miller, A; Oliver, J; Tervit, R; Niemann, H

    2001-07-01

    The successful production of embryos by nuclear transfer (NT) employing cultured somatic donor cells depends upon a variety of factors. The objective of the present study was to investigate the effects 1) of two different activation protocols, 2) the use of quiescent or nonquiescent donor cells (G(0) or G(1) of the cell cycle), and 3) passage number of donor cells on the relative abundance (RA) of eight specific mRNAs (DNA methyltransferase, DNMT; mammalian achaete-scute homologue, Mash2; glucose transporter-1, Glut-1; heat shock protein 70.1, Hsp; desmocollin II, Dc II; E-cadherin, E-cad; interferon tau, IF; insulin-like growth factor 2 receptor, Igf2r) in single blastocysts employing a semiquantitative reverse transcription-polymerase chain reaction assay. The results were compared with those for their in vitro (IVP)- and in vivo-generated noncloned counterparts. In experiment 1, employing either FBA (fusion before activation) or AFS (fusion and activation simultaneously) to generate NT blastocysts, Hsp mRNAs were not found in NT embryos from either protocol, whereas Hsp transcripts were detectable in IVP embryos. The relative abundance (RA) of IF transcripts was significantly increased in the AFS and IVP groups compared to the FBA treatment. In experiment 2, the use of either G(0) or G(1) donor cells to produce cloned embryos both significantly reduced the relative amount of DNMT transcripts and significantly increased the RA of Mash2 compared to the IVP embryos. In addition, IF transcript levels were significantly elevated in NT blastocysts employing G(1) donor cells for NT compared to IVP embryos and those generated using G(0) cells. In experiment 3, donor cells, either from passsage 5/6 or 8, were employed for NT. DNMT transcripts were significantly decreased, whereas Mash2 transcripts were significantly increased in both NT groups compared to their IVP counterparts. The amount of IF mRNA was significantly higher in P8-derived than in P5/6 and IVP embryos. In

  9. Rational design of avian metapneumovirus live attenuated vaccines by inhibiting viral messenger RNA cap methyltransferase

    Science.gov (United States)

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis, is a non-segmented negative-sense RNA virus belonging to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. aMPV is the causative agent of respiratory tract infection and ...

  10. INDUCTION OF INTERLEUKIN-1-BETA MESSENGER-RNA AFTER FOCAL CEREBRAL-ISCHEMIA IN THE RAT

    NARCIS (Netherlands)

    BUTTINI, M; SAUTER, A; BODDEKE, HWGM

    1994-01-01

    The expression of interleukin-1beta (IL-1beta) mRNA in the brain in response to cerebral ischaemia in rats was examined using in situ hybridization histochemistry. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent occlusion of the left middle cerebral artery (MCAO)

  11. Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

    Science.gov (United States)

    Ganguly, T C; Liu, Y; Hyde, J F; Hagenbuch, B; Meier, P J; Vore, M

    1994-01-01

    We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity. Images Figure 2 PMID:7945260

  12. Detection and Quantification of N (6)-Methyladenosine in Messenger RNA by TLC.

    Science.gov (United States)

    Bodi, Zsuzsanna; Fray, Rupert G

    2017-01-01

    The base-modified nucleotide, N (6)-methyladenosine, is a relatively abundant modification found in the mRNA of most higher eukaryotes. Methylation levels can change dependent upon environmental conditions, cell differentiation state, or following knockdown of members of the methylase complex, and it is often useful to directly measure and compare N (6)-methyladenosine levels between samples. Two dimensional chromatography of radiolabeled nucleotides, following specific nuclease treatments, provides a robust, sensitive, and reproducible assay for this modification.

  13. The selective elimination of messenger RNA underlies the mitosis-meiosis switch in fission yeast.

    Science.gov (United States)

    Yamamoto, Masayuki

    2010-01-01

    The cellular programs for meiosis and mitosis must be strictly distinguished but the mechanisms controlling the entry to meiosis remain largely elusive in higher organisms. In contrast, recent analyses in yeast have shed new light on the mechanisms underlying the mitosis-meiosis switch. In this review, the current understanding of these mechanisms in the fission yeast Schizosaccharomyces pombe is discussed. Meiosis-inducing signals in this microbe emanating from environmental conditions including the nutrient status converge on the activity of an RRM-type RNA-binding protein, Mei2. This protein plays pivotal roles in both the induction and progression of meiosis and has now been found to govern the meiotic program in a quite unexpected manner. Fission yeast contains an RNA degradation system that selectively eliminates meiosis-specific mRNAs during the mitotic cell cycle. Mmi1, a novel RNA-binding protein of the YTH-family, is essential for this process. Mei2 tethers Mmi1 and thereby stabilizes the transcripts necessary for the progression of meiosis.

  14. Models for solid-state transport: messenger RNA movement from nucleus to cytoplasm.

    Science.gov (United States)

    Agutter, P S

    1994-09-01

    This paper explores the idea that mRNAs are transported between their transcription and processing sites in the nucleus, and their translation and degradation sites in the cytoplasm, by a 'solid-state' process. The underlying assumption is that negligible quantities of mRNA and of mRNA precursors are in solution in vivo. Therefore, mRNA transport cannot be considered as movement in the aqueous phase of the cell. The main lines of experimental evidence supporting this 'solid-state' concept are summarized and related controversies are outlined. Three possible models for a solid-state transport mechanism are discussed: a direct transfer model, with receptors organized analogously to the components of a multienzyme complex; a motor-driven model, analogous to synaptic vesicle transport in axons; and an assembly-driven model which assumes net movement along a fibril resulting from differential activities at the poles. Qualitative evaluation indicates that each of these models has characteristic advantages and disadvantages. The possibility that other nucleocytoplasmic transport processes might operate by solid-state mechanisms is briefly discussed.

  15. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, Alan; Kariko, K

    1990-01-01

    of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36....... First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from...

  16. Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

    Directory of Open Access Journals (Sweden)

    Kheradpour Albert

    2008-05-01

    Full Text Available Abstract Background Methotrexate (MTX uptake is mediated by the reduced folate carrier (RFC. Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8, including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

  17. Messenger RNA expression of chicken CLOCK gene in the response to Campylobacter jejuni inoculation.

    Science.gov (United States)

    Liu, Xiaoyi; Liu, Liying; Zhang, Maozhi; Yang, Ning; Qi, Yukai; Sun, Yu; Li, Xianyao

    2015-09-01

    Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial gastroenteritis worldwide. Previous research has shown that circadian rhythm plays a critical role in host response to C. jejuni colonization. The CLOCK gene is one of the core genes regulating circadian rhythms and shows significant expression on 7 d post-C. jejuni inoculation. The objective of this study was to investigate temporal and spatial expression of chicken CLOCK gene post-C. jejuni inoculation. Cecal and splenic RNA were isolated from 2 distinct chicken breeds and used to compare the mRNA expression of CLOCK gene between inoculated and noninoculated chickens within each breed and between breeds within each of inoculated and noninoculated groups. Our results showed that the CLOCK gene was significantly down-regulated at 20 h postinoculation (hpi) in cecum and spleen in Jiningbairi chicken. CLOCK gene was significantly down-regulated at 4 and 16 hpi and up-regulated at 8 hpi in cecum and spleen in specific pathogen free white leghorn noninoculated chicken. The findings suggested that expression of CLOCK gene was significantly changed post C. jejuin inoculation. This change was affected by genetic background, tissue, and time points postinoculation. © 2015 Poultry Science Association Inc.

  18. Skeletal muscle microRNA and messenger RNA profiling in cofilin-2 deficient mice reveals cell cycle dysregulation hindering muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Sarah U Morton

    Full Text Available Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2 have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies.

  19. Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

    Science.gov (United States)

    Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-15

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

  20. Leishmania pifanoi: kinetics of messenger RNA expression during amastigote to promastigote transformation in vitro.

    Science.gov (United States)

    Campbell, S M; Rainey, P M

    1993-08-01

    Conditions have been developed which induce axenically grown Leishmania pifanoi amastigotes to transform into the promastigote stage in a highly reproducible fashion. Transformation was induced by a temperature shift from 31 to 22 degrees C, was inhibited by high cell concentration (> or = 40 x 10(6) cells/ml), and was unaffected by pH from 5.5-7.2. Morphologic transformation was first evident at 8 hr after induction, had occurred in > 50% of cells by 24 hr, and was > 90% complete by 48 hr. This system enabled study of the kinetics of mRNA expression during the transformation of Leishmania. The differentially expressed mRNAs for ATPase 1a and 1b, alpha- and beta-tubulin, P100/11E, Pro-1, and pLm 2, 7, 14, and 16 exhibited complex patterns of temporal expression, suggesting a highly regulated process. Differentiation on the biochemical level was evident within an hour and continued throughout the course of morphologic transformation. In addition, transformed L. pifanoi promastigotes in the plateau growth phase expressed genes characteristic of metacyclic promastigotes. Axenically cultured L. pifanoi should provide an excellent model for the study of differentiation in Leishmania.

  1. Compensatory evolution of a precursor messenger RNA secondary structure in the Drosophila melanogaster Adh gene

    Science.gov (United States)

    Chen, Ying; Stephan, Wolfgang

    2003-01-01

    Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (Adh) in diverse Drosophila species. In this study, the putative hairpin structure was evaluated systematically in Drosophila melanogaster by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in Drosophila melanogaster Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed. PMID:12972637

  2. Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting

    DEFF Research Database (Denmark)

    Hansen, Thomas Møller; Reihani, S Nader S; Oddershede, Lene B

    2007-01-01

    the Infectious Bronchitis Virus were used, differing by one base pair in the first stem. In Escherichia coli, these two pseudoknots caused frameshifting frequencies that differed by a factor of two. We used optical tweezers to unfold the pseudoknots. The pseudoknot giving rise to the highest degree...

  3. Emergence of the β-CASP ribonucleases: highly conserved and ubiquitous metallo-enzymes involved in messenger RNA maturation and degradation.

    Science.gov (United States)

    Dominski, Zbigniew; Carpousis, Agamemnon J; Clouet-d'Orval, Béatrice

    2013-01-01

    The β-CASP ribonucleases, which are found in the three domains of life, have in common a core of 460 residues containing seven conserved sequence motifs involved in the tight binding of two catalytic zinc ions. A hallmark of these enzymes is their ability to catalyze both endo- and exo-ribonucleolytic degradation. Exo-ribonucleolytic degradation proceeds in the 5' to 3' direction and is sensitive to the phosphorylation state of the 5' end of a transcript. Recent phylogenomic analyses have shown that the β-CASP ribonucleases can be partitioned into two major subdivisions that correspond to orthologs of eukaryal CPSF73 and bacterial RNase J. We discuss the known functions of the CPSF73 and RNase J orthologs, their association into complexes, and their structure as it relates to mechanism of action. Eukaryal CPSF73 is part of a large multiprotein complex that is involved in the maturation of the 3' end of RNA Polymerase II transcripts and the polyadenylation of messenger RNA. RNase J1 and J2 are paralogs in Bacillus subtilis that are involved in the degradation of messenger RNA and the maturation of non-coding RNA. RNase J1 and J2 co-purify as a heteromeric complex and there is recent evidence that they interact with other enzymes to form a bacterial RNA degradosome. Finally, we speculate on the evolutionary origin of β-CASP ribonucleases and on their functions in Archaea. Orthologs of CPSF73 with endo- and exo-ribonuclease activity are strictly conserved throughout the archaea suggesting a role for these enzymes in the maturation and/or degradation of messenger RNA. This article is part of a Special Issue entitled: RNA Decay mechanisms. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Melatonin, Noncoding RNAs, Messenger RNA Stability and Epigenetics—Evidence, Hints, Gaps and Perspectives

    Directory of Open Access Journals (Sweden)

    Rüdiger Hardeland

    2014-10-01

    Full Text Available Melatonin is a highly pleiotropic regulator molecule, which influences numerous functions in almost every organ and, thus, up- or down-regulates many genes, frequently in a circadian manner. Our understanding of the mechanisms controlling gene expression is actually now expanding to a previously unforeseen extent. In addition to classic actions of transcription factors, gene expression is induced, suppressed or modulated by a number of RNAs and proteins, such as miRNAs, lncRNAs, piRNAs, antisense transcripts, deadenylases, DNA methyltransferases, histone methylation complexes, histone demethylases, histone acetyltransferases and histone deacetylases. Direct or indirect evidence for involvement of melatonin in this network of players has originated in different fields, including studies on central and peripheral circadian oscillators, shift work, cancer, inflammation, oxidative stress, aging, energy expenditure/obesity, diabetes type 2, neuropsychiatric disorders, and neurogenesis. Some of the novel modulators have also been shown to participate in the control of melatonin biosynthesis and melatonin receptor expression. Future work will need to augment the body of evidence on direct epigenetic actions of melatonin and to systematically investigate its role within the network of oscillating epigenetic factors. Moreover, it will be necessary to discriminate between effects observed under conditions of well-operating and deregulated circadian clocks, and to explore the possibilities of correcting epigenetic malprogramming by melatonin.

  5. Bisphenol S alters embryonic viability, development, gallbladder size, and messenger RNA expression in chicken embryos exposed via egg injection.

    Science.gov (United States)

    Crump, Doug; Chiu, Suzanne; Williams, Kim L

    2016-06-01

    Amid concerns about the toxicological effects and environmental prevalence of bisphenol A (BPA), efforts to find suitable, safer replacement alternatives are essential. Bisphenol S (BPS) is a potential chemical substitute for BPA; however, few studies are available confirming that it has a more desirable ecotoxicological profile. In the present study, BPS was injected into the air cell of unincubated, fertilized chicken embryos at 6 concentrations ranging from 0 μg/g to 207 μg/g egg to determine effects on pipping success, development, hepatic messenger ribonucleic acid (mRNA) expression, thyroid hormone levels, and circulating bile acid concentrations. Concentrations of BPS increased in a dose-dependent manner in whole-embryo homogenates, and exposure to the highest dose, 207 μg/g, resulted in decreased pipping success (estimated median lethal dose  = 279 μg/g; 95% confidence interval = 161-486 μg/g). Exposure to BPS also reduced growth metrics including embryo mass and tarsus length, whereas the most pronounced phenotypic effect was the concentration-dependent, significant increase in gallbladder size at concentrations ≥52.8 μg/g. These adverse phenotypic outcomes were associated with the modulation of gene targets from a chicken ToxChip polymerase chain reaction array, which are involved with xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and the thyroid hormone pathway. Expression levels of 2 estrogen-responsive genes, apolipoprotein II and vitellogenin, were too low at the sampling time point assessed (i.e., pipping embryos) to quantify changes, and no effects were observed on circulating free thyroxine or bile acid concentrations. The present study provides novel, whole-animal toxicological data for a BPA replacement alternative that is not well characterized. Environ Toxicol Chem 2016;35:1541-1549. © 2015 SETAC.

  6. Plant serine/arginine-rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses.

    Science.gov (United States)

    Reddy, Anireddy S N; Shad Ali, Gul

    2011-01-01

    Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. Despite the widespread occurrence of AS in plants, the mechanisms that control splicing and the roles of splice variants generated from a gene are poorly understood. Studies on plant serine/arginine-rich (SR) proteins, a family of highly conserved proteins, suggest their role in both constitutive splicing and AS of pre-mRNAs. SR proteins have a characteristic domain structure consisting of one or two RNA recognition motifs at the N-terminus and a C-terminal RS domain rich in arginine/serine dipeptides. Plants have many more SR proteins compared to animals including several plant-specific subfamilies. Pre-mRNAs of plant SR proteins are extensively alternatively spliced to increase the transcript complexity by about six-fold. Some of this AS is controlled in a tissue- and development-specific manner. Furthermore, AS of SR pre-mRNAs is altered by various stresses, raising the possibility of rapid reprogramming of the whole transcriptome by external signals through regulation of the splicing of these master regulators of splicing. Most SR splice variants contain a premature termination codon and are degraded by up-frameshift 3 (UPF3)-mediated nonsense-mediated decay (NMD), suggesting a link between NMD and regulation of expression of the functional transcripts of SR proteins. Limited functional studies with plant SRs suggest key roles in growth and development and plant responses to the environment. Here, we discuss the current status of research on plant SRs and some promising approaches to address many unanswered questions about plant SRs.

  7. In situ changes in the relative abundance of human epidermal cytokine messenger RNA levels following exposure to the poison ivy/oak contact allergen urushiol.

    Science.gov (United States)

    Boehm, K D; Yun, J K; Strohl, K P; Trefzer, U; Häffner, A; Elmets, C A

    1996-06-01

    Abstract: Epidermal keratinocytes in culture have been shown to produce many cytokines, and their proteins have been identified in skin tissue samples. It has therefore been assumed that these cytokines are transcribed in vivo by the epidermis in response to contact allergens. In this report, in situ hybridization was used to detect the messenger RNAs for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) in samples of human skin prior to and at various times after application of urushiol, the immunogenic component of poison ivy/oak. In sensitive subjects, IL-1 alpha and TNF-alpha mRNAs showed a progressive increase in transcript levels that paralleled the clinical and histological features of the inflammatory process. The time-course of the IL-1 beta response differed from that of IL-1 alpha and TNF-alpha, in that there was an early (by 6 h after urushiol administration) elevation in IL-1 beta mRNA that occurred before there was evidence of inflammation and had returned to background levels by 72 h when the reaction had reached its peak. In contrast to urushiol-sensitive subjects, urushiol-anergic individuals did not exhibit an increase in IL-1 alpha, IL-1 beta or TNF-alpha mRNA levels. The data provide evidence for an in vivo role for epidermal IL-1 alpha, IL-1 beta and TNF-alpha transcription in the regulation of IL-1 beta and TNF-alpha polypeptide levels in the epidermis in response to this common contact allergen.

  8. In vitro Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications.

    Directory of Open Access Journals (Sweden)

    Meike-Kristin Abraham

    Full Text Available Stent angioplasty provides a minimally invasive treatment for atherosclerotic vessels. However, no treatment option for atherosclerosis-associated endothelial dysfunction, which is accompanied by a loss of CD39, is available, and hence, adverse effects like thromboembolism and restenosis may occur. Messenger RNA (mRNA-based therapy represents a novel strategy, whereby de novo synthesis of a desired protein is achieved after delivery of a modified mRNA to the target cells.Our study aimed to develop an innovative bioactive stent coating that induces overexpression of CD39 in the atherosclerotic vessel. Therefore, a modified CD39-encoding mRNA was produced by in vitro transcription. Different endothelial cells (ECs were transfected with the mRNA, and CD39 expression and functionality were analyzed using various assays. Furthermore, CD39 mRNA was immobilized using poly(lactic-co-glycolic-acid (PLGA, and the transfection efficiency in ECs was analyzed. Our data show that ECs successfully translate in vitro-generated CD39 mRNA after transfection. The overexpressed CD39 protein is highly functional in hydrolyzing ADP and in preventing platelet activation. Furthermore, PLGA-immobilized CD39 mRNA can be delivered to ECs without losing its functionality.In summary, we present a novel and promising concept for a stent coating for the treatment of atherosclerotic blood vessels, whereby patients could be protected against angioplasty-associated complications.

  9. Delivery of messenger RNA using poly(ethylene imine)-poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties.

    Science.gov (United States)

    Debus, Heiko; Baumhof, Patrick; Probst, Jochen; Kissel, Thomas

    2010-12-20

    Nucleic acid based therapies have so far mainly been focused on plasmid DNA (pDNA), small interfering RNA (siRNA), antisense and immunostimulatory oligonucleotides. Messenger RNA (mRNA) was the subject of only a few studies. The objective of this investigation was the preparation of new composite polyplexes with mRNA consisting of poly(ethylene imine) (PEI) and poly(ethylene imine)-poly(ethylene glycol)-copolymers (PEI-PEG) as blends. These complexes were designed to increase the stability of mRNA, to improve transfection efficiency and to reduce cytotoxicity. Hydrodynamic diameters of the polyplexes were measured by dynamic light scattering, polyplex stability was analyzed by gel retardation assay and transfection efficiency of luciferase (Luc) encoding mRNA was evaluated under in vitro conditions. Most of the polyplexes generated showed small particle sizes application of mRNA merit further investigation under in vitro and in vivo conditions. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA).

    Science.gov (United States)

    Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton; Karzai, A Wali

    2013-10-18

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.

  11. 肿瘤基因信使RNA可变剪接及其应用%Alternative splicing of tumor associated genes messenger RNA and application

    Institute of Scientific and Technical Information of China (English)

    张鑫桐; 岳文涛

    2014-01-01

    可变剪接作为基因的一种修饰方式,是真核细胞表达调控过程的重要因素。它使同一蛋白质编码基因能够产生多种转录本,极大地扩展了遗传信息的应用。在人类肿瘤细胞中前体信使RN A的可变剪接扮演着重要角色,一些重要基因通过可变剪接产生不同于正常细胞中的剪接异构体。这些肿瘤特异性剪接异构体的存在导致了肿瘤的发生、发展。深入探索肿瘤相关基因的可变剪接对肿瘤的诊断、治疗具有重要意义。%As a way of gene modification,alternative splicing is an important factor of eukaryotic gene expression and regulation.It makes various transcripts from one protein-coding gene,and greatly extends the genetic information.Alternative splicing of pre-messenger RNA plays an important role in tumor cells.By alter-native splicing,some important genes can generate splicing variants different from those in normal cells.The existence of tumor-specific splicing variants leads to the occurrence and progression of tumor.Therefore,explo-ration on the alternative splicing of tumor-associated genes may be of great significance in tumor diagnosis and treatment.

  12. Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons.

    Science.gov (United States)

    Stone, D M; Grillo, M; Margolis, F L; Joh, T H; Baker, H

    1991-09-08

    Expression of the dopaminergic phenotype in olfactory bulb (OB) juxtaglomerular neurons (constituting a population of periglomerular and external tufted cells) is dependent upon functional innervation by peripheral olfactory receptors. Loss of functional input in rodents, by either peripheral deafferentation or deprivation of odorant access, results in a profound decrease in the expression of juxtaglomerular tyrosine hydroxylase (TH). We have examined the effects of such treatments on the expression of the neurotransmitter biosynthetic enzyme glutamic acid decarboxylase (GAD), which is colocalized with TH in the majority of TH-containing juxtaglomerular neurons. Following either chemically induced OB deafferentation in adult mice or unilateral odor deprivation in neonatal rats, steady-state OB GAD messenger RNA levels remained essentially unchanged as assessed by Northern blot analysis 20-40 days after treatment. These results were confirmed by in situ hybridization analysis, which demonstrated a profound loss of juxtaglomerular TH messenger RNA but no accompanying decrease in regionally colocalized GAD message. Since GAD is found in nearly all dopaminergic OB cells, the preservation of juxtaglomerular GAD message implies that olfactory receptor neurons exert a differential transneuronal regulation of TH and GAD gene transcription.

  13. Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat.

    Science.gov (United States)

    Caboche, Jocelyne; Vernier, Philippe; Rogard, Monique; Julien, Jean-François; Mallet, Jacques; Besson, Marie-Jo

    1992-01-01

    Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also

  14. Regional age-related changes in neuronal nitric oxide synthase (nNOS, messenger RNA levels and activity in SAMP8 brain

    Directory of Open Access Journals (Sweden)

    Guidon Gérard

    2006-12-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8 mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS. To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx- levels, in three brain areas (n = 7 animals in each group. Calibrated reverse transcriptase (RT and real-time polymerase chain reaction (PCR and biochemical procedures were used. Results We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Conclusion Concomitant variations occurring in NO levels

  15. Translational Influence on Messenger Stability

    DEFF Research Database (Denmark)

    Eriksen, Mette

    , on messenger stability was examined. Depending on the translation initiation frequency, the chance of an initial ribosome trailing the RNA polymerase get better for better initiation sites, thus protecting transcription from termination by Rho. A polarity assay in which the activity of the downstream lac......-termination to be a global phenomena in gene regulation. The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments demonstrated that very fast...

  16. Involvement of second messengers in the signaling pathway of vitellogenesis-inhibiting hormone and their effects on vitellogenin mRNA expression in the whiteleg shrimp, Litopenaeus vannamei.

    Science.gov (United States)

    Bae, Sun-Hye; Okutsu, Tomoyuki; Tsutsui, Naoaki; Kang, Bong Jung; Chen, Hsiang-Yin; Wilder, Marcy N

    2017-05-15

    We incubated fragments of Litopenaeus vannamei ovary to investigate second messengers involved in the regulation of vitellogenin (vg) mRNA levels. The use of 100nM recombinant vitellogenesis-inhibiting hormone (VIH) (corresponding to recombinant L. vannamei sinus gland peptide-G: rLiv-SGP-G) significantly reduced vg mRNA expression in sub-adults after 8h incubation to less than 20% of the control. The concentration of intracellular cyclic guanosine monophosphate (cGMP) increased 3.2-fold relative to the control after 2h incubation with rLiv-SGP-G. However, it reached levels 18-fold relative to the control after 0.5h incubation with rLiv-SGP-G where 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) was also added. Moreover, vg mRNA expression was significantly reduced to less than 50% of the control after 24h incubation with 1μM A23187 (a calcium ionophore). Thus, rLiv-SGP-G and calcium ionophore reduced vg mRNA expression in in vitro-cultured ovary, and cGMP may be involved in the signaling pathway of VIH. Overall, the above results suggest that vg mRNA expression might be inhibited in vitro by increasing intracellular cGMP and Ca(2+) in L. vannamei ovary. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Consequence of nigrostriatal denervation and L-dopa therapy on the expression of glutamic acid decarboxylase messenger RNA in the pallidum.

    Science.gov (United States)

    Herrero, M T; Levy, R; Ruberg, M; Luquin, M R; Villares, J; Guillen, J; Faucheux, B; Javoy-Agid, F; Guridi, J; Agid, Y; Obeso, J A; Hirsch, E C

    1996-07-01

    To examine the consequences of nigrostriatal denervation and L-dopa treatment on the basal ganglia output system, we analyzed, by quantitative in situ hybridization, the messenger RNA coding for glutamic acid decarboxylase (Mr 67,000) (GAD67 mRNA) in pallidal cells from patients with Parkinson's disease (PD), monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) receiving or not receiving L-dopa, and their respective control subjects. In MPTP-treated monkeys, the expression of GAD67 mRNA was increased in cells from the internal pallidum, and this effect was abolished by L-dopa treatment. There were no differences in the levels of GAD67 mRNA between patients with PD, who were all treated with L-dopa, and control subjects. These results indicate that the level of GAD67 mRNA is increased in the cells of the internal pallidum after nigrostriatal dopaminergic denervation and that this increase can be reversed by L-dopa therapy.

  18. Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

    Science.gov (United States)

    Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

    2017-05-15

    Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection.

  19. Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.

    Directory of Open Access Journals (Sweden)

    Nour Eissa

    Full Text Available Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR. No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE in DSS-experimental murine colitis.Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle.Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh, β-actin (Actb, or β2-microglobulin (β2m showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp and eukaryotic translation elongation factor 2 (Eef2 were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used.This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.

  20. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

    Science.gov (United States)

    Pato, A; Eisenberg, G; Machlenkin, A; Margalit, A; Cafri, G; Frankenburg, S; Merims, S; Peretz, T; Lotem, M; Gross, G

    2015-11-01

    Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy. © 2015 British Society for Immunology.

  1. Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats.

    Science.gov (United States)

    Saini, R K; Manoj, P; Shetty, N P; Srinivasan, K; Giridhar, P

    2014-07-01

    In this study, the effects of iron depletion and repletion on biochemical and molecular indices of iron status were investigated in growing male Wistar rats. We hypothesized that iron from Moringa leaves could overcome the effects of iron deficiency and modulate the expression of iron-responsive genes better than conventional iron supplements. Iron deficiency was induced by feeding rats an iron-deficient diet for 10 weeks, whereas control rats were maintained on an iron-sufficient diet (35.0-mg Fe/kg diet). After the depletion period, animals were repleted with different source of iron, in combination with ascorbic acid. Iron deficiency caused a significant (P Moringa leaf was found to be superior compared with ferric citrate in overcoming the effects of iron deficiency in rats. These results suggest that changes in the relative expression of liver hepcidin messenger RNA can be used as a sensitive molecular marker for iron deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Overexpression of interleukin 2 receptor, thymidine kinase and immunoglobulin-associated alpha-1 messenger RNA in a clinical case of enzootic bovine leukosis.

    Science.gov (United States)

    Tawfeeq, Mohammad Monir; Tagawa, Michihito; Itoh, Yuuki; Sugimoto, Kazuya; Kobayashi, Yoshiyasu; Inokuma, Hisashi

    2012-09-01

    A 49-month-old Holstein cow with anorexia, tachypnea, enlarged peripheral lymph nodes, and difficulty standing up was suspected of bovine leukosis. Hematological examination revealed lymphocytosis with the presence of neoplastic cells. Increased total lactate dehydrogenase (LDH) activity, isozymes of LDH-2 and LDH-3 activities and thymidine kinase activity were observed. Cytological findings of fine needle aspiration of subiliac lymph nodes indicated lymphosarcoma. Histopathology and antibody analysis confirmed the diagnosis of enzootic bovine leukosis, a B-cell bovine lymphoma caused by bovine leukemia virus. Gene expressions known as biomarkers of hematopoietic neoplasia in human were also examined in the present case. Increased messenger RNA expression of interleukin 2 receptor, thymidine kinase, and immunoglobulin-associated alpha-1 was observed in the case animal.

  3. Relationship between expression of α-fetoprotein messenger RNA and some clinical parameters of human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    AIM To certify the relationship between AFP mRNA and some pathological parameters of he-patocellular carcinoma (HCC).METHOD We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffering from HCC by RT-PCR method.RESULTS The positive rate of AFP mRNA was 76.9% in the HCC tumor tissues, and 69.4% in the paratumor tissues from the HCC patients with severe cirrhosis. However, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFP mRNA expression was found in the related paratumor tissues.CONCLUSION The AFP protein was specially expressed by HCC cells and mutated hepatocytes. The AFP mRNA was positively related with cirrhosis, but no significant relationship was found between AFP mRNA and tumor size, capsule status and tumor metastasis.

  4. Erythropoietin messenger RNA levels in developing mice and transfer of /sup 125/I-erythropoietin by the placenta

    Energy Technology Data Exchange (ETDEWEB)

    Koury, M.J.; Bondurant, M.C.; Graber, S.E.; Sawyer, S.T.

    1988-07-01

    Erythropoietin (EP) mRNA was measured in normal and anemic mice during fetal and postnatal development. Normal fetal livers at 14 d of gestation contained a low level of EP mRNA. By day 19 of gestation, no EP mRNA was detected in normal or anemic fetal livers or normal fetal kidneys, but anemic fetal kidneys had low levels of EP mRNA. Newborn through adult stage mice responded to anemia by accumulating renal and hepatic EP mRNA. However, total liver EP mRNA was considerably less than that of the kidneys. Juvenile animals, 1-4 wk old, were hyperresponsive to anemia in that they produced more EP mRNA than adults. Moreover, nonanemic juveniles had readily measured renal EP mRNA, whereas the adult level was at the lower limit of detection. Because of the very low level of fetal EP mRNA, placental transfer of EP was evaluated. When administered to the pregnant mouse, /sup 125/I-EP was transferred in significant amounts to the fetuses. These results indicate that in mice the kidney is the main organ of EP production at all stages of postnatal development and that adult kidney may also play some role in providing EP for fetal erythropoiesis via placental transfer of maternal hormone.

  5. Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro.

    Science.gov (United States)

    Passos, J R S; Costa, J J N; da Cunha, E V; Silva, A W B; Ribeiro, R P; de Souza, G B; Barroso, P A A; Dau, A M P; Saraiva, M V A; Gonçalves, P B D; van den Hurk, R; Silva, J R V

    2016-01-01

    This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Alternative messenger RNA splicing of autophagic gene Beclin 1 in human B-cell acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Niu, Yu-Na; Liu, Qing-Qing; Zhang, Su-Ping; Yuan, Na; Cao, Yan; Cai, Jin-Yang; Lin, Wei-Wei; Xu, Fei; Wang, Zhi-Jian; Chen, Bo; Wang, Jian-Rong

    2014-01-01

    Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

  7. Expression of endogenous and exogenous growth hormone (GH) messenger (m) RNA in a GH-transgenic tilapia (Oreochromis niloticus).

    Science.gov (United States)

    Caelers, Antje; Maclean, Norman; Hwang, Gyulin; Eppler, Elisabeth; Reinecke, Manfred

    2005-02-01

    We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp beta-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 +/- 30 pg/microg total RNA but in transgenics only to 187 +/- 43 pg/microg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 +/- 2.5 pg/microg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 +/- 2.0 pg/microg total RNA in gills to 0.2 +/- 0.08 pg/microg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.

  8. Modeling microRNA-transcription factor networks in cancer.

    Science.gov (United States)

    Aguda, Baltazar D

    2013-01-01

    An increasing number of transcription factors (TFs) and microRNAs (miRNAs) is known to form feedback loops (FBLs) of interactions where a TF positively or negatively regulates the expression of a miRNA, and the miRNA suppresses the translation of the TF messenger RNA. FBLs are potential sources of instability in a gene regulatory network. Positive FBLs can give rise to switching behaviors while negative FBLs can generate periodic oscillations. This chapter presents documented examples of FBLs and their relevance to stem cell renewal and differentiation in gliomas. Feed-forward loops (FFLs) are only discussed briefly because they do not affect network stability unless they are members of cycles. A primer on qualitative network stability analysis is given and then used to demonstrate the network destabilizing role of FBLs. Steps in model formulation and computer simulations are illustrated using the miR-17-92/Myc/E2F network as an example. This example possesses both negative and positive FBLs.

  9. Acceptability and Accuracy of Cervical Cancer Screening Using a Self-Collected Tampon for HPV Messenger-RNA Testing among HIV-Infected Women in South Africa.

    Directory of Open Access Journals (Sweden)

    Paul C Adamson

    Full Text Available HIV increases women's risk for high-risk human papillomavirus (hrHPV infection and invasive cervical cancer. South Africa has a high HIV prevalence but low cervical cancer screening coverage. Self-collection of cervical specimens and hrHPV testing, including hrHPV messenger-RNA (mRNA testing, are methods aimed at increasing screening rates. However, data are limited on the acceptability and accuracy of tampon-based self-collection for hrHPV mRNA testing in HIV-infected women.We recruited 325 HIV-infected women seeking care at a government HIV clinic in Pretoria, South Africa. A clinician performed a pelvic examination and obtained an endocervical specimen. Study participants performed self-collection using a tampon. Both clinician- and self-collected specimens were tested for hrHPV mRNA. Acceptability of both collection methods was assessed, the prevalence of hrHPV mRNA in our study population was estimated, test positivity of the two collection methods were compared, and test agreement was assessed by calculating the κ-statistic, sensitivity, and specificity.Over 90% of women reported no difficulties self-collecting specimens and 82% were willing to perform the tampon-collection at home. Based on clinician-collection specimens, the prevalence of hrHPV mRNA in our study population was 36.7% (95% CI: 31.4%- 42.0%. There was no difference in test positivity between clinician-collection, 36.7%, and tampon-collection, 43.5% (p-value = 0.08. Using clinician-collection as the reference test, the sensitivity and specificity for hrHPV mRNA of tampon-collection were 77.4% (95% CI: 69.8-85.0% and 77.8% (95% CI: 71.9-83.6%, respectively.Tampon-based self-collection is acceptable to women and has similar hrHPV mRNA positivity rates as clinician-collection, but has reduced sensitivity and specificity compared to clinician-collection. The hrHPV mRNA prevalence in our study population is high, but similar to other high-risk populations, and highlights the

  10. Myocardial ischemic preconditioning in a porcine model leads to rapid changes in cardiac extracellular vesicle messenger RNA content

    Directory of Open Access Journals (Sweden)

    Kristina Svennerholm

    2015-09-01

    Conclusions: These findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production.

