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Sample records for hepatitis rna transcripts

  1. Alternative Processing of Hepatitis Delta Virus Antigenomic RNA Transcripts

    PubMed Central

    Nie, Xingcao; Chang, Jinhong; Taylor, John M.

    2004-01-01

    Intrinsic to the life cycle of hepatitis delta virus (HDV) is the fact that its RNAs undergo different forms of posttranscriptional RNA processing. Transcripts of both the genomic RNA and its exact complement, the antigenomic RNA, undergo ribozyme cleavage and RNA ligation. In addition, antigenomic RNA transcripts can undergo 5′ capping, 3′ polyadenylation, and even RNA editing by an adenosine deaminase. This study focused on the processing of antigenomic RNA transcripts. Two approaches were used to study the relationship between the events of polyadenylation, ribozyme cleavage, and RNA ligation. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing. We found that when a separate stable source of δAg-S, the small delta protein, was provided, the replication ability of the mutated RNA was restored. The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. The DNA constructs used were such that the RNA transcripts were antigenomic and began at the same 5′ site as the mRNA produced during RNA-directed HDV genome replication. A series of such constructs was assembled in order to test the relative abilities of the transcripts to undergo processing by polyadenylation or ribozyme cleavage at sites further 3′ on a multimer of HDV sequences. The findings from the two experimental approaches led to significant modifications in the rolling-circle model of HDV genome replication. PMID:15078932

  2. RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism.

    PubMed

    Aly, Hussein H; Suzuki, Junya; Watashi, Koichi; Chayama, Kazuaki; Hoshino, Shin-Ichi; Hijikata, Makoto; Kato, Takanobu; Wakita, Takaji

    2016-07-29

    Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism. PMID:27281821

  3. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  4. Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly

    PubMed Central

    Tsai, Kuen-Nan; Chong, Chin-Liew; Chou, Yu-Chi; Huang, Chien-Chiao; Wang, Yi-Ling; Wang, Shao-Win; Chen, Mong-Liang

    2015-01-01

    ABSTRACT The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBVG2335A expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes. IMPORTANCE Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients

  5. Divergent RNA transcription

    PubMed Central

    Naughton, Catherine; Corless, Samuel; Gilbert, Nick

    2013-01-01

    New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance. PMID:23863199

  6. Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

    PubMed

    Lim, Chun Shen; Brown, Chris M

    2016-09-01

    Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

  7. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.

    PubMed

    Kolykhalov, A A; Agapov, E V; Blight, K J; Mihalik, K; Feinstone, S M; Rice, C M

    1997-07-25

    More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease. PMID:9228008

  8. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  9. Transcriptional Regulation of Hepatic Lipogenesis

    PubMed Central

    Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

    2016-01-01

    Fatty acid and fat synthesis in liver is a highly regulated metabolic pathway critical for energy distribution. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcription level. Transcription factors, such as USF, SREBP-1c, LXR and ChREBP play critical roles in this process. Recently, insights have been gained into how various signaling pathways regulate these transcription factors. After feeding, high blood glucose and insulin induce lipogenic genes through several pathways, including DNA-PK, aPKC and Akt-mTOR. Various transcription factors and coregulators undergo specific modifications, such as phosphorylation, acetylation, or ubiquitination, which affect their function, stability, or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance. PMID:26490400

  10. Transcriptional Profiling and miRNA-Target Network Analysis Identify Potential Biomarkers for Efficacy Evaluation of Fuzheng-Huayu Formula-Treated Hepatitis B Caused Liver Cirrhosis

    PubMed Central

    Chen, Qilong; Wu, Feizhen; Wang, Mei; Dong, Shu; Liu, Yamin; Lu, Yiyu; Song, Yanan; Zhou, Qianmei; Liu, Ping; Luo, Yunquan; Su, Shibing

    2016-01-01

    Fuzheng-Huayu (FZHY) formula has been found to have a satisfactory effect on hepatitis B-caused cirrhosis (HBC) treatment. However, the efficacy evaluation of FZHY is often challenging. In this study, a randomized, double-blind and placebo-controlled trial was used to evaluate the therapeutic efficacy of FZHY in HBC treatment. In the trial, 35 medical indexes were detected, and 14 indexes had a statistically-significant difference before compared to after the trial. Importantly, the Child-Pugh score also demonstrated FZHY having therapeutic efficacy. Furthermore, the microRNA (miRNA) profiles of 12 serum samples were detected in FZHY groups, and 112 differential-expressed (DE) miRNAs were determined. Using predicted miRNA targets, 13 kernel miRNAs were identified from the established miRNA-target network. Subsequently, quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to validate the expression level of 13 identified miRNAs in the trials. The results showed that nine miRNAs have a statistically-significant difference before compared to after FZHY treatment. By means of a logistic regression model, a miRNA panel with hsa-miR-18a-5p, -326, -1182 and -193b-5p was established, and it can clearly improve the accuracy of the efficacy evaluation of FZHY. This study suggested that the particular miRNAs can act as potential biomarkers and obviously increase the diagnostic accuracy for drug evaluation in HBC treatment progression. PMID:27271613

  11. mRNA quality control goes transcriptional

    PubMed Central

    Kilchert, Cornelia; Vasiljeva, Lidia

    2013-01-01

    Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5′ capped, spliced and 3′ polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC (quality control) mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs. Recently, with the growing appreciation that mRNA processing occurs concomitantly with polII (RNA polymerase II) transcription, it has become evident that QC acts at the transcriptional level in addition to degrading aberrant RNAs. In the present review, we discuss mechanisms that allow cells to co-transcriptionally initiate the removal of RNAs as well as down-regulate transcription of transcripts where processing repeatedly fails. PMID:24256272

  12. Transcriptional regulation of hepatic stellate cells.

    PubMed

    Mann, Jelena; Mann, Derek A

    2009-07-01

    Hepatic stellate cell (HSC) activation is a process of cellular transdifferentiation in which, upon liver injury, the quiescent vitamin A storing perisinusoidal HSC is converted into a wound-healing myofibroblast and acquires potent pro-inflammatory and pro-fibrogenic activities. This remarkable phenotypic transformation is underpinned by changes in the expression of a vast number of genes. In this review we survey current knowledge of the transcription factors that either control HSC activation or which regulate specific fibrogenic functions of the activated HSC such as collagen expression, proliferation and resistance to apoptosis. PMID:19393271

  13. Molecular biology Mediating transcription and RNA export

    PubMed Central

    Rubin, Jonathan D.; Taatjes, Dylan J.

    2016-01-01

    The finding that the Mediator protein complex contributes to messenger RNA export from the nucleus in yeast adds to a growing list of roles for the complex in regulating transcriptional processes. PMID:26450052

  14. mRNA transcript therapy.