  11. Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy.

    Science.gov (United States)

    Herbison, A E; Augood, S J; McGowan, E M

    1992-08-01

    Evidence suggests that medial preoptic area (MPOA) neurones containing gamma-aminobutyric acid (GABA) are modulated directly by oestrogen. We have used an alkaline phosphatase-labelled antisense oligonucleotide probe to examine glutamic acid decarboxylase67 (GAD) mRNA expression within individual cells of the MPOA, diagonal band of Broca (DBB) and parietal cortex in rats killed at noon on each day of the oestrous cycle and after ovariectomy (n = 4-5). As a fall in extracellular GABA concentrations occurs in the MPOA on the afternoon of proestrus, the GAD67 mRNA content of cells was also examined in proestrous rats at 15:00h immediately prior to the preovulatory luteinising hormone (LH) surge. The MPOA was found to have an intermediate number of GAD67 mRNA-containing cells compared with the DBB and cortex (P less than 0.01) but expressed the lowest mean hybridisation signal (P less than 0.01). The parietal cortex had significantly fewer (P less than 0.01) GAD mRNA-containing cells than either the MPOA or DBB but these contained higher mean density of signal (P less than 0.01). The hybridisation signal for GAD mRNA was abolished by either ribonuclease pre-treatment or the use of excess non-labelled probe. No significant (P greater than 0.05) differences in GAD67 mRNA were detected in animals killed at noon throughout the oestrous cycle or after ovariectomy. On the afternoon of proestrus (15:00h) there was a significant 40% reduction in mean GAD67 mRNA content within cells of only the MPOA compared with noon (P less than 0.05). The numbers of cells in the MPOA expressing GAD67 mRNA were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

    Science.gov (United States)

    Yamamoto, M; Igarashi, T; Muramatsu, M; Fukagawa, M; Motokura, T; Ogata, E

    1989-01-01

    To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period. Images PMID:2493484

  13. Investigating RNA editing factors from trypanosome mitochondria

    Science.gov (United States)

    Aphasizheva, Inna; Zhang, Liye; Aphasizhev, Ruslan

    2016-01-01

    Mitochondrial U-insertion/deletion mRNA editing is carried out by two principal multiprotein assemblies, enzymatic RNA editing core (RECC) and RNA editing substrate binding (RESC) complexes, and a plethora of auxiliary factors. An integral part of mitochondrial gene expression, editing receives inputs from primary mRNA and gRNA precursor processing pathways, and generates substrates for mRNA polyadenylation and translation. Although nearly all RECC-embedded enzymes have been implicated in specific editing reactions, the majority of proteins that populate the RESC are also essential for generating edited mRNAs. However, lack of recognizable motifs in RESC subunits limits the prowess of bioinformatics in guiding biochemical experiments and elucidating their specific biological functions. In this chapter, we describe a generic workflow for investigating mitochondrial mRNA editing in Trypanosoma brucei and focus on several methods that proved instrumental is assigning definitive functions to editing factors lacking known signature sequences. PMID:27020893

  14. P-body loss is concomitant with formation of a messenger RNA storage domain in mouse oocytes.

    Science.gov (United States)

    Flemr, Matyas; Ma, Jun; Schultz, Richard M; Svoboda, Petr

    2010-05-01

    In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

  15. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

    OpenAIRE

    Yamamoto, M.; Igarashi, T; Muramatsu, M; Fukagawa, M.; Motokura, T.; Ogata, E

    1989-01-01

    To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaC...

  16. P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes1

    OpenAIRE

    Flemr, Matyas; Ma, Jun; Schultz, Richard M.; Svoboda, Petr

    2010-01-01

    In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. ...

  17. Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain.

    Science.gov (United States)

    Ferraguti, F; Zoli, M; Aronsson, M; Agnati, L F; Goldstein, M; Filer, D; Fuxe, K

    1990-01-01

    The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number

  18. A POSSIBLE CONTRIBUTION OF MESSENGER-RNA SECONDARY STRUCTURE TO TRANSLATION INITIATION EFFICIENCY IN LACTOCOCCUS-LACTIS

    NARCIS (Netherlands)

    VANDEGUCHTE, M; VANDERLENDE, T; KOK, J; VENEMA, G

    1991-01-01

    Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Muta

  19. Spatial distribution of CYP2B1/2 messenger RNA within the rat liver acinus following exposure to the inducers phenobarbital and dieldrin.

    Science.gov (United States)

    Dail, Mary B; Burgess, Shane C; Meek, Edward C; Wagner, Jennifer; Baravik, Jeffrey; Chambers, Janice E

    2007-09-01

    Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16beta-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.

  20. Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA.

    Science.gov (United States)

    Robert, F; Brakier-Gingras, L

    2001-02-01

    Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

  1. Correlation between PMP-22 messenger RNA expression and phenotype in hereditary neuropathy with liability to pressure palsies.

    Science.gov (United States)

    Schenone, A; Nobbio, L; Caponnetto, C; Abbruzzese, M; Mandich, P; Bellone, E; Ajmar, F; Gherardi, G; Windebank, A J; Mancardi, G

    1997-12-01

    Hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a deletion in chromosome 17p11.2, which includes the gene for the peripheral myelin protein 22 (PMP-22). A "gene dosage" effect is probably the mechanism underlying HNPP, but the amount of PMP-22 mRNA in sural nerves of HNPP patients is highly variable and the role of PMP-22 underexpression in impairing myelination has yet to be clarified. We have studied 6 genetically proven HNPP patients, to evaluate the relationship between PMP-22 mRNA levels, and clinical, neurophysiological, and neuropathological findings. Underexpression of PMP-22 mRNA correlates with disease severity and with mean axon diameter and g ratio, but not with myelin thickness, number of "tomacula," or nerve conduction parameters. Our findings further confirm that underexpression of PMP-22 is the main pathogenetic mechanism underlying the severity of clinical symptoms and signs in HNPP. Smaller axons in sural nerves of HNPP patients with lower PMP-22 levels suggests that underexpression of PMP-22 may also affect axon development.

  2. 5'-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events.

    Science.gov (United States)

    McCormick, J A; Lyons, V; Jacobson, M D; Noble, J; Diorio, J; Nyirenda, M; Weaver, S; Ester, W; Yau, J L; Meaney, M J; Seckl, J R; Chapman, K E

    2000-04-01

    Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 1(6) and 1(10)) were expressed in all tissues examined, together present in 77-87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 1(7). Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predominant exon 1(10), suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

  3. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

    Science.gov (United States)

    Kim, H Y; Hanley, M R

    1999-06-30

    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  4. Is the expression of γ-glutamyl transpeptidase messenger RNA an indicator of biological behavior in recurrent hepatocellular carcinoma?

    Institute of Scientific and Technical Information of China (English)

    I-Shyan Sheen; Kuo-Shyang Jeng; Yi-Chun Tsai

    2003-01-01

    AIM: To investigate the correlation between gammaglutamyl transpeptidase (γ-GTP) expression in the primary HCC and post-resection recurrence and its biological behaviors.METHODS: Forty consecutive patients having curative resection for HCC were included in this study. The primers for reverse-transcription polymerase chain reaction (RTPCR) were corresponding to the 5'-noncoding human γ-GTP mRNA of fetal liver (type A), HepG2 cells (type B),and placenta (type C). Both the cancer and non-cancerous tissues of the resected liver were analyzed. The correlations between the expression of γ-GTP and the clinicopathological variables and outcomes (recurrence and survival) were studied.RESULTS: Those with type B γ-GTP mRNA in cancer had significant higher recurrence rate than those without it (63.6 % vs 14.3 %). Both those with type B in cancer and in non-cancer died significantly more than those without it (45.5 % vs 0 % and 53.6 % vs 0 %, respectively). By multivariate analysis, the significant predictors of recurrence included high serum AFP (P=0.0108), vascular permeation (P=0.0084), and type B γ-GTP mRNA in non-cancerous liver (P=0.0107). The significant predictors of postrecurrence death included high serum AFP (P=0.0141),vascular permeation (P=0.0130), and daughter nodules (P=0.0053). As to the manifestations (recurrent number ≥2, recurrent extent≥2 segments, extra-hepatic metastasis, and death) in recurrent patients, there were no statistical significant differences between those with type B in the primary tumor and those without it. The difference between those with type B in non-cancerous liver and those without it also was not significant.CONCLUSION: Patients of HCC with type B γ-GTP mRNA both in cancer and in non-cancerous tissue had a worse outcome, earlier recurrence, and more post-recurrence death.

  5. Two insulin-like growth factor I messenger RNAs are expressed in human liver.

    OpenAIRE

    Rotwein, P

    1986-01-01

    Through use of a synthetic oligonucleotide probe, human insulin-like growth factor I (IGF-I) cDNA clones were isolated from a liver library. Two types of cDNAs were defined by restriction enzyme analysis and DNA sequencing. Both encode IGF-I precursors of either 195 or 153 amino acids. The two predicted protein precursors are identical from their amino terminus to a lysine residue 16 codons beyond the IGF-I sequence, and then they diverge. Both cDNAs predict additional unique carboxyl-termina...

  6. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Krochko, J E; Pramanik, S K; Bewley, J D

    1992-05-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media

  7. MicroRNA and messenger RNA analyses of mesenchymal stem cells derived from teeth and the Wharton jelly of umbilical cord.

    Science.gov (United States)

    Chen, Hua-Chien; Lee, Yun-Shien; Sieber, Martin; Lu, Huan-Ting; Wei, Pei-Cih; Wang, Chao-Nin; Peng, Hsiu-Huei; Chao, An-Shine; Cheng, Po-Jen; Chang, Shuenn-Dyh; Chen, Shu-Jen; Wang, Tzu-Hao

    2012-04-10

    Microarray analyses of transcriptomes have been used to characterize mesenchymal stem cells (MSCs) of various origins. MicroRNAs (miRNAs) are short, nonprotein-coding RNAs involved in post-transcriptional gene inhibition in a variety of tissues, including cancer cells and MSCs. This study has integrated the use of miRNA and mRNA expression profiles to analyze human MSCs derived from Wharton's jelly (WJ) of the umbilical cord, milk teeth (MT), and adult wisdom teeth (AT). Because both miRNA and mRNA expression in MT and AT MSCs were so similar, they were combined together as tooth MSCs for comparison with WJ MSCs. Twenty-five genes that were up-regulated in tooth MSCs and 41 genes that were up-regulated in WJ MSCs were identified by cross-correlating miRNA and mRNA profiles. Functional network analysis show that tooth MSCs signature genes, represented by SATB2 and TNFRSF11B, are involved in ossification, bone development, and actin cytoskeleton organization. In addition, 2 upregulated genes of tooth MSCs-NEDD4 and EMP1-have been shown to be involved in neuroectodermal differentiation. The signature genes of WJ MSCs, represented by KAL1 and PAPPA, are involved in tissue development, regulation of cell differentiation, and bone morphogenetic protein signaling pathways. In conclusion, the combined interrogation of miRNA and mRNA expression profiles in this study proved useful in extracting reliable results from a genome-wide comparison of multiple types of MSCs. Subsequent functional network analysis provided further functional insights about these MSCs.

  8. Translational Influence on Messenger Stability

    DEFF Research Database (Denmark)

    Eriksen, Mette

    , on messenger stability was examined. Depending on the translation initiation frequency, the chance of an initial ribosome trailing the RNA polymerase get better for better initiation sites, thus protecting transcription from termination by Rho. A polarity assay in which the activity of the downstream lac......A in the lac operon was measured, demonstrated that Rho dependent transcriptional pre-termination correlated with ribosome translation initiation frequency. It was further shown that Rho terminates RNA polymerases during transcription on messengers normally considered stable, suggesting Rho transcriptional pre...... pulls out the nascent transcript. Finally this thesis also focussed on the cell stress caused by an artificial and very strong RNA psudoknot used in a fusion gene. The technique of ribosomal foot printing and whole transcriptome deep sequencing was employed to facilitate an overview of the changes...

  9. Transforming growth factor-beta messenger RNA and protein in murine colitis

    DEFF Research Database (Denmark)

    Whiting, C V; Williams, A M; Claesson, Mogens Helweg

    2001-01-01

    Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-beta transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-beta1 and TGF-beta3 mRNAs, but both were expressed significantly...... TGF-beta. By ELISA, very low levels (0-69 pg/mg) of soluble total or active TGF-beta were detected in hypotonic tissue lysates. TGF-beta1 and TGF-beta3 are produced by SCID mouse colon and transcription is increased in the colitis caused by transplantation of CD4+ T-cells, but this does not result...

  10. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

    Directory of Open Access Journals (Sweden)

    Jesse E. Jun

    2013-09-01

    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  11. Onapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium.

    Science.gov (United States)

    Rekawiecki, Robert; Kowalik, Magdalena K; Kotwica, Jan

    2015-08-01

    The aim of this study was to examine whether progesterone (P(4)) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P4, ZK299, or RU486 at a dose of 10(-4), 10(-5), or 10(-6) M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P(4) (10(-4) M; P < 0.01) and RU486 (10(-6); P < 0.001) and was decreased by ZK299 (10(-5); P < 0.05). In contrast, PGRB mRNA was decreased by the all P(4) (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10(-4) M; P < 0.01 and 10(-5) M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10(-5) M; P < 0.001). PGRB mRNA was decreased by P(4) (10(-4) and 10(-5) M; P < 0.001), ZK299 (10(-4) and 10(-5) M; P < 0.001), and RU486 (10(-4) M; P < 0.01 and 10(-6) M; P < 0.001) and was increased by ZK299 (10(-6) M; P < 0.001) and RU486 (10(-5) M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10(-4) M; P < 0.05 and 10(-5) M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10(-5) M; P < 0.05 and 10(-5) M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10(-5) M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.

  12. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress.

    Science.gov (United States)

    Datta, Riddhi; Kumar, Deepak; Sultana, Asma; Hazra, Saptarshi; Bhattacharyya, Dipto; Chattopadhyay, Sharmila

    2015-12-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress.

  13. A carvacrol-thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger RNA levels of tight junction proteins in jejunal mucosa of weaning piglets.

    Science.gov (United States)

    Wei, H-K; Xue, H-X; Zhou, Z X; Peng, J

    2017-02-01

    Recent studies indicate that intestinal oxidative stress and microbiota imbalance is involved in weaning-induced intestinal dysfunction in piglets. We have investigated the effect of feeding a carvacrol-thymol blend supplemented diet on intestinal redox status, selected microbial populations and the intestinal barrier in weaning piglets. The piglets (weaned at 21 days of age) were randomly allocated to two groups with six pens per treatment and 10 piglets per pen. At weaning day (21 days of age), six piglets were sacrificed before weaning to serve as the preweaning group. The weaned group was fed with a basal diet, while the weaned-CB group was fed with the basal diet supplemented with 100 mg/kg carvacrol-thymol (1 : 1) blend for 14 days. On day 7 post-weaning, six piglets from each group were sacrificed to determine intestinal redox status, selected microbial populations, messenger RNA (mRNA) transcript levels of proinflammatory cytokines and biomarkers of intestinal barrier function. Weaning resulted in intestinal oxidative stress, indicated by the increased concentration of reactive oxygen species and thiobarbituric acid-reactive substances present in the intestine. Weaning also reduced the population of Lactobacillus genus and increased the populations of Enterococcus genus and Escherichia coli in the jejunum, and increased mRNA levels of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 (IL-6). In addition, decreased mRNA levels of zonula occludens and occludin in the jejunal mucosa and increased plasma diamine oxidase concentrations indicated that weaning induced dysfunction of the intestinal barrier. On day 7 post-weaning, supplementation with the carvacrol-thymol blend restored weaning-induced intestinal oxidative stress. Compared with the weaned group, the weaned-CB group had an increased population of Lactobacillus genus but reduced populations of Enterococcus genus and E. coli in the jejunum and decreased mRNA levels of TNF-α. The

  14. The Changes of TGF-α, TGF-β1 and Basic FGF Messenger RNA Expression in Rabbit Cornea after Photorefractive Keratectomy

    Institute of Scientific and Technical Information of China (English)

    Yisheng Zhong; Yingrning Zhou; Jingcai Lian; Wen Ye; Kangsun Wang; Feng Cheng

    2000-01-01

    Objective: To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-αα(TGF-αα), transforming growth factor-β1 (TGF-β1)and basic fibroblast growth factor (bFGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK).Methods: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2na and 3ra month after PRK, and the expressions of TGF-αα , TGF-βi and bFGF mRNA were detected with in situ hybridization.Results: The corneal haze formed at the 1st month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3ra month after ablation. TGF-αα mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-βl and bGFG mRNA were expressed by both corneal epitheliurn and stroma. The capacities for cornea tissue expression of three growth factors mRNA increased after PRK, and the peaks appeared on the 1s, 2na month. The extent for expressions of three growth factors related proportionally to the haze formation.Conclusion: Three growth factors took part in promoting corneal wound healing after PRK, and might contribute to corneal haze formation and development.

  15. INTERLEUKIN-4 PREVENTS THE INDUCTION OF G-CSF MESSENGER-RNA IN HUMAN ADHERENT MONOCYTES IN RESPONSE TO ENDOTOXIN AND IL-1 STIMULATION

    NARCIS (Netherlands)

    VELLENGA, E; DOKTER, W; DEWOLF, JTM; VANDEVINNE, B; ESSELINK, MT; HALIE, MR

    Human recombinant interleukin-4 (IL-4) was studied for its effects on the expression of granulocyte-colony stimulating factor (G-CSF) mRNA in human adherent monocytes in the absence and presence of endotoxin and interleukin 1 (IL-1). IL-4 (15 ng/ml) did not induce G-CSF transcripts in monocytes but

  16. Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus.

    Science.gov (United States)

    Sukjumlong, S; Persson, E; Dalin, A-M; Janson, V; Sahlin, L

    2009-06-01

    The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.

  17. Connecting RNA Processing to Abiotic Environmental Response in Arabidopsis: the role of a polyadenylation factor

    Science.gov (United States)

    Li, Q. Q.; Xu, R.; Hunt, A. G.; Falcone, D. L.

    Plants are constantly challenged by numerous environmental stresses both biotic and abiotic It is clear that plants have evolved to counter these stresses using all but limited means We recently discovered the potential role of a messenger RNA processing factor namely the Arabidopsis cleavage and polyadenylation specificity factor 30 kDa subunit AtCPSF30 when a mutant deficient in this factor displayed altered responses to an array of abiotic stresses This AtCPSF30 mutant named oxt6 exhibited an elevated tolerance to oxidative stress Microarray experiments of oxt6 and its complemented lines revealed an altered gene expression profile among which were antioxidative defense genes Interestingly the same gene encoding AtCPSF30 can also be transcribed into a large transcript that codes for a potential splicing factor Both protein products have a domain for RNA binding and a calmodulin binding domain activities of which have been confirmed by biochemical assays Surprisingly binding of AtCPSF30 to calmodulin inhibits the RNA-binding activity of the protein Mutational analysis shows that a small part of the protein is responsible for calmodulin binding and point mutations in this region abolished both RNA binding activity and the inhibition of this activity by calmodulin Analyses of the potential splicing factor are on going and the results will be presented The interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through

  18. Influences of Sex, Incubation Temperature, and Environmental Quality on Gonadal Estrogen and Androgen Receptor Messenger RNA Expression in Juvenile American Alligators (Alligator mississippiensis)1

    Science.gov (United States)

    Moore, Brandon C.; Milnes, Matthew R.; Kohno, Satomi; Katsu, Yoshinao; Iguchi, Taisen; Guillette, Louis J.

    2009-01-01

    Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5°C expressed greater AR levels than females incubated at 30°C; dimorphic expression was not observed in animals incubated at 32°C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants. PMID:19759368

  19. Increased messenger RNA levels of the antagonist thyroid hormone receptor erbA-alpha 2 and decreased levels of erbA-alpha 1 and erbA-beta 1 receptor messenger RNAs in neoplastic rodent cells.

    Science.gov (United States)

    Too, C K; Guernsey, D L

    1992-04-15

    Nothern blot analysis of total RNA from the mouse C3H/10T1/2 cell line indicated that the erbA alpha gene transcribed three mRNA species of similar sizes (2.6, 5.5, 6.6 kilobases) as found in rodents. The 2.6-kilobase mRNA (erbA-alpha 2) was approximately 7- to 8-fold more abundant than either the 5.5- (erbA-alpha 1) or 6.6-kilobase species. The expression of the erbA-alpha 2 transcript increased 3- to 30-fold when "normal" mouse or rat cells were growth arrested by concluence. Triiodothyronine, at a concentration of 1 nM, had no effect on the levels of the erbA-alpha mRNA species in confluent cells nor on the levels of erbA-alpha 2 in proliferative normal or transformed C3H/10T1/2 cells. In log-phase growing cells there was a 2.5- to 5-fold increase in the relative expression of erbA-alpha 2 mRNA in transformed mouse C3H/10T1/2 cells, transformed cloned rat embryo fibroblasts (CREF), transformed rat embryo fibroblasts (REF), and a transformed temperature-sensitive rat mutant cell line (ts7E) when compared with their non-transformed counterparts. In contrast to the elevation of erbA-alpha 2 in transformed cells, erbA-alpha 1 and erbA-beta 1 mRNAs decreased in transformed mouse and rat cell lines. In conclusion, it is suggested that the increased levels of the erbA-alpha 2 transcript and the decreased levels of erbA-alpha 1 and erbA-beta 1 in neoplastic cells may account for the loss of thyroid hormone regulation of inducible pathways and decreased nuclear triiodothyronine binding as previously reported.

  20. Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

    Science.gov (United States)

    Eissa, Nour; Kermarrec, Laëtitia; Hussein, Hayam; Bernstein, Charles N.; Ghia, Jean-Eric

    2017-01-01

    2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn’s disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes. PMID:28186172

  1. Transmembrane Signalling: Membrane messengers

    Science.gov (United States)

    Cockroft, Scott L.

    2017-05-01

    Life has evolved elaborate means of communicating essential chemical information across cell membranes. Inspired by biology, two new artificial mechanisms have now been developed that use synthetic messenger molecules to relay chemical signals into or across lipid membranes.

  2. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

    Science.gov (United States)

    Rouvier, E; Luciani, M F; Mattéi, M G; Denizot, F; Golstein, P

    1993-06-15

    To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

  3. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus Saimiri gene

    Energy Technology Data Exchange (ETDEWEB)

    Rouvier, E.; Luciani, M.F.; Golstein, P. (Centre d' Immunologie INSERM-CNRS de Marseille-Luminy, Marseille (France)); Mattei, M.G. (INSERM U242, Marseille (France)); Denizot, F. (Centre de Sequencage d' ADN, Marseille (France))

    1993-06-15

    To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that was named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival. 69 refs., 5 figs.

  4. Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide.

    Directory of Open Access Journals (Sweden)

    Diana P Inchaustegui Gil

    Full Text Available The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.

  5. Is tRNA binding or tRNA mimicry mandatory for translation factors?

    Science.gov (United States)

    Kristensen, Ole; Laurberg, Martin; Liljas, Anders; Selmer, Maria

    2002-02-01

    tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

  6. High-fat diet-induced reduction of peroxisome proliferator-activated receptor-γ coactivator-1α messenger RNA levels and oxidative capacity in the soleus muscle of rats with metabolic syndrome.

    Science.gov (United States)

    Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Takeda, Isao; Tsuda, Kinsuke; Ishihara, Akihiko

    2012-02-01

    Animal models of type 2 diabetes exhibit reduced peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) messenger RNA (mRNA) levels, which are associated with decreased oxidative capacity, in skeletal muscles. In contrast, animal models with metabolic syndrome show normal PGC-1α mRNA levels. We hypothesized that a high-fat diet decreases PGC-1α mRNA levels in skeletal muscles of rats with metabolic syndrome, reducing muscle oxidative capacity and accelerating metabolic syndrome or inducing type 2 diabetes. We examined mRNA levels and fiber profiles in the soleus muscles of rats with metabolic syndrome (SHR/NDmcr-cp [cp/cp]; CP) fed a high-fat diet. Five-week-old CP rats were assigned to a sedentary group (CP-N) that was fed a standard diet (15.1 kJ/g, 23.6% protein, 5.3% fat, and 54.4% carbohydrates) or a sedentary group (CP-H) that was fed a high-fat diet (21.6 kJ/g, 23.6% protein, 34.9% fat, and 25.9% carbohydrates) and were housed for 10 weeks. Body weight, energy intake, and systolic blood pressure were higher in the CP-H group than in the CP-N group. Nonfasting glucose, triglyceride, total cholesterol, and leptin levels were higher in the CP-H group than in the CP-N group. There was no difference in insulin levels between the CP-N and CP-H groups. Muscle PGC-1α mRNA levels and succinate dehydrogenase activity were lower in the CP-H group than in the CP-N group. We concluded that a high-fat diet reduces PGC-1α mRNA levels and oxidative capacity in skeletal muscles and accelerates metabolic syndrome.

  7. A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer.

    Science.gov (United States)

    Castle, Philip E; Dockter, Janel; Giachetti, Cristina; Garcia, Francisco A R; McCormick, Mary Kay; Mitchell, Amy L; Holladay, E Blair; Kolk, Daniel P

    2007-05-01

    To evaluate carcinogenic human papillomavirus (HPV) mRNA for E6 and E7 mRNA detection on clinical specimens to identify women with cervical precancer and cancer. We evaluated a prototype assay that collectively detects oncogenes E6/E7 mRNA for 14 carcinogenic HPV genotypes on a sample of liquid cytology specimens (n=531), masked to clinical data and to the presence of HPV genotypes detected by PGMY09/11 L1 consensus primer PCR assay. We found an increasing likelihood of testing positive for carcinogenic HPV E6/E7 mRNA with increasing severity of cytology (P(Trend) E6/E7 mRNA. Overall, fewer specimens tested positive for carcinogenic HPV E6/E7 mRNA than for carcinogenic HPV DNA (PE6/E7 mRNA improved the association of positive test results with cervical precancer and cancer by reducing the number of test positives in women without precancer without reducing clinical sensitivity for cervical precancer and cancer compared with detection of carcinogenic HPV E6/E7 mRNA using a lower positive cutpoint by the same assay and with detection of carcinogenic HPV DNA. We found that carcinogenic HPV E6/E7 mRNA is a potentially useful biomarker for detection of cervical precancer and cancer and warrants further evaluation.

  8. PTB and TIAR binding to insulin mRNA 3′- and 5′UTRs; implications for insulin biosynthesis and messenger stability

    Directory of Open Access Journals (Sweden)

    Rikard G. Fred

    2016-09-01

    Conclusions: These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

  9. Tumor gene mutations and messenger RNA expression: correlation with clinical response to icotinib hydrochloride in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    REN Guan-jun; ZHAO Yuan-yuan; ZHU Yu-jia; XIAO Yi; XU Jia-sen; SHAN Bin; ZHANG Li

    2011-01-01

    Background Molecular targeted drugs is now widely used in non-small cell lung cancer (NSCLC) clinical treatment.lcotinib hydrochloride is a new type of oral epidermal growth factor receptor (EGFR) tyresine kinase inhibitors (EGFR-TKIs). In this study, we examined the role of EGFR, K-RAS, B-RAF somatic mutations and EGFR mRNAexpression in tumor specimens from advanced NSCLC patients as predicators of the efficacy of icotinib hydrochlodde.Methods We analyzed tumor paraffin-embedded specimens, which were obtained from 14 of 40 patients with advanced NSCLC who enrolled in the stage Ⅰ clinical trial of icotinib hydrochloride. Somatic mutations were evaluated by mutant-enriched liquidchip (MEL) technology, and EGFR mRNA expression was measured by branched DNA liquidchip (MBL) technology.Results In the 14 specimens, seven patients showed EGFR mutations, exon 19 deletion (3/7) and exon 21 point mutation (4/7); and two patients showed K-RAS mutation. No mutations in EGFR exon 20. or B-RAF were detected. In patients with EGFR mutation, one patient developed progress disease (PD), three patients had stable disease (SD), two patients had partial responses (PR) and one patient had a complete response (CR). In patients with wild-type EGFR, four patients had PD, three patients acquired SD, and none had PR/CR (P=0.0407). EGFR mutations were associated with better progress-free survival (PFS) (141 days vs. 61 days) but without a statistically significant difference (P=0.8597), and median overall survival (OS) (≥449 days vs. 140 days). EGFR mRNA expression levels were evaluated (three high, eight moderate, one low, and two that can not be measured due to insufficient tumor tissue) and no statistically significant relationships was observed with response, PFS or OS.Conclusions The EGFR mutation rate was consistent with that reported in the Asian population, so the MEL technology is reliable for measuring EGFR mutation with high throughput and rapidity. EGFR exon 19 deletions and

  10. Geodesy at Mercury with MESSENGER

    Science.gov (United States)

    Smith, David E.; Zuber, Maria t.; Peale, Stanley J.; Phillips, Roger J.; Solomon, Sean C.

    2006-01-01

    In 2011 the MESSENGER (MErcury Surface, Space ENvironment, GEochemistry, and Ranging) spacecraft will enter Mercury orbit and begin the mapping phase of the mission. As part of its science objectives the MESSENGER mission will determine the shape and gravity field of Mercury. These observations will enable the topography and the crustal thickness to be derived for the planet and will determine the small libration of the planet about its axis, the latter critical to constraining the state of the core. These measurements require very precise positioning of the MESSENGER spacecraft in its eccentric orbit, which has a periapsis altitude as low as 200 km, an apoapsis altitude near 15,000 km, and a closest approach to the surface varying from latitude 60 to about 70 N. The X-band tracking of MESSENGER and the laser altimetry are the primary data that will be used to measure the planetary shape and gravity field. The laser altimeter, which has an expected range of 1000 to 1200 km, is expected to provide significant data only over the northern hemisphere because of MESSENGER's eccentric orbit. For the southern hemisphere, radio occultation measurements obtained as the spacecraft passes behind the planet as seen from Earth and images obtained with the imaging system will be used to provide the long-wavelength shape of the planet. Gravity, derived from the tracking data, will also have greater resolution in the northern hemisphere, but full global models for both topography and gravity will be obtained at low harmonic order and degree. The limiting factor for both gravity and topography is expected to be knowledge of the spacecraft location. Present estimations are that in a combined tracking, altimetry, and occultation solution the spacecraft position uncertainty is likely to be of order 10 m. This accuracy should be adequate for establishing an initial geodetic coordinate system for Mercury that will enable positioning of imaged features on the surface, determination of

  11. Expression of alpha-fetoprotein messenger RNA in BEL-7404 human hepatoma cells and effect of L-4-oxalysine on the expression

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    AIM To investigate alpha-fetoprotein (AFP) mRNA expression in BEL-7404 human hepatoma cells and the effect of L-4-oxalysine (OXL) on the expression.METHODS BEl-7404 human hepatoma cells were maintained in RPMI 1640 media. Human AFP cDNA probe was labelled with digoxigenin-11-dUTP by the random primer labelling method. The expression of AFP mRNA in Bel-7404 cells was determined by an in situ hybridization technique with digoxigenin-labelled human AFP cDNA probe. The positive intensities of AFP mRNA in cells were analyzed by microspectrophotometer and expressed as absorbance at 470nm. For the experiment with OXL, cells were incubated with various concentrations of the agent for 72h.RESULTS Essentially all the hepatoma cells contained AFP mRNA in the cytoplasm, although in various amounts. The specificity of the hybridization reaction was confirmed by control experiments in which the use of RNase-treated BEL-7404 cells, non-AFP-producing cells (HL-60 human leukemia cells) or a nonspecific cDNA probe resulted in negative hybridization. When the cells were treated with OXL (25, 50mg/L), the content of AFP mRNA in the cytoplasm was decreased with the inhibition percentages of 34.3% and 70.1%, respectively (P<0.05).CONCLUSION AFP mRNA was expressed in BEL-7404 human hepatoma cells and OXL suppressed AFP mRNA expression in the cells.

  12. Changes in messenger RNA of pancreatic enzymes and intestinal cholecystokinin after a 7-day bile-pancreatic juice diversion from the proximal small intestine in rats.

    Science.gov (United States)

    Hara, H; Ochi, Y; Kasai, T

    1997-06-01

    We have previously demonstrated the bile-pancreatic juice (BPJ)-independent stimulation of pancreatic enzyme secretion in chronic BPJ-diverted rats. Pancreatic and intestinal adaptation to 7-day BPJ diversion was next examined. Pancreatic enzyme mRNA and cholecystokinin mRNA in the jejunal mucosa were measured in rats with BPJ diverted into the ileum (PBD rats) in comparison with the figures for rats with BPJ returned to the duodenum (normal rats) or laparotomized (Intact) rats under well-nourished conditions. Amylase mRNA in the pancreas was lower and trypsinogen plus chymotrypsinogen mRNA was higher in the PBD rats than in the intact rats. The change in pancreatic mRNA was similar to that in the specific activities of the enzymes after a chronic BPJ diversion. This finding suggests that these pancreatic enzymes were regulated by the mRNA level. The portal concentration of cholecystokinin in the postabsorptive period (exogenously non-stimulated status) was 4-fold higher in the PBD group than in the normal and intact groups. Cholecystokinin mRNA in the jejunal mucosa of PBD rats was somewhat higher than that of intact rats. These results suggest that intestinal cholecystokinin was predominantly increased at the translational or later stage by chronic BPJ diversion.

  13. Messengers of new physics

    Energy Technology Data Exchange (ETDEWEB)

    Rodejohann, W. [Max-Planck-Institut fuer Kernphysik, Postfach 103980, D-69029 Heidelberg (Germany); Shiozawa, M. [Kamioka Observatory, Institute for Cosmic Ray Research, University of Tokyo, Higashi-Mozumi, Kamioka-cho, Hida-shi, Gifu 506-1205 (Japan)

    2011-08-15

    The talks of the parallel session 'Messengers of new physics' of the NOW2010 workshop are summarized. Topics under discussion included models of neutrino mass and mixing, charged lepton flavor violation and neutrinos, neutrinos and the LHC, future large-volume detectors, neutrinos and dark matter, neutrino properties and interactions beyond the Standard Model, leptogenesis, etc.

  14. Regulation of corticotropin releasing hormone receptor type 1 messenger RNA level in Y-79 retinoblastoma cells: potential implications for human stress response and immune/inflammatory reaction

    Directory of Open Access Journals (Sweden)

    N. C. Vamvakopoulos

    1996-01-01

    Full Text Available We report the regulation of type 1 receptor mRNA in Y-79 human retinoblastoma cells, grown in the absence or presence of pharmacological levels of phorbol esters, forskolin, glucocorticoids and their combinations. To control for inducibility and for assessing the sensitivity of the Y-79 system to glucocorticoids, corticotropin releasing hormone mRNA levels were measured in parallel. All treatments stimulated corticotropin releasing hormone receptor type 1 gene expression relative to baseline. A weak suppression of corticotropin releasing hormone mRNA level was observed during dexamethasone treatment. The cell line expressed ten-fold excess of receptor to ligand mRNA under basal conditions. The findings predict the presence of functional phorbol ester, cyclic AMP and glucocorticoid response elements in the promoter region of corticotropin releasing hormone receptor type 1 gene and support a potential role for its product during chronic stress and immune/inflammatory reaction.

  15. Regulation of corticotropin releasing hormone receptor type 1 messenger RNA level in Y-79 retinoblastoma cells: potential implications for human stress response and immune/inflammatory reaction

    OpenAIRE

    Vamvakopoulos, N C; Sioutopoulou, T. O.; Mamuris, Z.; Marcoulatos, P.; Avgerinos, P. C.

    1996-01-01

    We report the regulation of type 1 receptor mRNA in Y-79 human retinoblastoma cells, grown in the absence or presence of pharmacological levels of phorbol esters, forskolin, glucocorticoids and their combinations. To control for inducibility and for assessing the sensitivity of the Y-79 system to glucocorticoids, corticotropin releasing hormone mRNA levels were measured in parallel. All treatments stimulated corticotropin releasing hormone receptor type 1 gene expression relative to baseline....

  16. Role of dopamine in the plasticity of glutamic acid decarboxylase messenger RNA in the rat frontal cortex and the nucleus accumbens.

    Science.gov (United States)

    Rétaux, S; Trovero, F; Besson, M J

    1994-12-01

    The modulatory role of dopamine (DA) on the expression of mRNA encoding the large isoform of glutamic acid decarboxylase (GAD67), the biosynthesis enzyme of gamma aminobutyric acid (GABA), was examined in GABA neurons of two structures innervated by DA neurons originating from the ventral tegmental area (VTA): the medial frontal cortex (MFC) and the nucleus accumbens (NAcc). A bilateral electrolytic lesion of VTA was performed in rats to produce a DA denervation of both the MFC and NAcc. The efficacy of VTA lesions was verified by measurement of locomotor activity and by immunohistochemical detection of tyrosine hydroxylase in the mesencephalon. GAD67 mRNA was detected by in situ hybridization histochemistry using a 35S-labelled cDNA probe. Densitometric analysis of GAD67 mRNA hybridization signals revealed in VTA-lesioned rats a significant decrease (-24%) in GAD67 mRNA levels in the prelimbic area of the MFC and no significant effect in the anterior cingulate area or the frontoparietal cortex. Single cell analyses by computer-assisted grain counting showed that the decrease in GAD67 mRNA levels in prelimbic MFC was due to a change in GAD67 mRNA expression in a subpopulation of GABA interneurons located in the deep cortical layers (V-VI). By contrast, in the NAcc of VTA-lesioned rats, GAD67 mRNA levels were significantly increased in the anterior part and in the core but were unchanged in the shell part. These results suggest that in two target structures of VTA DA neurons, GAD67 mRNA expression is, in normal conditions, under a tonic stimulatory and a tonic inhibitory DA control in the MFC and the NAcc respectively. A schematic diagram is proposed for functional interactions between these structures.

  17. Identification of messenger RNA of fetoplacental source in maternal plasma of women with normal pregnancies and pregnancies with intrauterine growth restriction

    Directory of Open Access Journals (Sweden)

    Paola Andrea Ayala Ramírez

    2012-09-01

    Full Text Available Normal 0 21 false false false ES-CO X-NONE X-NONE Objetivo: cuantificar RNA (ácido ribonucleico específico de placenta en el plasma de mujeres con embarazos con fetos con Restricción de Crecimiento Intrauterino (RCIU y gestantes con embarazos normales.    Materiales y métodos: se estudiaron 8 mujeres con embarazos con fetos con RCIU y 18 mujeres con embarazos sin complicaciones, en el tercer trimestre de embarazo. Se cuantificó el RNA total libre en plasma materno por espectrofotometría y la expresión del gen Lactógeno Placentario Humano (hPL a nivel de RNA mensajero (RNAm por medio de la técnica Reacción en Cadena de la Polimerasa en Tiempo Real (qPCR-RT.   Resultados: se logró detectar RNA en plasma de origen fetoplacentario en el 100% de las gestantes. No se encontraron diferencias estadísticamente significativas entre los valores de RNA total extraído de plasma (p=0,5975 ni en la expresión del RNAm del gen hPL (p=0,5785 entre casos y controles.   Conclusión: es posible detectar RNAm de origen fetoplacentario en plasma materno durante el embarazo.  

  18. The Adh-related gene of Drosophila melanogaster is expressed as a functional dicistronic messenger RNA: multigenic transcription in higher organisms.

    Science.gov (United States)

    Brogna, S; Ashburner, M

    1997-01-01

    Essentially all eukaryotic cellular mRNAs are monocistronic, and are usually transcribed individually. Two tandemly arranged Drosophila genes, alcohol dehydrogenase (Adh) and Adh-related (Adhr), are transcribed as a dicistronic transcript. From transcripts initiated from the Adh promoter, two classes of mRNA are accumulated, one is monocistronic and encodes Adh alone, the other is dicistronic and includes the open reading frames of both Adh and Adhr. The dicistronic transcript is found in polysomes and the Adhr protein product is detected by antibody staining. We present evidence that the accumulation of the dicistronic mRNA is controlled at the level of the 3' end processing. PMID:9155028

  19. Effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating Holstein cows

    DEFF Research Database (Denmark)

    Røjen, Betina Amdisen; Poulsen, Søren Brandt; Theil, Peter Kappel

    2011-01-01

    To test the hypothesis that dietary N concentrations affect gut epithelial urea transport by modifying the expression of urea transporter B (UT-B) and aquaporins (AQP), the mRNA expression and protein abundance of UT-B and AQP3, AQP7, AQP8, and AQP10 were investigated in ruminal papillae from 9...... lactating dairy cows. Ruminal papillae were harvested from cows fed low N (12.9% crude protein) and high N (17.1% crude protein) diets in a crossover design with 21-d periods. The mRNA expression was determined by real-time reverse transcription-PCR and protein abundance by immunoblotting. The mRNA...... expression of UT-B was not affected by dietary treatment, whereas mRNA expression of AQP3, 7, and 10 were greater in the high N compared with the low N fed cows. Using peptide-derived rabbit antibodies to cow AQP3, 7, and 8, immunoblotting revealed bands of approximately 27, 27, and 24 kDa in ruminal...

  20. Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1.

    Directory of Open Access Journals (Sweden)

    Judy T Tellam

    2012-12-01

    Full Text Available Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a

  1. Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells.

    Science.gov (United States)

    Ponsaerts, Peter; Van den Bosch, Glenn; Cools, Nathalie; Van Driessche, Ann; Nijs, Griet; Lenjou, Marc; Lardon, Filip; Van Broeckhoven, Christine; Van Bockstaele, Dirk R; Berneman, Zwi N; Van Tendeloo, Viggo F I

    2002-08-15

    Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

  2. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

    DEFF Research Database (Denmark)

    Andersen, Christian Brix Folsted; Ballut, Lionel; Johansen, Jesper Sanderhoff;

    2006-01-01

    In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric e...

  3. Detection of Messenger RNA for Gonadotropin-Releasing Hormone (GnRH) but not for GnRH Receptors in Rat Pancreas

    Institute of Scientific and Technical Information of China (English)

    王雷; 谢莉萍; 黄威权; 张荣庆

    2001-01-01

    Although gonadotropin-releasing hormone (GnRH), GnRH-like molecule, and GnRH receptor (GnRH-R) have been reported to exist in several tissues other than brain or anterior pituitary, there are no reports concerning GnRH or GnRH-R gene expression in a normal pancreatic gland. In order to define the production of GnRH as well as GnRH-R in the pancreatic gland, we examined their gene expression in various developmental stages of rat pancreas using the reverse transcriptase-polymerase chain reaction (RT-PCR).GnRH mRNA transcripts were found in pancreas of male and female rats at different ages, expressing at about the same level, whereas GnRH-R mRNA transcripts could not be detected in any rat pancreatic gland samples. These results suggest a possible biological role of GnRH in rodent pancreas.

  4. A REMOTE HEALTH MONITORING MESSENGER

    Directory of Open Access Journals (Sweden)

    Sharmili Minu.DH

    2013-02-01

    Full Text Available Health is an important factor of every human being. Remote health monitoring messenger is needed for the people to reduce their inconvenience in travel to hospitals due to ailing health. Ill-patientrequires accurate decision to be taken immediately in critical situations, so that life-protecting and lifesaving therapy can be properly applied. In recent years, sensors are used in each and every fast developing application for designing the miniaturized system which is much easier for people use. A remote health monitoring messenger informs the doctor about the patient condition through wireless media such as Global System for Mobile communication. The system specifically deals with the signal conditioning and data acquisition of heart beat, temperature, and blood pressure of human body. The Heart beat sensor is used to read the patient’s beats per minute (bpm and temperature sensor to measure the body temperature of patient externally and pressure sensor to measure the level of pressure in blood. Signals obtained from sensors are fed into the microcontroller for processing and medicine is prescribed as first aid for patient to control the parameters through visual basic. A message is then sent to the doctor for further actions to be taken for treatment of patient after first aid. The system has a very good response time and it is cost effective.

  5. Characterizing the transcriptome upon depletion of RNA processing factors

    DEFF Research Database (Denmark)

    Herudek, Jan

    nucleus and is responsible for the proper processing and decay of a wide range of RNA molecules. Notably, the RNA exosome complex associates with a plethora of co-factors and activators that assist in the recognition of specific RNA substrates. Although many exosome partners have been characterized...... this method with CRISPR/Cas9 genome editing to pursue rapid depletion of endogenous protein. Applying this technology, I aim to study dynamics of the RNA decay machinery and obtain a deeper understanding of recently characterized ncRNAs....

  6. An energy-rich diet enhances expression of Na(+)/H(+) exchanger isoform 1 and 3 messenger RNA in rumen epithelium of goat.

    Science.gov (United States)

    Yang, W; Shen, Z; Martens, H

    2012-01-01

    Rumen epithelial Na(+)/H(+) exchanger (NHE) catalyzes the exchange of extracellular Na(+) for intracellular H(+). Thus, it is of importance in the maintenance of Na and pH homeostasis of rumen epithelial cells. We have tested the hypothesis that an increase in energy and protein intake induces alterations of NHE isoform 1, 2, and 3 (NHE1, NHE1, and NHE3, respectively) mRNA abundance in the rumen epithelium of goats. Goats (n = 26) were randomly allocated to 2 experiments (n = 16 in Exp. 1, and n = 10 in Exp. 2) and fed either peanut straw ad libitum [PNS, n = 8 in Exp. 1, and n = 5 in Exp. 2; 600 kJ of ME/(kg(0.75)·d)] or PNS + concentrate [CF, n = 8 in Exp. 1, and n = 5 in Exp. 2; 1,000 kJ of ME/(kg(0.75)·d)] for 42 d. Concentrate (400 g/d) was given daily (0800 to 1700 h) in 4 equal portions at 3-h intervals. In Exp. 1, the goats were euthanized 2 h after the last portion of concentrate was fed, and in Exp. 2, the goats were euthanized after a fasting period of 16 h. In Exp. 1, goats in the CF treatment exhibited a greater ruminal short-chain fatty acid (SCFA) concentration (140.6 ± 1.30 mM) compared with those in the PNS treatment (114.3 ± 3.11 mM; P goats in the CF treatment than for those in the PNS treatment. However, in Exp. 2, 16 h of fasting abolished differences in ruminal SCFA concentration, pH, and NHE mRNA expression between goats in the CF and PNS treatments. In both Exp. 1 and 2, a positive correlation was observed between ruminal SCFA concentration and expression of mRNA in NHE1 and NHE3, whereas expression was negatively correlated with ruminal pH. In in vitro studies with isolated rumen epithelial cells from goats fed dried grass, exposure to pH of 6.8 or to 20 mM SCFA increased (P diet-dependent rumen epithelial NHE1 and NHE3 expression is probably related to ruminal SCFA concentration and pH, but that is not the case with NHE2.

  7. Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver

    Directory of Open Access Journals (Sweden)

    Ľudmila Pašková

    2016-01-01

    Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT, with methotrexate (MTX, the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA in male Lewis rats. The experiment included healthy controls (CO, arthritic animals (AA, AA given N-f-5HT (AA-N-f-5HT, and AA given MTX (AA-MTX. N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1β in plasma and IL-1β mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX.

  8. Dynamical Messengers for Gauge Mediation

    Energy Technology Data Exchange (ETDEWEB)

    Hook, Anson; Torroba, Gonzalo; /SLAC /Stanford U., Phys. Dept.

    2011-08-17

    We construct models of indirect gauge mediation where the dynamics responsible for breaking supersymmetry simultaneously generates a weakly coupled subsector of messengers. This provides a microscopic realization of messenger gauge mediation where the messenger and hidden sector fields are unified into a single sector. The UV theory is SQCD with massless and massive quarks plus singlets, and at low energies it flows to a weakly coupled quiver gauge theory. One node provides the primary source of supersymmetry breaking, which is then transmitted to the node giving rise to the messenger fields. These models break R-symmetry spontaneously, produce realistic gaugino and sfermion masses, and give a heavy gravitino.

  9. MESSENGER: Exploring Mercury's Magnetosphere

    Science.gov (United States)

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. Mercury's magnetosphere is unique in many respects. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. For this reason there are no closed dri-fi paths for energetic particles and, hence, no radiation belts; the characteristic time scales for wave propagation and convective transport are short possibly coupling kinetic and fluid modes; magnetic reconnection at the dayside magnetopause may erode the subsolar magnetosphere allowing solar wind ions to directly impact the dayside regolith; inductive currents in Mercury's interior should act to modify the solar In addition, Mercury's magnetosphere is the only one with its defining magnetic flux tubes rooted in a planetary regolith as opposed to an atmosphere with a conductive ionosphere. This lack of an ionosphere is thought to be the underlying reason for the brevity of the very intense, but short lived, approx. 1-2 min, substorm-like energetic particle events observed by Mariner 10 in Mercury's magnetic tail. In this seminar, we review what we think we know about Mercury's magnetosphere and describe the MESSENGER science team's strategy for obtaining answers to the outstanding science questions surrounding the interaction of the solar wind with Mercury and its small, but dynamic magnetosphere.

  10. Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.

    Science.gov (United States)

    Frohman, M A; Downs, T R; Chomczynski, P; Frohman, L A

    1989-10-01

    We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    Science.gov (United States)

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

  12. microRNA Decay: Refining microRNA Regulatory Activity.

    Science.gov (United States)

    Pepin, Genevieve; Gantier, Michael P

    2016-01-01

    MicroRNAs (miRNAs) are short 19-25 nucleotide RNA molecules that impact on most biological processes by regulating the efficiency of messenger RNA (mRNA) translation. To date, most research activities have been focused on the control of miRNA expression and its functional consequences. Nonetheless, much remains unknown about the mechanisms affecting the level of specific miRNAs in the cell, a critical feature impacting their regulatory activity. This review focuses on the factors that regulate the abundance of miRNAs, including synthesis, post-transcriptional modifications, nucleases, target binding, and secretion.

  13. MESSENGER'S First Flyby of Mercury

    Science.gov (United States)

    Slavin, James A.

    2008-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. An overview of the MESSENGER mission and its January 14th close flyby of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER'S first flyby on January 14th, 2008 will be discussed with an emphasis on the magnetic field and charged particle measurements.

  14. Peptide primary messengers in plants

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The peptide primary messengers regulate embryonic development,cell growth and many other activities in animal cells. But recent evidence verified that peptide primary messengers are also involved in plant defense responses, the recognition between pollen and stigma and keep the balance between cell proliferation and differentiations in shoot apical meristems. Those results suggest that plants may actually make wide use of peptide primary messengers, both in embryonic development and late life when they rally their cells to defend against pathogens and insect pests. The recent advance in those aspects is reviewed.

  15. The MESSENGER Spacecraft

    Science.gov (United States)

    Leary, James C.; Conde, Richard F.; Dakermanji, George; Engelbrecht, Carl S.; Ercol, Carl J.; Fielhauer, Karl B.; Grant, David G.; Hartka, Theodore J.; Hill, Tracy A.; Jaskulek, Stephen E.; Mirantes, Mary A.; Mosher, Larry E.; Paul, Michael V.; Persons, David F.; Rodberg, Elliot H.; Srinivasan, Dipak K.; Vaughan, Robin M.; Wiley, Samuel R.

    2007-08-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft was designed and constructed to withstand the harsh environments associated with achieving and operating in Mercury orbit. The system can be divided into eight subsystems: structures and mechanisms (e.g., the composite core structure, aluminum launch vehicle adapter, and deployables), propulsion (e.g., the state-of-the-art titanium fuel tanks, thruster modules, and associated plumbing), thermal (e.g., the ceramic-cloth sunshade, heaters, and radiators), power (e.g., solar arrays, battery, and controlling electronics), avionics (e.g., the processors, solid-state recorder, and data handling electronics), software (e.g., processor-supported code that performs commanding, data handling, and spacecraft control), guidance and control (e.g., attitude sensors including star cameras and Sun sensors integrated with controllers including reaction wheels), radio frequency telecommunications (e.g., the spacecraft antenna suites and supporting electronics), and payload (e.g., the science instruments and supporting processors). This system architecture went through an extensive (nearly four-year) development and testing effort that provided the team with confidence that all mission goals will be achieved.

  16. The Messenger Mission to Mercury

    CERN Document Server

    Domingue, D. L

    2007-01-01

    NASA’s MESSENGER mission, launched on 3 August, 2004 is the seventh mission in the Discovery series. MESSENGER encounters the planet Mercury four times, culminating with an insertion into orbit on 18 March 2011. It carries a comprehensive package of geophysical, geological, geochemical, and space environment experiments to complete the complex investigations of this solar-system end member, which begun with Mariner 10. The articles in this book, written by the experts in each area of the MESSENGER mission, describe the mission, spacecraft, scientific objectives, and payload. The book is of interest to all potential users of the data returned by the MESSENGER mission, to those studying the nature of the planet Mercury, and by all those interested in the design and implementation of planetary exploration missions.

  17. CKD患者尿沉渣中CCN2/CCN3mRNA检测的临床意义探讨%Investigation of messenger RNA expression of CCN2/CCN3 in the urinary sediments in patients with chronic kidney disease

    Institute of Scientific and Technical Information of China (English)

    陈珑; 高民; 刘丹; 倪海峰; 曹玉涵; 刘宏; 张建东; 刘必成

    2013-01-01

    Objective: To study the messenger RNA ( mRNA ) expression in urinary sediment emerged as a promising tool to monitor renal damage in patients with chronic kidney diseases ( CKDs ). We investigated whether urinary mRNA of connective tissue growth factor ( CCN2 ) and nephroblastoma overexpressed ( CCN3 ), two molecules involved in the pathogenesis of glomerulosclerosis, has some clinical insights into the management of nondiabetic CKDs. Methods: A total of 35 patients with nondiabetic CKDs were enrolled in our study, as well as 12 healthy controls. mRNA expression of CCN2 and CCN3 were measured by realtime PCR. There were 17 out of those 35 patients who received renal biopsy. Glomerular histological changes were also analyzed on PAS stained slides of those biopsied patients. Results: Urinary mRNA of both CCN2 and CCN3 were distinctively greater in CKD patients compared to healthy controls. However, urinary mRNA level of neither of those two markers was differently distributed among CKD patients with variable stages of proteinuria. Nevertheless, urinary mRNA of CCN3, but not CCN2 was shown to inversely correlate to the degree of glomerular histological changes. Furthermore, ratio of urinary mRNA of CCN2 to CCN3 had an even stronger correlation efficient with glomerulosclerosis. Conclusion: In summary, our study demonstrates that urinary mRNA of CCN2/3 correlated to the degree of glomerular histological changes in non-diabetic CKDs patients, suggesting that this measurement may be a useful noninvasive tool for evaluating patients with nondiabetic CKDs.%目的:探讨慢性肾脏疾病(CKD)患者尿沉渣中CCN2(结缔组织生长因子,CTGF)和CCN3(肾母细胞瘤过度表达因子,NOV)mRNA检测及其在监测肾脏损伤中的意义.方法:研究包括35名非糖尿病的CKD患者和12名健康成人.尿沉渣中CCN2/CCN3 mRNA的检测采用Real time-PCR方法.35例CKD患者中17例患者接受了肾活检,这些肾活检组织的肾小球病变程度经PAS染色

  18. RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

    Science.gov (United States)

    Zhang, Heping; Li, Jiancheng; Cheng, Wenfang; Liu, D I; Chen, Cheng; Wang, Xiaoying; Lu, Xujing; Zhou, Xifa

    2015-09-01

    The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.

  19. Temporal changes in glial fibrillary acidic protein messenger RNA and [{sup 3}H]PK11195 binding in relation to imidazoline-I{sub 2}-receptor and {alpha}{sub 2}-adrenoceptor binding in the hippocampus following transient global forebrain ischaemia in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Craven, J.A.; Gundlach, A.L.; Conway, E.L. [The University of Melbourne, Clinical Pharmacology and Therapeutics Unit (Australia); Department of Medicine, Austin and Repatriation Medical Centre (Australia)

    1997-10-24

    Immunohistochemical studies have demonstrated that following global forebrain ischaemia the selective neuronal loss that occurs in the CA1 pyramidal cell layer of the hippocampus is accompanied by a reactive astrocytosis, characterized by increases in glial fibrillary acidic protein, and activation of microglia. In this study the spatial changes in glial fibrillary acidic protein messenger RNA levels in the hippocampus have been mapped four, eight, 12, 16 and 20 days following 10 min of global forebrain ischaemia in the rat and related to changes in [{sup 3}H]PK11195 binding to peripheral benzodiazepine receptors, a putative marker of activated microglia. Recent studies have suggested that the imidazoline-I{sub 2}-receptor, one of a class of non-adrenergic receptors related to, but structurally and functionally distinct from {alpha}{sub 2}-adrenoceptors, may have a functional role in controlling the expression of glial fibrillary acidic protein. To explore this possibility further we have also mapped changes in imidazoline-I{sub 2}-receptor and {alpha}{sub 2}-adrenoceptor binding sites. Following transient ischaemia there was a marked, biphasic increase in glial fibrillary acidic protein messenger RNA levels throughout the vulnerable CA1 region of the hippocampus, peaking four days after ischaemia and then increasing gradually during the remainder of the study period. There was also a sustained increase in [{sup 3}H]PK11195 binding, however, in contrast to the initial increase in glial fibrillary acidic protein messenger RNA levels that peaked four days after ischaemia the density of [{sup 3}H]PK11195 binding increased rapidly in all strata of the CA1 region over the first eight days and then increased more slowly throughout days 12 to 20. Despite the marked increase in glial fibrillary acidic protein messenger RNA levels there was no concomitant alteration in imidazoline-I{sub 2}-receptor binding sites detected using the specific radioligand, [{sup 3}H]2

  20. UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Yi-Hsiu Chen

    Full Text Available The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

  1. Changes in mRNA levels for brain-derived neurotrophic factor after wheel running in rats selectively bred for high- and low-aerobic capacity.

    Science.gov (United States)

    Groves-Chapman, Jessica L; Murray, Patrick S; Stevens, Kristin L; Monroe, Derek C; Koch, Lauren G; Britton, Steven L; Holmes, Philip V; Dishman, Rod K

    2011-11-24

    We evaluated levels of exercise-induced brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) within the hippocampal formation in rats selectively bred for 1) high intrinsic (i.e., untrained) aerobic capacity (High Capacity Runners, HCR), 2) low intrinsic aerobic capacity (Low Capacity Runners, LCR), and 3) unselected Sprague-Dawley (SD) rats with or without free access to running wheels for 3 weeks. The specific aim of the study was to determine whether a dose-response relationship exists between cumulative running distance and levels of BDNF mRNA. No additional treatments or behavioral manipulations were used. HCR, LCR, and SD rats were grouped by strain and randomly assigned to sedentary or activity (voluntary access to activity wheel) conditions. Animals were killed after 21 days of exposure to the assigned conditions. Daily running distances (mean ± standard deviation meters/day) during week three were: HCR (4726 ± 3220), SD (2293 ± 3461), LCR (672 ± 323). Regardless of strain, levels of BDNF mRNA in CA1 were elevated in wheel runners compared to sedentary rats and this difference persisted after adjustment for age (p=0.040). BDNF mRNA was not affected by intrinsic aerobic capacity and was not related to total running distance. The results support that BDNF mRNA expression is increased by unlimited access to activity wheel running for 3 weeks but is not dependent upon accumulated running distance.

  2. Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

    Science.gov (United States)

    Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

    2015-01-01

    Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by 'trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics.

  3. [Pathophysiological implications of the chemical messengers].

    Science.gov (United States)

    Blázquez Fernández, Enrique

    2009-01-01

    To maintain a physical organization and a different composition of its surroundings environment, living beings use a great part of the energy that they produce. Vital processes require an elevated number of reactions which are regulated and integrated by chemical messengers. They use autocrine, paracrine, endocrine and synaptic signals through receptors of cell surface, nuclear or associated with ionic chanels, enzymes, trimeric G proteins and to intracellular kinases. Through these mechanisms pheromones play an important role in the relationships between different individuals, and hormones are able to regulate the integrative functions of our organism. In the nervous system, neurotransmitters, neuromodulators, sensors and receptors between other messengers, play functions of great relevance, while growth factors stimulate cell proliferation and cytokines have many effects but the most important is the ones related with the control of the immflamatory process. Alterations of these messengers permit us a better understanding of the diseases and possibly of its treatments in a near future. Modifications of the expression of genes from the nuclear and mitochondrial genomas are responsible of monogenic, polygenic and mitochondrial diseases, while alterations in the activities of dopamine and serotonin neurotransmitters are related with schizophrenia, Parkinson disease and depression, respectively. Other example is the hyperthyroidism of the Graves-Bassedow disease due to the competitive interference of the LATS immunoglobulin with TSH at the level of the folicular cells producing thyroid hormones Twenty five years ago in the reviews on the mechanisms of insulin action, there was presentations in which the insulin receptor was located in the plasma membrane of the target cells while in the cytoplasm only a big interrogative was observed, that at present is replaced by chemical mediators cascades responsible of the multiple effects of insulin. This finding is similar to

  4. Temporal changes in the expression of brain-derived neurotrophic factor mRNA in the ventromedial nucleus of the hypothalamus of the developing rat brain.

    Science.gov (United States)

    Sugiyama, Nobuhiro; Kanba, Shigenobu; Arita, Jun

    2003-07-04

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which is important for the growth, differentiation, and survival of neurons during development. We have performed a detailed mapping of BDNF mRNA in the neonatal rat brain using a quantitative in situ hybridization technique. At postnatal day (PND) 4, hypothalamic structures showed only modest expression of BDNF mRNA, with the exception of the ventromedial nucleus (VMN), where expression was higher than that detected in the hippocampus. Abundant BDNF mRNA was also found in the bed nucleus of the anterior commissure, retrosplenial granular cortex, and the posteroventral part of the medial amygdaloid nucleus. Messenger RNAs encoding other neurotrophins, including nerve growth factor (NGF) and neurotrophin-3 (NT-3) and the BDNF receptor trkB, were not selectively localized in neonatal VMN. During subsequent developmental stages, BDNF mRNA expression in the VMN changed dynamically, peaking at PND 4 and falling to minimal levels in the adult brain. In contrast, the low levels of BDNF mRNA observed in the CA3 region of the hippocampus increased to adult levels following PND 10. As the VMN undergoes sexual differentiation, we compared BDNF, NGF, NT-3, and trkB mRNA expression in the VMN in males and females at embryonic day 20 and PND 4, but found no differences between them. These results suggest that localized and high level expression of BDNF mRNA in the neonatal VMN plays an important role in its neural organization and functional development.

  5. The yeast mitochondrial RNA polymerase specificity factor, MTF1, is similar to bacterial sigma factors.

    Science.gov (United States)

    Jang, S H; Jaehning, J A

    1991-11-25

    We have purified the protein that confers selective promoter recognition on the core subunit of the yeast mitochondrial RNA polymerase. The N-terminal sequence of the 43-kDa specificity factor identified it as the product of the MTF1 gene described by Lisowsky and Michaelis (1988). We confirmed that MTF1 encoded the specificity factor by analyzing extracts from a yeast strain bearing a disruption of the gene. The extracts contained normal levels of core RNA polymerase but lacked selective transcription activity; adding the purified 43-kDa protein restored selective transcription. Comparison of the MTF1 protein sequence to the family of bacterial sigma factors has revealed striking similarity to domains identified with--10 promoter recognition, promoter melting, and holoenzyme stability.

  6. On the role of the Escherichia coli RNA polymerase sigma factor in T4 phage development.

    Science.gov (United States)

    Zograff, Y N

    1981-01-01

    The rpoD800 mutation causing the temperature sensitivity of Escherichia coli RNA polymerase sigma factor has been used to demonstrate that the bacterial sigma factor is necessary throughout T4 phage development. In T4-infected RpoD800 mutant cells RNA synthesis is equally depressed at restrictive temperature at early and late stages of infection. The results show the bacterial sigma factor to be necessary for the synthesis of all RNA types in infected cells.

  7. RNA epigenetics

    OpenAIRE

    Liu, Nian; Pan, Tao

    2014-01-01

    Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modifi...

  8. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    Science.gov (United States)

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  9. Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

    Science.gov (United States)

    Tsukamoto, H; Boado, R J; Pardridge, W M

    1996-01-01

    The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors. PMID:8675694

  10. Increased micro-RNA 29b in the aged brain correlates with the reduction of insulin-like growth factor-1 and fractalkine ligand.

    Science.gov (United States)

    Fenn, Ashley M; Smith, Kristen M; Lovett-Racke, Amy E; Guerau-de-Arellano, Mireia; Whitacre, Caroline C; Godbout, Jonathan P

    2013-12-01

    Microglia develop an inflammatory phenotype during normal aging. The mechanism by which this occurs is not well understood, but might be related to impairments in several key immunoregulatory systems. Here we show that micro-RNA (miR)-29a and miR-29b, 2 immunoregulatory micro-RNAs, were increased in the brain of aged BALB/c mice compared with adults. Insulin-like growth factor-1 (IGF-1) and fractalkine ligand (CX3CL1) are negative modulators of microglial activation and were identified as targets of miR-29a and miR-29b using luciferase assay and primary microglia transfection. Indeed, higher expression of miR-29b in the brain of aged mice was associated with reduced messenger RNA (mRNA) levels of IGF-1 and CX3CL1. Parallel to these results in mice, miR-29a and miR-29b were also markedly increased in cortical brain tissue of older individuals (mean, 77 years) compared with middle-aged adults (mean, 45 years). Moreover, increased expression of miR-29b in human cortical tissue was negatively correlated with IGF-1 and CX3CL1 expression. Collectively, these data indicate that an age-associated increase in miR-29 corresponded with the reduction of 2 important regulators of microglia, IGF-1 and CX3CL1. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Yi; Seo, Hyuk-Soo; Blobel, Günter; Hoelz, André (Rockefeller)

    2010-07-23

    The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 {angstrom} resolution. Rae1 forms a seven-bladed {beta}-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an {approx} 50-{angstrom}-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 {beta}-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1 {center_dot} Nup98{sup GLEBS} surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1 {center_dot} Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.

  12. Effect of escitalopram versus placebo on GRα messenger RNA expression in peripheral blood cells of healthy individuals with a family history of depression - a secondary outcome analysis from the randomized AGENDA trial

    DEFF Research Database (Denmark)

    Knorr, Ulla; Koefoed, Pernille; Gluud, Christian

    2016-01-01

    to receive daily tablets of escitalopram 10 mg versus placebo for 4 weeks. GRα mRNA expression levels in peripheral blood were measured using reverse transcription polymerase chain reaction. Results Four weeks of intervention with escitalopram decreased the relative change from baseline in the expression...... of GRα mRNA compared with placebo (p = 0.002). Conclusion These findings from a randomized trial suggest that a 4-week escitalopram administration to healthy participants results in a decrease in GRα mRNA expression levels in peripheral blood compared with inert placebo. The decrease in GRα m...

  13. Cotranscriptional Recruitment of RNA Exosome Cofactors Rrp47p and Mpp6p and Two Distinct Trf-Air-Mtr4 Polyadenylation (TRAMP) Complexes Assists the Exonuclease Rrp6p in the Targeting and Degradation of an Aberrant Messenger Ribonucleoprotein Particle (mRNP) in Yeast*

    Science.gov (United States)

    Stuparevic, Igor; Mosrin-Huaman, Christine; Hervouet-Coste, Nadège; Remenaric, Mateja; Rahmouni, A. Rachid

    2013-01-01

    The cotranscriptional mRNA processing and packaging reactions that lead to the formation of export-competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p, which is recruited cotranscriptionally in association with Nrd1p following Rho action. Here we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p, Mpp6p as well as the Trf-Air-Mtr4 polyadenylation (TRAMP) components Trf4p, Trf5p, and Air2p contribute significantly by stimulating the degradation process upon their cotranscriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p, which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs. PMID:24047896

  14. The RNA polymerase II elongation complex.

    Science.gov (United States)

    Aso, T; Conaway, J W; Conaway, R C

    1995-11-01

    The initiation stage of transcription by RNA polymerase II has long been regarded as the primary site for regulation of eukaryotic gene expression. Nevertheless, a growing body of evidence reveals that the RNA polymerase II elongation complex is also a major target for regulation. Biochemical studies are implicating an increasing number of transcription factors in the regulation of elongation, and these transcription factors are being found to function by a diverse collection of mechanisms. Moreover, unexpected features of the structure and catalytic mechanism of RNA polymerase II are forcing a reconsideration of long-held views on the mechanics of some of the most basic aspects of polymerase function. In this review, we will describe recent insights into the structures and functions of RNA polymerase II and the transcription factors that control its activity during the elongation stage of eukaryotic messenger RNA synthesis.

  15. Astroparticles: Messengers from Outer Space

    Science.gov (United States)

    Desiati, Paolo

    2016-07-01

    Since Galileo pointed a spyglass toward the sky, 400 years ago, observations empowered by man-made instrumentation have provided us with an enormous leap in the knowledge of how the Universe functions. More and more powerful optical telescopes made it possible for us to reach the farthest corners of space. At the same time, the advances in microphysics and the discovery of the electromagnetic spectrum, made it possible to directly look at the Universe in a way that our eyes cannot see. The discoveries of the intimate structure of matter, of subatomic particles and of how they interact with each other, have led astronomers to use the smallest objects in Nature to observe the farthest reaches of the otherwise invisible Universe. Not unlike Galileo, today we observe Outer Space with visible light and beyond, across the entire electromagnetic spectrum, from long wavelength radio waves to short wavelength gamma rays. But also with instruments detecting cosmic rays (the atomic nuclei we know on Earth) neutrinos (neutral subatomic particles that interact very weakly with matter) and gravitational waves (perturbations of spacetime predicted by General Relativity). Each cosmic messenger provides us with a unique piece of information about their source and the history of their journey to us. Modern astrophysics has the challenging goal to collect as much information as possible from all those messengers, to reconstruct the story of the Universe and how it became what it is today. This journey started with the unsettling discovery that we are only one minuscule dot in the immensity of the Universe and yet we are able to observe objects that are far in space and time. This journey is yet to complete its course, and the more we advance our knowledge, the more we need to understand. This interdisciplinary talk provides an overview of this journey and the future perspectives.

  16. A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Chao-Feng Huang

    Full Text Available Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs. In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA. Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation.

  17. MESSENGER: Exploring the Innermost Planet

    Science.gov (United States)

    Solomon, S. C.