    PubMed

    Weissman, Drew

    2015-02-01

    mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery. PMID:25359562

  15. FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes*

    PubMed Central

    Singh, Brijesh Kumar; Sinha, Rohit Anthony; Zhou, Jin; Xie, Sherwin Ying; You, Seo-Hee; Gauthier, Karine; Yen, Paul Michael

    2013-01-01

    Hepatic gluconeogenesis is a concerted process that integrates transcriptional regulation with hormonal signals. A major regulator is thyroid hormone (TH), which acts through its nuclear receptor (TR) to induce the expression of the hepatic gluconeogenic genes, phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC). Forkhead transcription factor FoxO1 also is an important regulator of these genes; however, its functional interactions with TR are not known. Here, we report that TR-mediated transcriptional activation of PCK1 and G6PC in human hepatic cells and mouse liver was FoxO1-dependent and furthermore required FoxO1 deacetylation by the NAD+-dependent deacetylase, SirT1. siRNA knockdown of FoxO1 decreased, whereas overexpression of FoxO1 increased, TH-dependent transcriptional activation of PCK1 and G6PC in cultured hepatic cells. FoxO1 siRNA knockdown also decreased TH-mediated transcription in vivo. Additionally, TH was unable to induce FoxO1 deacetylation or hepatic PCK1 gene expression in TH receptor β-null (TRβ−/−) mice. Moreover, TH stimulated FoxO1 recruitment to the PCK1 and G6PC gene promoters in a SirT1-dependent manner. In summary, our results show that TH-dependent deacetylation of a second metabolically regulated transcription factor represents a novel mechanism for transcriptional integration of nuclear hormone action with cellular energy status. PMID:23995837

  16. Transcriptional profiling of Dictyostelium with RNA sequencing

    PubMed Central

    Miranda, Edward Roshan; Rot, Gregor; Toplak, Marko; Santhanam, Balaji; Curk, Tomaz; Shaulsky, Gad; Zupan, Blaz

    2014-01-01

    Summary Transcriptional profiling methods have been utilized in the analysis of various biological processes in Dictyostelium. Recent advances in high-throughput sequencing have increased the resolution and the dynamic range of transcriptional profiling. Here we describe the utility of RNA-sequencing with the Illumina technology for production of transcriptional profiles. We also describe methods for data mapping and storage as well as common and specialized tools for data analysis, both online and offline. PMID:23494306

  17. RNA-guided transcriptional regulation

    DOEpatents

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  18. Anti-Transcription Factor RNA Aptamers as Potential Therapeutics

    PubMed Central

    Mondragón, Estefanía

    2016-01-01

    Transcription factors (TFs) are DNA-binding proteins that play critical roles in regulating gene expression. These proteins control all major cellular processes, including growth, development, and homeostasis. Because of their pivotal role, cells depend on proper TF function. It is, therefore, not surprising that TF deregulation is linked to disease. The therapeutic drug targeting of TFs has been proposed as a frontier in medicine. RNA aptamers make interesting candidates for TF modulation because of their unique characteristics. The products of in vitro selection, aptamers are short nucleic acids (DNA or RNA) that bind their targets with high affinity and specificity. Aptamers can be expressed on demand from transgenes and are intrinsically amenable to recognition by nucleic acid-binding proteins such as TFs. In this study, we review several natural prokaryotic and eukaryotic examples of RNAs that modulate the activity of TFs. These examples include 5S RNA, 6S RNA, 7SK, hepatitis delta virus-RNA (HDV-RNA), neuron restrictive silencer element (NRSE)-RNA, growth arrest-specific 5 (Gas5), steroid receptor RNA activator (SRA), trophoblast STAT utron (TSU), the 3′ untranslated region of caudal mRNA, and heat shock RNA-1 (HSR1). We then review examples of unnatural RNA aptamers selected to inhibit TFs nuclear factor-kappaB (NF-κB), TATA-binding protein (TBP), heat shock factor 1 (HSF1), and runt-related transcription factor 1 (RUNX1). The field of RNA aptamers for DNA-binding proteins continues to show promise. PMID:26509637

  19. A novel real-time reverse transcription-polymerase chain reaction assay with partially double-stranded linear DNA probe for sensitive detection of hepatitis C viral RNA.

    PubMed

    Liu, Tianfu; Wan, Zhenzhou; Liu, Jia; Zhang, Lingyi; Zhou, Yanheng; Lan, Ke; Hu, Yihong; Zhang, Chiyu

    2016-10-01

    The detection and quantification of HCV RNA is very helpful for the management and treatment of HCV related diseases. Detection of low HCV viral load is a great challenge in HCV RNA detection. Here, we developed a novel real-time RT-PCR assay with partially double-stranded linear DNA probe which can detect all HCV genotypes and improve the detection performance. The novel assay has a wide linear dynamic range of HCV RNA quantification (1×10(2)-1×10(11)IU/ml) and a limit of detection of 78IU/ml. The assay exhibits an excellent reproducibility with 2.52% and 1.33% coefficients of variations, for inter- and intra-assays, respectively. To evaluate the viability of the assay, a comparison with a commercial HCV RNA detection kit was performed using 106 serum samples. The lineared correlation coefficient between the novel assay and the commercial HCV RNA detection kit was 0.940. Meanwhile, the deviation between the two methods was tolerable. Therefore, the novel real-time RT-PCR assay was applicable for laboratory diagnosis and monitoring of HCV infection. PMID:27451264

  20. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  1. A bridge to transcription by RNA polymerase.

    PubMed

    Kaplan, Craig D; Kornberg, Roger D

    2008-01-01

    A comprehensive survey of single amino-acid substitution mutations critical for RNA polymerase function published in Journal of Biology supports a proposed mechanism for polymerase action in which movement of the polymerase 'bridge helix' promotes transcriptional activity in cooperation with a critical substrate-interaction domain, the 'trigger loop'. PMID:19090964

  2. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool. PMID:22726432

  3. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7

    PubMed Central

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  4. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

    PubMed

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  5. The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome

    SciTech Connect

    Greco-Stewart, Valerie S.; Miron, Paul; Abrahem, Abrahem; Pelchat, Martin . E-mail: mpelchat@uottawa.ca

    2007-01-05

    The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.

  6. Structural analysis of hepatitis C RNA genome using DNA microarrays

    PubMed Central

    Martell, María; Briones, Carlos; de Vicente, Aránzazu; Piron, María; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gómez, Jordi

    2004-01-01

    Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5′ non-coding region (5′NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions. PMID:15247323

  7. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  8. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts.

    PubMed

    Turowski, Tomasz W; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-07-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5' peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3'-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3'-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5'-exonuclease Rat1. PMID:27206856

  9. Seasonal changes in hepatic progesterone receptor mRNA, estrogen receptor mRNA, and vitellogenin mRNA in the painted turtle, Chrysemys picta.

    PubMed

    Custodia-Lora, Noemí; Callard, Ian P

    2002-10-01

    Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species. PMID:12392693

  10. Transcription and translation in an RNA world

    PubMed Central

    Taylor, William R

    2006-01-01

    The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). If such a replicase makes a reverse complementary copy of any sequence (including itself), in a simple RNA world, there is no mechanism to prevent self-hybridization. It is proposed that this can be avoided through the synthesis of a parallel complementary copy. The logical consequences of this are pursued and developed in a computer simulation, where the behaviour of the parallel copy is compared to the conventional reverse complementary copy. It is found that the parallel copy is more efficient at higher temperatures (up to 90°C). A model for the ribopolymerase, based on the core of the large subunit (LSU) of the ribosome, is described. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have ‘poisoned’ the ribopolymerase by cross-reacting with the nucleoside-triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes, it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of aminoacylated tRNA-like products. Using base-pairing interactions, some of these molecules might cross-link two ribopolymerases, giving rise to a precursor of the modern ribosome. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors. PMID:17008216

  11. Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing.

    PubMed

    Saldi, Tassa; Cortazar, Michael A; Sheridan, Ryan M; Bentley, David L

    2016-06-19

    Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II. The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes the current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes. PMID:27107644

  12. Elevated hepatic apolipoprotein A-I transcription is associated with diet-induced hyperalphalipoproteinemia in rabbits.

    PubMed

    Schwab, D A; Rea, T J; Hanselman, J C; Bisgaier, C L; Krause, B R; Pape, M E

    2000-03-24

    Past studies have shown that a high saturated fatty acid diet containing coconut oil elevates plasma HDL cholesterol and apolipoprotein A-I (apoA-1) in rabbits through a mechanism involving increased synthesis. We have extended those studies by investigating expression of the hepatic apolipoprotein A-I gene and other lipid related genes in that model. Rabbits fed a diet containing 14% coconut oil for 4 weeks showed HDL-C elevations of 170% to 250% over chow-fed controls with peak differences occurring at 1 week. Plasma apoA-I levels were also increased over this time frame (160% to 180%) reflecting the HDL-C changes. After 4 weeks, there were no differences in plasma VLDL-C or LDL-C levels in chow versus coconut oil-fed rabbits. Hepatic levels of apoA-I mRNA in coconut oil-fed animals were elevated 150% after 4 weeks compared to chow-fed controls; hepatic mRNA levels for ten other genes either decreased slightly (apoB, LCAT, hepatic lipase, albumin, ACAT, and HMG CoA reductase) or were unchanged (CETP, apoE, LDL-receptor, and acyl CoA oxidase). Nuclear run-on transcription assays revealed that coconut oil feeding for 4 weeks caused a 220% increase in hepatic apoA-I transcription rate compared to controls; no change was observed for CETP and apoE. Treatment of cultured rabbit liver cells with various saturated fatty acids and sera from chow-fed and coconut oil-fed rabbits did not alter apoA-I mRNA levels as observed in vivo. These data demonstrate that coconut oil elevates plasma HDL-C and apoA-I by increasing hepatic apoA-I transcription while expression of other genes involved in lipid metabolism are reduced or unchanged in response to coconut oil feeding. PMID:10809165

  13. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

    PubMed Central

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription. PMID:25764111

  14. Coupling pre-mRNA processing to transcription on the RNA factory assembly line

    PubMed Central

    Lee, Kuo-Ming; Tarn, Woan-Yuh

    2013-01-01

    It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression. PMID:23392244

  15. TFIIH plays an essential role in RNA polymerase I transcription.

    PubMed

    Iben, Sebastian; Tschochner, Herbert; Bier, Mirko; Hoogstraten, Deborah; Hozák, Pavel; Egly, Jean Marc; Grummt, Ingrid

    2002-05-01

    TFIIH is a multisubunit protein complex that plays an essential role in nucleotide excision repair and transcription of protein-coding genes. Here, we report that TFIIH is also required for ribosomal RNA synthesis in vivo and in vitro. In yeast, pre-rRNA synthesis is impaired in TFIIH ts strains. In a mouse, part of cellular TFIIH is localized within the nucleolus and is associated with subpopulations of both RNA polymerase I and the basal factor TIF-IB. Transcription systems lacking TFIIH are inactive and exogenous TFIIH restores transcriptional activity. TFIIH is required for productive but not abortive rDNA transcription, implying a postinitiation role in transcription. The results provide a molecular link between RNA polymerase I transcription and transcription-coupled repair of active ribosomal RNA genes. PMID:12015980

  16. Specific HDV RNA-templated transcription by pol II in vitro.

    PubMed Central

    Filipovska, J; Konarska, M M

    2000-01-01

    RNA polymerase II is implicated in the RNA-templated RNA synthesis during replication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA template nor protein factor requirements for this process are well defined. We have developed an in vitro transcription system based on HeLa cell nuclear extract (NE), in which a segment of antigenomic RNA corresponding to the left-hand tip region of the HDV rod-like structure serves as a template for efficient and highly specific RNA synthesis. Accumulation of the unique RNA product is highly sensitive to alpha-amanitin in HeLa NE and only partially sensitive to this drug in NE from PMG cells that contain an allele of the alpha-amanitin-resistant subunit of pol II, strongly suggesting pol II involvement in this reaction. Detailed analysis of the RNA product revealed that it represents a chimeric molecule composed of a newly synthesized transcript covalently attached to the 5' half of the RNA template. Selection of the start site for transcription is remarkably specific and depends on the secondary structure of the RNA template, rather than on its primary sequence. Some features of this reaction resemble the RNA cleavage-extension process observed for pol II-arrested complexes in vitro. A possible involvement of the described reaction in HDV replication is discussed. PMID:10668797

  17. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    SciTech Connect

    Ness, Gene C.; Edelman, Jeffrey L.; Brooks, Patricia A.

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  18. Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery

    PubMed Central

    Hampsey, Michael

    1998-01-01

    Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions. PMID:9618449

  19. Inhibition of RNA polymerase III transcription by BRCA1.

    PubMed

    Veras, Ingrid; Rosen, Eliot M; Schramm, Laura

    2009-04-01

    RNA polymerase III (RNA pol III) transcribes structural RNAs involved in RNA processing (U6 snRNA) and translation (tRNA), thereby regulating the growth rate of cells. Proper initiation by RNA pol III requires the transcription factor TFIIIB. Gene-external U6 snRNA transcription requires TFIIIB consisting of Bdp1, TBP, and Brf2. Transcription from the gene-internal tRNA promoter requires TFIIIB composed of Bdp1, TBP, and Brf1. TFIIIB is a target of tumor suppressors, including PTEN, ARF, p53, and RB, and RB-related pocket proteins. Breast cancer susceptibility gene 1 (BRCA1) tumor suppressor plays a role in DNA repair, cell cycle regulation, apoptosis, genome integrity, and ubiquitination. BRCA1 has a conserved amino-terminal RING domain, an activation domain 1 (AD1), and an acidic carboxyl-terminal domain (BRCA1 C-terminal region). In Saccharomyces cerevisiae, TFIIB interacts with the BRCA1 C-terminal region domain of Fcp1p, an RNA polymerase II phosphatase. The TFIIIB subunits Brf1 and Brf2 are structurally similar to TFIIB. Hence, we hypothesize that RNA pol III may be regulated by BRCA1 via the TFIIB family members Brf1 and Brf2. Here we report that: (1) BRCA1 inhibits both VAI (tRNA) and U6 snRNA RNA pol III transcription; (2) the AD1 of BRCA1 is responsible for inhibition of U6 snRNA transcription, whereas the RING domain and AD1 of BRCA1 are required for VAI transcription inhibition; and (3) overexpression of Brf1 and Brf2 alleviates inhibition of U6 snRNA and VAI transcription by BRCA1. Taken together, these data suggest that BRCA1 is a general repressor of RNA pol III transcription. PMID:19361418