    2011-12-01

    One of Earth's closest planetary neighbors, Mercury remained comparatively unexplored for the more than three decades that followed the three flybys of the innermost planet by the Mariner 10 spacecraft in 1974-75. Mariner 10 imaged 45% of Mercury's surface at about 1 km/pixel average resolution, confirmed Mercury's anomalously high bulk density and implied large fractional core size, discovered Mercury's internal magnetic field, documented that H and He are present in the planet's tenuous exosphere, and made the first exploration of Mercury's magnetosphere and solar wind environment. Ground-based astronomers later reported Na, K, and Ca in Mercury's exosphere; the presence of deposits in the floors of polar craters having radar characteristics best matched by water ice; and strong evidence from the planet's forced libration amplitude that Mercury has a fluid outer core. Spacecraft exploration of Mercury resumed with the selection for flight, under NASA's Discovery Program, of the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission. Launched in 2004, MESSENGER flew by the innermost planet three times in 2008-2009 en route to becoming the first spacecraft to orbit Mercury in March of this year. MESSENGER's first chemical remote sensing measurements of Mercury's surface indicate that the planet's bulk silicate fraction differs from those of the other inner planets, with a low-Fe surface composition intermediate between basalts and ultramafic rocks and best matched among terrestrial rocks by komatiites. Moreover, surface materials are richer in the volatile constituents S and K than predicted by most planetary formation models. Global image mosaics and targeted high-resolution images (to resolutions of 10 m/pixel) reveal that Mercury experienced globally extensive volcanism, including large expanses of plains emplaced as flood lavas and widespread examples of pyroclastic deposits likely emplaced during explosive eruptions of volatile

  18. The Mercury exosphere after MESSENGER

    Science.gov (United States)

    Killen, Rosemary; McClintock, William; Vervack, Ronald; Merkel, Aimee; Burger, Matthew; Cassidy, Timothy; Sarantos, Menelaos

    2016-07-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft observed sodium, calcium and magnesium emisison in Mercury's exosphere on a near-daily basis for >16 Mercury years. The MASCS observations showed that calcium in Mercury's exosphere is persistently concentrated in the dawn hemisphere and is of extreme temperature (>50,000 K). The column abundance varies seasonally, and is extremely repeatable each Mercury year. In addition, the calcium exhibits a persistent maximum not at perihelion but 20° after perihelion, an enhancement that was shown to be coincident with the probable intersection of Mercury's orbit with a dust stream originating at Comet Encke. Any mechanism producing the Mercurian Ca exosphere must explain the facts that the Ca is extremely hot, that it is seen almost exclusively on the dawnside of the planet, and that its content varies seasonally, not sporadically. Energization of the Ca atoms was suggested to originate through dissociation of Ca-bearing molecules ejected by meteoritic impacts. Magnesium was also observed on a daily basis throughout the MESSENGER orbital phase. Mg has its own spatial and temporal pattern, peaking at mid-morning instead of early morning like Ca, and exhibiting a warm thermal profile, about 5000 K, unlike the extreme temperature of Ca which is an order of magnitude hotter. Although Mercury's sodium exosphere has been observed from the ground for many decades, the MASCS observations showed that, like calcium, the sodium exosphere is dominated by seasonal variations, not sporadic variations. However a conundrum exists as to why ground-based observations show highly variable high-latitude variations that eluded the MASCS. The origin of a persistent south polar enhancement has not been explained. The more volatile element, Na, is again colder, about 1200 K, but not thermally accommodated to the surface temperature. A

  19. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release.

    Science.gov (United States)

    Zimmermann, Herbert

    2016-03-01

    Extracellular nucleotides, and ATP in particular, are cellular signal substances involved in the control of numerous (patho)physiological mechanisms. They provoke nucleotide receptor-mediated mechanisms in select target cells. But nucleotides can considerably expand their range of action. They function as primary messengers in intercellular communication by stimulating the release of other extracellular messenger substances. These in turn activate additional cellular mechanisms through their own receptors. While this applies also to other extracellular messengers, its omnipresence in the vertebrate organism is an outstanding feature of nucleotide signaling. Intercellular messenger substances released by nucleotides include neurotransmitters, hormones, growth factors, a considerable variety of other proteins including enzymes, numerous cytokines, lipid mediators, nitric oxide, and reactive oxygen species. Moreover, nucleotides activate or co-activate growth factor receptors. In the case of hormone release, the initially paracrine or autocrine nucleotide-mediated signal spreads through to the entire organism. The examples highlighted in this commentary suggest that acting as ubiquitous triggers of intercellular messenger release is one of the major functional roles of extracellular nucleotides. While initiation of messenger release by nucleotides has been unraveled in many contexts, it may have been overlooked in others. It can be anticipated that additional nucleotide-driven messenger functions will be uncovered with relevance for both understanding physiology and development of therapy.

  20. Modus operandi of the bacterial RNA polymerase containing the sigma54 promoter-specificity factor.

    Science.gov (United States)

    Wigneshweraraj, Sivaramesh; Bose, Daniel; Burrows, Patricia C; Joly, Nicolas; Schumacher, Jörg; Rappas, Mathieu; Pape, Tillmann; Zhang, Xiaodong; Stockley, Peter; Severinov, Konstantin; Buck, Martin

    2008-05-01

    Bacterial sigma (sigma) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative sigma factor sigma(54) confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the sigma(54) factor makes with the bacterial transcription machinery.

  1. MESSENGER'S First and Second Flybys of Mercury

    Science.gov (United States)

    Slavin, James A.

    2009-01-01

    The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only approximately 1000 km above the surface. An overview of the MESSENGER mission and its January 14th and October 6th, 2008 close flybys of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER will be discussed with an emphasis on the magnetic field and charged particle measurements.

  2. Attenuation of diacylglycerol second messengers

    Energy Technology Data Exchange (ETDEWEB)

    Bishop, W.R.; Ganong, B.R.; Bell, R.M.

    1986-05-01

    Diacylglycerol(DAG) derived from phosphatidylinositol activates protein kinase C in agonist-stimulated cells. At least two pathways may contribute to the attenuation of the DAG signal: (1) phosphorylation to phosphatidic acid(PA) by DAG kinase(DGK), and (2) deacylation by DAG and monoacylglycerol lipases. A number of DAG analogs were tested as substrates and inhibitors of partially purified pig brain DGK. Two analogs were potent inhibitors in vitro, 1-monooleoylglycerol(MOG,K/sub I/ = 91 ..mu..M) and diotanoylethyleneglycol (diC/sub 8/EG, K/sub I/ = 58 ..mu..M). These compounds were tested in human platelets. DiC/sub 8/EG inhibited (70 - 100%) (/sup 32/P/sub i/) incorporation into PA in thrombin-stimulated platelets. Under these conditions the DAG signal was somewhat long-lived but was still metabolized, presumably by the lipase pathway. MOG treatment elevated DAG levels up to 4-fold in unstimulated platelets. The DAG formed was in a pool where it did not activate protein kinase C. Thrombin-stimulation of MOG-treated platelets resulted in DAG levels 10-fold higher than control platelets. This appears to be due to the inability of these platelets to metabolize agonist-linked DAG via the lipase pathway. The development of specific inhibitors of DAG kinase and DAG lipase, in conjunction with mass quantification of DAG levels as used here, will provide further insights into the regulation of DAG second messengers.

  3. [Effect of Escherichia coli mutation affecting the RNA polymerase sigma factor on phage T4 development].

    Science.gov (United States)

    Zograf, Iu N

    1982-01-01

    The bacterial RNA polymerase sigma factor is necessary throughout T4 development. T4 can develop in the E. coli RpoD800 mutant cells only at permissive temperature. RNA synthesis in T4-infected mutant cells remains temperature-sensitive throughout infection as in uninfected mutant bacteria. This shows that bacterial sigma factor is necessary for all types of RNA synthesis in infected E. coli. The data obtained suggest also that active sigma factor is necessary for early, but not for late T4 DNA replication.

  4. Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

    Science.gov (United States)

    Palmer, Adam C; Egan, J Barry; Shearwin, Keith E

    2011-01-01

    Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

  5. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

    1996-01-01

    were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl...

  6. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

    1996-01-01

    -tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay...

  7. On messengers and metastability in gauge mediation

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Emilian, E-mail: emilian.dudas@cpht.polytechnique.f [Centre de Physique Theorique, Ecole Polytechnique and CNRS, F-91128 Palaiseau (France); Laboratoire de Physique Theorique, Universite de Paris-Sud, Bat. 210, F-91405 Orsay (France); Lavignac, Stephane [Institut de Physique Theorique, CEA-Saclay, F-91191 Gif-sur-Yvette Cedex (France); Parmentier, Jeanne [Centre de Physique Theorique, Ecole Polytechnique and CNRS, F-91128 Palaiseau (France); Institut de Physique Theorique, CEA-Saclay, F-91191 Gif-sur-Yvette Cedex (France)

    2011-04-04

    One notoriously difficult problem in perturbative gauge mediation of supersymmetry breaking via messenger fields is the generic presence of a phenomenologically unacceptable vacuum with messenger vevs, with a lower energy than the desired ('MSSM') vacuum. We investigate the possibility that quantum corrections promote the latter to the ground state of the theory, and find that this is indeed feasible. For this to happen, the couplings of the messengers to the goldstino superfield must be small, and this implies an additional suppression of the MSSM soft terms with respect to the supersymmetry breaking scale. This in turn sets a lower limit on the masses of the messengers and of the supersymmetry breaking fields, which makes both sectors inaccessible at colliders. Contrary to other scenarios like direct gauge mediation, gaugino masses are unsuppressed with respect to scalar masses.

  8. Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes

    NARCIS (Netherlands)

    Mokry, M.; Hatzis, P.; Schuijers, J.; Lansu, N.; Ruzius, F.P.; Clevers, H.; Cuppen, E.

    2012-01-01

    Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by

  9. Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes

    NARCIS (Netherlands)

    Mokry, Michal; Hatzis, Pantelis; Schuijers, Jurian; Lansu, Nico; Ruzius, Frans-Paul; Clevers, Hans; Cuppen, Edwin

    2012-01-01

    Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RN

  10. Mechanical forces and their second messengers in stimulating cell growth in vitro

    Science.gov (United States)

    Vandenburgh, Herman H.

    1992-01-01

    Mechanical forces play an important role in modulating the growth of a number of different tissues including skeletal muscle, smooth muscle, cardiac muscle, bone, endothelium, epithelium, and lung. As interest increases in the molecular mechanisms by which mechanical forces are transduced into growth alterations, model systems are being developed to study these processes in tissue culture. This paper reviews the current methods available for mechanically stimulating tissue cultured cells. It then outlines some of the putative 'mechanogenic' second messengers involved in altering cell growth. Not surprisingly, many mechanogenic second messengers are the same as those involved in growth factor-induced cell growth. It is hypothesized that from an evolutionary standpoint, some second messenger systems may have initially evolved for unicellular organisms to respond to physical forces such as gravity and mechanical perturbation in their environment. As multicellular organisms came into existence, they appropriated these mechanogenic second messenger cascades for cellular regulation by growth factors.

  11. The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

    Institute of Scientific and Technical Information of China (English)

    Wenxing Liang; Changbao Li; Fang Liu; Hongling Jiang; Shuyu Li; Jiaqiang Sun; Xiaoyan Wu; Chuanyou Li

    2009-01-01

    Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3' end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We re-port here that AtCAFla and AtCAFlb, putative Arabidopsis homologs of the yeast CAFI gene, partially complement the growth defect of the yeast cafl mutant in the presence of caffeine or at high temperatures. The expression of At-CAFla and AtCAFlb is induced by multiple stress-related hormones and stimuli. Both AtCAFIa and AtCAFIb show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA inser-tion mutants disrupting the expression of AtCAFla and/or AtCAFlb are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAFI proteins are involved in regulated mRNA deadenylation in vivo. Interest-ingly, the single and double mutants of AtCAFla and AtCAFlb show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAFla show elevated expression of PR1 and PR2 and increased resis-tance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

  12. A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis.

    Science.gov (United States)

    Schnapp, A; Pfleiderer, C; Rosenbauer, H; Grummt, I

    1990-09-01

    Control of mouse ribosomal RNA synthesis in response to extracellular signals is mediated by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. Factor TIF-IA interacts with RNA polymerase I (pol I), thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other auxiliary transcription initiation factors, designated TIF-IB, TIF-IC and UBF. With regard to several criteria, the growth-dependent factor TIF-IA behaves like a bacterial sigma factor: (i) it associates physically with pol I, (ii) it is required for initiation of transcription, (iii) it is present in limiting amounts and (iv) under certain salt conditions, it is chromatographically separable from the polymerase. In addition, evidence is presented that dephosphorylation of pol I abolishes in vitro transcription initiation from the ribosomal gene promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The involvement of both a regulatory factor and post-translational modification of the transcribing enzyme provides an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to adapt ribosome synthesis rapidly to a variety of extracellular signals.

  13. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  14. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  15. Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA.

    Science.gov (United States)

    Fu, Yan; Bannach, Oliver; Chen, Hao; Teune, Jan-Hendrik; Schmitz, Axel; Steger, Gerhard; Xiong, Liming; Barbazuk, W Brad

    2009-05-01

    Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

  16. Transcription factor binding sites are highly enriched within microRNA precursor sequences

    Directory of Open Access Journals (Sweden)

    Piriyapongsa Jittima

    2011-12-01

    Full Text Available Abstract Background Transcription factors are thought to regulate the transcription of microRNA genes in a manner similar to that of protein-coding genes; that is, by binding to conventional transcription factor binding site DNA sequences located in or near promoter regions that lie upstream of the microRNA genes. However, in the course of analyzing the genomics of human microRNA genes, we noticed that annotated transcription factor binding sites commonly lie within 70- to 110-nt long microRNA small hairpin precursor sequences. Results We report that about 45% of all human small hairpin microRNA (pre-miR sequences contain at least one predicted transcription factor binding site motif that is conserved across human, mouse and rat, and this rises to over 75% if one excludes primate-specific pre-miRs. The association is robust and has extremely strong statistical significance; it affects both intergenic and intronic pre-miRs and both isolated and clustered microRNA genes. We also confirmed and extended this finding using a separate analysis that examined all human pre-miR sequences regardless of conservation across species. Conclusions The transcription factor binding sites localized within small hairpin microRNA precursor sequences may possibly regulate their transcription. Transcription factors may also possibly bind directly to nascent primary microRNA gene transcripts or small hairpin microRNA precursors and regulate their processing. Reviewers This article was reviewed by Guillaume Bourque (nominated by Jerzy Jurka, Dmitri Pervouchine (nominated by Mikhail Gelfand, and Yuriy Gusev.

  17. Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Kulshina, Nadia; Baird, Nathan J.; Ferré-D' Amaré, Adrian R.; (UWASH); (FHCRC)

    2009-12-03

    The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

  18. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  19. New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel.

    Science.gov (United States)

    Zenkin, Nikolay; Yuzenkova, Yulia

    2015-06-25

    Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.

  20. New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel

    Directory of Open Access Journals (Sweden)

    Nikolay Zenkin

    2015-06-01

    Full Text Available Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.

  1. Messenger Ribonucleic Acid Synthesis and Degradation in Escherichia coli During Inhibition of Translation

    Science.gov (United States)

    Pato, Martin L.; Bennett, Peter M.; Von Meyenburg, Kaspar

    1973-01-01

    Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3′) portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5′) portion of the mRNA, is subsequently degraded. PMID:4583248

  2. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  3. Kidney growth in normal and diabetic mice is not affected by human insulin-like growth factor binding protein-1 administration

    NARCIS (Netherlands)

    V. Cingel-Ristic; B.F. Schrijvers; A.K. van Vliet (Arlène); R. Rasch; V.K. Han; S.L.S. Drop (Stenvert); A. Flyvbjerg (Allan)

    2005-01-01

    textabstractInsulin-like growth factor I (IGF-I) accumulates in the kidney following the onset of diabetes, initiating diabetic renal hypertrophy. Increased renal IGF-I protein content, which is not reflected in messenger RNA (mRNA) levels, suggests that renal IGF-I accumulation is

  4. Direct regulation of rRNA transcription by fibroblast growth factor 2.

    Science.gov (United States)

    Sheng, Zhi; Liang, Yanping; Lin, Chih-Yin; Comai, Lucio; Chirico, William J

    2005-11-01

    Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to approximately 34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

  5. Higgs mass from neutrino-messenger mixing

    CERN Document Server

    Byakti, Pritibhajan; Mummidi, V Suryanarayana; Vempati, Sudhir K

    2016-01-01

    The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, $A_t$, relaxing these constraints. The detailed survey of these models \\cite{Byakti:2013ti,Evans:2013kxa} so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 15 of them can lead to Higgs mass within the observed value without raising the sfermion masses s...

  6. RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To inhibit the expression of vascular endothelial growth factor (VEGF) in colon cancer cell line by RNA interference (RNAi). METHODS: Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamineTM 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting. RESULTS: We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively. CONCLUSION: The expression of VEGF can be inhibited by RNA interference in HT29 cells.

  7. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    DEFF Research Database (Denmark)

    Düring, Louis; Thorsen, Michael; Petersen, Darima

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin...... architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6¿¿ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature. This change in U6...

  8. Construction Strategy and Affecting Factors of Positive-Strand RNA Virus Infectious Clone

    Institute of Scientific and Technical Information of China (English)

    SHEN Shichuan; YUE Kuizhong

    2008-01-01

    Construction of infectious clones by full-length eDNA is basic and key for recovering RNA virus and is core of reverse genetics. In this article, basic consideration and key technology were viewed and factors affecting infectivity of clones were also summarized. Some research advances were briefly introduced about positive-strand RNA viruses infectious clones. Finally, this article also reviewed the application of infectious clones.

  9. Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

    Science.gov (United States)

    Tjaden, G; Aguanno, A; Kumar, R; Benincasa, D; Gubits, R M; Yu, H; Dolan, K P

    1990-01-01

    Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment. Images PMID:2160599

  10. Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II

    OpenAIRE

    Elsby, Laura M.; O'Donnell, Amanda J M; Green, Laura M.; Sharrocks, Andrew D.; Roberts, Stefan G. E.

    2006-01-01

    The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on th...

  11. Stimulation of Pol III-dependent 5S rRNA and U6 snRNA gene expression by AP-1 transcription factors.

    Science.gov (United States)

    Ahuja, Richa; Kumar, Vijay

    2017-07-01

    RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. © 2017 Federation of European Biochemical Societies.

  12. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  13. MicroRNA gene polymorphisms and environmental factors increase patient susceptibility to hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Yin-Hung Chu

    Full Text Available BACKGROUND: Micro RNAs (miRNAs are small RNA fragments that naturally exist in the human body. Through various physiological mechanisms, miRNAs can generate different functions for regulating RNA protein levels and balancing abnormalities. Abnormal miRNA expression has been reported to be highly related to several diseases and cancers. Single-nucleotide polymorphisms (SNPs in miRNAs have been reported to increase patient susceptibility and affect patient prognosis and survival. We adopted a case-control research design to verify the relationship between miRNAs and hepatocellular carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: A total of 525 subjects, including 377 controls and 188 hepatocellular carcinoma patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and real-time PCR were used to analyze miRNA146a (rs2910164, miRNA149 (rs2292832, miRNA196 (rs11614913, and miRNA499 (rs3746444 genetic polymorphisms between the control group and the case group. The results indicate that people who carry the rs3746444 CT or CC genotypes may have a significantly increased susceptibility to hepatocellular carcinoma (adjusted odds ratio [AOR] = 2.84, 95% confidence interval [CI] = 1.88-4.30. In addition, when combined with environmental risk factors, such as smoking and alcohol consumption, interaction effects were observed between gene polymorphisms and environmental factors (odds ratio [OR] = 4.69, 95% CI = 2.52-8.70; AOR = 3.38, 95% CI = 1.68-6.80. CONCLUSIONS: These results suggest that a significant association exists between miRNA499 SNPs and hepatocellular carcinoma. Gene-environment interactions of miRNA499 polymorphisms, smoking, and alcohol consumption might alter hepatocellular carcinoma susceptibility.

  14. Having it both ways: transcription factors that bind DNA and RNA.

    Science.gov (United States)

    Cassiday, Laura A; Maher, L James

    2002-10-01

    Multifunctional proteins challenge the conventional 'one protein-one function' paradigm. Here we note apparent multifunctional proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors that bind double-stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA-binding proteins. Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT-1, TRA-1, bicoid, the bacterial sigma(70) subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle. Little is known concerning the biological roles of these alternative protein-nucleic acid interactions, and even less is known concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us to identify artificial RNA sequences that competitively inhibit a DNA-binding transcription factor not known to have a natural RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA-binding proteins is more common than currently appreciated.

  15. MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

    DEFF Research Database (Denmark)

    Düring, Louis; Thorsen, Michael; Petersen, Darima;

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

  16. Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

    Institute of Scientific and Technical Information of China (English)

    MinZHANG; XinZHANG; Chun-xueBAI; JieCHEN; MinQWEI

    2004-01-01

    AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

  17. Mapping interactions between mRNA export factors in living cells.

    Directory of Open Access Journals (Sweden)

    I-Fang Teng

    Full Text Available The TREX complex couples nuclear mRNA processing events with subsequent export to the cytoplasm. TREX also acts as a binding platform for the mRNA export receptor Nxf1. The sites of mRNA transcription and processing within the nucleus have been studied extensively. However, little is known about where TREX assembly takes place and where Nxf1 is recruited to TREX to form the export competent mRNP. Here we have used sensitized emission Förster resonance energy transfer (FRET and fluorescence lifetime imaging (FLIM-FRET, to produce a spatial map in living cells of the sites for the interaction of two TREX subunits, Alyref and Chtop, with Nxf1. Prominent assembly sites for export factors are found in the vicinity of nuclear speckles in regions known to be involved in transcription, splicing and exon junction complex formation highlighting the close coupling of mRNA export with mRNP biogenesis.

  18. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I.

    Science.gov (United States)

    Mayer, Andreas; Schreieck, Amelie; Lidschreiber, Michael; Leike, Kristin; Martin, Dietmar E; Cramer, Patrick

    2012-04-01

    During transcription elongation, RNA polymerase II (Pol II) binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for cotranscriptional recruitment of the Paf1 complex (D. L. Lindstrom et al., Mol. Cell. Biol. 23:1368-1378, 2003; Z. Zhang, J. Fu, and D. S. Gilmour, Genes Dev. 19:1572-1580, 2005). Here we report a new role of the Spt5 CTR in the recruitment of 3' RNA-processing factors. Chromatin immunoprecipitation (ChIP) revealed that the Spt5 CTR is required for normal recruitment of pre-mRNA cleavage factor I (CFI) to the 3' ends of Saccharomyces cerevisiae genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detected occupancy peaks of CFI subunits around 100 nucleotides downstream of the polyadenylation (pA) sites of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro but is not required for pA site recognition and transcription termination in vivo.

  19. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Wang Jianing

    2011-05-01

    Full Text Available Abstract Background The human coagulation trigger tissue factor (TF is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. Methods The specific small interfering RNA (siRNA designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. Results TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. Conclusions Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

  20. Perturbation of MicroRNA-370/Lin-28 homolog A/nuclear factor kappa B regulatory circuit contributes to the development of hepatocellular carcinoma.

    Science.gov (United States)

    Xu, Wen-Ping; Yi, Min; Li, Qian-Qian; Zhou, Wei-Ping; Cong, Wen-Ming; Yang, Yuan; Ning, Bei-Fang; Yin, Chuan; Huang, Zhao-Wei; Wang, Jian; Qian, Hui; Jiang, Cai-Feng; Chen, Yue-Xiang; Xia, Chun-Yan; Wang, Hong-Yang; Zhang, Xin; Xie, Wei-Fen

    2013-12-01

    MicroRNA 370 (miR-370) is located within the DLK1/DIO3 imprinting region on human chromosome 14, which has been identified as a cancer-associated genomic region. However, the role of miR-370 in malignances remains controversial. Here, we report that miR-370 was repressed in human hepatocellular carcinoma (HCC) tissues and hepatoma cell lines. Using gain-of-function and loss-of-function experiments, we demonstrated that miR-370 inhibited the malignant phenotype of HCC cells in vitro. Overexpression of miR-370 inhibited growth and metastasis of HCC cells in vivo. Moreover, the RNA-binding protein, LIN28A, was identified as a direct functional target of miR-370, which, in turn, blocked the biogenesis of miR-370 by binding to its precursor. LIN28A also mediated the suppressive effects of miR-370 on migration and invasion of HCC cells by post-transcriptionally regulating RelA/p65, which is an important effector of the canonical nuclear factor kappa B (NF-κB) pathway. Interleukin-6 (IL-6), a well-known NF-κB downstream inflammatory molecule, reduced miR-370 but increased LIN28A levels in HCC. Furthermore, miR-370 levels were inversely correlated with LIN28A and IL-6 messenger RNA (mRNA) levels, whereas LIN28A mRNA expression was positively correlated with IL-6 expression in human HCC samples. Interestingly, reduction of miR-370 expression was associated with the development of HCC in rats, as well as with aggressive tumor behavior and short survival in HCC patients. These data demonstrate the involvement of a novel regulatory circuit consisting of miR-370, LIN28A, RelA/p65 and IL-6 in HCC progression. Manipulating this feedback loop may have beneficial effect in HCC treatment. © 2013 by the American Association for the Study of Liver Diseases.

  1. Small interference RNA targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice model

    Institute of Scientific and Technical Information of China (English)

    KONG Yi-chun; SUN Bei; ZHAO Kan-xing; HAN Mei; WANG Yu-chuan

    2013-01-01

    Background The mechanism of retinal neovascularization is not understood completely.Many growth factors are involved in the process of retinal neovascularization,such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF),which are the representatives of angiogenic and antiangiogenic molecules respectively.Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization.The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).Methods In vitro,cultured EOMA cells were transfected with VEGF-siRNA (psi-HITM/EGFPNEGF siRNA) and LipofectamineTM 2000 for 24,48,and 72 hours,respectively.Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting.In vivo,OIR model mice were established,the mice (C57BL/6J) received an intra-vitreal injection of 1 μl of mixture of psi-HITM/EGFPNEGF siRNA and Lipofectamine 2000.Expressions of retinal VEGF and PEDF protein were measured by Western blotting,retinal neovascularization was observed by fluorescein angiography,and quantified.Results In vitro psi-HITM/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression.In vivo,with decreased VEGF and VEGF-PEDF ratio,significant attenuation of neovascular tufts,avascular regions,tortuous,and dilated blood vessels were observed in the interfered animals.Conclusions VEGF plays an important role in OIR,and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo,accompanied by the downregulation of VEGF-PEDF ratio,and simultaneous attenuation of retinal neovascularization was also observed.These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression

  2. Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Hammer, Niels A; Nielsen, Jacob

    2004-01-01

    Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). T...

  3. Analyses of in vivo interactions between transcription factors and the archaeal RNA polymerase.

    Science.gov (United States)

    Walker, Julie E; Santangelo, Thomas J

    2015-09-15

    Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation.

  4. Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

    Science.gov (United States)

    de Longevialle, Andéol Falcon; Small, Ian D; Lurin, Claire

    2010-07-01

    Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

  5. MESSENGER at Mercury: Early Orbital Operations

    Science.gov (United States)

    McNutt, Ralph L., Jr; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; Philips, Roger J.; Prockter, Louise M.; Slavin, James A.; Zuber, M. T.; Finnegan, Eric J.; Grant, David G.

    2013-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, launched in August 2004 under NASA's Discovery Program, was inserted into orbit about the planet Mercury in March 2011. MESSENGER's three flybys of Mercury in 2008-2009 marked the first spacecraft visits to the innermost planet since the Mariner 10 flybys in 1974-1975. The unprecedented orbital operations are yielding new insights into the nature and evolution of Mercury. The scientific questions that frame the MESSENGER mission led to the mission measurement objectives to be achieved by the seven payload instruments and the radio science experiment. Interweaving the full set of required orbital observations in a manner that maximizes the opportunity to satisfy all mission objectives and yet meet stringent spacecraft pointing and thermal constraints was a complex optimization problem that was solved with a software tool that simulates science observations and tracks progress toward meeting each objective. The final orbital observation plan, the outcome of that optimization process, meets all mission objectives. MESSENGER's Mercury Dual Imaging System is acquiring a global monochromatic image mosaic at better than 90% coverage and at least 250 m average resolution, a global color image mosaic at better than 90% coverage and at least 1 km average resolution, and global stereo imaging at better than 80% coverage and at least 250 m average resolution. Higher-resolution images are also being acquired of targeted areas. The elemental remote sensing instruments, including the Gamma-Ray and Neutron Spectrometer and the X-Ray Spectrometer, are being operated nearly continuously and will establish the average surface abundances of most major elements. The Visible and Infrared Spectrograph channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer is acquiring a global map of spectral reflectance from 300 to 1450 nm wavelength at a range of incidence and emission

  6. MESSENGER at Mercury: Early orbital operations

    Science.gov (United States)

    McNutt, Ralph L.; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.; Phillips, Roger J.; Prockter, Louise M.; Slavin, James A.; Zuber, Maria T.; Finnegan, Eric J.; Grant, David G.; MESSENGER Team

    2014-01-01

    The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, launched in August 2004 under NASA's Discovery Program, was inserted into orbit about the planet Mercury in March 2011. MESSENGER's three flybys of Mercury in 2008-2009 marked the first spacecraft visits to the innermost planet since the Mariner 10 flybys in 1974-1975. The unprecedented orbital operations are yielding new insights into the nature and evolution of Mercury. The scientific questions that frame the MESSENGER mission led to the mission measurement objectives to be achieved by the seven payload instruments and the radio science experiment. Interweaving the full set of required orbital observations in a manner that maximizes the opportunity to satisfy all mission objectives and yet meet stringent spacecraft pointing and thermal constraints was a complex optimization problem that was solved with a software tool that simulates science observations and tracks progress toward meeting each objective. The final orbital observation plan, the outcome of that optimization process, meets all mission objectives. MESSENGER's Mercury Dual Imaging System is acquiring a global monochromatic image mosaic at better than 90% coverage and at least 250 m average resolution, a global color image mosaic at better than 90% coverage and at least 1 km average resolution, and global stereo imaging at better than 80% coverage and at least 250 m average resolution. Higher-resolution images are also being acquired of targeted areas. The elemental remote sensing instruments, including the Gamma-Ray and Neutron Spectrometer and the X-Ray Spectrometer, are being operated nearly continuously and will establish the average surface abundances of most major elements. The Visible and Infrared Spectrograph channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer is acquiring a global map of spectral reflectance from 300 to 1450 nm wavelength at a range of incidence and emission

  7. Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

    DEFF Research Database (Denmark)

    Dabelsteen, S; Wandall, H H; Grøn, B

    1997-01-01

    Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m...

  8. siRNA epidermal growth factor receptor silencing in U251 glioma cells

    Institute of Scientific and Technical Information of China (English)

    Chunsheng Kang; Zhiyong Zhang; Zhifan Jia; Qiang Huang; Guangxiu Wang; Mingzhe Qiu; Peiyu Pu

    2008-01-01

    BACKGROUND: Dicer, a large multidomain ribonuclease, is responsible for processing double-stranded RNAs (dsRNAs) to 20-bp-long small interfering RNAs (siRNAs), which act as effectors during RNA interference (RNAi). OBJECTIVE: To observe the efficacy of siRNA cocktails generated by recombinant human Dicer on the down-regulation of epidermal growth factor receptor (EGFR) expression in human glioma cells. DESIGN, TIME AND SETTING: The following in vitro experiment was performed at the Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute. MATERIALS: Mini-RNA isolation kit, human placenta complimentary DNA (cDNA) was produced by Tiangen Biotech (Beijing, China), human glioblastoma U251-MG cells were produced by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. METHODS: A PCR product from the human EGFR, which corresponded to the tyrosine kinase domain of the 3'-end fragment, was used as the T7-promotor for in vitro transcription, siRNA cocktails were generated by in vitro dicing of double stranded RNA. A total of 500, 250 and 125 μg siRNA cocktails were transiently transfected into U251 glioma cells through the use of the GeneSilencer. MAIN OUTCOME MEASURE: Expression of EGFR was detected by real-time PCR. RESULTS: The total PCR product of the human EGFR, corresponding to the tyrosine kinase domain, is approximately 680 bp in length. The PCR transcriptants included GCC leader sequences and a T7 promoter sequence, with a fragment of EGFR cDNA at the center. The T7 promoter was prepared for in vitro transcription of dsRNA. After dicing for 24 hours, the 21-nt siRNA cocktails were verified by 4% agarose gel. The difference between threshold cycle of a sample assay and threshold cycle of the corresponding endogenous reference (△ Ct) among parental U251 cells and cells transfected with different doses of siRNA cocktails were determined to be 3.06, 7.35, and 10

  9. Diet and lifestyle factors associated with miRNA expression in colorectal tissue

    Directory of Open Access Journals (Sweden)

    Slattery ML

    2016-12-01

    Full Text Available Martha L Slattery,1 Jennifer S Herrick,1 Lila E Mullany,1 John R Stevens,2 Roger K Wolff1 1Department of Internal Medicine, The University of Utah, Salt Lake City, 2Department of Mathematics and Statistics, Utah State University, Logan, UT, USA Abstract: MicroRNAs (miRNAs are small non-protein-coding RNA molecules that regulate gene expression. Diet and lifestyle factors have been hypothesized to be involved in the regulation of miRNA expression. In this study it was hypothesized that diet and lifestyle factors are associated with miRNA expression. Data from 1,447 cases of colorectal cancer to evaluate 34 diet and lifestyle variables using miRNA expression in normal colorectal mucosa as well as for differential expression between paired carcinoma and normal tissue were used. miRNA data were obtained using an Agilent platform. Multiple comparisons were adjusted for using the false discovery rate q-value. There were 250 miRNAs differentially expressed between carcinoma and normal colonic tissue by level of carbohydrate intake and 198 miRNAs differentially expressed by the level of sucrose intake. Of these miRNAs, 166 miRNAs were differentially expressed for both carbohydrate intake and sucrose intake. Ninety-nine miRNAs were differentially expressed by the level of whole grain intake in normal colonic mucosa. Level of oxidative balance score was associated with 137 differentially expressed miRNAs between carcinoma and paired normal rectal mucosa. Additionally, 135 miRNAs were differentially expressed in colon tissue based on recent NSAID use. Other dietary factors, body mass index, waist and hip circumference, and long-term physical activity levels did not alter miRNA expression after adjustment for multiple comparisons. These results suggest that diet and lifestyle factors regulate miRNA level. They provide additional support for the influence of carbohydrate, sucrose, whole grains, NSAIDs, and oxidative balance score on colorectal cancer risk

  10. The role of the DEAD-box RNA helicase DDX3 in mRNA metabolism.

    Science.gov (United States)

    Soto-Rifo, Ricardo; Ohlmann, Théophile

    2013-01-01

    DDX3 belongs to the DEAD-box proteins, a large family of ATP-dependent RNA helicases that participate in all aspects of RNA metabolism. Human DDX3 is a component of several messenger ribonucleoproteins that are found in the spliceosome, the export and the translation initiation machineries but also in different cytoplasmic mRNA granules. DDX3 has been involved in several cellular processes such as cell cycle progression, apoptosis, cancer, innate immune response, and also as a host factor for viral replication. Interestingly, not all these functions require the catalytic activities of DDX3 and thus, the precise roles of this apparently multifaceted protein remain largely obscure. The aim of this review is to provide a rapid and critical overview of the structure and functions of DDX3 with a particular emphasis on its role during mRNA metabolism.

  11. The interaction between bacterial transcription factors and RNA polymerase during the transition from initiation to elongation.

    Science.gov (United States)

    Yang, Xiao; Lewis, Peter J

    2010-01-01

    There are three stages of transcription: initiation, elongation and termination, and traditionally there has been a clear distinction between the stages. The specificity factor sigma is completely released from bacterial RNA polymerase after initiation, and then recycled for another round of transcription. Elongation factors then associate with the polymerase followed by termination factors (where necessary). These factors dissociate prior to initiation of a new round of transcription. However, there is growing evidence suggesting that sigma factors can be retained in the elongation complex. The structure of bacterial RNAP in complex with an essential elongation factor NusA has recently been published, which suggested rather than competing for the major σ binding site, NusA binds to a discrete region on RNAP. A model was proposed to help explain the way in which both factors could be associated with RNAP during the transition from transcription initiation to elongation.