  20. Nascent RNA transcripts facilitate the formation of G-quadruplexes

    PubMed Central

    Shrestha, Prakash; Xiao, Shan; Dhakal, Soma; Tan, Zheng; Mao, Hanbin

    2014-01-01

    Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls. PMID:24829453

  1. Dysregulation of RNA polymerase I transcription during disease.

    PubMed

    Hannan, K M; Sanij, E; Rothblum, L I; Hannan, R D; Pearson, R B

    2013-01-01

    Transcription of the ribosomal RNA genes by the dedicated RNA polymerase I enzyme and subsequent processing of the ribosomal RNA are fundamental control steps in the synthesis of functional ribosomes. Dysregulation of Pol I transcription and ribosome biogenesis is linked to the etiology of a broad range of human diseases. Diseases caused by loss of function mutations in the molecular constituents of the ribosome, or factors intimately associated with RNA polymerase I transcription and processing are collectively termed ribosomopathies. Ribosomopathies are generally rare and treatment options are extremely limited tending to be more palliative than curative. Other more common diseases are associated with profound changes in cellular growth such as cardiac hypertrophy, atrophy or cancer. In contrast to ribosomopathies, altered RNA polymerase I transcriptional activity in these diseases largely results from dysregulated upstream oncogenic pathways or by direct modulation by oncogenes or tumor suppressors at the level of the RNA polymerase I transcription apparatus itself. Ribosomopathies associated with mutations in ribosomal proteins and ribosomal RNA processing or assembly factors have been covered by recent excellent reviews. In contrast, here we review our current knowledge of human diseases specifically associated with dysregulation of RNA polymerase I transcription and its associated regulatory apparatus, including some cases where this dysregulation is directly causative in disease. We will also provide insight into and discussion of possible therapeutic approaches to treat patients with dysregulated RNA polymerase I transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols. PMID:23153826

  2. Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice

    PubMed Central

    Wang, Joseph Che-Yen; Nickens, David G.; Lentz, Thomas B.; Loeb, Daniel D.; Zlotnick, Adam

    2014-01-01

    Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well. PMID:25034253

  3. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  4. Transcription factor trapping by RNA in gene regulatory elements

    PubMed Central

    Sigova, Alla A.; Abraham, Brian J.; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M.; Eric Guo, Yang; Jangi, Mohini; Giallourakis, Cosmas C.; Sharp, Phillip A.; Young, Richard A.

    2016-01-01

    Transcription factors (TFs) bind specific sequences in promoter-proximal and distal DNA elements in order to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA-binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF YY1 binds to both gene regulatory elements and also to their associated RNA species genome-wide. Reduced transcription of regulatory elements diminishes YY1 occupancy whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive feedback loop that contributes to the stability of gene expression programs. PMID:26516199

  5. Transcription factor trapping by RNA in gene regulatory elements.

    PubMed

    Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

    2015-11-20

    Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. PMID:26516199

  6. Replication of hepatitis delta virus RNA in mice after intramuscular injection of plasmid DNA.

    PubMed Central

    Polo, J M; Lim, B; Govindarajan, S; Lai, M M

    1995-01-01

    To establish a readily manipulable small-animal system for the study of human hepatitis delta virus (HDV) replication in vivo, plasmid DNAs containing head-to-tail cDNA dimers of HDV were inoculated intramuscularly into mice. Genomic-sense HDV RNA was detected in the injected muscle within 1 week and increased to substantial levels by week 7 postinjection. The intramuscular accumulation of HDV RNA was determined to be the direct result of viral RNA replication by three lines of evidence: (i) injected tissues also accumulated antigenomic-sense HDV RNA, (ii) plasmid DNA that synthesized primary transcripts of antigenomic sense also led to the accumulation of genomic-sense HDV RNA, and (iii) injection of a cDNA dimer defective in antigenomic RNA cleavage failed to produce detectable HDV RNA in muscle. Immunohistochemical analysis of injected muscle demonstrated the presence and nuclear localization of hepatitis delta antigen in myocytes. Finally, sera from DNA-injected mice contained antibodies specific for delta antigen, indicating the induction of an immunological response to the intracellularly expressed antigen. These findings demonstrated the ability of HDV RNA to replicate in skeletal muscle and provide a useful system for the study of HDV replication, delta antigen processing, and its presentation to the immune system in vivo. Furthermore, this system offers an efficiently replicating RNA as a potential vehicle for in vivo gene transfer. PMID:7609095

  7. mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

    PubMed Central

    Jerome, J F; Jaehning, J A

    1986-01-01

    We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708

  8. TATA-binding protein and transcription factor IIB induce transcript slipping during early transcription by RNA polymerase II.

    PubMed

    Gilman, Benjamin; Drullinger, Linda F; Kugel, Jennifer F; Goodrich, James A

    2009-04-01

    To better understand the mechanism of steps in early transcription by RNA polymerase II (pol II), we investigated the molecular determinants of transcript slipping within complexes assembled on promoters containing a pre-melted transcription bubble from -9 to +3. Transcript slippage occurs when an RNA transcript contains a repetitive sequence that allows the transcript to slip back and pair with the template strand of the DNA at a new register before transcription continues. We established the contributions of individual transcription factors, DNA elements, and RNA length to slipping on a heteroduplex template using a highly purified human pol II transcription system. We found that transcripts slip at a very defined point in the transcription reaction, after pol II completes phosphodiester bond synthesis at register +5. This point is set by the position of the polymerase active site on the DNA template, as opposed to the length of the transcript, as well as by a repetitive CUCU sequence that must occur from +2 to +5. Interestingly, slipping at this juncture is induced by TATA-binding protein and transcription factor IIB and requires a TATA box but not a transcription factor IIB recognition sequence. We propose a model in which transcribing complexes, upon completing phosphodiester bond synthesis at register +5, enter one of two branches in which they either complete productive synthesis of the transcript or undergo multiple rounds of transcript slipping. PMID:19193635

  9. Regulated post-transcriptional RNA cleavage diversifies the eukaryotic transcriptome

    PubMed Central

    Mercer, Tim R.; Dinger, Marcel E.; Bracken, Cameron P.; Kolle, Gabriel; Szubert, Jan M.; Korbie, Darren J.; Askarian-Amiri, Marjan E.; Gardiner, Brooke B.; Goodall, Gregory J.; Grimmond, Sean M.; Mattick, John S.

    2010-01-01

    The complexity of the eukaryotic transcriptome is generated by the interplay of transcription initiation, termination, alternative splicing, and other forms of post-transcriptional modification. It was recently shown that RNA transcripts may also undergo cleavage and secondary 5′ capping. Here, we show that post-transcriptional cleavage of RNA contributes to the diversification of the transcriptome by generating a range of small RNAs and long coding and noncoding RNAs. Using genome-wide histone modification and RNA polymerase II occupancy data, we confirm that the vast majority of intraexonic CAGE tags are derived from post-transcriptional processing. By comparing exonic CAGE tags to tissue-matched PARE data, we show that the cleavage and subsequent secondary capping is regulated in a developmental-stage- and tissue-specific manner. Furthermore, we find evidence of prevalent RNA cleavage in numerous transcriptomic data sets, including SAGE, cDNA, small RNA libraries, and deep-sequenced size-fractionated pools of RNA. These cleavage products include mRNA variants that retain the potential to be translated into shortened functional protein isoforms. We conclude that post-transcriptional RNA cleavage is a key mechanism that expands the functional repertoire and scope for regulatory control of the eukaryotic transcriptome. PMID:21045082

  10. A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing

    PubMed Central

    Diebel, Kevin W.; Claypool, David J.; van Dyk, Linda F.

    2014-01-01

    Canonical RNA polymerase III (pol III) type 2 promoters contain a single A and B box and are well documented for their role in tRNA and SINE transcription in eukaryotic cells. The genome of Murid herpesvirus 4 (MuHV-4) contains eight polycistronic tRNA-microRNA encoded RNA (TMER) genes that are transcribed from a RNA pol III type 2-like promoter containing triplicated A box elements. Here, we demonstrate that the triplicated A box sequences are required in their entirety to produce functional MuHV-4 miRNAs. We also identify that these RNA pol III type 2-like promoters are conserved in eukaryotic genomes. Human and mouse predicted tRNA genes containing these promoters also show enrichment of alternative RNA pol III transcription termination sequences and are predicted to give rise to longer tRNA primary transcripts. PMID:24747015

  11. Kinetic models of the interference of gene transcription to ncRNA and mRNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-06-01

    The experiments indicate that the transcription of genes into ncRNA can positively or negatively interfere with transcription into mRNA. We propose two kinetic models describing this effect. The first model is focused on the ncRNA-induced chromatin modification facilitating the transcription of the downstream gene into mRNA. The second model includes the competition between the transcription into ncRNA and the binding of activator to a regulatory site of the downstream gene transcribed into mRNA. Our analysis based on the mean-field kinetic equations and Monte Carlo simulations shows the likely dependences of the transcription rate on RNA polymerase concentration in situations with different rate-limiting steps. Our models can also be used to scrutinize the dependence of the transcription rate on other kinetic parameters. Our kinetic Monte Carlo simulations show that the first model predicts stochastic bursts in the mRNA formation provided that the transcription into ncRNA is slow, while the second model predicts in addition anti-phase stochastic bursts in the mRNA and ncRNA formation provided that that the protein attachment to and detachment from a regulatory site is slow.

  12. Basic mechanism of transcription by RNA polymerase II

    PubMed Central

    Svetlov, Vladimir; Nudler, Evgeny

    2012-01-01

    RNA polymerase II-like enzymes carry out transcription of genomes in Eukaryota, Archaea, and some viruses. They also exhibit fundamental similarity to RNA polymerases from bacteria, chloroplasts, and mitochondria. In this review we take an inventory of recent studiesilluminating different steps of basic transcription mechanism, likely common for most multi-subunit RNA polymerases. Through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible reaction pathway for the two-metal nucleotidyl transfer mechanism, and to evaluate the way catalysis can be linked to translocation in the mechano-chemical cycle catalyzed by RNA polymerase II. PMID:22982365

  13. Transcriptional and Post-Transcriptional Regulation of RNA Levels in Maize Mitochondria.

    PubMed Central

    Finnegan, PM; Brown, GG

    1990-01-01

    Relatively little is known about the mechanisms that govern the expression of plant mitochondrial genomes. We have addressed this problem by analyzing the transcriptional activity of different regions of the maize mitochondrial genome using both in vivo and isolated mitochondrial pulse-labeling systems. The regions examined included the protein genes atpA, atp6, and coxII, the 26S, 18S, and 5S rRNA genes, and sequences surrounding the rRNA genes. The rRNAs were found to be transcribed at rates fivefold to 10-fold higher than the protein genes. These rate differences are comparable with the differences in abundance of these species in the total or steady-state RNA population. Pulse-labeled RNA unexpectedly detected transcription of all regions examined, including approximately 21 kilobases of presumed noncoding sequences flanking the rRNA genes for which stable transcripts were not detected. The results obtained with RNA labeled for short pulses in vivo and in isolated mitochondria were similar, suggesting that isolated mitochondria provide a faithful run-on transcription assay. Our results indicate that the absence in total RNA of transcripts homologous to a given region of maize mitochondrial DNA does not necessarily exclude transcriptional activity of that region and that both transcriptional and post-transcriptional processes play important roles in maize mitochondrial genome expression. PMID:12354946

  14. Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a genomic-length template.

    PubMed Central

    Yokomori, K; Banner, L R; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, m

  15. Direct Characterization of Transcription Elongation by RNA Polymerase I.

    PubMed

    Ucuncuoglu, Suleyman; Engel, Krysta L; Purohit, Prashant K; Dunlap, David D; Schneider, David A; Finzi, Laura

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  16. Identification of protein coding regions in RNA transcripts

    PubMed Central

    Tang, Shiyuyun; Lomsadze, Alexandre; Borodovsky, Mark

    2015-01-01

    Massive parallel sequencing of RNA transcripts by next-generation technology (RNA-Seq) generates critically important data for eukaryotic gene discovery. Gene finding in transcripts can be done by statistical (alignment-free) as well as by alignment-based methods. We describe a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. The algorithm parameters are estimated by unsupervised training which makes unnecessary manually curated preparation of training sets. We demonstrate that (i) the unsupervised training is robust with respect to the presence of transcripts assembly errors and (ii) the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting translation initiation sites in modelled as well as in assembled transcripts compares favourably to other existing methods. PMID:25870408

  17. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  18. Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication

    PubMed Central

    Liu, Helene Minyi; Aizaki, Hideki; Machida, Keigo; Ou, J.-H. James; Lai, Michael M. C.

    2012-01-01

    Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome. PMID:22937067

  19. Divergent RNA transcription: a role in promoter unwinding?

    PubMed

    Naughton, Catherine; Corless, Samuel; Gilbert, Nick

    2013-01-01

    New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance. PMID:23863199

  20. The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA.

    PubMed

    Li, You; Yamane, Daisuke; Masaki, Takahiro; Lemon, Stanley M

    2015-09-01

    Hepatitis C virus (HCV) is an unusual RNA virus that has a striking capacity to persist for the remaining life of the host in the majority of infected individuals. In order to persist, HCV must balance viral RNA synthesis and decay in infected cells. In this Review, we focus on interactions between the positive-sense RNA genome of HCV and the host RNA-binding proteins and microRNAs, and describe how these interactions influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. Furthermore, we discuss how these processes affect hepatitis C pathogenesis and therapeutic strategies against HCV. PMID:26256788

  1. A modular strategy for engineering orthogonal chimeric RNA transcription regulators

    PubMed Central

    Takahashi, Melissa K.; Lucks, Julius B.

    2013-01-01

    Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry. PMID:23761434

  2. Dynamic visualization of transcription and RNA subcellular localization in zebrafish.

    PubMed

    Campbell, Philip D; Chao, Jeffrey A; Singer, Robert H; Marlow, Florence L

    2015-04-01

    Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes. PMID:25758462

  3. Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability

    PubMed Central

    Yan, Caifeng; Chen, Jinfeng; Chen, Nuoqi

    2016-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability. PMID:26935028

  4. Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.

    PubMed

    Pilsl, Michael; Merkl, Philipp E; Milkereit, Philipp; Griesenbeck, Joachim; Tschochner, Herbert

    2016-01-01

    RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S. cerevisiae were performed using either complex in vitro systems reconstituted from more or less purified fractions or genetic analyses. While providing many valuable insights these strategies did not always discriminate between direct and indirect effects in transcription initiation and termination, when mutated forms of Pol I subunits or transcription factors were investigated. Therefore, a well-defined minimal system was developed which allows to reconstitute highly efficient promoter-dependent Pol I initiation and termination of transcription. Transcription can be initiated at a minimal promoter only in the presence of recombinant core factor and extensively purified initiation competent Pol I. Addition of recombinant termination factors triggers transcriptional pausing and release of the ternary transcription complex. This minimal system represents a valuable tool to investigate the direct impact of (lethal) mutations in components of the initiation and termination complexes on the mechanism and regulation of rRNA synthesis. PMID:27576713

  5. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  6. Transcriptional termination in mammals: Stopping the RNA polymerase II juggernaut.