  12. Analysis of RNA metabolism in fission yeast

    DEFF Research Database (Denmark)

    Wise, Jo Ann; Nielsen, Olaf

    2017-01-01

    Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles.......Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

  13. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline;

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...... with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion...... extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors....

  14. Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion.

    Science.gov (United States)

    Sundaramoorthy, Sriramkumar; Vázquez-Novelle, María Dolores; Lekomtsev, Sergey; Howell, Michael; Petronczki, Mark

    2014-11-18

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

  15. The Energy Messenger, Number 1, Volume 4

    Energy Technology Data Exchange (ETDEWEB)

    Stancil, J. [ed.

    1995-01-01

    `The Energy Messenger` is a Department of Energy publication on energy activities of interest to American Indians. The first issue of 1995 (in a magazine format) includes articles on: tribes winning grants to develop energy resources, recruiting of internships for DOE, information about Title XXVI-Indian Energy Resources, American Indian Heritage Month, tribal perspective on DOE actions, joint ventures between tribes and the DOE, and brief description of recent DOE activities.

  16. Environmental Contaminants and microRNA Regulation: Transcription Factors as Regulators of Toxicant-Altered microRNA Expression

    Science.gov (United States)

    Sollome, James; Martin, Elizabeth; Sethupathy, Praveen; Fry, Rebecca C.

    2016-01-01

    MicroRNAs (miRNAs) regulate gene expression by binding mRNA transcripts and inhibiting translation and/or inducing degradation of the associated transcripts. Expression levels of miRNAs have been shown to be altered in response to environmental toxicants, thus impacting cellular function and influencing disease risk. Transcription factors (TFs) are known to be altered in response to environmental toxicants and play a critical role in the regulation of miRNA expression. To date, environmentally-responsive TFs that are important for regulating miRNAs remain understudied. In a state-of-the-art analysis, we utilized in silico bioinformatic analysis to characterize potential transcriptional regulators of environmentally-responsive miRNAs. Using the miRStart database, genomic sequences of promoter regions for all available human miRNAs (n=847) were identified and promoter regions were defined as −1000/+500 base pairs from the transcription start site. Subsequently, the promoter region sequences of environmentally-responsive miRNAs (n=128) were analyzed using enrichment analysis to determine overrepresented TF binding sites (TFBS). While most (56/73) TFs differed across environmental contaminants, a set of 17 TFs was enriched for promoter binding among miRNAs responsive to numerous environmental contaminants. Of these, one TF was common to miRNAs altered by the majority of environmental contaminants, namely SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 3 (SMARCA3). These identified TFs represent candidate common transcriptional regulators of miRNAs perturbed by environmental toxicants. PMID:27292125

  17. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response

    Science.gov (United States)

    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. PMID:27252702

  18. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  19. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

    Directory of Open Access Journals (Sweden)

    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  20. Trans-acting factors governing acetylcholinesterase mRNA metabolism in neurons

    Directory of Open Access Journals (Sweden)

    Lucas M. Bronicki

    2012-03-01

    Full Text Available The most characterized function of acetylcholinesterase (AChE is to terminate cholinergic signaling at neuron-neuron and neuro-muscular synapses. In addition, AChE is causally or casually implicated in neuronal development, stress-response, cognition and neurodegenerative diseases. Given the importance of AChE, many studies have focused on identifying the molecular mechanisms that govern its expression. Despite these efforts, post-transcriptional control of AChE mRNA expression is still relatively unclear. Here, we review the trans-acting factors and cis-acting elements that are known to control AChE pre-mRNA splicing, mature mRNA stability and translation. Moreover, since the Hu/ELAV family of RNA-binding proteins (RBPs have emerged in recent years as ‘master’ post-transcriptional regulators, we discuss the possibility that predominantly neuronal ELAVs (nELAVs play multiple roles in regulating splicing, stability, localization and translation of AChE mRNA.

  1. Progress of Targeting Transforming Growth Factor-β1 Small Interfering RNA in Liver Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Xuan Zhou; Xue-feng Yang

    2014-01-01

    Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells (HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix (ECM). Transforming growth factor-beta (TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference (RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA (siRNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 siRNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 siRNA in liver fibrosis.

  2. Mining functional elements in messenger RNAs: overview, challenges, and perspectives

    Directory of Open Access Journals (Sweden)

    Firoz eAhmed

    2011-11-01

    Full Text Available Eukaryotic messenger RNA contains not only protein-coding regions but also a plethora of functional cis-elements that influence or coordinate a number of regulatory aspects of gene expression, such as mRNA stability, splicing forms, and translation rates. Understanding the rules that apply to each of these element types (e.g., whether the element is defined by primary or higher-order structure allows for the discovery of novel mechanisms of gene expression as well as the design of transcripts with controlled expression. Bioinformatics plays a major role in creating databases and finding non-evident patterns governing each type of eukaryotic functional element. Much of what we currently know about mRNA regulatory elements in eukaryotes is derived from microorganism and animal systems, with the particularities of plant systems lagging behind. In this review, we provide a general introduction to the most well-known eukaryotic mRNA regulatory motifs (splicing regulatory elements, internal ribosome entry sites, iron-responsive elements, AU-rich elements, zipcodes, and polyadenylation signals and describe available bioinformatics resources (databases and analysis tools to analyze eukaryotic transcripts in search of functional elements, focusing on recent trends in bioinformatics methods and tool development. We also discuss future directions in the development of better computational tools based upon current knowledge of these functional elements. Improved computational tools would advance our understanding of the processes underlying gene regulations. We encourage plant bioinformaticians to turn their attention to this subject to help identify novel mechanisms of gene expression regulation using RNA motifs that have potentially evolved or diverged in plant species.

  3. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: lxuhelen@163.com [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: liuzhiguo@fmmu.edu.cn [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  4. Nonsense-mediated mRNA decay among coagulation factor genes

    Directory of Open Access Journals (Sweden)

    Shirin Shahbazi

    2016-04-01

    Full Text Available Objective(s: Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulation factors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulation factor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulation factors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD. There are many pieces of evidence of decay among coagulation factor genes. However, the hemophilia gene (F8 does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade.

  5. Potential RNA polymerase II-induced interactions of transcription factor TFIIB.

    Science.gov (United States)

    Malik, S; Lee, D K; Roeder, R G

    1993-10-01

    The ubiquitous transcription factor TFIIB is required for initiation by RNA polymerase II and serves as a target of some regulatory factors. The carboxy-terminal portion of TFIIB contains a large imperfect direct repeat reminiscent of the structural organization of the TATA-binding component (TBP) of TFIID, as well as sequence homology to conserved regions of bacterial sigma factors. The present study shows that the carboxy-terminal portion of TFIIB, like that of TBP, is folded into a compact protease-resistant core. The TFIIB core, unlike the TBP core, is inactive in transcription but retains structural features that enable it to form a complex with promoter-bound TFIID. The protease-susceptible amino terminus appears to contain components responsible for direct interaction with RNA polymerase II (in association with TFIIF) either on the promoter (in association with TFIID) or independently. In addition, core TFIIB (but not intact TFIIB) extends the footprint of TBP on promoter DNA, suggesting that TFIIB has a cryptic DNA-binding potential. These results are consistent with a model in which TFIIB, in a manner functionally analogous to that of bacterial sigma factors, undergoes an RNA polymerase II-dependent conformational change with resultant DNA interactions during the pathway leading to a functional preinitiation complex.

  6. VDR mRNA overexpression is associated with worse prognostic factors in papillary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    June Young Choi

    2017-03-01

    Full Text Available The purpose of this study was to assess the relationship between vitamin D receptor gene (VDR expression and prognostic factors in papillary thyroid cancer (PTC. mRNA sequencing and somatic mutation data from The Cancer Genome Atlas (TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find cutoff and patients were split into low and high VDR group. Logistic regression, Kaplan–Meier analysis, differentially expressed gene (DEG test and pathway analysis were performed to assess the differences between two VDR groups. VDR mRNA expression was elevated in PTC than that in normal thyroid tissue. VDR expressions were high in classic and tall-cell variant PTC and lateral neck node metastasis was present. High VDR group was also associated with classic and tall cell subtype, AJCC stage IV and lower recurrence-free survival. DEG test reveals that 545 genes were upregulated in high VDR group. Thyroid cancer-related pathways were enriched in high VDR group in pathway analyses. VDR mRNA overexpression was correlated with worse prognostic factors such as subtypes of papillary thyroid carcinoma that are known to be worse prognosis, lateral neck node metastasis, advanced stage and recurrence-free survival.

  7. Messenger Plus!2.0——MSN Messenger 5.0的新伴侣

    Institute of Scientific and Technical Information of China (English)

    宁宏睿

    2003-01-01

    使用MSN Messenger 的用户,想要实现保存聊天记录等功能肯定都会安装Mesenger Plus!或者Messenger Help!这样的插件程序。由于Microsoft将Windows Messenger升级到MSN Messenger 5以后,编程接口发生了变化,Messenger Plus!的老版本无法使用,让MSN Messenger 5的用户感到很不方便。经过数个月的等待,Messenger Plus!(后文简称Plus!)终于推出了最新版本,完全支持Windows Messenger和MSN Messenger 5。

  8. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.

    Directory of Open Access Journals (Sweden)

    Zhong Hua

    Full Text Available MicroRNAs (miRNAs are a class of 20-24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle. We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false

  9. Effect of siRNA interference on nerve growth factor in intervertebral disc inflammation rats

    Institute of Scientific and Technical Information of China (English)

    Ming-Lei Lang; Ai-Lin Qin; Jian-Min Li; Peng Fu

    2014-01-01

    Objective:To investigate the inhibition effect of siRNA interference onNGF induced by inflammatory factorIL-6, andIL-1 so as to provide novel targets for clinical treatment of discogenic low back pain.Methods:The intervertebral disc nucleus and annulus fibrosus cells of rats were separated .The cells were co-cultured with different concentrations(10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L) ofIL-6 andIL-1β.TheNGF- siRNA was leaded into the co-cultured cells with its import ability assessed by flow cytometry instrument tests,before and after which theNGF mRNA expression was detected by real-timeQ-PCR and theNGF content was detected byELISA.Results:Flow cytometry instrument test results showed that theNGF-siRNA cell conversion rate was99.8%.Real-timeQ-PCR detection results showed that compared with negative control group, theNGF mRNA expression of co-cultured cells treated by10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/LIL-6 andIL-1β were respectively raised3.4,3.7,4.7, 3.7 times which were all significantly down-regulated after the import ofNGF- siRNA.EILSA detection results showed that compared with negative control group, theNGF content of co-cultured medium treated by10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/LI-L6 andIL-1β were respectively raised2.9,3.3,4.5,7.4 times which were all significantly decreased after the import ofNGF- siRNA.Conclusions:These molecular biological results suggest that inflammatory factorIL-6 andIL-1β could stimulateNGF on intervertebral disc cells in vitro culture model and its efficiency is concentration dependent, while siRNA interference can inhibit the stimulation effect ofIL-6 andIL-1β on intervertebral disc cell, which provides a new targets for the clinical treatment of discogenic low back pain.

  10. Multi-Messenger Astronomy: White Dwarf Binaries, LISA and GAIA

    Science.gov (United States)

    Bueno, Michael; Breivik, Katelyn; Larson, Shane L.

    2017-01-01

    The discovery of gravitational waves has ushered in a new era in astronomy. The low-frequency band covered by the future LISA detector provides unprecedented opportunities for multi-messenger astronomy. With the Global Astrometric Interferometer for Astrophysics (GAIA) mission, we expect to discover about 1,000 eclipsing binary systems composed of a WD and a main sequence star - a sizeable increase from the approximately 34 currently known binaries of this type. In advance of the first GAIA data release and the launch of LISA within the next decade, we used the Binary Stellar Evolution (BSE) code simulate the evolution of White Dwarf Binaries (WDB) in a fixed galaxy population of about 196,000 sources. Our goal is to assess the detectability of a WDB by LISA and GAIA using the parameters from our population synthesis, we calculate GW strength h, and apparent GAIA magnitude G. We can then use a scale factor to make a prediction of how many multi- messenger sources we expect to be detectable by both LISA and GAIA in a galaxy the size of the Milky Way. We create binaries 10 times to ensure randomness in distance assignment and average our results. We then determined whether or not astronomical chirp is the difference between the total chirp and the GW chirp. With Astronomical chirp and simulations of mass transfer and tides, we can gather more information about the internal astrophysics of stars in ultra-compact binary systems.

  11. Gene Ranking of RNA-Seq Data via Discriminant Non-Negative Matrix Factorization.

    Science.gov (United States)

    Jia, Zhilong; Zhang, Xiang; Guan, Naiyang; Bo, Xiaochen; Barnes, Michael R; Luo, Zhigang

    2015-01-01

    RNA-sequencing is rapidly becoming the method of choice for studying the full complexity of transcriptomes, however with increasing dimensionality, accurate gene ranking is becoming increasingly challenging. This paper proposes an accurate and sensitive gene ranking method that implements discriminant non-negative matrix factorization (DNMF) for RNA-seq data. To the best of our knowledge, this is the first work to explore the utility of DNMF for gene ranking. When incorporating Fisher's discriminant criteria and setting the reduced dimension as two, DNMF learns two factors to approximate the original gene expression data, abstracting the up-regulated or down-regulated metagene by using the sample label information. The first factor denotes all the genes' weights of two metagenes as the additive combination of all genes, while the second learned factor represents the expression values of two metagenes. In the gene ranking stage, all the genes are ranked as a descending sequence according to the differential values of the metagene weights. Leveraging the nature of NMF and Fisher's criterion, DNMF can robustly boost the gene ranking performance. The Area Under the Curve analysis of differential expression analysis on two benchmarking tests of four RNA-seq data sets with similar phenotypes showed that our proposed DNMF-based gene ranking method outperforms other widely used methods. Moreover, the Gene Set Enrichment Analysis also showed DNMF outweighs others. DNMF is also computationally efficient, substantially outperforming all other benchmarked methods. Consequently, we suggest DNMF is an effective method for the analysis of differential gene expression and gene ranking for RNA-seq data.

  12. Integrative transcriptome analysis identifies deregulated microRNA-transcription factor networks in lung adenocarcinoma.

    Science.gov (United States)

    Cinegaglia, Naiara C; Andrade, Sonia Cristina S; Tokar, Tomas; Pinheiro, Maísa; Severino, Fábio E; Oliveira, Rogério A; Hasimoto, Erica N; Cataneo, Daniele C; Cataneo, Antônio J M; Defaveri, Júlio; Souza, Cristiano P; Marques, Márcia M C; Carvalho, Robson F; Coutinho, Luiz L; Gross, Jefferson L; Rogatto, Silvia R; Lam, Wan L; Jurisica, Igor; Reis, Patricia P

    2016-05-17

    Herein, we aimed at identifying global transcriptome microRNA (miRNA) changes and miRNA target genes in lung adenocarcinoma. Samples were selected as training (N = 24) and independent validation (N = 34) sets. Tissues were microdissected to obtain >90% tumor or normal lung cells, subjected to miRNA transcriptome sequencing and TaqMan quantitative PCR validation. We further integrated our data with published miRNA and mRNA expression datasets across 1,491 lung adenocarcinoma and 455 normal lung samples. We identified known and novel, significantly over- and under-expressed (p ≤ 0.01 and FDR≤0.1) miRNAs in lung adenocarcinoma compared to normal lung tissue: let-7a, miR-10a, miR-15b, miR-23b, miR-26a, miR-26b, miR-29a, miR-30e, miR-99a, miR-146b, miR-181b, miR-181c, miR-421, miR-181a, miR-574 and miR-1247. Validated miRNAs included let-7a-2, let-7a-3, miR-15b, miR-21, miR-155 and miR-200b; higher levels of miR-21 expression were associated with lower patient survival (p = 0.042). We identified a regulatory network including miR-15b and miR-155, and transcription factors with prognostic value in lung cancer. Our findings may contribute to the development of treatment strategies in lung adenocarcinoma.

  13. MicroRNA-297a regulates vascular calcification by targeting fibroblast growth factor 23

    Directory of Open Access Journals (Sweden)

    Shouhua Zheng

    2016-12-01

    Full Text Available Objective(s: Vascular calcification is one the major characteristics in patients with various types of chronic inflammatory disorders. MiRNAs have been shown to be involved in many normal biological functions as well as diseases; however, their role in vascular calcification has not received much attention. Materials and Methods: In the current study, we built a vascular calcification rat model using vitamin D3 plus nicotine and analyzed miRNA expression profile by miRNA chip assay. Potential target of one selected miRNA with sharpest variation in expression were predicted by both PicTar and TargetScan. The impact of the selected miRNA on the expression of the potential target on both mRNA and protein levels were measured by RT-PCR and Western blot, respectively. Results: Our results identified 16 dysregulated miRNAs, among which miR-297a showed the sharpest variation. Further analysis focusing on miR-297a revealed that fibroblast growth factor 23 (FGF23 was a potential target of miR297a. Measurement of FGF23 and its regulator Klotho on both mRNA and protein levels demonstrated that FGF23 was significantly increased while Klotho was decreased in rats with vascular calcification. Conclusion: Our results indicated that FGF23 was target of miR-297a and decreased miR-297a in vascular calcification lead to the increase of FGF23, which together with Klotho might enhance vascular calcification. The findings of this study could provide valuable information for the understanding of mechanisms underlying miR-dependent vascular calcification as well as potential treatment target for the disease.

  14. Increased N6-methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia.

    Science.gov (United States)

    Yang, Ying; Huang, Wei; Huang, Jing-Tao; Shen, Fan; Xiong, Jun; Yuan, Er-Feng; Qin, Shan-shan; Zhang, Ming; Feng, Yu-Qi; Yuan, Bi-Feng; Liu, Song-Mei

    2016-04-13

    Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N(6)-methyladenosine (m(6)A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m(6)A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m(6)A-selective-binding protein (YTHDF2). We found that m(6)A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m(6)A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 - 8.853, p = 0.023). Moreover, m(6)A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = -0.288, p = 0.038; non-progressive motility: r = -0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m(6)A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m(6)A contents in sperm RNA.

  15. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Fukumitsu, Hidefumi, E-mail: hfukumi@gifu-pu.ac.jp [Laboratory of Molecular Biology, Department of Biofunctional Analysis, Gifu Pharmaceutical University, Daigakunishi 1-25-4, Gifu 501 1196 (Japan); Soumiya, Hitomi; Furukawa, Shoei [Laboratory of Molecular Biology, Department of Biofunctional Analysis, Gifu Pharmaceutical University, Daigakunishi 1-25-4, Gifu 501 1196 (Japan)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  16. Altered expression of circulating microRNA in plasma of patients with primary osteoarthritis and in silico analysis of their pathways.

    Directory of Open Access Journals (Sweden)

    Verónica M Borgonio Cuadra

    Full Text Available OBJECTIVE: To analyze a set of circulating microRNA (miRNA in plasma from patients with primary Osteoarthritis (OA and describe the biological significance of altered miRNA in OA based on an in silico analysis of their target genes. METHODS: miRNA expression was analyzed using TaqMan Low Density Arrays and independent assays. The search for potential messenger RNA (mRNA targets of the differentially expressed miRNA was performed by means of the miRWalk and miRecords database; we conducted the biological relevance of the predicted miRNA targets by pathway analysis with the Reactome and DAVID databases. RESULTS: We measured the expression of 380 miRNA in OA; 12 miRNA were overexpressed under the OA condition (p value, ≤0.05; fold change, >2. These results were validated by the detection of some selected miRNA by quantitative PCR (qPCR. In silico analysis showed that target messenger RNA (mRNA were potentially regulated by these miRNA, including genes such as SMAD1, IL-1B, COL3A, VEGFA, and FGFR1, important in chondrocyte maintenance and differentiation. Some metabolic pathways affected by the miRNA: mRNA ratio are signaling Bone morphogenetic proteins (BMP, Platelet-derived growth factor (PDGF, and Nerve growth factor (NGF, these latter two involved in the process of pain. CONCLUSIONS: We identified 12 miRNA in the plasma of patients with primary OA. Specific miRNA that are altered in the disease could be released into plasma, either due to cartilage damage or to an inherent cellular mechanism. Several miRNA could regulate genes and pathways related with development of the disease; eight of these circulating miRNA are described, to our knowledge, for first time in OA.

  17. H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein

    DEFF Research Database (Denmark)

    Runge, Steffen; Nielsen, Finn Cilius; Nielsen, Jacob;

    2000-01-01

    attachment sites are clustered within a 700-nucleotide segment encoded by exons 4 and 5. This 3'-terminal segment targets H19 RNA to lamellipodia and perinuclear regions in dispersed fibroblasts where IMP is also localized. The results suggest that IMP participates in H19 RNA localization and provides a link...

  18. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Science.gov (United States)

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  19. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Adrian Valli

    Full Text Available RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  20. Regulatory coordination of clustered microRNAs based on microRNA-transcription factor regulatory network

    Directory of Open Access Journals (Sweden)

    Wang Jin

    2011-12-01

    Full Text Available Abstract Background MicroRNA (miRNA is a class of small RNAs of ~22nt which play essential roles in many crucial biological processes and numerous human diseases at post-transcriptional level of gene expression. It has been revealed that miRNA genes tend to be clustered, and the miRNAs organized into one cluster are usually transcribed coordinately. This implies a coordinated regulation mode exerted by clustered miRNAs. However, how the clustered miRNAs coordinate their regulations on large scale gene expression is still unclear. Results We constructed the miRNA-transcription factor regulatory network that contains the interactions between transcription factors (TFs, miRNAs and non-TF protein-coding genes, and made a genome-wide study on the regulatory coordination of clustered miRNAs. We found that there are two types of miRNA clusters, i.e. homo-clusters that contain miRNAs of the same family and hetero-clusters that contain miRNAs of various families. In general, the homo-clustered as well as the hetero-clustered miRNAs both exhibit coordinated regulation since the miRNAs belonging to one cluster tend to be involved in the same network module, which performs a relatively isolated biological function. However, the homo-clustered miRNAs show a direct regulatory coordination that is realized by one-step regulation (i.e. the direct regulation of the coordinated targets, whereas the hetero-clustered miRNAs show an indirect regulatory coordination that is realized by a regulation comprising at least three steps (e.g. the regulation on the coordinated targets by a miRNA through a sequential action of two TFs. The direct and indirect regulation target different categories of genes, the former predominantly regulating genes involved in emergent responses, the latter targeting genes that imply long-term effects. Conclusion The genomic clustering of miRNAs is closely related to the coordinated regulation in the gene regulatory network. The pattern of

  1. A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing

    Directory of Open Access Journals (Sweden)

    Benoit Moindrot

    2015-07-01

    Full Text Available X-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.

  2. Detecting heterogeneity in single-cell RNA-Seq data by non-negative matrix factorization

    Science.gov (United States)

    Zhu, Xun; Ching, Travers; Pan, Xinghua; Weissman, Sherman M.

    2017-01-01

    Single-cell RNA-Sequencing (scRNA-Seq) is a fast-evolving technology that enables the understanding of biological processes at an unprecedentedly high resolution. However, well-suited bioinformatics tools to analyze the data generated from this new technology are still lacking. Here we investigate the performance of non-negative matrix factorization (NMF) method to analyze a wide variety of scRNA-Seq datasets, ranging from mouse hematopoietic stem cells to human glioblastoma data. In comparison to other unsupervised clustering methods including K-means and hierarchical clustering, NMF has higher accuracy in separating similar groups in various datasets. We ranked genes by their importance scores (D-scores) in separating these groups, and discovered that NMF uniquely identifies genes expressed at intermediate levels as top-ranked genes. Finally, we show that in conjugation with the modularity detection method FEM, NMF reveals meaningful protein-protein interaction modules. In summary, we propose that NMF is a desirable method to analyze heterogeneous single-cell RNA-Seq data. The NMF based subpopulation detection package is available at: https://github.com/lanagarmire/NMFEM. PMID:28133571

  3. Mercury's Na Exosphere from MESSENGER Data

    Science.gov (United States)

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. El; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-01-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UWS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft :0 infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The portion of the hot/cold source appears to be highly variable.

  4. Mercury's Na Exosphere from MESSENGER Data

    Science.gov (United States)

    Killen, Rosemary M.; Burger, M. H.; Cassidy, T. A.; Sarantos, M.; Vervack, R. J.; McClintock, W. El; Merkel, A. W.; Sprague, A. L.; Solomon, S. C.

    2012-01-01

    MESSENGER entered orbit about Mercury on March 18, 2011. Since then, the Ultraviolet and Visible Spectrometer (UWS) channel of MESSENGER's Mercury Atmospheric and Surface Composition Spectrometer (MASCS) has been observing Mercury's exosphere nearly continuously. Daily measurements of Na brightness were fitted with non-uniform exospheric models. With Monte Carlo sampling we traced the trajectories of a representative number of test particles, generally one million per run per source process, until photoionization, escape from the gravitational well, or permanent sticking at the surface removed the atom from the simulation. Atoms were assumed to partially thermally accommodate on each encounter with the surface with accommodation coefficient 0.25. Runs for different assumed source processes are run separately, scaled and co-added. Once these model results were saved onto a 3D grid, we ran lines of sight from the MESSENGER spacecraft :0 infinity using the SPICE kernels and we computed brightness integrals. Note that only particles that contribute to the measurement can be constrained with our method. Atoms and molecules produced on the nightside must escape the shadow in order to scatter light if the excitation process is resonant-light scattering, as assumed here. The aggregate distribution of Na atoms fits a 1200 K gas, with a PSD distribution, along with a hotter component. Our models constrain the hot component, assumed to be impact vaporization, to be emitted with a 2500 K Maxwellian. Most orbits show a dawnside enhancement in the hot component broadly spread over the leading hemisphere. However, on some dates there is no dawn/dusk asymmetry. The portion of the hot/cold source appears to be highly variable.

  5. Stress-induced nuclear RNA degradation pathways regulate yeast bromodomain factor 2 to promote cell survival.

    Directory of Open Access Journals (Sweden)

    Kevin Roy

    2014-09-01

    Full Text Available Bromodomain proteins are key regulators of gene expression. How the levels of these factors are regulated in specific environmental conditions is unknown. Previous work has established that expression of yeast Bromodomain factor 2 (BDF2 is limited by spliceosome-mediated decay (SMD. Here we show that BDF2 is subject to an additional layer of post-transcriptional control through RNase III-mediated decay (RMD. We found that the yeast RNase III Rnt1p cleaves a stem-loop structure within the BDF2 mRNA to down-regulate its expression. However, these two nuclear RNA degradation pathways play distinct roles in the regulation of BDF2 expression, as we show that the RMD and SMD pathways of the BDF2 mRNA are differentially activated or repressed in specific environmental conditions. RMD is hyper-activated by salt stress and repressed by hydroxyurea-induced DNA damage while SMD is inactivated by salt stress and predominates during DNA damage. Mutations of cis-acting signals that control SMD and RMD rescue numerous growth defects of cells lacking Bdf1p, and show that SMD plays an important role in the DNA damage response. These results demonstrate that specific environmental conditions modulate nuclear RNA degradation pathways to control BDF2 expression and Bdf2p-mediated gene regulation. Moreover, these results show that precise dosage of Bromodomain factors is essential for cell survival in specific environmental conditions, emphasizing their importance for controlling chromatin structure and gene expression in response to environmental stress.

  6. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s).

    OpenAIRE

    Barik, S

    1992-01-01

    Extracts made from human respiratory syncytial virus (RSV)-infected Hep-2 cells synthesized mRNAs encoded by all known viral genes. In contrast, RSV ribonucleoproteins purified from infected cells failed to transcribe in vitro; transcription was restored by addition of a cytoplasmic extract of uninfected Hep-2 cells, demonstrating that a cellular factor(s) has a role in RSV gene expression. Quantitation of the individual gene mRNAs transcribed in vitro revealed polarity of transcription of th...

  7. The Dawn of Multi-Messenger Astronomy

    CERN Document Server

    Santander, Marcos

    2016-01-01

    The recent discoveries of high-energy astrophysical neutrinos and gravitational waves have opened new windows of exploration to the Universe. Combining neutrino observations with measurements of electromagnetic radiation and cosmic rays promises to unveil the sources responsible for the neutrino emission and to help solve long-standing problems in astrophysics such as the origin of cosmic rays. Neutrino observations may also help localize gravitational-wave sources, and enable the study of their astrophysical progenitors. In this work we review the current status and future plans for multi-messenger searches of neutrino sources.

  8. Gravitational Waves and Multi-Messenger Astronomy

    Science.gov (United States)

    Centrella, Joan M.

    2010-01-01

    Gravitational waves are produced by a wide variety of sources throughout the cosmos, including the mergers of black hole and neutron star binaries/compact objects spiraling into central black holes in galactic nuclei, close compact binaries/and phase transitions and quantum fluctuations in the early universe. Observing these signals can bring new, and often very precise, information about their sources across vast stretches of cosmic time. In this talk we will focus on thee opening of this gravitational-wave window on the universe, highlighting new opportunities for discovery and multi-messenger astronomy.

  9. The role of RNA-binding factor AUF1 in regulated gene expression and modulation of tumorigenesis%RNA 结合因子 AUF1调控基因表达及其在肿瘤发生中的双重调控作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨颖卓; 康鹏; 高杰; 徐春林; 王诗美(综述); 吴霞(审校)

    2014-01-01

    Turn-over of messenger ribonucleic acid ( mRNA) is a major control point in gene expres-sion.In mammals,many mRNAs encode inflammatory cytokines ,oncoproteins,and G-protein-coupled receptors are destabilized by the presence of AU -rich elements ( AREs ) in their 3′-untranslated regions .Association of ARE-binding proteins(AUBPs)with these mRNAs promotes rapid mRNA degradation .ARE/poly(U)-binding factor 1(AUF1),one of the best-characterized AUBPs,binds to many ARE-mRNAs and assembles other fac-tors to recruit the mRNA degradation machinery .Most studies support an mRNA -destabilizing role for AUF1,al-though other findings suggest additional functions for this factor .However,several lines of evidence also support a role for AUF1 in the initiation and/or development of cancer .Many AUF1-targeted transcripts encode products that control pro-or anti-oncogenic processes .Numerous signaling pathways alter the composition of this AUF 1 complex of proteins to affect changes in ARE -mRNA degradation rates .This review briefly describes the roles of mRNA decay in gene expression in general and ARE -mediated decay ( AMD) in particular ,with a focus on AUF1 and the different modes of regulation that govern AUF 1 involvement in AMD.In the end,we discuss how changes in AUF1 isoform distribution,subcellular localization,and post-translational protein modifications can influence the metabolism of targeted mRNAs .%信使RNA( Messenger ribonucleic acid ,mRNA)不断更新在基因表达中具有重要作用,而且编码一些重要蛋白。本文详细阐述了RNA结合因子1(RNA-binding factor 1,AUF1)使mRNA稳定或去稳定,在转录后水平调控基因表达的可能的分子机制,归纳了AUF1各个亚型蛋白在基因表达中的独特作用的相关研究进展,并探讨了AUF1表达水平的改变在肿瘤发生中的双重调控作用。认识到AUF1发挥生物学效应的分子机制以及作用的具体信号通路,能够帮助我们揭开更多的生命谜题。

  10. Dynamic nucleocytoplasmic shuttling of an Arabidopsis SR splicing factor: role of the RNA-binding domains.

    Science.gov (United States)

    Rausin, Glwadys; Tillemans, Vinciane; Stankovic, Nancy; Hanikenne, Marc; Motte, Patrick

    2010-05-01

    Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytoplasmic shuttling properties in specific cell types. Comparison of transient ectopic- and stable tissue-specific expression highlights the advantages of both approaches for nuclear protein dynamic studies. By site-directed mutagenesis of RSZp22 RNA-binding sequences, we show that functional RNA recognition motif RNP1 and zinc-knuckle are dispensable for the exclusive protein nuclear localization and speckle-like distribution. Fluorescence resonance energy transfer imaging also revealed that these motifs are implicated in RSZp22 molecular interactions. Furthermore, the RNA-binding motif mutants are defective for their export through the CRM1/XPO1/Exportin-1 receptor pathway but retain nucleocytoplasmic mobility. Moreover, our data suggest that CRM1 is a putative export receptor for mRNPs in plants.

  11. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia

    Science.gov (United States)

    AZIZI, ZAHRA; RAHGOZAR, SOHEILA; MOAFI, ALIREZA; DABAGHI, MOHAMMAD; NADIMI, MOTAHAREH

    2015-01-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BAALC and MRD1 were measured in bone marrow samples of 28 new diagnosed childhood ALL patients and 13 children without cancer. Minimal residual disease (MRD) was measured one year after the initiation of the chemotherapy using the RT-qPCR method. The high level expression of BAALC had a significant association with the pre-B-ALL subtype, leukocytosis and positive MRD after one year of treatment in leukemic patients. In addition, a positive correlation between BAALC and MDR1 mRNA expression was shown in this group. In conclusion, to the best of our knowledge, the increase of BAALC expression as a poor prognostic factor for childhood ALL is shown for the first time. Additionally, the correlation between BAALC and MDR1 in mRNA expression levels can aid for an improved understanding of the mechanism through which BAALC may function in ALL and multidrug resistance. PMID:26137238

  12. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia.

    Science.gov (United States)

    Azizi, Zahra; Rahgozar, Soheila; Moafi, Alireza; Dabaghi, Mohammad; Nadimi, Motahareh

    2015-05-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BAALC and MRD1 were measured in bone marrow samples of 28 new diagnosed childhood ALL patients and 13 children without cancer. Minimal residual disease (MRD) was measured one year after the initiation of the chemotherapy using the RT-qPCR method. The high level expression of BAALC had a significant association with the pre-B-ALL subtype, leukocytosis and positive MRD after one year of treatment in leukemic patients. In addition, a positive correlation between BAALC and MDR1 mRNA expression was shown in this group. In conclusion, to the best of our knowledge, the increase of BAALC expression as a poor prognostic factor for childhood ALL is shown for the first time. Additionally, the correlation between BAALC and MDR1 in mRNA expression levels can aid for an improved understanding of the mechanism through which BAALC may function in ALL and multidrug resistance.

  13. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  14. Interaction between viral RNA silencing suppressors and host factors in plant immunity.

    Science.gov (United States)

    Nakahara, Kenji S; Masuta, Chikara

    2014-08-01

    To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host-pathogen interactions. By analogy of the steps in virus-host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus-host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus-plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.

    Science.gov (United States)

    Teferi, Wondimagegnehu M; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole; Evans, David H

    2013-04-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

  16. Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly.

    Science.gov (United States)

    Murota, Yukiko; Ishizu, Hirotsugu; Nakagawa, Shinichi; Iwasaki, Yuka W; Shibata, Shinsuke; Kamatani, Miharu K; Saito, Kuniaki; Okano, Hideyuki; Siomi, Haruhiko; Siomi, Mikiko C

    2014-07-10

    PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Yb Integrates piRNA Intermediates and Processing Factors into Perinuclear Bodies to Enhance piRISC Assembly

    Directory of Open Access Journals (Sweden)

    Yukiko Murota

    2014-07-01

    Full Text Available PIWI-interacting RNAs (piRNAs direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.

  18. Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M Tanaka

    2012-02-24

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

  19. MESSENGER observations of Mercury's magnetic field structure

    Science.gov (United States)

    Johnson, Catherine L.; Purucker, Michael E.; Korth, Haje; Anderson, Brian J.; Winslow, Reka M.; Al Asad, Manar M. H.; Slavin, James A.; Alexeev, Igor. I.; Phillips, Roger J.; Zuber, Maria T.; Solomon, Sean C.

    2012-12-01

    We present a baseline, time-averaged model for Mercury's magnetosphere, derived from MESSENGER Magnetometer data from 24 March to 12 December 2011, comprising the spacecraft's first three Mercury years in orbit around the innermost planet. The model, constructed under the approximation that the magnetospheric shape can be represented as a paraboloid of revolution, includes two external (magnetopause and magnetotail) current systems and an internal (dipole) field and allows for reconnection. We take advantage of the geometry of the orbital Magnetometer data to estimate all but one of the model parameters, and their ranges, directly from the observations. These parameters are then used as a priori constraints in the paraboloid magnetospheric model, and the sole remaining parameter, the dipole moment, is estimated as 190 nT RM3 from a grid search. We verify that the best fit dipole moment is insensitive to changes in the other parameters within their determined ranges. The model provides an excellent first-order fit to the MESSENGER observations, with a root-mean-square misfit of less than 20 nT globally. The results show that the magnetopause field strength ranges from 10% to 50% of the dipole field strength at observation locations on the dayside and at nightside latitudes north of 60°N. Globally, the residual signatures observed to date are dominated by the results of magnetospheric processes, confirming the dynamic nature of Mercury's magnetosphere.

  20. A mathematical analysis of second messenger compartmentalization.

    Science.gov (United States)

    Chen, Wen; Levine, Herbert; Rappel, Wouter-Jan

    2008-12-10

    Intracellular compartmentalization of second messengers can lead to microdomains of elevated concentration that are thought to be involved in ensuring signaling specificity. Most experimental evidence for this compartmentalization involves the second messenger adenosine monophosphate (cAMP), which is degraded by phosphodiesterases (PDEs). One possible way of creating these compartments, supported by recent experiments, is to spatially separate the source of cAMP from regions of elevated PDE concentration. To quantify this possibility, we study here a simplified geometry in two dimensions (2D) and in three dimensions (3D), containing a cAMP point source and regions with different degradation constants. Using the symmetry of our geometry, we are able to derive steady state solutions for the cAMP concentration as a function of the system parameters. Furthermore, we show, using analytics as well as direct numerical simulations, that for physiologically relevant time scales the steady state solution has been reached. Our results indicate that elevating the degradation constant throughout the cell, except for a small microdomain surrounding the source, requires an unphysiologically high cellular PDE concentration. On the other hand, a tight spatial relationship of localized PDEs with the cAMP source can result in functional microdomains while maintaining a physiologically plausible cellular PDE concentration.

  1. RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes.

    Directory of Open Access Journals (Sweden)

    Pénélope Legros

    2014-11-01

    Full Text Available Condensin-mediated chromosome condensation is essential for genome stability upon cell division. Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s direct(s the accumulation of condensin remains unclear. Here we show in fission yeast that condensin becomes strikingly enriched at RNA Pol III-transcribed genes when Swd2.2 and Sen1, two factors involved in the transcription process, are simultaneously deleted. Sen1 is an ATP-dependent helicase whose orthologue in Saccharomyces cerevisiae contributes both to terminate transcription of some RNA Pol II transcripts and to antagonize the formation of DNA:RNA hybrids in the genome. Using two independent mapping techniques, we show that DNA:RNA hybrids form in abundance at Pol III-transcribed genes in fission yeast but we demonstrate that they are unlikely to faciliate the recruitment of condensin. Instead, we show that Sen1 forms a stable and abundant complex with RNA Pol III and that Swd2.2 and Sen1 antagonize both the interaction of RNA Pol III with chromatin and RNA Pol III-dependent transcription. When Swd2.2 and Sen1 are lacking, the increased concentration of RNA Pol III and condensin at Pol III-transcribed genes is accompanied by the accumulation of topoisomerase I and II and by local nucleosome depletion, suggesting that Pol III-transcribed genes suffer topological stress. We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1. Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.

  2. Factors affecting reproducibility between genome-scale siRNA-based screens

    Science.gov (United States)

    Barrows, Nicholas J.; Le Sommer, Caroline; Garcia-Blanco, Mariano A.; Pearson, James L.

    2011-01-01

    RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage. In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies. PMID:20625183

  3. Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

    Science.gov (United States)

    Zhang, Wei; Qiao, Haishi; Lv, Yuanzi; Wang, Jingjing; Chen, Xiaoqing; Hou, Yayi; Tan, Renxiang; Li, Erguang

    2014-01-01

    Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5'-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.

  4. Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex

    DEFF Research Database (Denmark)

    Brown, S; Blumenthal, T

    1976-01-01

    (C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results...... by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly...

  5. Direct Regulation of rRNA Transcription by Fibroblast Growth Factor 2

    OpenAIRE

    Sheng, Zhi; Liang, Yanping; Lin, Chih-Yin; Comai, Lucio; Chirico, William J

    2005-01-01

    Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to ∼34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may...

  6. Endonucleolysis in the turnover of insulin-like growth factor II mRNA

    DEFF Research Database (Denmark)

    Nielsen, F C; Christiansen, Jan

    1992-01-01

    between a putative hairpin and a phylogenetically conserved guanosine-rich region which forms a stable higher order RNA structure in the presence of K+. We suggest that endonucleolysis is the initial step in IGF-II mRNA decay and that this event may participate in the post-transcriptional regulation......The overlapping transcription units constituting the rat insulin-like growth factor II (IGF-II) locus generate multiple mRNAs by using different promoters. Three promoters have been identified, giving rise to 4.6-, 3.8-, and 3.6-kilobase mRNAs. The latter, originating from promoter P3, is the most...... abundant IGF-II mRNA in the rat liver cell-line BRL-3A. Moreover, a non-polyadenylated 1.2-kilobase (kb) transcript and a 1.8-kb tail fragment are prominent transcripts at steady-state. In this study, we show that the 1.8-kb tail fragment is uncapped and sediments as a 30 S ribonucleoprotein particle...

  7. Micro-RNA-126 Reduces the Blood Thrombogenicity in Diabetes Mellitus via Targeting of Tissue Factor.

    Science.gov (United States)

    Witkowski, Marco; Weithauser, Alice; Tabaraie, Termeh; Steffens, Daniel; Kränkel, Nicolle; Witkowski, Mario; Stratmann, Bernd; Tschoepe, Diethelm; Landmesser, Ulf; Rauch-Kroehnert, Ursula

    2016-06-01

    Diabetes mellitus involves vascular inflammatory processes and is a main contributor to cardiovascular mortality. Notably, heightened levels of circulating tissue factor (TF) account for the increased thrombogenicity and put those patients at risk for thromboembolic events. Here, we sought to investigate the role of micro-RNA (miR)-driven TF expression and thrombogenicity in diabetes mellitus. Plasma samples of patients with diabetes mellitus were analyzed for TF protein and activity as well as miR-126 expression before and after optimization of the antidiabetic treatment. We found low miR-126 levels to be associated with markedly increased TF protein and TF-mediated thrombogenicity. Reduced miR-126 expression was accompanied by increased vascular inflammation as evident from the levels of vascular adhesion molecule-1 and fibrinogen, as well as leukocyte counts. With optimization of the antidiabetic treatment miR-126 levels increased and thrombogenicity was reduced. Using a luciferase reporter system, we demonstrated miR-126 to directly bind to the F3-3'-untranslated region, thereby reducing TF expression both on mRNA and on protein levels in human microvascular endothelial cells as well as TF mRNA and activity in monocytes. Circulating miR-126 exhibits antithrombotic properties via regulating post-transcriptional TF expression, thereby impacting the hemostatic balance of the vasculature in diabetes mellitus. © 2016 The Authors.

  8. N-ethylmaleimide-sensitive factor siRNA improves cardiac function following myocardial infarction in rats.

    Science.gov (United States)

    Zhou, Y; Liu, Y; Yang, S X; Wang, Z

    2015-08-14

    This study examined the effects of N-ethylmaleimide-sensitive factor (NSF) small interfering RNA (siRNA) on cardiac function following myocardial infarction (MI) in rats. Thirty-six adult Sprague Dawley rats were randomly divided into three equivalent groups. An acute MI model was established by ligating the anterior descending branch of the left coronary artery and confirmed by electrocardiogram. Recombinant NSF-siRNA adenovirus (experimental), negative adenovirus (control), and normal saline were injected near the infarcted area of the left ventricle in each respective group. The left ventricular ejection fraction (LVEF) was measured with a noninvasive ultrasonic cardiogram. Left ventricular end-diastolic pressure (LVEDP) and the maximum rate of rise in left ventricular pressure (+dp/dt max) were measured using the BL-420 Biological Functional Experimental System. Hearts were sectioned and stained with 2,3,5,-triphenyl tetrazolium chloride (TTC) to observe the MI area. Two weeks after surgery, LVEF in the experimental group (46.0 ± 7.5%) was higher than control (34.0 ± 6.0%) and saline (37.5 ± 4.5%) group LVEFs (P function two weeks after MI, but had no impact on the MI area.

  9. Oligodendrocyte transcription factor 1 mRNA and protein expression in organotypic rat brain slices

    Institute of Scientific and Technical Information of China (English)

    Hong Cui; Lijun Yang; Dezhuang Huang; Wandong Zhang; Weijuan Han; Yanqing Yao; Wenxing Jiang

    2010-01-01

    Numerous studies have confirmed that oligodendrocyte transcription factor 1 (Olig-1) is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown.In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxia/ischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

  10. ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions.

    Science.gov (United States)

    Edelmann, Franziska T; Niedner, Annika; Niessing, Dierk

    2015-01-01

    Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions.

  11. Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export* ♦

    OpenAIRE

    Zheng, Chao; Fasken, Milo B.; Marshall, Neil J.; Brockmann, Christoph; Rubinson, Max E.; Wente, Susan R.; Corbett, Anita H.; Stewart, Murray

    2010-01-01

    Following transcription, mRNA is processed, packaged into messenger ribonucleoprotein (mRNP) particles, and transported through nuclear pores (NPCs) to the cytoplasm. At the NPC cytoplasmic face, Dbp5 mediates mRNP remodeling and mRNA export factor dissociation, releasing transcripts for translation. In Saccharomyces cerevisiae, the conserved poly(A) RNA-binding protein, Nab2, facilitates NPC targeting of transcripts and also modulates poly(A) tail length. Dbp5 removes Nab2 from mRNPs at the ...

  12. ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56.

    Science.gov (United States)

    Taniguchi, Ichiro; Ohno, Mutsuhito

    2008-01-01

    Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

  13. Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

    Science.gov (United States)

    Hashikawa, Naoya; Ogawa, Takumi; Sakamoto, Yusuke; Ogawa, Mami; Matsuo, Yumi; Zamami, Yoshito; Hashikawa-Hobara, Narumi

    2015-08-01

    Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test.

  14. Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth

    Science.gov (United States)

    Koch, Sylvia; Garcia Gonzalez, Omar; Assfalg, Robin; Schelling, Adrian; Schäfer, Patrick; Scharffetter-Kochanek, Karin; Iben, Sebastian

    2014-01-01

    Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients. PMID:24781187

  15. DCLK1 regulates pluripotency and angiogenic factors via microRNA-dependent mechanisms in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Sripathi M Sureban

    Full Text Available Stem cell pluripotency, angiogenesis and epithelial-mesenchymal transition (EMT have been shown to be significantly upregulated in pancreatic ductal adenocarcinoma (PDAC and many other aggressive cancers. The dysregulation of these processes is believed to play key roles in tumor initiation, progression, and metastasis, and is contributory to PDAC being the fourth leading cause of cancer-related deaths in the US. The tumor suppressor miRNA miR-145 downregulates critical pluripotency factors and oncogenes and results in repressed metastatic potential in PDAC. Additionally, the miR-200 family regulates several angiogenic factors which have been linked to metastasis in many solid tumors. We have previously demonstrated that downregulation of DCLK1 can upregulate critical miRNAs in both in vitro and in vivo cancer models and results in downregulation of c-MYC, KRAS, NOTCH1 and EMT-related transcription factors. A recent report has also shown that Dclk1 can distinguish between normal and tumor stem cells in Apc (min/+ mice and that ablation of Dclk1(+ cells resulted in regression of intestinal polyps without affecting homeostasis. Here we demonstrate that the knockdown of DCLK1 using poly(lactide-co-glycolide-encapsulated-DCLK1-siRNA results in AsPC1 tumor growth arrest. Examination of xenograft tumors revealed, (a increased miR-145 which results in decreased pluripotency maintenance factors OCT4, SOX2, NANOG, KLF4 as well as KRAS and RREB1; (b increased let-7a which results in decreased pluripotency factor LIN28B; and (c increased miR-200 which results in decreased VEGFR1, VEGFR2 and EMT-related transcription factors ZEB1, ZEB2, SNAIL and SLUG. Specificity of DCLK1 post-transcriptional regulation of the downstream targets of miR-145, miR-200 and let-7a was accomplished utilizing a luciferase-based reporter assay. We conclude that DCLK1 plays a significant master regulatory role in pancreatic tumorigenesis through the regulation of multiple tumor

  16. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

    Science.gov (United States)

    Weber, Gregor G; Kortmann, Jens; Narberhaus, Franz; Klose, Karl E

    2014-09-30

    Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

  17. The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

    Directory of Open Access Journals (Sweden)

    Tamara Kakoschke

    Full Text Available To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin.

  18. Theory of high-energy messengers

    CERN Document Server

    Dermer, Charles D

    2016-01-01

    Knowledge of the distant high-energy universe comes from photons, ultra-high energy cosmic rays (UHECRs), high-energy neutrinos, and gravitational waves. The theory of high-energy messengers reviewed here focuses on the extragalactic background light at all wavelengths, cosmic rays and magnetic fields in intergalactic space, and neutrinos of extragalactic origin. Comparisons are drawn between the intensities of photons and UHECRs in intergalactic space, and the high-energy neutrinos recently detected with IceCube at about the Waxman-Bahcall flux. Source candidates for UHECRs and high-energy neutrinos are reviewed, focusing on star-forming and radio-loud active galaxies. HAWC and Advanced LIGO are just underway, with much anticipation.

  19. Gauge Mediation Models with Adjoint Messengers

    CERN Document Server

    Gogoladze, Ilia; Shafi, Qaisar; Un, Cem Salih

    2016-01-01

    We present a class of models in the framework of gauge mediation supersymmetry breaking where the messenger fields transform in the adjoint representation of the Standard Model gauge symmetry. To avoid unacceptably light right-handed sleptons in the spectrum we introduce a non-zero U(1)_B-L D-term. This leads to an additional contribution to the soft supersymmetry breaking mass terms which makes the right-handed slepton masses compatible with the current experimental bounds. We show that in this framework the observed 125 GeV Higgs boson mass can be accommodated with the sleptons accessible at the LHC, while the squarks and gluinos lie in the multi-TeV range. We also discuss the issue of the fine-tuning and show that the desired relic dark matter abundance can also be accommodated.

  20. [Irisin: a messenger from the gods?].

    Science.gov (United States)

    Moreno, María; Moreno-Navarrete, José María; Fernández-Real, José Manuel

    2014-01-01

    Due to the need to understand the basis of the metabolic benefits of exercise, irisin was discovered a few years ago. This cytokine, secreted by skeletal muscle due to exercise, should have positive effects on energetic metabolism. In particular, it could act as a messenger on white adipose tissue, modifying its phenotype into the beige adipocyte, and increasing its thermogenic capacity. Since it was described, there have been numerous studies led to depict its function, with the aim of determining if irisin could become a therapeutic target in the context of diseases associated with a caloric excess, such as obesity and diabetes. In this review, the irisin discovery is summarized, along with its in vitro and in vivo effects described up until now. Copyright © 2013 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  1. Theory of high-energy messengers

    Science.gov (United States)

    Dermer, Charles D.

    2016-05-01

    Knowledge of the distant high-energy universe comes from photons, ultra-high energy cosmic rays (UHECRs), high-energy neutrinos, and gravitational waves. The theory of high-energy messengers reviewed here focuses on the extragalactic background light at all wavelengths, cosmic rays and magnetic fields in intergalactic space, and neutrinos of extragalactic origin. Comparisons are drawn between the intensities of photons and UHECRs in intergalactic space, and the high-energy neutrinos recently detected with IceCube at about the Waxman-Bahcall flux. Source candidates for UHECRs and high-energy neutrinos are reviewed, focusing on star-forming and radio-loud active galaxies. HAWC and Advanced LIGO are just underway, with much anticipation.

  2. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  3. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

    Science.gov (United States)

    Tabib-Salazar, Aline; Liu, Bing; Doughty, Philip; Lewis, Richard A; Ghosh, Somadri; Parsy, Marie-Laure; Simpson, Peter J; O'Dwyer, Kathleen; Matthews, Steve J; Paget, Mark S

    2013-06-01

    RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

  4. The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome

    Science.gov (United States)

    Crickard, John B.; Lee, Jaehyoun; Lee, Tae-Hee

    2017-01-01

    Abstract RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone–DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the ‘jaws’ of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA–histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone–DNA interactions. PMID:28379497

  5. Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases.

    Science.gov (United States)

    Elseviers, D; Gallagher, P; Hoffman, A; Weinberg, B; Schwartz, I

    1982-10-01

    A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.

  6. Endogenous Arabidopsis messenger RNAs transported to distant tissues.

    Science.gov (United States)

    Thieme, Christoph J; Rojas-Triana, Monica; Stecyk, Ewelina; Schudoma, Christian; Zhang, Wenna; Yang, Lei; Miñambres, Miguel; Walther, Dirk; Schulze, Waltraud X; Paz-Ares, Javier; Scheible, Wolf-Rüdiger; Kragler, Friedrich

    2015-03-23

    The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants? Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.

  7. Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

    Science.gov (United States)

    Denegri, Marco; Chiodi, Ilaria; Corioni, Margherita; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2001-01-01

    Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename “stress-induced SNBs.” The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580–725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts. PMID:11694584

  8. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    Science.gov (United States)

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  9. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

  10. Transforming growth factor-β1 short hairpin RNA inhibits renal allograft fibrosis

    Institute of Scientific and Technical Information of China (English)

    YIN Zhi-kang; WU Xiao-hou; XIA Yu-guo; LUO Chun-li

    2011-01-01

    Background Transforming growth factor-β1 (TGF-β1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-β1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects.Methods A Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group T (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200μg of TGF-β1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-β1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively.Results Plasmid transfection significantly inhibited the expression of TGF-β1, as well as that of its target gene, type I collagen (P <0.05 and P <0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-β1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P <0.05 and P<0.01, respectively).Conclusions This study suggests that transfection of a TGF-β1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation

  11. Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

    Science.gov (United States)

    Yuan, P Q; Grånäs, C; Källström, L; Yu, J; Huhman, K; Larhammar, D; Albers, H E; Johnson, A E

    1997-05-01

    The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the

  12. MAGIA²: from miRNA and genes expression data integrative analysis to microRNA-transcription factor mixed regulatory circuits (2012 update).

    Science.gov (United States)

    Bisognin, Andrea; Sales, Gabriele; Coppe, Alessandro; Bortoluzzi, Stefania; Romualdi, Chiara

    2012-07-01

    MAGIA(2) (http://gencomp.bio.unipd.it/magia2) is an update, extension and evolution of the MAGIA web tool. It is dedicated to the integrated analysis of in silico target prediction, microRNA (miRNA) and gene expression data for the reconstruction of post-transcriptional regulatory networks. miRNAs are fundamental post-transcriptional regulators of several key biological and pathological processes. As miRNAs act prevalently through target degradation, their expression profiles are expected to be inversely correlated to those of the target genes. Low specificity of target prediction algorithms makes integration approaches an interesting solution for target prediction refinement. MAGIA(2) performs this integrative approach supporting different association measures, multiple organisms and almost all target predictions algorithms. Nevertheless, miRNAs activity should be viewed as part of a more complex scenario where regulatory elements and their interactors generate a highly connected network and where gene expression profiles are the result of different levels of regulation. The updated MAGIA(2) tries to dissect this complexity by reconstructing mixed regulatory circuits involving either miRNA or transcription factor (TF) as regulators. Two types of circuits are identified: (i) a TF that regulates both a miRNA and its target and (ii) a miRNA that regulates both a TF and its target.

  13. Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development.

    Science.gov (United States)

    Siemer, Corinna; Smiljakovic, Tatjana; Bhojwani, Monika; Leiding, Claus; Kanitz, Wilhelm; Kubelka, Michal; Tomek, Wolfgang

    2009-12-01

    Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

  14. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

    OpenAIRE

    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don

    2001-01-01

    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian...

  15. Z' factor including siRNA design quality parameter in RNAi screening experiments.

    Science.gov (United States)

    Mazur, Sławomir; Kozak, Karol

    2012-05-01

    RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z' factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z' factor, introduced by Zhang et al., ( 1) is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describe a new extension of the Z' factor, which integrates bioinformatics RNAi non-target compounds for screening quality assessment. Currently presented Z' factor is based on positive and negative control, which may not be sufficient for RNAi experiments including oligonucleotides (oligo) with lack of knock-down. This paper proposes an algorithm which extends existing algorithm by using additional controls generetaed from on-target analysis.

  16. Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo

    NARCIS (Netherlands)

    M. van Kleffens (Marjolein); C.A.H. Groffen; N.F. Dits (Natasja); D.J. Lindenbergh-Kortleve (Dicky); A.G.P. Schuller (Alwin); S.L. Bradshaw; J.E. Pintar; E.C. Zwarthoff (Ellen); S.L.S. Drop (Stenvert); J.W. van Neck (Han)

    1999-01-01

    textabstractThe insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of

  17. Alternative pre-mRNA splicing in Drosophila spliceosomal assembly factor RNP-4F during development.

    Science.gov (United States)

    Fetherson, Rebecca A; Strock, Stephen B; White, Kristen N; Vaughn, Jack C

    2006-04-26

    The 5'- and 3'-UTR regions in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation. Here we report the results of a systematic study of alternative splicing in rnp-4f, which encodes a Drosophila spliceosomal assembly factor. We show that most of the nine introns are constitutively spliced, but several patterns of alternative splicing are observed in two pre-mRNA regions including the 5'-UTR. Intron V is shown to be of recent evolutionary origin and is infrequently spliced, resulting in generation of an in-frame stop codon and a predicted truncated protein lacking a nuclear localization signal, so that alternative splicing regulates its subcellular localization. Intron 0, located in the 5'-UTR, is subject to three different splicing decisions in D. melanogaster. Northern analysis of poly(A+) mRNAs reveals two differently sized rnp-4f mRNA isoforms in this species. A switch in relative isoform abundance occurs during mid-embryo stages, when the larger isoform becomes more abundant. This isoform is shown to represent intron 0 unspliced mRNA, whereas the smaller transcript represents the product of alternative splicing. Comparative genomic analysis predicts that intron 0 is present in diverse Drosophila species. Intron 0 splicing results in loss of an evolutionarily conserved stem-loop constituting a potential cis-regulatory element at the 3'-splice site. A model is proposed for the role of this element both in 5'-UTR alternative splicing decisions and in RNP-4F translational modulation. Preliminary evidences in support of our model are discussed.

  18. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    Science.gov (United States)

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  19. RNA Export through the NPC in Eukaryotes.

    Science.gov (United States)

    Okamura, Masumi; Inose, Haruko; Masuda, Seiji

    2015-03-20

    In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.

  20. Calcium in Mercury's Exosphere: Modeling MESSENGER Data

    Science.gov (United States)

    Burger, M. H.; Killen, R. M.; McClintock, W. E.; Merkel, A. W.; Vervack, R. J.; Sarantos, M.; Sprague, A. L.

    2011-12-01

    Mercury is surrounded by a surface-bounded exosphere known to contain hydrogen, sodium, potassium, calcium, and magnesium. Because the exosphere is collisionless, its composition represents a balance of active source and loss processes. The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft has made high-spatial-resolution observations of sodium, calcium, and magnesium near Mercury's surface and in the extended, anti-sunward direction. The most striking feature of these data is the substantial differences among species, which was detected during three close flybys of the planet and has been persistantly present during MESSENGER's orbital phase. Our modeling demonstrates that these differences are not because of post-ejection dynamics such as differences in photo-ionization rate and radiation pressure, but rather result from differences in the source mechanisms and regions on the surface from which each species is ejected. The observations of calcium have revealed a strong dawn/dusk asymmetry, with the abundance over the dawn hemisphere substantially greater than that on the dusk side. To understand this asymmetry, we use a Monte Carlo model of Mercury's exosphere that we developed to track the motions of exospheric neutrals under the influence of gravity and radiation pressure. In this model, Ca atoms can be ejected directly from the surface or produced by ejection of CaO followed by dissociation to produce Ca and O. Particles are removed from the system if they stick to the surface or escape from the model region of interest (within 15 Mercury radii). Photoionization reduces the final weighting given to each particle when simulating the Ca radiance. Data from the flybys are consistent with a high temperature (~1-2 x 104 K) source of atomic Ca concentrated over the dawn hemisphere. Such a high temperature resutls from dissociation of CaO in a near

  1. Mercury: A Synthesis from MESSENGER's Extended Mission

    Science.gov (United States)

    Solomon, S. C.; Nittler, L. R.; McNutt, R. L.

    2012-12-01

    Launched in 2004, MESSENGER flew by Mercury three times in 2008-2009 en route to becoming the first spacecraft to orbit the solar system's innermost planet in March 2011. Orbital observations over the subsequent 21 months have provided the first global view of this nearby but heretofore little studied world. MESSENGER's chemical remote sensing measurements of Mercury's surface indicate that the planet's bulk silicate fraction, low in Fe and high in Mg, differs from those of the other inner planets. Moreover, surface materials are richer in the moderately volatile constituents S and K than predicted by most current models for inner planet formation. Global image mosaics and targeted high-resolution images reveal that Mercury experienced globally extensive volcanism, with large expanses of plains emplaced as flood lavas and widespread examples of pyroclastic deposits likely emplaced during explosive eruptions of volatile-bearing magmas. Bright deposits within impact craters host fresh-appearing, rimless depressions or hollows, often with high-reflectance interiors and halos and likely formed through processes involving the geologically recent loss of volatiles. The large-scale deformational history of Mercury, although dominated by near-global contractional deformation as first seen by Mariner 10, is more complex than first appreciated, with numerous examples of extensional deformation that accompanied impact crater and basin modification. Mercury's magnetic field is dominantly dipolar, but the field is axially symmetric and equatorially asymmetric, a geometry that poses challenges to dynamo models for field generation. The interaction between the solar wind and Mercury's magnetosphere, among the most dynamic in the solar system, serves both to replenish the exosphere and space weather the planet's surface. Plasma ions of planetary origin are seen throughout the sampled volume of Mercury's magnetosphere, with maxima in heavy-ion fluxes in the planet's magnetic

  2. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  3. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

    Science.gov (United States)

    Dupuy, Bruno; Raffestin, Stéphanie; Matamouros, Susana; Mani, Nagraj; Popoff, Michel R; Sonenshein, Abraham L

    2006-05-01

    The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

  4. Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

    DEFF Research Database (Denmark)

    Askou, Anne Louise; Aagaard, Lars; Kostic, Corinne

    2015-01-01

    Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters......-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor...... by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new...

  5. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  6. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    OpenAIRE

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M.; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among th...

  7. Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.

    Science.gov (United States)

    Gajewski, John P; Arnold, Jamie J; Salminen, Tiina S; Kaguni, Laurie S; Cameron, Craig E

    2016-01-01

    Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism.

  8. Circadian transcription factor BMAL1 regulates innate immunity against select RNA viruses.

    Science.gov (United States)

    Majumdar, Tanmay; Dhar, Jayeeta; Patel, Sonal; Kondratov, Roman; Barik, Sailen

    2017-02-01

    BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1(-/-) mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1(-/-)mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

  9. Dimerization of the yeast eukaryotic translation initiation factor 5A requires hypusine and is RNA dependent.

    Science.gov (United States)

    Gentz, Petra M; Blatch, Gregory L; Dorrington, Rosemary A

    2009-02-01

    Post-translational modification of the highly conserved K51 residue of the Saccharomyces cerevisiae eukaryotic translation initiation factor 5A (eIF5A) to form hypusine, is essential for its many functions including the binding of specific mRNAs. We characterized hypusinated yeast eIF5A by size-exclusion chromatography and native PAGE, showing that the protein exists as a homodimer. A K51R mutant, which was not functional in vivo eluted as a monomer and inhibition of hypusination abolished dimerization. Furthermore, treatment of dimeric eIF5A with RNase A resulted in disruption of the dimer, leading us to conclude that RNA binding is also required for dimerization of eIF5A. We present a model of dimerization, based on the Neurospora crassa structural analogue, HEX-1.

  10. Expression of P450c17 messenger ribonucleic acid in postmenopausal human ovary tissues.

    Science.gov (United States)

    José, M; Puche, C; Cabero, A; Cabero, L; Meseguer, A

    1999-03-01

    To investigate the expression of the P450c17 gene in postmenopausal human ovaries compared with normal cycling ovaries. Prospective nonrandomized clinical research study. Servei de Medicina Reproductiva and Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospitals Vall d'Hebron, Barcelona, Spain. Six premenopausal women and four postmenopausal women undergoing bilateral oophorectomy for nonovarian gynecologic disease. Extraction of 10 mL of peripheral venous blood for hormone measurements. Extraction of RNA from surgically removed ovaries for Northern blot, ribonuclease protection, and reverse transcriptase polymerase chain reaction Southern blot assays. Definition of the reproductive cycle state of each patient and determination of the level of P450c17 gene expression in all samples with the use of the semiquantitative reverse transcriptase polymerase chain reaction Southern blot assay. P450c17 messenger RNA levels in postmenopausal ovaries varied considerably between samples. Although the levels were similar to those detected in the early follicular phase, one of the samples had levels as high as those observed in the late follicular phase. Although the degree varied from one sample to another, all the postmenopausal ovaries studied expressed the P450c17 gene at the messenger RNA level. In a sample from a patient with endometrial adenocarcinoma, the level was as high as the levels observed in the late follicular phase.

  11. Messenger Observations of Mercury's Bow Shock and Magnetopause

    Science.gov (United States)

    Slavin J. A.; Acuna, M. H.; Anderson, B. J.; Benna, M.; Gloeckler, G.; Krimigis, S. M.; Raines, M.; Schriver, D.; Travnicek, P.; Zurbuchen, T. H.

    2008-01-01

    The MESSENGER spacecraft made the first of three flybys of Mercury on January 14.2008 (1). New observations of solar wind interaction with Mercury were made with MESSENGER'S Magnetometer (MAG) (2.3) and Energetic Particle and Plasma Spectrometer (EPPS) - composed of the Energetic Particle Spectrometer (EPS) and Fast Imaging Plasma Spectrometer (FIPS) (3,4). These MESSENGER observations show that Mercury's magnetosphere has a large-scale structure that is distinctly Earth-like, but it is immersed in a comet-like cloud of planetary ions [5]. Fig. 1 provides a schematic view of the coupled solar wind - magnetosphere - neutral atmosphere - solid planet system at Mercury.

  12. Long noncoding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit.

    Science.gov (United States)

    Puvvula, Pavan Kumar; Desetty, Rohini Devi; Pineau, Pascal; Marchio, Agnés; Moon, Anne; Dejean, Anne; Bischof, Oliver

    2014-11-19

    Cellular senescence is a stable cell cycle arrest that limits the proliferation of pre-cancerous cells. Here we demonstrate that scaffold-attachment-factor A (SAFA) and the long noncoding RNA PANDA differentially interact with polycomb repressive complexes (PRC1 and PRC2) and the transcription factor NF-YA to either promote or suppress senescence. In proliferating cells, SAFA and PANDA recruit PRC complexes to repress the transcription of senescence-promoting genes. Conversely, the loss of SAFA-PANDA-PRC interactions allows expression of the senescence programme. Accordingly, we find that depleting either SAFA or PANDA in proliferating cells induces senescence. However, in senescent cells where PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes, PANDA depletion leads to an exit from senescence. Together, our results demonstrate that PANDA confines cells to their existing proliferative state and that modulating its level of expression can cause entry or exit from senescence.

  13. Mercury's Seasonal Sodium Exosphere: MESSENGER Orbital Observations

    Science.gov (United States)

    Cassidy, Timothy A.; Merkel, Aimee W.; Burger, Matthew H.; Killen, Rosemary M.; McClintock, William E.; Vervack, Ronald J., Jr.; Sarantos, Menelaos

    2014-01-01

    The Mercury Atmospheric and Surface Composition Spectrometer (MASCS) Ultraviolet and Visible Spectrometer (UVVS) on the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft now orbiting Mercury provides the first close-up look at the planet's sodium exosphere. UVVS has observed the exosphere from orbit almost daily for over 10 Mercury years. In this paper we describe and analyze a subset of these data: altitude profiles taken above the low-latitude dayside and south pole. The observations show spatial and temporal variations, but there are no obvious year-to-year variations in most of the observations. We do not see the episodic variability reported by some ground-based observers. We used these altitude profiles to make estimates of sodium density and temperature. The bulk of the exosphere, at about 1200 K, is much warmer than Mercury's surface. This value is consistent with some ground-based measurements and suggests that photon-stimulated desorption is the primary ejection process. We also observe a tenuous energetic component but do not see evidence of the predicted thermalized (or partially thermalized) sodium near Mercury's surface temperature. Overall we do not see the variable mixture of temperatures predicted by most Monte Carlo models of the exosphere.

  14. Detection of the expression of cellular fetal hemoglobin gamma chain messenger RNA(HbF-γmRNA)in maternal circulation for non-invasive prenatal diagnosis%应用原位PCR检测母体循环中胎儿遗传信息HbF γ mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    汤冬玲; 周新; 李艳; 郑芳; 李从荣

    2010-01-01

    目的 原位PCR是一种直接在细胞或组织材料标本上原位扩增目的DNA或RNA片段,并在原位检测扩增产物,从而进行细胞内特定核酸序列检出及定位的分子技术.胎儿血红蛋白(fetal hemoglobin,Hbr)(α2γ2)是胎儿红细胞中一种主要的胞浆蛋白,依据其特有的发育规律,拟从转录水平的角度,采用高灵敏度的反转录聚合酶链反应(RT-PCR)结合原位杂交技术,深入探讨胎儿血红蛋白γ mRNA在孕妇外周血中的表达机理.方法 取EDTA抗凝的孕妇静脉血5 ml.PBS稀释后,经淋巴细胞分离液分离获取单个核细胞层.PBS洗3次,离心后将沉淀涂片在经多聚赖氨酸处理的栽玻片上,10%缓冲中性甲醛固定,PBS洗3次,蛋白酶K消化,PBS洗3次,气干.用地高辛标记的脱氧三磷酸尿苷掺入的原位RT-PCR的方法进行检测,经25轮PCR循环后,用碱性磷酸酶标记的抗地高辛抗体检测扩增产物,显色镜检,分析36例孕妇外周血中胎儿血红蛋白γ基因的表达情况.结果 靶序列的原位RT-PCR结果显示,36例孕妇外周血中有30例标本中可定位检测到胞核、胞浆内的蓝紫色的阳性信号,符合率为83.33%,在孕妇外周血和初产妇的脐血的有核红细胞的胞浆内均可检测到DIG标记的HbF-γ mRNA蓝紫色信号,而正常人外周血则无此信号.结论 原位RT-PCR将PCR技术的高效扩增与细胞定位相结合,在细胞原位检测低拷贝mRNA,具有敏感性高、特异性强的特点,能从分子水平上定位定量检测HbF-γ mRNA的表达,该基因仅在孕妇外周血中表达,未孕女性未见其表达,提示HbF-γ mRNA可能为孕妇外周血中的一种新的靶标,为无创性产前诊断提供线索和思路.

  15. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Directory of Open Access Journals (Sweden)

    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  16. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding.

    Science.gov (United States)

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1.