    PubMed

    Proudfoot, Nick J

    2016-06-10

    Terminating transcription is a highly intricate process for mammalian protein-coding genes. First, the chromatin template slows down transcription at the gene end. Then, the transcript is cleaved at the poly(A) signal to release the messenger RNA. The remaining transcript is selectively unraveled and degraded. This induces critical conformational changes in the heart of the enzyme that trigger termination. Termination can also occur at variable positions along the gene and so prevent aberrant transcript formation or intentionally make different transcripts. These may form multiple messenger RNAs with altered regulatory properties or encode different proteins. Finally, termination can be perturbed to achieve particular cellular needs or blocked in cancer or virally infected cells. In such cases, failure to terminate transcription can spell disaster for the cell. PMID:27284201

  7. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  8. Traffic into silence: endomembranes and post-transcriptional RNA silencing

    PubMed Central

    Kim, Yun Ju; Maizel, Alexis; Chen, Xuemei

    2014-01-01

    microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs. PMID:24668229

  9. Nascent transcription affected by RNA polymerase IV in Zea mays.

    PubMed

    Erhard, Karl F; Talbot, Joy-El R B; Deans, Natalie C; McClish, Allison E; Hollick, Jay B

    2015-04-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  10. Nascent Transcription Affected by RNA Polymerase IV in Zea mays

    PubMed Central

    Erhard, Karl F.; Talbot, Joy-El R. B.; Deans, Natalie C.; McClish, Allison E.; Hollick, Jay B.

    2015-01-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3ʹ-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  11. Dissecting chemical interactions governing RNA polymerase II transcriptional fidelity.

    PubMed

    Kellinger, Matthew W; Ulrich, Sébastien; Chong, Jenny; Kool, Eric T; Wang, Dong

    2012-05-16

    Maintaining high transcriptional fidelity is essential to life. For all eukaryotic organisms, RNA polymerase II (Pol II) is responsible for messenger RNA synthesis from the DNA template. Three key checkpoint steps are important in controlling Pol II transcriptional fidelity: nucleotide selection and incorporation, RNA transcript extension, and proofreading. Some types of DNA damage significantly reduce transcriptional fidelity. However, the chemical interactions governing each individual checkpoint step of Pol II transcriptional fidelity and the molecular basis of how subtle DNA base damage leads to significant losses of transcriptional fidelity are not fully understood. Here we use a series of "hydrogen bond deficient" nucleoside analogues to dissect chemical interactions governing Pol II transcriptional fidelity. We find that whereas hydrogen bonds between a Watson-Crick base pair of template DNA and incoming NTP are critical for efficient incorporation, they are not required for efficient transcript extension from this matched 3'-RNA end. In sharp contrast, the fidelity of extension is strongly dependent on the discrimination of an incorrect pattern of hydrogen bonds. We show that U:T wobble base interactions are critical to prevent extension of this mismatch by Pol II. Additionally, both hydrogen bonding and base stacking play important roles in controlling Pol II proofreading activity. Strong base stacking at the 3'-RNA terminus can compensate for loss of hydrogen bonds. Finally, we show that Pol II can distinguish very subtle size differences in template bases. The current work provides the first systematic evaluation of electrostatic and steric effects in controlling Pol II transcriptional fidelity. PMID:22509745

  12. piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire

    PubMed Central

    Senti, Kirsten-André; Jurczak, Daniel; Sachidanandam, Ravi; Brennecke, Julius

    2015-01-01

    PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. PMID:26302790

  13. piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire.

    PubMed

    Senti, Kirsten-André; Jurczak, Daniel; Sachidanandam, Ravi; Brennecke, Julius

    2015-08-15

    PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. PMID:26302790

  14. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements.

    PubMed

    Gosline, Sara J C; Gurtan, Allan M; JnBaptiste, Courtney K; Bosson, Andrew; Milani, Pamela; Dalin, Simona; Matthews, Bryan J; Yap, Yoon S; Sharp, Phillip A; Fraenkel, Ernest

    2016-01-12

    MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems. PMID:26748710

  15. The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes

    SciTech Connect

    Wang, Horng-Dar; Johnson, D.L.; Yuh, Chio-Hwa

    1995-12-01

    This report decribes the mechanism by which the hepatitis B virus X gene product induces RNA polymerase III genes. The RNA pol III transcription system serves as model for understanding the mechanism of X in the transactivation of cellular genes in both Drosophila and rat cell lines. 53 refs., 7 figs., 1 tab.

  16. Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle.

    PubMed Central

    Polo, J M; Jeng, K S; Lim, B; Govindarajan, S; Hofman, F; Sangiorgi, F; Lai, M M

    1995-01-01

    Hepatitis delta virus (HDV) is hepatotropic and frequently causes fulminant hepatitis in both human and nonhuman primate hosts. To understand the molecular basis of HDV tissue tropism and the mechanism of pathogenesis, transgenic mice in which replication-competent HDV dimeric RNA is expressed under the control of either liver-specific or universal transcriptional promoters were developed. The expressed RNA replicated efficiently in the liver and several tissues of nonhepatic origin. Surprisingly, maximal replication of HDV RNA occurred in skeletal muscle and was almost 100-fold greater than in the liver. These findings suggest that the hepatotropism of HDV is most likely a receptor-mediated restriction and that muscle-specific factors may facilitate HDV RNA replication. No evidence of cytopathology was apparent in most of the tissues examined, including the liver, supporting the contention that hepatocellular disease is not mediated by direct cytopathological effects associated with HDV RNA replication and gene expression. However, mild muscle atrophy in some of the transgenic mice was noted. Delta antigen was detected in the nuclei of myocytes. Only the small form, not the large form, of delta antigen was detected, suggesting that the RNA editing event which causes the conversion of delta antigen did not occur in transgenic mice. Furthermore, the 0.8-kb antigenomic RNA species, which is postulated to be the mRNA for delta antigen, was not detected in mice. The preferential replication of HDV RNA in skeletal muscle suggests that HDV RNA replication can be facilitated by certain muscle-specific factors. PMID:7609056

  17. Transcriptional regulation of human small nuclear RNA genes

    PubMed Central

    Jawdekar, Gauri W.; Henry, R. William

    2009-01-01

    The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated. PMID:18442490

  18. MicroRNA and Transcriptional Crosstalk in Myelinating Glia

    PubMed Central

    Svaren, John

    2014-01-01

    Several recent studies have addressed the important role of microRNA in regulation of differentiation of myelinating glia. While Schwann cells and oligodendrocytes in the peripheral and central nervous systems, respectively, exhibit significant morphological and regulatory differences, some aspects of transcriptional and microRNA regulation are shared between these two cell types. This review focuses on the intersection of microRNAs with transcriptional regulation in Schwann cell and oligodendrocyte differentiation. In particular, several microRNAs have been shown to modulate expression of critical transcription factors, and in turn, the regulation of microRNA expression is enmeshed within transcriptional networks that coordinate both coding gene and noncoding RNA profiles of myelinating cells. These hubs of regulation control both myelin gene expression as well as the cell cycle transitions of Schwann cells and oligodendrocytes as they terminally differentiate. In addition, some studies have begin to highlight the combinatorial effects of different microRNAs that establish the narrow range of gene regulation required for efficient and stable myelin formation. Overall, the integration of microRNA and transcriptional aspects will help elucidate mechanistic control of the myelination process. PMID:24979526