  17. Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

    Science.gov (United States)

    Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

    2006-04-28

    Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

  18. Upregulation of RNA Processing Factors in Poorly Differentiated Lung Cancer Cells.

    Science.gov (United States)

    Geles, Kenneth G; Zhong, Wenyan; O'Brien, Siobhan K; Baxter, Michelle; Loreth, Christine; Pallares, Diego; Damelin, Marc

    2016-04-01

    Intratumoral heterogeneity in non-small cell lung cancer (NSCLC) has been appreciated at the histological and cellular levels, but the association of less differentiated pathology with poor clinical outcome is not understood at the molecular level. Gene expression profiling of intact human tumors fails to reveal the molecular nature of functionally distinct epithelial cell subpopulations, in particular the tumor cells that fuel tumor growth, metastasis, and disease relapse. We generated primary serum-free cultures of NSCLC and then exposed them to conditions known to promote differentiation: the air-liquid interface (ALI) and serum. The transcriptional network of the primary cultures was associated with stem cells, indicating a poorly differentiated state, and worse overall survival of NSCLC patients. Strikingly, the overexpression of RNA splicing and processing factors was a prominent feature of the poorly differentiated cells and was also observed in clinical datasets. A genome-wide analysis of splice isoform expression revealed many alternative splicing events that were specific to the differentiation state of the cells, including an unexpectedly high frequency of events on chromosome 19. The poorly differentiated cells exhibited alternative splicing in many genes associated with tumor progression, as exemplified by the preferential expression of the short isoform of telomeric repeat-binding factor 1 (TERF1), also known as Pin2. Our findings demonstrate the utility of the ALI method for probing the molecular mechanisms that underlie NSCLC pathogenesis and provide novel insight into posttranscriptional mechanisms in poorly differentiated lung cancer cells.

  19. Upregulation of RNA Processing Factors in Poorly Differentiated Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kenneth G. Geles

    2016-04-01

    Full Text Available Intratumoral heterogeneity in non–small cell lung cancer (NSCLC has been appreciated at the histological and cellular levels, but the association of less differentiated pathology with poor clinical outcome is not understood at the molecular level. Gene expression profiling of intact human tumors fails to reveal the molecular nature of functionally distinct epithelial cell subpopulations, in particular the tumor cells that fuel tumor growth, metastasis, and disease relapse. We generated primary serum-free cultures of NSCLC and then exposed them to conditions known to promote differentiation: the air-liquid interface (ALI and serum. The transcriptional network of the primary cultures was associated with stem cells, indicating a poorly differentiated state, and worse overall survival of NSCLC patients. Strikingly, the overexpression of RNA splicing and processing factors was a prominent feature of the poorly differentiated cells and was also observed in clinical datasets. A genome-wide analysis of splice isoform expression revealed many alternative splicing events that were specific to the differentiation state of the cells, including an unexpectedly high frequency of events on chromosome 19. The poorly differentiated cells exhibited alternative splicing in many genes associated with tumor progression, as exemplified by the preferential expression of the short isoform of telomeric repeat-binding factor 1 (TERF1, also known as Pin2. Our findings demonstrate the utility of the ALI method for probing the molecular mechanisms that underlie NSCLC pathogenesis and provide novel insight into posttranscriptional mechanisms in poorly differentiated lung cancer cells.

  20. Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes.

    Science.gov (United States)

    Takano, Katsura; Yamasaki, Hiroshi; Kawabe, Kenji; Moriyama, Mitsuaki; Nakamura, Yoichi

    2012-01-01

    Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

  1. Transcription factor KLF4 regulates microRNA-544 that targets YWHAZ in cervical cancer.

    Science.gov (United States)

    Mao, Langyong; Zhang, Yan; Deng, Xiaolong; Mo, Wenjuan; Yu, Yao; Lu, Hong

    2015-01-01

    The deregulation of microRNAs has been demonstrated in various tumor processes. Here, we report that microRNA-544 (miR-544) is decreased in cervical cancer tissues compared with normal cervical tissues. To identify the mechanisms involved in miR-544 deregulation, we studied the regulation of miR-544 expression at the transcriptional level. We first identified the transcriptional start site of miR-544 by 5' rapid amplification of cDNA ends and subsequently determined the miR-544 promoter. We discovered that the transcription factor Krueppel-like factor 4 (KLF4) is involved in the transcriptional regulation of miR-544 through interaction with the miR-544 promoter. In addition, we found that miR-544 directly targets the YWHAZ oncogene and functions as a tumor suppressor in cervical cancer cells. miR-544 is involved in cell cycle regulation and suppresses cervical cancer cell proliferation, colony formation, migration and invasion in a manner associated with YWHAZ downregulation. In summary, our findings demonstrate that KLF4 upregulates miR-544 transcription by activating the miR-544 promoter and that miR-544 functions as a tumor suppressor by targeting YWHAZ. Therefore, miR-544 may be a potential novel therapeutic target and prognostic marker for cervical cancer.

  2. Dissecting role of regulatory factors in NF-κB pathway with siRNA

    Institute of Scientific and Technical Information of China (English)

    Jun GUO; Yu-cai FU; Carlos R BECERRA

    2005-01-01

    NF-κB, a family of related transcription factors, has been a focus of intense scientific research during the past decade.Multiple stimuli, both extracellular and intracellular, lead to its activation.The NF-κB pathway regulates expression of a diverse array of genes involved in different biological processes.Various pathological states are characterized by the dysregulated NF-κB pathway.Recently,NF-κB activation has been connected with multiple aspects of oncogenesis and serves as an important mechanism to regulate cell survival in response to chemotherapy by activating different genes that inhibit apoptosis.Several methods of inhibiting NF-κB activation, such as antisense oligonucleotides, proteosome inhibitors and RNA interference (RNAi) are currently under investigation.RNAi represents a powerful tool to better define the role of specific genes in different signal transduction pathways and has recently been used to define the function of genes that regulate the NF-κB pathway.This review discusses the emerging role of RNAi to dissect the function of regulatory factors in the NF-κB pathway and its potential use as a targeted therapy.

  3. Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming.

    Directory of Open Access Journals (Sweden)

    Masamitsu Konno

    Full Text Available Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2 splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming.

  4. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    Energy Technology Data Exchange (ETDEWEB)

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  5. Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

    Directory of Open Access Journals (Sweden)

    John Martin

    Full Text Available Transforming Growth Factor Beta-1 (TGF-β1 is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-β1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-β1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-β1 3'Untranslated Region (3'UTR. Two different 3'UTR lengths have been reported for TGF-β1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was up-regulated following auto-induction by TGF-β1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-β1 3'UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-β1 synthesis, while direct targeting of TGF-β1 was shown in separate experiments, in which miR-744 decreased TGF-β1 3'UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGF-β1 which, given the pleiotropic nature of cellular responses to TGF-β1, is potentially widely significant.

  6. Expression of brain-derived neurotrophic factor mRNA in rat hippocampus after treatment with antipsychotic drugs.

    Science.gov (United States)

    Bai, Ou; Chlan-Fourney, Jennifer; Bowen, Rudy; Keegan, David; Li, Xin-Min

    2003-01-01

    Typical and atypical antipsychotic drugs, though both effective, act on different neurotransmitter receptors and are dissimilar in some clinical effects and side effects. The typical antipsychotic drug haloperidol has been shown to cause a decrease in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in neuronal cell survival, differentiation, and neuronal connectivity. However, it is still unknown whether atypical antipsychotic drugs similarly regulate BDNF expression. We examined the effects of chronic (28 days) administration of typical and atypical antipsychotic drugs on BDNF mRNA expression in the rat hippocampus using in situ hybridization. Quantitative analysis revealed that the typical antipsychotic drug haloperidol (1 mg/kg) down-regulated BDNF mRNA expression in both CA1 (P BDNF mRNA expression in CA1, CA3, and dentate gyrus regions of the rat hippocampus compared with their respective controls (P BDNF mRNA expression in rat hippocampus.

  7. A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

    Science.gov (United States)

    Finkelstein, A; Kostrub, C F; Li, J; Chavez, D P; Wang, B Q; Fang, S M; Greenblatt, J; Burton, Z F

    1992-01-30

    RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

  8. Expected Geochemical and Mineralogical Properties of Meteorites from Mercury: Inferences from Messenger Data

    Science.gov (United States)

    McCubbin, F. M.; McCoy, T. J.

    2016-01-01

    Meteorites from the Moon, Mars, and many types of asteroid bodies have been identified among our global inventory of meteorites, however samples of Mercury and Venus have not been identified. The absence of mercurian and venusian meteorites could be attributed to an inability to recognize them in our collections due to a paucity of geochemical information for Venus and Mercury. In the case of mercurian meteorites, this possibility is further supported by dynamical calculations that suggest mercurian meteorites should be present on Earth at a factor of 2-3 less than meteorites from Mars [1]. In the present study, we focus on the putative mineralogy of mercurian meteorites using data obtained from the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft, which has provided us with our first quantitative constraints on the geochemistry of planet Mercury. We have used the MESSENGER data to compile a list of mineralogical and geochemical characteristics that a meteorite from Mercury is likely to exhibit.

  9. RNA granules

    OpenAIRE

    Anderson, Paul; Kedersha, Nancy

    2006-01-01

    Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationshi...

  10. Topicality and impact in social media: diverse messages, focused messengers.

    Directory of Open Access Journals (Sweden)

    Lilian Weng

    Full Text Available We have a limited understanding of the factors that make people influential and topics popular in social media. Are users who comment on a variety of matters more likely to achieve high influence than those who stay focused? Do general subjects tend to be more popular than specific ones? Questions like these demand a way to detect the topics hidden behind messages associated with an individual or a keyword, and a gauge of similarity among these topics. Here we develop such an approach to identify clusters of similar hashtags in Twitter by detecting communities in the hashtag co-occurrence network. Then the topical diversity of a user's interests is quantified by the entropy of her hashtags across different topic clusters. A similar measure is applied to hashtags, based on co-occurring tags. We find that high topical diversity of early adopters or co-occurring tags implies high future popularity of hashtags. In contrast, low diversity helps an individual accumulate social influence. In short, diverse messages and focused messengers are more likely to gain impact.

  11. Topicality and impact in social media: diverse messages, focused messengers.

    Science.gov (United States)

    Weng, Lilian; Menczer, Filippo

    2015-01-01

    We have a limited understanding of the factors that make people influential and topics popular in social media. Are users who comment on a variety of matters more likely to achieve high influence than those who stay focused? Do general subjects tend to be more popular than specific ones? Questions like these demand a way to detect the topics hidden behind messages associated with an individual or a keyword, and a gauge of similarity among these topics. Here we develop such an approach to identify clusters of similar hashtags in Twitter by detecting communities in the hashtag co-occurrence network. Then the topical diversity of a user's interests is quantified by the entropy of her hashtags across different topic clusters. A similar measure is applied to hashtags, based on co-occurring tags. We find that high topical diversity of early adopters or co-occurring tags implies high future popularity of hashtags. In contrast, low diversity helps an individual accumulate social influence. In short, diverse messages and focused messengers are more likely to gain impact.

  12. RNA-dependent RNA polymerase (NIb) of the potyviruses is an avirulence factor for the broad-spectrum resistance gene Pvr4 in Capsicum annuum cv. CM334.

    Science.gov (United States)

    Kim, Saet-Byul; Lee, Hye-Young; Seo, Seungyeon; Lee, Joo Hyun; Choi, Doil

    2015-01-01

    Potyviruses are one of the most destructive viral pathogens of Solanaceae plants. In Capsicum annuum landrace CM334, a broad-spectrum gene, Pvr4 is known to be involved in resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and Potato virus Y (PVY). However, a potyvirus avirulence factor against Pvr4 has not been identified. To identify the avirulence factor corresponding to Pvr4 in potyviruses, we performed Agrobacterium-mediated transient expressions of potyvirus protein coding regions in potyvirus-resistant (Pvr4) and -susceptible (pvr4) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV, PepSMV, or PVY was expressed in Pvr4-bearing pepper leaves in a genotype-specific manner. In contrast, HR was not observed when the NIb of Tobacco etch virus (TEV), a virulent potyvirus, was expressed in Pvr4-bearing pepper leaves. Our results clearly demonstrate that NIbs of PepMoV, PepSMV, and PVY serve as avirulence factors for Pvr4 in pepper plants.

  13. Expression of messenger RNAs for glutamic acid decarboxylase, preprotachykinin, cholecystokinin, somatostatin, proenkephalin and neuropeptide Y in the adult rat superior colliculus.

    Science.gov (United States)

    Harvey, A R; Heavens, R P; Yellachich, L A; Sirinathsinghji, D J

    2001-01-01

    The mammalian superior colliculus is an important subcortical integrator of sensorimotor behaviours. It is multi-layered, each layer containing specific neuronal types and possessing distinct input/output relationships. Here we use in situ hybridisation methods to map the distribution of seven neurotransmitters/neuromodulator systems in adult rat superior colliculus. Coronal sections were probed for preprotachykinin, cholecystokinin, somatostatin, proenkephalin, neuropeptide Y and the enzymes glutamic acid decarboxylase and choline acetyltransferase, markers for GABA and acetylcholine respectively. Cells expressing glutamic acid decarboxylase messenger RNA were the most abundant, the highest density being found in the superficial layers. Many cells containing proprotachykinin messenger RNA were found in stratum zonale and the upper two-thirds of stratum griseum superficiale; cells were also located in deeper tectal laminae, particularly caudomedially. Most cholecystokinin messenger RNA expressing cells were located in the superficial layers with a prominent band in the middle third of stratum griseum superficiale. Cells expressing moderate to high levels of somatostatin messenger RNA formed a dense band in the lower third of stratum griseum superficiale/upper stratum opticum; two less distinct tiers of labelling were seen in deeper layers. These in situ hybridisation data reveal three distinct sub-laminae in rat stratum griseum superficiale. Cells expressing moderate to low levels of proenkephalin messenger RNA were located in lower stratum griseum superficiale/upper stratum opticum and intermediate laminae. A cluster of enkephalinergic cells was located medially in the deep tectal laminae. Expression of neuropeptide Y messenger RNA was relatively low and mostly confined to cells in stratum griseum superficiale and stratum opticum. No choline acetyltransferase messenger RNA was detected. This in situ analysis of seven different neurotransmitters

  14. Destabilization of survival factor MEF2D mRNA by neurotoxin in models of Parkinson's disease.

    Science.gov (United States)

    Wang, Bao; Cai, Zhibiao; Lu, Fangfang; Li, Chen; Zhu, Xiaofei; Su, Linna; Gao, Guodong; Yang, Qian

    2014-09-01

    Progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) is an important pathological feature in Parkinson's disease (PD). Loss of transcription factor myocyte enhancer factor 2D (MEF2D), a key neuronal survival factor, has been shown to underlie the loss of DA neurons in SNc and the pathogenic process of PD. It is known that PD-associated neurotoxins reduce the level of MEF2D protein to trigger neuronal death. Although neurotoxins clearly destabilize MEF2D by post-translational mechanisms, it is not known whether regulation of MEF2D mRNA contributes to neurotoxin-induced decrease in MEF2D protein. In this work, we showed that MPP(+), the toxic metabolite of MPTP, caused a significant decrease in the half-life and total level of MEF2D mRNA in a DA neuronal cell line, SN4741 cells. Quantitative PCR analysis of the SNc DA neurons captured by immune-laser capture microdissection showed that exposure to MPTP led to a marked reduction in the level of MEF2D mRNA in SNc DA neurons compared to controls. Down-regulation of MEF2D mRNA alone reduced the viability of SN4741 cells and sensitized the cells to MPP(+)-induced toxicity. These results suggest that destabilization and reduction in MEF2D mRNA is in part responsible for neurotoxin-induced decrease in MEF2D protein and neuronal viability. Myocyte enhancer factor 2D (MEF2D) plays an important role in neuronal survival. How MEF2D mRNA is deregulated under toxic stress is unclear. We found that PD-associated neurotoxins destabilize MEF2D mRNA and reduce its level in vitro and in vivo. Reduction in MEF2D mRNA is sufficient to sensitize model cells to neurotoxin-induced toxicity, suggesting that destabilization of MEF2D mRNA is part of the mechanism by which neurotoxins trigger deregulation of neuronal survival.

  15. Viral factors reveal a role for REF/Aly in nuclear RNA stability.

    Science.gov (United States)

    Stubbs, Sarah H; Hunter, Olga V; Hoover, Ashley; Conrad, Nicholas K

    2012-04-01

    TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In Saccharomyces cerevisiae, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV noncoding polyadenylated nuclear (PAN) RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation, and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independently of their export and support a broader conservation of RNA quality control mechanisms from yeast to humans.

  16. Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson’s disease rats

    Institute of Scientific and Technical Information of China (English)

    Shuju Wang; Jianqiao Fang; Jun Ma; Yanchun Wang; Shaorong Liang; Dan Zhou; Guojie Sun

    2013-01-01

    Acupuncture for the treatment of Parkinson's disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu (GV16) and Taichong (LR3) acupoints in rat models of Parkinson's disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson's disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson's disease. Thus, electroacupuncture may be useful in the treatment of Parkinson's disease.

  17. Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats.

    Science.gov (United States)

    Wang, Shuju; Fang, Jianqiao; Ma, Jun; Wang, Yanchun; Liang, Shaorong; Zhou, Dan; Sun, Guojie

    2013-02-25

    Acupuncture for the treatment of Parkinson's disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu (GV16) and Taichong (LR3) acupoints in rat models of Parkinson's disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson's disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson's disease. Thus, electroacupuncture may be useful in the treatment of Parkinson's disease.

  18. Specificity Protein (Sp) Transcription Factors and Metformin Regulate Expression of the Long Non-coding RNA HULC

    Science.gov (United States)

    There is evidence that specificity protein 1 (Sp1) transcription factor (TF) regulates expression of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) cells. RNA interference (RNAi) studies showed that among several lncRNAs expressed in HepG2, SNU-449 and SK-Hep-1...

  19. A petunia ocs element binding factor, PhOBF1, plays an important role in antiviral RNA silencing

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

  20. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...

  1. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF-P ...

  2. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

    Science.gov (United States)

    Radoshitzky, Sheli R; Pegoraro, Gianluca; Chī, Xi Olì; D Ng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C; Cooper, Christopher L; Courier, Duane; Langan, David P; Underwood, Knashka; Kuehl, Kathleen A; Sun, Mei G; Caì, Yíngyún; Yú, Shu Qìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H; Bavari, Sina

    2016-03-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

  3. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

    Directory of Open Access Journals (Sweden)

    Sheli R Radoshitzky

    2016-03-01

    Full Text Available Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2 expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

  4. MicroRNA-24 Regulates Osteogenic Differentiation via Targeting T-Cell Factor-1

    Directory of Open Access Journals (Sweden)

    Weigong Zhao

    2015-05-01

    Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.

  5. Targeted protein footprinting: where different transcription factors bind to RNA polymerase.

    Science.gov (United States)

    Traviglia, S L; Datwyler, S A; Yan, D; Ishihama, A; Meares, C F

    1999-11-30

    Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

  6. Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II.

    Science.gov (United States)

    Sopta, M; Burton, Z F; Greenblatt, J

    1989-10-05

    RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II. Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites. The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors.

  7. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation......, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  8. Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σAntA

    Directory of Open Access Journals (Sweden)

    Ryan F. Seipke

    2014-02-01

    Full Text Available Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF RNA polymerase sigma factor named σAntA which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σAntA controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σAntA is highly conserved in antimycin producing strains and the −10 and −35 elements at the σAntA regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σAntA proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.

  9. MicroRNA and transcription factor mediated regulatory network for ovarian cancer: regulatory network of ovarian cancer.

    Science.gov (United States)

    Ying, Huanchun; Lv, Jing; Ying, Tianshu; Li, Jun; Yang, Qing; Ma, Yuan

    2013-10-01

    A better understanding on the regulatory interactions of microRNA (miRNA) target genes and transcription factor (TF) target genes in ovarian cancer may be conducive for developing early diagnosis strategy. Thus, gene expression data and miRNA expression data were downloaded from The Cancer Genome Atlas in this study. Differentially expressed genes and miRNAs were selected out with t test, and Gene Ontology enrichment analysis was performed with DAVID tools. Regulatory interactions were retrieved from miRTarBase, TRED, and TRANSFAC, and then networks for miRNA target genes and TF target genes were constructed to globally present the mechanisms. As a result, a total of 1,939 differentially expressed genes were identified, and they were enriched in 28 functions, among which cell cycle was affected to the most degree. Besides, 213 differentially expressed miRNAs were identified. Two regulatory networks for miRNA target genes and TF target genes were established and then both were combined, in which E2F transcription factor 1, cyclin-dependent kinase inhibitor 1A, cyclin E1, and miR-16 were the hub genes. These genes may be potential biomarkers for ovarian cancer.

  10. Predicting sequence and structural specificities of RNA binding regions recognized by splicing factor SRSF1

    Directory of Open Access Journals (Sweden)

    Wang Xin

    2011-12-01

    Full Text Available Abstract Background RNA-binding proteins (RBPs play diverse roles in eukaryotic RNA processing. Despite their pervasive functions in coding and noncoding RNA biogenesis and regulation, elucidating the sequence specificities that define protein-RNA interactions remains a major challenge. Recently, CLIP-seq (Cross-linking immunoprecipitation followed by high-throughput sequencing has been successfully implemented to study the transcriptome-wide binding patterns of SRSF1, PTBP1, NOVA and fox2 proteins. These studies either adopted traditional methods like Multiple EM for Motif Elicitation (MEME to discover the sequence consensus of RBP's binding sites or used Z-score statistics to search for the overrepresented nucleotides of a certain size. We argue that most of these methods are not well-suited for RNA motif identification, as they are unable to incorporate the RNA structural context of protein-RNA interactions, which may affect to binding specificity. Here, we describe a novel model-based approach--RNAMotifModeler to identify the consensus of protein-RNA binding regions by integrating sequence features and RNA secondary structures. Results As an example, we implemented RNAMotifModeler on SRSF1 (SF2/ASF CLIP-seq data. The sequence-structural consensus we identified is a purine-rich octamer 'AGAAGAAG' in a highly single-stranded RNA context. The unpaired probabilities, the probabilities of not forming pairs, are significantly higher than negative controls and the flanking sequence surrounding the binding site, indicating that SRSF1 proteins tend to bind on single-stranded RNA. Further statistical evaluations revealed that the second and fifth bases of SRSF1octamer motif have much stronger sequence specificities, but weaker single-strandedness, while the third, fourth, sixth and seventh bases are far more likely to be single-stranded, but have more degenerate sequence specificities. Therefore, we hypothesize that nucleotide specificity and

  11. Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

    OpenAIRE

    Bogenhagen, D F

    1993-01-01

    Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody ...

  12. MESSENGER Measurements of Mercury's Magnetic Field during the First Flyby

    Science.gov (United States)

    Slavin, James A.; Boardsen, S. A.; Acuna, M. H.; Anderson, B. J.; Johnson, C. L.; Korth, H.; Krimigis, S. M.; McNutt, R. L., Jr.; Purucker, M. E.; Solomon, S. C.

    2008-01-01

    On 14 January 2008 the MESSENGER spacecraft will encounter Mercury for the first time. Depending upon the solar wind conditions, this initial flyby will return Magnetometer measurements of Mercury's magnetic field over a time interval lasting between - 30 md 60 min. Closest approach for MESSENGER is targeted for an altitude of 200 km as compared with the 707 krn and 327 km attained by Mariner 10 on 29 March 1974 and 16 March 1975, respectively. Furthermore, the differences in the MESSENGER and Mariner 10 encounter trajectories, with respect both to magnetospheric and body-fixed coordinates are highly complementary and expected to lead to significant improvements in our knowledge of Mercury's magnetic field. We present an overview of the MESSENGER magnetic field observations, an initial subtraction of the magnetic fields attributable to magnetospheric current systems from the total measured magnetic field, and an improved model of Mercury's intrinsic magnetic field. We also discuss the expected advances afforded by the two additional MESSENGER flybys, which occur in October 2008 and September 2009, as well as the orbital phase that will begin in March 201 1.

  13. MicroRNA: Potential Targets for the Development of Novel Drugs?

    OpenAIRE

    Wei WU

    2012-01-01

    MicroRNA (miRNA) is an endogenous non-protein coding small RNA molecule that negatively regulates gene expression by the degradation of messenger RNA (mRNA) or the suppression of mRNA translation. miRNA plays important roles in physiologic processes such as cellular development, differentiation, proliferation, apoptosis, and stem cell self-renewal. Studies show that deregulation of miRNA expression is closely associated with tumorigenicity, invasion, and metastasis. The functionality of aberr...

  14. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    Science.gov (United States)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

  15. Elongation Factor 1β' Gene from Spodoptera exigua: Characterization and Function Identification through RNA Interference

    Directory of Open Access Journals (Sweden)

    Li-Na Zhao

    2012-06-01

    Full Text Available Elongation factor (EF is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1β' from Spodoptera exigua (SeEF-1β', its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1β' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1β' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1β' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1β' was suppressed. The results demonstrate that SeEF-1β' is a key gene in transcription in S. exigua.

  16. Comparing the MicroRNA spectrum between serum and plasma.

    Directory of Open Access Journals (Sweden)

    Kai Wang

    Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that regulate various biological processes, primarily through interaction with messenger RNAs. The levels of specific, circulating miRNAs in blood have been shown to associate with various pathological conditions including cancers. These miRNAs have great potential as biomarkers for various pathophysiological conditions. In this study we focused on different sample types' effects on the spectrum of circulating miRNA in blood. Using serum and corresponding plasma samples from the same individuals, we observed higher miRNA concentrations in serum samples compared to the corresponding plasma samples. The difference between serum and plasma miRNA concentration showed some associations with miRNA from platelets, which may indicate that the coagulation process may affect the spectrum of extracellular miRNA in blood. Several miRNAs also showed platform dependent variations in measurements. Our results suggest that there are a number of factors that might affect the measurement of circulating miRNA concentration. Caution must be taken when comparing miRNA data generated from different sample types or measurement platforms.

  17. Over-expression of corticotropin-releasing factor mRNA in inferior olivary neurons of rolling mouse Nagoya.

    Science.gov (United States)

    Sawada, Kazuhiko; Kawano, Michihiro; Tsuji, Hiroshi; Sakata-Haga, Hiromi; Hisano, Setsuji; Fukui, Yoshihiro

    2003-10-01

    Expression of corticotropin-releasing factor (CRF) mRNA was examined in the inferior olivary nucleus (ION) of an ataxic mutant, rolling mouse Nagoya (RMN) by semi-quantitative in situ hybridization. The most marked difference in the level of CRF mRNA signals between RMN and non-ataxic littermates (control mice) was observed in the beta-subnucleus and ventrolateral protrusion of the ION. The level of signals in these subnuclei was about twofold higher in RMN than in the controls. Signal levels in the dorsal nucleus, principal nucleus and subnucleus A were slightly but significantly higher in RMN than in the controls. In the other subnuclei, there were no differences in signal level between RMN and controls. These results suggest a region-related over-expression of CRF mRNA in the ION of RMN. This may be responsible for the increased sensitivity of some Purkinje cells to glutamate, resulting in ataxic symptoms of RMN.

  18. Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors

    Science.gov (United States)

    Cole, Michael D.; Cowling, Victoria H.

    2009-01-01

    Methylation of the mRNA 5′ guanosine cap is essential for efficient gene expression. The 5′methyl cap binds to eIF4E, which is the first step in the recruitment of mRNA to the 40S ribosomal subunit. To investigate whether mRNA cap methylation is regulated in a gene-specific manner, we established a method to detect the relative level of cap methylation on specific mRNAs. We found that two transcription factors, c-Myc and E2F1, induce cap methylation of their transcriptional target genes, and therefore, c-Myc and E2F1 upregulate gene expression by simultaneously inducing transcription and promoting translation. c-Myc-induced cap methylation is greater than transcriptional induction for the majority of its target genes, indicating that this is a major mechanism by which Myc regulates gene expression. PMID:19137018

  19. Structural features of the tmRNA-ribosome interaction.

    Science.gov (United States)

    Bugaeva, Elizaveta Y; Surkov, Serhiy; Golovin, Andrey V; Ofverstedt, Lars-Göran; Skoglund, Ulf; Isaksson, Leif A; Bogdanov, Alexey A; Shpanchenko, Olga V; Dontsova, Olga A

    2009-12-01

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  20. Transcription factor-microRNA-target gene networks associated with ovarian cancer survival and recurrence.

    Science.gov (United States)

    Delfino, Kristin R; Rodriguez-Zas, Sandra L

    2013-01-01

    The identification of reliable transcriptome biomarkers requires the simultaneous consideration of regulatory and target elements including microRNAs (miRNAs), transcription factors (TFs), and target genes. A novel approach that integrates multivariate survival analysis, feature selection, and regulatory network visualization was used to identify reliable biomarkers of ovarian cancer survival and recurrence. Expression profiles of 799 miRNAs, 17,814 TFs and target genes and cohort clinical records on 272 patients diagnosed with ovarian cancer were simultaneously considered and results were validated on an independent group of 146 patients. Three miRNAs (hsa-miR-16, hsa-miR-22*, and ebv-miR-BHRF1-2*) were associated with both ovarian cancer survival and recurrence and 27 miRNAs were associated with either one hazard. Two miRNAs (hsa-miR-521 and hsa-miR-497) were cohort-dependent, while 28 were cohort-independent. This study confirmed 19 miRNAs previously associated with ovarian cancer and identified two miRNAs that have previously been associated with other cancer types. In total, the expression of 838 and 734 target genes and 12 and eight TFs were associated (FDR-adjusted P-value cancer survival and recurrence, respectively. Functional analysis highlighted the association between cellular and nucleotide metabolic processes and ovarian cancer. The more direct connections and higher centrality of the miRNAs, TFs and target genes in the survival network studied suggest that network-based approaches to prognosticate or predict ovarian cancer survival may be more effective than those for ovarian cancer recurrence. This study demonstrated the feasibility to infer reliable miRNA-TF-target gene networks associated with survival and recurrence of ovarian cancer based on the simultaneous analysis of co-expression profiles and consideration of the clinical characteristics of the patients.

  1. Transcription factor-microRNA-target gene networks associated with ovarian cancer survival and recurrence.

    Directory of Open Access Journals (Sweden)

    Kristin R Delfino

    Full Text Available The identification of reliable transcriptome biomarkers requires the simultaneous consideration of regulatory and target elements including microRNAs (miRNAs, transcription factors (TFs, and target genes. A novel approach that integrates multivariate survival analysis, feature selection, and regulatory network visualization was used to identify reliable biomarkers of ovarian cancer survival and recurrence. Expression profiles of 799 miRNAs, 17,814 TFs and target genes and cohort clinical records on 272 patients diagnosed with ovarian cancer were simultaneously considered and results were validated on an independent group of 146 patients. Three miRNAs (hsa-miR-16, hsa-miR-22*, and ebv-miR-BHRF1-2* were associated with both ovarian cancer survival and recurrence and 27 miRNAs were associated with either one hazard. Two miRNAs (hsa-miR-521 and hsa-miR-497 were cohort-dependent, while 28 were cohort-independent. This study confirmed 19 miRNAs previously associated with ovarian cancer and identified two miRNAs that have previously been associated with other cancer types. In total, the expression of 838 and 734 target genes and 12 and eight TFs were associated (FDR-adjusted P-value <0.05 with ovarian cancer survival and recurrence, respectively. Functional analysis highlighted the association between cellular and nucleotide metabolic processes and ovarian cancer. The more direct connections and higher centrality of the miRNAs, TFs and target genes in the survival network studied suggest that network-based approaches to prognosticate or predict ovarian cancer survival may be more effective than those for ovarian cancer recurrence. This study demonstrated the feasibility to infer reliable miRNA-TF-target gene networks associated with survival and recurrence of ovarian cancer based on the simultaneous analysis of co-expression profiles and consideration of the clinical characteristics of the patients.

  2. The translation initiation factor DAP5 promotes IRES-driven translation of p53 mRNA.

    Science.gov (United States)

    Weingarten-Gabbay, S; Khan, D; Liberman, N; Yoffe, Y; Bialik, S; Das, S; Oren, M; Kimchi, A

    2014-01-30

    Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Δ40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Δ40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Δ40p53 protein levels and the subsequent transcriptional activation of the 14-3-3σ gene, a known target of Δ40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.

  3. Significance of changes in transforming growth factor-β mRNA levels in autogenous vein grafts

    Institute of Scientific and Technical Information of China (English)

    尤文俊; 萧明第; 袁忠祥

    2004-01-01

    Background This study was designed to investigate changes in mRNA levels of transforming growth factor-β(TGF-β), collagen Ⅰ, and collagen Ⅲ in autogenous vein grafts. Methods Twenty-four New Zealand rabbits were randomly divided into 4 groups with 6 rabbits each. The external jugular veins of the New Zealand rabbits were harvested and grafted into the ipsilateral carotid artery. All rabbits were fed with a standard diet. After the operation, the rabbits were sacrificed at 1, 2, 3, or 4 weeks. TGF-β, collagen Ⅰ, and collagen Ⅲ mRNA levels in the venous grafts were measured by semiquantitative methods at every time point. The contralateral external jugular veins were also harvested and analyzed as controls. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples for potential variations in mRNA content. In order to observe the expression of TGF-β protein, immunohistochemical SABC methods were used. Results One week postoperation, the mRNA level of TGF-β was upregulated to 1.73±0.19 in the vein graft and 1.21±0.16 in the control vein (P<0.01). High mRNA levels were maintained until week 4 postoperation. The mRNA levels of collagen Ⅰ and collagen Ⅲ were also significantly increased to 2.18±0.21 versus 1.12±0.24 and 1.08±0.13 versus 0.83±0.12, respectively (P<0.05). Immunohistochemical staining revealed a higher density of TGF-β expression in the vein grafts.Conclusions An uninterrupted increase in mRNA levels of TGF-β, collagen Ⅰ, and collagen Ⅲ is observed in autogenous vein grafts. This increase may be the major cause of intimal hyperplasia, sclerosis, and even graft failure.

  4. Screening the key microRNAs and transcription factors in prostate cancer based on microRNA functional synergistic relationships.

    Science.gov (United States)

    Feng, Fan; Wu, Jitao; Gao, Zhenli; Yu, Shengqiang; Cui, Yuanshan

    2017-01-01

    Prostate cancer (PC) is a common neoplasm, and metastatic PC remains incurable. The study aims to screen key microRNAs (miRNAs) and transcription factors (TFs) involved in PC.The miRNA expression profile dataset (GSE45604) was downloaded from Gene Expression Omnibus database, including 50 PC and 10 normal specimens. Differentially expressed miRNAs (DEmiRNAs) were identified through limma package in R, and DEmiRNA-DEmiRNA co-regulation network was constructed based on the number of co-regulated target genes. Functional enrichment analysis of co-regulated target genes was performed using clusterProfiler package in R, and miRNA interactions sharing at least 1 functional term were used to construct a DEmiRNA-DEmiRNA functional synergistic network (MFSN). Based on Transcriptional Regulatory Element Database, cancer-related TFs which were co-regulated by DEmiRNAs were utilized to construct a DEmiRNA-TF regulation network.A total of 66 DEmiRNAs were identified, including 7 up-regulated miRNAs with 18,642 target genes and 59 down-regulated miRNAs with 130,694 target genes. Then, the DEmiRNA-DEmiRNA co-regulation network was constructed, including 66 DEmiRNAs and 2024 co-regulation relationships. In MFSN, hsa-miR-1184, hsa-miR-1207-5p, and hsa-miR-24 had significant functional synergistic relationships. The DEmiRNA-TF network contained 6 up-regulated DEmiRNAs and 4 of them were highlighted, as hsa-miR-1184, hsa-miR-1207-5p, hsa-miR-182, and hsa-miR-183. In subnetwork of the 4 miRNAs, peroxisome proliferative activated receptor, alpha (PPARA) and cyclic AMP-responsive element modulator (CREM) were the critical regulated TFs.Four up-regulated miRNAs (hsa-miR-1207-5p, hsa-miR-1184, hsa-miR-182, and hsa-miR-183) and 2 TFs (PPARA and CREM) were identified as key regulators in PC progression. The above 4 miRNAs might participate in PC progression by targeting PPARA and CREM.