  19. Falling for the dark side of transcription: Nab2 fosters RNA polymerase III transcription

    PubMed Central

    Reuter, L. Maximilian; Sträßer, Katja

    2016-01-01

    ABSTRACT RNA polymerase III (RNAPIII) synthesizes diverse, small, non-coding RNAs with many important roles in the cellular metabolism. One of the open questions of RNAPIII transcription is whether and how additional factors are involved. Recently, Nab2 was identified as the first messenger ribonucleoprotein particle (mRNP) biogenesis factor with a function in RNAPIII transcription. PMID:27049816

  20. Falling for the dark side of transcription: Nab2 fosters RNA polymerase III transcription.

    PubMed

    Reuter, L Maximilian; Sträßer, Katja

    2016-05-26

    RNA polymerase III (RNAPIII) synthesizes diverse, small, non-coding RNAs with many important roles in the cellular metabolism. One of the open questions of RNAPIII transcription is whether and how additional factors are involved. Recently, Nab2 was identified as the first messenger ribonucleoprotein particle (mRNP) biogenesis factor with a function in RNAPIII transcription. PMID:27049816

  1. Numbers and Organization of RNA Polymerases, Nascent Transcripts, and Transcription Units in HeLa Nuclei

    PubMed Central

    Jackson, Dean A.; Iborra, Francisco J.; Manders, Erik M.M.; Cook, Peter R.

    1998-01-01

    Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15,000 transcripts were elongated by polymerase I, and ∼75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in ∼2400 sites with diameters of ∼80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a “cloud” of loops around a site; each site, or transcription “factory,” would contain ∼30 active polymerases and associated transcripts. PMID:9614191

  2. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    2007-04-06

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

  3. In vivo dynamics of RNA polymerase II transcription

    PubMed Central

    Darzacq, Xavier; Shav-Tal, Yaron; de Turris, Valeria; Brody, Yehuda; Shenoy, Shailesh M; Phair, Robert D; Singer, Robert H

    2016-01-01

    We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min−1, much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo. PMID:17676063

  4. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

    PubMed Central

    Kassavetis, G A; Riggs, D L; Negri, R; Nguyen, L H; Geiduschek, E P

    1989-01-01

    Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes. Images PMID:2668737

  5. The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

    PubMed

    Curtil, Claire; Enache, Liviu S; Radreau, Pauline; Dron, Anne-Gaëlle; Scholtès, Caroline; Deloire, Alexandre; Roche, Didier; Lotteau, Vincent; André, Patrice; Ramière, Christophe

    2014-03-01

    Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled. PMID:24297698

  6. A heteromeric transcription factor required for mammalian RNA polymerase II.

    PubMed Central

    Kitajima, S; Tanaka, Y; Kawaguchi, T; Nagaoka, T; Weissman, S M; Yasukochi, Y

    1990-01-01

    A general transcription factor, FC, essential for specific initiation of in vitro transcription by mammalian RNA polymerase II was identified and a procedure developed to purify it to near homogeneity from HeLa cell nuclei. Purified FC is composed of two polypeptides of apparent molecular masses 80 kDa and 30 kDa, on SDS-PAGE, and has a native size of 280 kDa estimated by gel filtration column. Both polypeptides were shown to be essential for reconstituting in vitro transcription activity. Biochemical analysis showed that the 80 kDa and 30 kDa components were present in a 1:1 molar ratio. FC was also demonstrated to interact directly or indirectly with purified RNA polymerase II. Similarities between FC and transcription factors reported by others from human, rat or Drosophila cells are discussed. Images PMID:2395645

  7. Two control regions for eukaryotic tRNA gene transcription.

    PubMed Central

    DeFranco, D; Schmidt, O; Söll, D

    1980-01-01

    Two Drosophila tRNALys genes with identical coding sequences were shown to transcribe with very different efficiences in nuclear extracts from Xenopus oocytes. The use of recombinant plasmids in which the 5'-flanking sequences of these genes were either "switched" or replaced by defined pBR322 sequences revealed two control regions for tRNA gene transcription. An internal control region comprising the mature tRNA coding sequence (and possibly its 3'-flanking sequences) is sufficient for transcription initiation, and an external control region comprising the 5'-flanking sequences represses this transcription. All transcripts have short leader sequences. Altered precursor tRNAs transcribed from truncated tRNALys genes (missing a single base pair in the acceptor stem) are not processed well in vitro. Images PMID:6774336

  8. Swinger RNA self-hybridization and mitochondrial non-canonical swinger transcription, transcription systematically exchanging nucleotides.

    PubMed

    Seligmann, Hervé

    2016-06-21

    Stem-loop hairpins punctuate mitochondrial post-transcriptional processing. Regulation of mitochondrial swinger transcription, transcription producing RNAs matching the mitogenome only assuming systematic exchanges between nucleotides (23 bijective transformations along 9 symmetric exchanges X<>Y, e.g. A<>G, and 14 asymmetric exchanges X>Y>Z>X, e.g. A>G>C>A) remains unknown. Does swinger RNA self-hybridization regulate swinger, as regular, transcription? Groups of 8 swinger transformations share canonical self-hybridization properties within each group, group 0 includes identity (regular) transcription. The human mitogenome has more stem-loop hairpins than randomized sequences for all groups. Group 2 transformations reveal complementarity of the light strand replication origin (OL) loop and a neighboring tRNA gene, detecting the longtime presumed OL/tRNA homology. Non-canonical G=U pairings in hairpins increases with swinger RNA detection. These results confirm biological relevancy of swinger-transformed DNA/RNA, independently of, and in combination with, previously detected swinger DNA/RNA and swinger peptides. Swinger-transformed mitogenomes include unsuspected multilayered information. PMID:27079465

  9. Recent advances in understanding transcription termination by RNA polymerase II

    PubMed Central

    Loya, Travis J.; Reines, Daniel

    2016-01-01

    Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery. A greater appreciation for the coupling of termination to RNA processing and metabolism has been recognized. In addition to serving as an essential step at the end of the transcription cycle, termination is involved in the regulation of a broad range of cellular processes. More recently, a role for termination in pervasive transcription, non-coding RNA regulation, genetic stability, chromatin remodeling, the immune response, and disease has come to the fore. Interesting mechanistic questions remain, but the last several years have resulted in significant insights into termination and an increasing recognition of its biological importance. PMID:27408690

  10. Impact of template backbone heterogeneity on RNA polymerase II transcription

    PubMed Central

    Xu, Liang; Wang, Wei; Zhang, Lu; Chong, Jenny; Huang, Xuhui; Wang, Dong

    2015-01-01

    Variations in the sugar component (ribose or deoxyribose) and the nature of the phosphodiester linkage (3′-5′ or 2′-5′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eukaryotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity remain elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quantitatively investigated pol II transcriptional behaviors through different template backbone variants. We revealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phosphodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA. PMID:25662224