  5. Gauge Mediation with Gauge Messengers in SU(5)

    CERN Document Server

    Matos, Luis

    2010-01-01

    The inclusion of gauge messengers in models of gauge mediation allows for more general predictions that those described by the framework of general gauge mediation. Motivated by this, we explore some models of gauge mediation with gauge messengers in SU(5) GUTs. In most previous attempts of building viable models where gauge messengers play a role in determining the soft terms, squark and/or slepton masses turned out to be tachyonic. The objective of this paper is to address this problem and propose two possible solutions, one of which has a natural realization in the solution of the doublet-triplet problem. Another interesting result is that in these models the association of SUSY breaking with the breaking of the GUT group provides a simple mechanism that can explain why $SU(5)\\rightarrow SU(3)\\times SU(2) \\times U(1)$ is preferred over other symmetry breaking patterns.

  6. ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth

    DEFF Research Database (Denmark)

    Zhao, Jian; Yuan, Xuejun; Frödin, Morten;

    2003-01-01

    Phosphorylation of transcription factors by mitogen-activated protein kinase (MAPK) cascades links cell signaling with the control of gene expression. Here we show that growth factors induce rRNA synthesis by activating MAPK-dependent signaling cascades that target the RNA polymerase I-specific t...

  7. Rapid induction of hepatocyte growth factor mRNA after administration of gomisin A, a lignan component of shizandra fruits.

    Science.gov (United States)

    Shiota, G; Yamada, S; Kawasaki, H

    1996-11-01

    Gomisin A (Go), a lignan component of shizandra fruits, protects the liver from injury by acetaminophen (AAP). One of its possible mechanisms is supposed to be related to the suppression of lipid peroxidation. Since hepatocyte growth factor (HGF) was reported to prevent hepatotoxin-induced liver damage, we tested HGF as the intermediary of Go effects. Simultaneous analyses of HGF mRNA expression and liver histology in rats were performed at 6 and 24 hr after treatment with AAP, Go or both. HGF mRNA rapidly expressed at 6 hr after Go treatment, while no HGF mRNA was observed at 6 hr after AAP treatment. Induction of HGF mRNA at 24 hr was observed after treatment with AAP or AAP plus Go. Histological findings indicate that massive necrosis and vacuolization in the liver of rats treated with both AAP and Go were reduced in comparison with rats treated with AAP only. These data suggest that Go rapidly induces HGF mRNA through different mechanisms from AAP-induced liver injury.

  8. Trans and cis factors affecting A-to-I RNA editing efficiency of a noncoding editing target in C. elegans.

    Science.gov (United States)

    Washburn, Michael C; Hundley, Heather A

    2016-05-01

    Adenosine-to-inosine RNA editing by ADARs affects thousands of adenosines in an organism's transcriptome. However, adenosines are not edited at equal levels nor do these editing levels correlate well with ADAR expression levels. Therefore, additional mechanisms are utilized by the cell to dictate the editing efficiency at a given adenosine. To examine cis-and trans-acting factors that regulate A-to-I editing levels specifically in neural cells, we utilized the model organism Caenorhabditis elegans We demonstrate that a double-stranded RNA (dsRNA) binding protein, ADR-1, inhibits editing in neurons, which is largely masked when examining editing levels from whole animals. Furthermore, expression of ADR-1 and mRNA expression of the editing target can act synergistically to regulate editing efficiency. In addition, we identify a dsRNA region within the Y75B8A.83' UTR that acts as acis-regulatory element by enhancing ADR-2 editing efficiency. Together, this work identifies mechanisms that regulate editing efficiency of noncoding A-to-I editing sites, which comprise the largest class of ADAR targets.

  9. GAMETOPHYTIC FACTOR 1, Involved in Pre-mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Man Liu; Li Yuan; Nai-You Liu; Dong-Qiao Shi; Jie Liu; Wei-Cai Yang

    2009-01-01

    RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homoiogs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogeneeis and embryogenesis in plant.

  10. How MESSENGER Meshes Simulations and Games with Citizen Science

    Science.gov (United States)

    Hirshon, B.; Chapman, C. R.; Edmonds, J.; Goldstein, J.; Hallau, K. G.; Solomon, S. C.; Vanhala, H.; Weir, H. M.; Messenger Education; Public Outreach (Epo) Team

    2010-12-01

    How MESSENGER Meshes Simulations and Games with Citizen Science In the film The Last Starfighter, an alien civilization grooms their future champion—a kid on Earth—using a video game. As he gains proficiency in the game, he masters the skills he needs to pilot a starship and save their civilization. The NASA MESSENGER Education and Public Outreach (EPO) Team is using the same tactic to train citizen scientists to help the Science Team explore the planet Mercury. We are building a new series of games that appear to be designed primarily for fun, but that guide players through a knowledge and skill set that they will need for future science missions in support of MESSENGER mission scientists. As players score points, they gain expertise. Once they achieve a sufficiently high score, they will be invited to become participants in Mercury Zoo, a new program being designed by Zooniverse. Zooniverse created Galaxy Zoo and Moon Zoo, programs that allow interested citizens to participate in the exploration and interpretation of galaxy and lunar data. Scientists use the citizen interpretations to further refine their exploration of the same data, thereby narrowing their focus and saving precious time. Mercury Zoo will be designed with input from the MESSENGER Science Team. This project will not only support the MESSENGER mission, but it will also add to the growing cadre of informed members of the public available to help with other citizen science projects—building on the concept that engaged, informed citizens can help scientists make new discoveries. The MESSENGER EPO Team comprises individuals from the American Association for the Advancement of Science (AAAS); Carnegie Academy for Science Education (CASE); Center for Educational Resources (CERES) at Montana State University (MSU) - Bozeman; National Center for Earth and Space Science Education (NCESSE); Johns Hopkins University Applied Physics Laboratory (JHU/APL); National Air and Space Museum (NASM); Science

  11. HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia

    Directory of Open Access Journals (Sweden)

    Mishra Ritu

    2012-06-01

    Full Text Available Abstract Background HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. Methods We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. Results HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor–associated factor 3 TRAF3 is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3 and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. Conclusion HIV-1 Tat protein can modulate TRAF3 expression through

  12. Increased N 6 -methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia

    OpenAIRE

    Ying Yang; Wei Huang; Jing-Tao Huang; Fan Shen; Jun Xiong; Er-Feng Yuan; Shan-shan Qin; Ming Zhang; Yu-Qi Feng; Bi-Feng Yuan; Song-Mei Liu

    2016-01-01

    Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N 6-methyladenosine (m6A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m6A contents in sperm RNA, and rea...

  13. Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor Maelstrom

    Directory of Open Access Journals (Sweden)

    Naoki Matsumoto

    2015-04-01

    Full Text Available PIWI-interacting RNAs (piRNAs protect the genome from transposons in animal gonads. Maelstrom (Mael is an evolutionarily conserved protein, composed of a high-mobility group (HMG domain and a MAEL domain, and is essential for piRNA-mediated transcriptional transposon silencing in various species, such as Drosophila and mice. However, its structure and biochemical function have remained elusive. Here, we report the crystal structure of the MAEL domain from Drosophila melanogaster Mael, at 1.6 Å resolution. The structure reveals that the MAEL domain has an RNase H-like fold but lacks canonical catalytic residues conserved among RNase H-like superfamily nucleases. Our biochemical analyses reveal that the MAEL domain exhibits single-stranded RNA (ssRNA-specific endonuclease activity. Our cell-based analyses further indicate that ssRNA cleavage activity appears dispensable for piRNA-mediated transcriptional transposon silencing in Drosophila. Our findings provide clues toward understanding the multiple roles of Mael in the piRNA pathway.

  14. Discovery and characterization of the first non-coding RNA that regulates gene expression,micF RNA:A historical perspective

    Institute of Scientific and Technical Information of China (English)

    Nicholas; Delihas

    2015-01-01

    The first evidence that RNA can function as a regulator of gene expression came from experiments with prokaryotes in the 1980 s. It was shown that Escherichia coli micF isan independent gene,has its own promoter,and encodes a small non-coding RNA that base pairs with and inhibits translation of a target messenger RNA in response to environmental stress conditions. The mic F RNA was isolated,sequenced and shown to be a primary transcript. In vitro experiments showed binding to the target ompF mR NA. Secondary structure probing revealed an imperfect micF RNA/ompF RNA duplex interaction and the presence of a non-canonical base pair. Several transcription factors,including OmpR,regulate micF transcription in response to environmental factors. micF has also been found in other bacterial species,however,recently Gerhart Wagner and J?rg Vogel showed pleiotropic effects and found micF inhibits expression of multiple target mR NAs; importantly,one is the global regulatory gene lrp. In addition,micF RNA was found to interact with its targets in different ways; it either inhibits ribosome binding or induces degradation of the message. Thus the concept and initial experimental evidence that RNA can regulate gene expression was born with prokaryotes.

  15. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

    Science.gov (United States)

    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki

    2016-01-01

    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

  16. A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

    Science.gov (United States)

    Gedamu, L; Iatrou, K; Dixon, G H

    1978-06-01

    Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more

  17. RNA Sequencing Analysis Reveals Transcriptomic Variations in Tobacco (Nicotiana tabacum Leaves Affected by Climate, Soil, and Tillage Factors

    Directory of Open Access Journals (Sweden)

    Bo Lei

    2014-04-01

    Full Text Available The growth and development of plants are sensitive to their surroundings. Although numerous studies have analyzed plant transcriptomic variation, few have quantified the effect of combinations of factors or identified factor-specific effects. In this study, we performed RNA sequencing (RNA-seq analysis on tobacco leaves derived from 10 treatment combinations of three groups of ecological factors, i.e., climate factors (CFs, soil factors (SFs, and tillage factors (TFs. We detected 4980, 2916, and 1605 differentially expressed genes (DEGs that were affected by CFs, SFs, and TFs, which included 2703, 768, and 507 specific and 703 common DEGs (simultaneously regulated by CFs, SFs, and TFs, respectively. GO and KEGG enrichment analyses showed that genes involved in abiotic stress responses and secondary metabolic pathways were overrepresented in the common and CF-specific DEGs. In addition, we noted enrichment in CF-specific DEGs related to the circadian rhythm, SF-specific DEGs involved in mineral nutrient absorption and transport, and SF- and TF-specific DEGs associated with photosynthesis. Based on these results, we propose a model that explains how plants adapt to various ecological factors at the transcriptomic level. Additionally, the identified DEGs lay the foundation for future investigations of stress resistance, circadian rhythm and photosynthesis in tobacco.

  18. A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

    Directory of Open Access Journals (Sweden)

    Hao Gong

    2011-09-01

    Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

  19. Limits to Mercury's magnesium exosphere from MESSENGER second flyby observations

    Science.gov (United States)

    Sarantos, Menelaos; Killen, Rosemary M.; McClintock, William E.; Todd Bradley, E.; Vervack, Ronald J.; Benna, Mehdi; Slavin, James A.

    2011-12-01

    The discovery measurements of Mercury's exospheric magnesium, obtained by the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) probe during its second Mercury flyby, are modeled to constrain the source and loss processes for this neutral species. Fits to a Chamberlain exosphere reveal that at least two source temperatures are required to reconcile the distribution of magnesium measured far from and near the planet: a hot ejection process at the equivalent temperature of several tens of thousands of degrees K, and a competing, cooler source at temperatures as low as 400 K. For the energetic component, our models indicate that the column abundance that can be attributed to sputtering under constant southward interplanetary magnetic field conditions is at least a factor of five less than the rate dictated by the measurements. Although highly uncertain, this result suggests that another energetic process, such as the rapid dissociation of exospheric MgO, may be the main source of the distant neutral component. If meteoroid and micrometeoroid impacts eject mainly molecules, the total amount of magnesium at altitudes exceeding ˜100 km is found to be consistent with predictions by impact vaporization models for molecule lifetimes of no more than two minutes. Though a sharp increase in emission observed near the dawn terminator region can be reproduced if a single meteoroid enhanced the impact vapor at equatorial dawn, it is much more likely that observations in this region, which probe heights increasingly near the surface, indicate a reservoir of volatile Mg being acted upon by lower-energy source processes.

  20. Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is a bona fide substrate for Legionelle pneumophila effector glucosyltransferases

    DEFF Research Database (Denmark)

    Tzivelekidis, Tina; Jank, Thomas; Pohl, Corinna;

    2011-01-01

    selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNAPhe and GTP was enhanced 150 and 590-fold......, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNAHis and Phe-tRNAPhe) we could show that aminoacylation is crucial for Lgtcatalyzed glucosylation. Aminoacyl-tRNA had no effect...

  1. Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

    Directory of Open Access Journals (Sweden)

    M Andrea Markus

    Full Text Available Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

  2. Insulin-like growth factor II mRNA, peptides, and receptors in a thoracopulmonary malignant small round cell tumor

    DEFF Research Database (Denmark)

    Nielsen, F C; Orskov, C; Haselbacher, G;

    1994-01-01

    Insulin-like growth factor-(IGF) II and IGF-I and IGF-II/mannose 6-phosphate receptors were expressed in a thoracopulmonary malignant small round cell tumor (MSRCT) from a 14-year-old boy. Northern analysis showed that the MSRCT expresses multiple IGF-II mRNA of 6.0, 4.8, 4.2, and 2.2 kilobase from...

  3. Hormone and metabolic factors associated with leptin mRNA expression in pre- and postmenopausal women.

    Science.gov (United States)

    Fajardo, Martha E; Malacara, Juan M; Martínez-Rodríguez, Herminia G; Barrera-Saldaña, Hugo A

    2004-06-01

    Recent information has extended leptin's action, beyond the control of appetite, to various sites of metabolic regulation. To better understand leptin's role we studied its production in subcutaneous and visceral fat compartments before and after menopause. During elective abdominal surgery, biopsies of subcutaneous and omental tissues were taken from 20 women at pre- (BMI 28.4 +/- 4.5 kg/m2) and 10 at postmenopause (BMI 30.6 +/- 7.7 kg/m2). In both groups serum leptin levels were similar, and highly correlated with BMI. In subcutaneous adipose tissue, leptin mRNA expression was significantly higher in pre- than in postmenopausal women (50.4 +/- 20.5 amol/microg total RNA versus 34.5 +/- 24.9 amol/microg total RNA, respectively). Leptin mRNA expression in subcutaneous tissue was independently correlated with fasting glucose (R = 0.89, P < 0.006) at premenopause, and with serum estradiol (R = 0.77, P < 0.04) at postmenopause. Leptin mRNA expression in visceral fat was correlated with DHEAS (R = 0.86, P < 0.001), at premenopause. These results indicate that in both compartments, leptin production is sensitive to different but overlapping stimuli, conveying information about energy availability to central and peripheral sites under different conditions of estrogen exposure.

  4. Incidence of and factors associated with false positives in laboratory diagnosis of norovirus infection by amplification of the RNA-dependent RNA polymerase gene.

    Directory of Open Access Journals (Sweden)

    Fang-Ru Lin

    Full Text Available BACKGROUND: Conventional reverse transcription-polymerase chain reaction (RT-PCR amplification of the RNA-dependent RNA polymerase (RdRp gene remains a used method for the rapid detection of norovirus (NV in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated. METHODS/PRINCIPAL FINDINGS: After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6% samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91-97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17-37.92, P = .003 and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21-24.73, P = .027 as significant and independent factors associated with false positives. CONCLUSION: Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.

  5. Estrogen and exercise interact to regulate brain-derived neurotrophic factor mRNA and protein expression in the hippocampus.

    Science.gov (United States)

    Berchtold, N C; Kesslak, J P; Pike, C J; Adlard, P A; Cotman, C W

    2001-12-01

    We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain-derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up-regulation is reduced in the absence of estrogen, in a time-dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short-term (3 weeks) estrogen deprivation. However, long-term estrogen-deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long-term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well-being, particularly in women.

  6. Impacts of microRNA gene polymorphisms on the susceptibility of environmental factors leading to carcinogenesis in oral cancer.

    Directory of Open Access Journals (Sweden)

    Yin-Hung Chu

    Full Text Available BACKGROUND: MicroRNAs (miRNAs have been regarded as a critical factor in targeting oncogenes or tumor suppressor genes in tumorigenesis. The genetic predisposition of miRNAs-signaling pathways related to the development of oral squamous cell carcinoma (OSCC remains unresolved. This study examined the associations of polymorphisms with four miRNAs with the susceptibility and clinicopathological characteristics of OSCC. METHODOLOGY/PRINCIPAL FINDINGS: A total of 895 male subjects, including 425 controls and 470 male oral cancer patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and real-time PCR were used to analyze miRNA146a, miRNA196, miRNA499 and miRNA149 genetic polymorphisms between the control group and the case group. This study determined that a significant association of miRNA499 with CC genotype, as compared to the subjects with TT genotype, had a higher risk (AOR = 4.52, 95% CI = 1.24-16.48 of OSCC. Moreover, an impact of those four miRNAs gene polymorphism on the susceptibility of betel nut and tobacco consumption leading to oral cancer was also revealed. We found a protective effect between clinical stage development (AOR = 0.58, 95% CI = 0.36-0.94 and the tumor size growth (AOR = 0.47, 95% CI = 0.28-0.79 in younger patients (age<60. CONCLUSIONS: Our results suggest that genetic polymorphism of miRNA499 is associated with oral carcinogenesis, and the interaction of the miRNAs genetic polymorphism and environmental carcinogens is also related to an increased risk of oral cancer in Taiwanese.

  7. APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo

    Science.gov (United States)

    Snyder, Elizabeth M.; McCarty, Christopher; Mehalow, Adrienne; Svenson, Karen L.; Murray, Stephen A.; Korstanje, Ron; Braun, Robert E.

    2017-01-01

    Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney. PMID:28069890

  8. Estrogen regulates the development of brain-derived neurotrophic factor mRNA and protein in the rat hippocampus.

    Science.gov (United States)

    Solum, Derek T; Handa, Robert J

    2002-04-01

    During development, estrogen has a variety of effects on morphological and electrophysiological properties of hippocampal neurons. Brain-derived neurotrophic factor (BDNF) also plays an important role in the survival and differentiation of neurons during development. We examined the effects of gonadectomy with and without estrogen replacement on the mRNA and protein of BDNF and its receptor, trkB, during early postnatal development of the rat hippocampus. We used immunocytochemistry to demonstrate that estrogen receptor alpha (ERalpha) and BDNF were localized to the same cells within the developing hippocampus. BDNF and ERalpha were colocalized in pyramidal cells of the CA3 subregion and to a lesser extent in CA1. To determine whether BDNF mRNA was regulated by estrogen during development, we gonadectomized male rat pups at postnatal day 0 (P0) and examined mRNA and protein levels from P0 to P25 using real-time reverse transcription-PCR and Western blot analysis. After gonadectomy, BDNF mRNA levels are significantly reduced on P7, but after treatment of gonadectomized animals with estradiol benzoate on P0, levels at all ages were similar to those in intact animals. BDNF mRNA changes after gonadectomy are accompanied by an increase in the levels of BDNF protein, which were reduced by estrogen treatment at P0. We also examined the effect of postnatal estrogen treatment on trkB. There were no significant changes in trkB mRNA or protein in gonadectomized or estrogen-replaced animals. These results suggest that a direct interaction may exist between ERalpha and BDNF to alter hippocampal physiology during development in the rat.

  9. APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

    Science.gov (United States)

    Snyder, Elizabeth M; McCarty, Christopher; Mehalow, Adrienne; Svenson, Karen L; Murray, Stephen A; Korstanje, Ron; Braun, Robert E

    2017-04-01

    Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.

  10. Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

    Science.gov (United States)

    Mizushima, Shusei; Takagi, Soichi; Ono, Tamao; Atsumi, Yusuke; Tsukada, Akira; Saito, Noboru; Shimada, Kiyoshi

    2009-12-01

    This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

  11. Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry.

    Science.gov (United States)

    Kultas-Ilinsky, K; Leontiev, V; Whiting, P J

    1998-07-01

    In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and

  12. Factors influencing a low rate of hepatitis C viral RNA clearance in heroin users from Southern China

    Institute of Scientific and Technical Information of China (English)

    ShengHan Lai; Jin-Bing Zhang; Wei Liu; Jie Chen; Xiao-Fang Yu

    2008-01-01

    AIM: To study the virological and host factors influencing hepatitis C infection outcomes in heroin users in southern China.METHODS: HCV RNA and associated factors were analyzed among 347 heroin users from Guangxi Zhuang Autonomous Region, southern China who were hepatitis C virus (HCV) EIA positive for two or more consecutive visits.RESULTS: Using the COBAS AMPLICOR HCV TEST, a remarkably low HCV RNA negative rate of 8.6% was detected. After multivariate logistic regression analysis, HCV RNA clearance was significantly associated with the presence of HBsAg (OR = 8.436, P < 0.0001), the lack of HIV-1 infection (OR = 0.256, P = 0.038) and age younger than 25 (OR = 0.400, P = 0.029).CONCLUSION: Our study suggests HCV infection among Chinese heroin users results in high levels of viral persistence even amidst factors previously found to enhance viral clearance. Prospective studies of a possible genetic component within the Chinese population and the pathogenicity of non-genotype 1 HCV infections are needed.

  13. To Be Connected or Not To Be Connected? Mobile Messenger Overload, Fatigue, and Mobile Shunning.

    Science.gov (United States)

    Shin, Jaewook; Shin, Mincheol

    2016-10-01

    With the increased adoption of mobile devices, mobile communication is all around us and we are connected anywhere, anytime. Mobile communication in general and mobile messenger service in particular make interpersonal communication in Korea so frequent and convenient. However, being connected too much anywhere and anytime via mobile messenger service appears to lead an increasing number of people to feel fatigue and to decrease mobile communication under some conditions. Based on a sample of 334 respondents, this study empirically investigated the relationships among commercial, noncommercial mobile messenger overload, mobile messenger fatigue, relational self-concept, and mobile shunning behavior. The findings show that (a) the effect of noncommercial mobile messenger overload is stronger than that of commercial mobile messenger overload in increasing mobile messenger fatigue although both positively affect mobile messenger fatigue, (b) relational self-concept has moderating effects on the relationship between mobile messenger overload and mobile messenger fatigue, and that (c) mobile messenger fatigue triggers mobile communicators' shunning behavior through which the communicators increase their intention to avoid mobile communication, to change their mobile phone numbers, and to subscribe to dual number service on one mobile device. When confronted with mobile messenger fatigue caused by mobile messenger overload, mobile messaging service users are likely to shun their mobile communication. Being constantly and conveniently connected appears to be a blessing in disguise.

  14. The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae.

    Science.gov (United States)

    Sabharwal, Dharmesh; Song, Tianyan; Papenfort, Kai; Wai, Sun Nyunt

    2015-01-01

    Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5' region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5' region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.

  15. Neuronal chemokines : Versatile messengers in central nervous system cell interaction

    NARCIS (Netherlands)

    de Haas, A. H.; van Weering, H. R. J.; de Jong, E. K.; Boddeke, H. W. G. M.; Biber, K. P. H.

    2007-01-01

    Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemoki

  16. Uncovering MicroRNA and Transcription Factor Mediated Regulatory Networks in Glioblastoma

    OpenAIRE

    Jingchun Sun; Xue Gong; Benjamin Purow; Zhongming Zhao

    2012-01-01

    Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in humans. Recent studies revealed that patterns of microRNA (miRNA) expression in GBM tissue samples are different from those in normal brain tissues, suggesting that a number of miRNAs play critical roles in the pathogenesis of GBM. However, little is yet known about which miRNAs play central roles in the pathology of GBM and their regulatory mechanisms of action. To address this issue, in this study, we systematically ...

  17. Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

    OpenAIRE

    Gick, G G; Zeytin, F N; BRAZEAU, P.; Ling, N C; Esch, F S; Bancroft, C

    1984-01-01

    A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer c...

  18. Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1

    OpenAIRE

    Roche, Rebecca I.

    2005-01-01

    Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1 By Rebecca I. Roche, MS Dr. W.R. Huckle, Chairman Department of Biomedical Sciences and Pathobiology (ABSTRACT) Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, the formation of new blood vessels. sFlt-1, a secreted form of the signal-transducing VEGF receptor Flt-1, can inhibit cellular responses to VEGF both in vitro and in viv...

  19. Interaction of Eukaryote Initiator Methionyl-tRNA with the Eukaryote Equivalent of Bacterial Elongation Factor T and Guanosine Triphosphate

    Science.gov (United States)

    Richter, Dietmar; Lipmann, Fritz; Tarragó, Adela; Allende, Jorge E.

    1971-01-01

    The initiator tRNA, methionyl-tRNAiMet, of yeast and wheat germ forms relatively unstable ternary complexes with their corresponding elongation factors T and GTP. Such complexes can be demonstrated only with fast separation techniques such as Sephadex G-50 and Millipore filtration, but not with the slow Sephadex G-100 method, although both techniques yield stable ternary complexes with all other aminoacyl-tRNAs, including the internal Met-tRNAmMet. To bind yeast-initiating Met-tRNAiMet to ribosomes, initiation factors present in a ribosomal wash fraction from yeast are needed. PMID:5288767

  20. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J;

    1999-01-01

    Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins.......5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development....

  1. Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 徐永健; 张珍祥; 许淑云; 倪望; 陈士新

    2004-01-01

    Summary: In order to investigate the effect of nuclear factor-kappa B (NF-κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. The NF-κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBa protein expression was measured by Western blot.RT-PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF-κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF-κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups (P<0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups (P<0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration-dependent manner in hypoxia. In conclusion, NF-κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF-κB activation can decrease the VEGF mRNA expression. h is suggested that the activation of NF-κB is involved in the VEGFmRNA expression of HPASMCs under hypoxia.

  2. Exosomes and the kidney: blaming the messenger.

    Science.gov (United States)

    Fang, Doreen Yp; King, Hamish W; Li, Jordan Yz; Gleadle, Jonathan M

    2013-01-01

    Exosomes are membrane-bound vesicles of endosomal origin, present in a wide range of biological fluids, including blood and urine. They range between 30 and 100 nm in diameter, and consist of a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNA). Exosomes can act as extracellular vehicles by which cells communicate, through the delivery of their functional cargo to recipient cells, with many important biological, physiological and pathological implications. The exosome release pathway contributes towards protein secretion, antigen presentation, pathogen transfer and cancer progression. Exosomes and exosome-mediated signalling have been implicated in disease processes such as atherosclerosis, calcification and kidney diseases. Circulating levels of exosomes and extracellular vesicles can be influenced by the progression of renal disease. Advances in methods for purification and analysis of exosomes are leading to potential diagnostic and therapeutic avenues for kidney diseases. This review will focus on biophysical properties and biogenesis of exosomes, their pathophysiological roles and their potential as biomarkers and therapeutics in kidney diseases.

  3. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States); Rebecchi, Mario, E-mail: Mario.rebecchi@SBUmed.org [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States)

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  4. tRNA(Pro) -mediated downregulation of elongation factor P is required for mgtCBR expression during Salmonella infection.

    Science.gov (United States)

    Nam, Daesil; Choi, Eunna; Shin, Dongwoo; Lee, Eun-Jin

    2016-10-01

    Bacterial ribosome requires elongation factor P to translate fragments harbouring consecutive proline codons. Given the abundance of ORFs with potential EF-P regulated sites, EF-P was assumed to be constitutively expressed. Here, we report that the intracellular pathogen Salmonella enterica serovar Typhimurium decreases efp mRNA levels during course of infection. We determined that the decrease in efp mRNA is triggered by low levels of charged tRNA(Pro) , a condition that Salmonella experiences when inside a macrophage phagosome. Surprisingly, downregulation of EF-P selectively promotes expression of the virulence mgtC gene and contributes to Salmonella's ability to survive inside macrophages. The decrease in EF-P levels induces ribosome stalling at the consecutive proline codons of the mgtP open reading frame in the mgtCBR leader RNA, and thus allows formation of a stem-loop structure promoting transcription of the mgtC gene. The substitution of proline codons in the mgtP gene eliminates EF-P-mediated mgtC expression and thus Salmonella's survival inside macrophages. Our findings indicate that Salmonella benefits virulence genes by decreasing EF-P levels and inducing the stringent response inside host.

  5. RNA interference against transcription elongation factor SII does not support its role in transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Mackinnon-Roy, Christine; Stubbert, Lawton J; McKay, Bruce C

    2011-01-10

    RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.

  6. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  7. Expression of nerve growth factor mRNA in splenic lymphocytes of bronchial asthma rats and its influencing actors

    Institute of Scientific and Technical Information of China (English)

    Jihong Dai; Yonghong Wang; Haixia He

    2008-01-01

    BACKGROUND: Previous research has proved that nerve growth factor (NGF) participates in the onset of asthma by the induction of neurogenic inflammation.OBJECTIVE: To investigate the effect of interleukin-13 (IL-13) and interferon-γ(IFN-γ) on the expression of NGF mRNA in the splenic lymphocytes of bronchial asthma rats.DESIGN, TIME AND SETTING: The experiment, a completely randomized study based on cellular immunology, was performed in the Laboratory of Neurology in Chongqing Medical University and the Department of Clinical Pharmacy in College of Clinical Medicine, Chongqing Medical University (Chongqing, China) from January 2006 to April 2007.MATERIALS: Four adult male Wistar rats were used in this study. Rat IL-13, IFN-γprobe and the total RNA extraction kit were produced by Shanghai Sangon Biological Technology & Services Co., Ltd (China). The NGF ELISA kit was a product of Wuhan Boster Bioengineering Co., Ltd (China). A Du-70 automatic UV spectrophotometer was produced by Beckman Company (USA).METHODS: Rats were subjected to 1-mL intraperitoneal injections each containing 100 mg of ovalbumin, and were sensitized by using antigen solution, which was sensitized with 5×109 Bacillus pertussis and 100 mg aluminum hydroxide powder. Four rats were challenged with 1% ovalbumin using an ultrasonic nebulizer for 60 minutes to establish an asthmatic model. After rats were anesthetized, splenic lymphocytes were isolated and cultured in medium, which was supplemented with IL-13 or IFN-γ, for 0, 12, 24 or 48 hours. A parallel study was conducted with cultured splenic lymphocytes, which were divided into a control group, an IL-13 group and an IFN-γ group. Culture medium was added with different concentrations of IL-13 (10, 50, 100 μg/L) and IFN-γ (1, 10, 50 μg/L); 24 hours later, all samples were harvested.MAIN OUTCOME MEASURES: The expression levels of NGF mRNA were detected by reverse transcription-polymerase chain reaction.RESULTS: In the control group, the

  8. Characterization of a "TRAMP-like" co-factor of the human RNA exosome

    DEFF Research Database (Denmark)

    Christensen, Marianne Skovgaard; Kristiansen, Maiken Søndergaard; Lubas, Michal Szymon

    Genome-wide studies in yeast, plants and humans have revealed numerous new transcripts in what was previously thought to be silent DNA or junk DNA. One class of non-coding transcript discovered recently is the PROMoter uPstream Transcripts (PROMPTs), which is only seen upon depletion of the RNA e......, serving to degrade PROMPTs in a core exosome independent manner....

  9. Transcriptome of common bean (Phaseolus vulgaris) through RNA-seq: nodulation, symbiotic nitrogen fixation, transcription factors

    Science.gov (United States)

    Phaseolus vulgaris (common bean) is one of the most important grain legumes for direct human consumption. It comprises 50% of the grain legumes consumed worldwide and is important as a primary source of dietary protein in developing countries. We have performed next generation sequencing (RNA-seq) o...

  10. Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli.

    Science.gov (United States)

    Knutsson Jenvert, Rose-Marie; Holmberg Schiavone, Lovisa

    2005-02-01

    Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

  11. Delivery of dsRNA for RNAi in insects: an overview and future directions.

    Science.gov (United States)

    Yu, Na; Christiaens, Olivier; Liu, Jisheng; Niu, Jinzhi; Cappelle, Kaat; Caccia, Silvia; Huvenne, Hanneke; Smagghe, Guy

    2013-02-01

    RNA interference (RNAi) refers to the process of exogenous double-stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi-based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.

  12. Role of LncRNA-activated by transforming growth factor beta in the progression of hepatitis C virus-related liver fibrosis.

    Science.gov (United States)

    Fu, Na; Niu, Xuemin; Wang, Yang; Du, Huijuan; Wang, Baoyu; Du, Jinghua; Li, Ya; Wang, Rongqi; Zhang, Yuguo; Zhao, Suxian; Sun, Dianxing; Qiao, Liang; Nan, Yuemin

    2016-08-01

    Long non-coding RNA (LncRNA)-activated by transforming growth factor-beta (LncRNA-ATB) is a key regulator of transforming growth factor-beta (TGF-β) signaling pathway, and is positively correlated with the development of liver cirrhosis and vascular invasion of hepatocellular carcinoma (HCC). However, the role of LncRNA-ATB in hepatitis C virus (HCV)-related liver fibrosis remains largely unknown. In the present study, we confirmed a high expression level of LncRNA-ATB in the liver tissues and plasma samples of patients with HCV-related hepatic fibrosis, and the plasma level of LncRNA-ATB was significantly correlated with liver fibrosis stages. Furthermore, increased expression level of LncRNA-ATB was also present in activated hepatic stellate cells (HSCs), and knockdown of LncRNA-ATB inhibited the expression of alpha-smooth muscle actin (α-SMA) and alpha-1 type I collagen (Col1A1). LncRNA-ATB was found to share the common miRNA responsive element of miR-425-5p with TGF-β type II receptor (TGF-βRII) and SMAD2. Ectopic expression of LncRNA-ATB in HSCs could upregulate the protein expression of TGF-βRII and SMAD2 by inhibiting the endogenous miR-425-5p. Moreover, overexpression of miR-425-5p could partly abrogate the expression of TGF-βRII and SMAD2 induced by LncRNA-ATB. Hence, we conclude that LncRNA-ATB promotes HCV-induced liver fibrogenesis by activating HSCs and increasing collagen I production through competitively binding to miR-425-5p. LncRNA-ATB may be a novel diagnostic biomarker and a potential therapeutic target for HCV-related hepatic fibrosis.

  13. A guanosine quadruplex and two stable hairpins flank a major cleavage site in insulin-like growth factor II mRNA

    DEFF Research Database (Denmark)

    Christiansen, Jan; Kofod, M; Nielsen, F C

    1994-01-01

    Insulin-like growth factor II (IGF-II) mRNAs are cleaved by an endonucleolytic event in a conserved part of their 3' untranslated region that is predicted to exhibit a complex higher-order RNA structure. In the present study, we have examined the putative secondary structures of in vitro...... is not sequestered in stable RNA structures, thus allowing interactions with RNA or proteins at posttranscriptional stages of IGF-II expression....

  14. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    Science.gov (United States)

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

  15. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    Science.gov (United States)

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  16. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1...

  17. Determination of the role of DDX3 a factor involved in mammalian RNAi pathway using an shRNA-expression library.

    Directory of Open Access Journals (Sweden)

    Vivi Kasim

    Full Text Available RNA interference (RNAi is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs. In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals.

  18. Determination of the role of DDX3 a factor involved in mammalian RNAi pathway using an shRNA-expression library.

    Science.gov (United States)

    Kasim, Vivi; Wu, Shourong; Taira, Kazunari; Miyagishi, Makoto

    2013-01-01

    RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this <