  11. RNA interference directed to CDK2 inhibits HIV-1 transcription.

    PubMed

    Ammosova, Tatyana; Berro, Reem; Kashanchi, Fatah; Nekhai, Sergei

    2005-10-25

    We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target. PMID:16085226

  12. Patterns and regulation of ribosomal RNA transcription in Borrelia burgdorferi

    PubMed Central

    2011-01-01

    Background Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r) RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. Results RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNAAla); tRNAIle; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK)-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a relBbu deletion mutant unable to generate (p)ppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. Conclusions We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate. PMID:21251259

  13. Functionally related transcripts have common RNA motifs for specific RNA-binding proteins in trypanosomes

    PubMed Central

    Noé, Griselda; De Gaudenzi, Javier G; Frasch, Alberto C

    2008-01-01

    Background Trypanosomes mostly control gene expression by post-transcriptional events such as modulation of mRNA stability and translational efficiency. These mechanisms involve RNA-binding proteins (RBPs), which associate with transcripts to form messenger ribonucleoprotein (mRNP) complexes. Results In this study, we report the identification of mRNA targets for Trypanosoma cruzi U-rich RBP 1 (TcUBP1) and T. cruzi RBP 3 (TcRBP3), two phylogenetically conserved proteins among Kinetoplastids. Co-immunoprecipitated RBP-associated RNAs were extracted from mRNP complexes and binding of RBPs to several targets was confirmed by independent experimental assays. Analysis of target transcript sequences allowed the identification of different signature RNA motifs for each protein. Cis-elements for RBP binding have a stem-loop structure of 30–35 bases and are more frequently represented in the 3'-untranslated region (UTR) of mRNAs. Insertion of the correctly folded RNA elements to a non-specific mRNA rendered it into a target transcript, whereas substitution of the RNA elements abolished RBP interaction. In addition, RBPs competed for RNA-binding sites in accordance with the distribution of different and overlapping motifs in the 3'-UTRs of common mRNAs. Conclusion Functionally related transcripts were preferentially associated with a given RBP; TcUBP1 targets were enriched in genes encoding proteins involved in metabolism, whereas ribosomal protein-encoding transcripts were the largest group within TcRBP3 targets. Together, these results suggest coordinated control of different mRNA subsets at the post-transcriptional level by specific RBPs. PMID:19063746

  14. Post-transcriptional RNA Regulons Affecting Cell Cycle and Proliferation

    PubMed Central

    Blackinton, Jeff G.

    2014-01-01

    The cellular growth cycle is initiated and maintained by punctual, yet agile, regulatory events involving modifications of cell cycle proteins as well as coordinated gene expression to support cyclic checkpoint decisions. Recent evidence indicates that post-transcriptional partitioning of messenger RNA subsets by RNA-binding proteins help physically localize, temporally coordinate, and efficiently translate cell cycle proteins. This dynamic organization of mRNAs encoding cell cycle components contributes to the overall economy of the cell cycle consistent with the post-transcriptional RNA regulon model of gene expression. This review examines several recent studies demonstrating the coordination of mRNA subsets encoding cell cycle proteins during nuclear export and subsequent coupling to protein synthesis, and discusses evidence for mRNA coordination of p53 targets and the DNA damage response pathway. We consider how these observations may connect to upstream and downstream post-transcriptional coordination and coupling of splicing, export, localization, and translation. Published examples from yeast, nematode, insect, and mammalian systems are discussed, and we consider genetic evidence supporting the conclusion that dysregulation of RNA regulons may promote pathogenic states of growth such as carcinogenesis. PMID:24882724

  15. Basic Mechanisms in RNA Polymerase I Transcription of the Ribosomal RNA Genes

    PubMed Central

    Goodfellow, Sarah J.; Zomerdijk, Joost C. B. M.

    2013-01-01

    RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity. PMID:23150253

  16. Characterization of CRISPR RNA transcription by exploiting stranded metatranscriptomic data.

    PubMed

    Ye, Yuzhen; Zhang, Quan

    2016-07-01

    CRISPR-Cas systems are bacterial adaptive immune systems, each typically composed of a locus of cas genes and a CRISPR array of spacers flanked by repeats. Processed transcripts of CRISPR arrays (crRNAs) play important roles in the interference process mediated by these systems, guiding targeted immunity. Here we developed computational approaches that allow us to characterize the expression of many CRISPRs in their natural environments, using community RNA-seq (metatranscriptomic) data. By exploiting public human gut metatranscriptomic data sets, we studied the expression of 56 repeat-sequence types of CRISPRs, revealing that most CRISPRs are transcribed in one direction (producing crRNAs). In rarer cases, including a type II system associated with Bacteroides fragilis, CRISPRs are transcribed in both directions. Type III CRISPR-Cas systems were found in the microbiomes, but metatranscriptomic reads were barely found for their CRISPRs. We observed individual-level variation of the crRNA transcription, and an even greater transcription of a CRISPR from the antisense strand than the crRNA strand in one sample. The orientations of CRISPR expression implicated by metatranscriptomic data are largely in agreement with prior predictions for CRISPRs, with exceptions. Our study shows the promise of exploiting community RNA-seq data for investigating the transcription of CRISPR-Cas systems. PMID:27190232

  17. Bijective transformation circular codes and nucleotide exchanging RNA transcription.

    PubMed

    Michel, Christian J; Seligmann, Hervé

    2014-04-01

    The C(3) self-complementary circular code X identified in genes of prokaryotes and eukaryotes is a set of 20 trinucleotides enabling reading frame retrieval and maintenance, i.e. a framing code (Arquès and Michel, 1996; Michel, 2012, 2013). Some mitochondrial RNAs correspond to DNA sequences when RNA transcription systematically exchanges between nucleotides (Seligmann, 2013a,b). We study here the 23 bijective transformation codes ΠX of X which may code nucleotide exchanging RNA transcription as suggested by this mitochondrial observation. The 23 bijective transformation codes ΠX are C(3) trinucleotide circular codes, seven of them are also self-complementary. Furthermore, several correlations are observed between the Reading Frame Retrieval (RFR) probability of bijective transformation codes ΠX and the different biological properties of ΠX related to their numbers of RNAs in GenBank's EST database, their polymerization rate, their number of amino acids and the chirality of amino acids they code. Results suggest that the circular code X with the functions of reading frame retrieval and maintenance in regular RNA transcription, may also have, through its bijective transformation codes ΠX, the same functions in nucleotide exchanging RNA transcription. Associations with properties such as amino acid chirality suggest that the RFR of X and its bijective transformations molded the origins of the genetic code's machinery. PMID:24565870

  18. Characterization of CRISPR RNA transcription by exploiting stranded metatranscriptomic data

    PubMed Central

    Ye, Yuzhen; Zhang, Quan

    2016-01-01

    CRISPR–Cas systems are bacterial adaptive immune systems, each typically composed of a locus of cas genes and a CRISPR array of spacers flanked by repeats. Processed transcripts of CRISPR arrays (crRNAs) play important roles in the interference process mediated by these systems, guiding targeted immunity. Here we developed computational approaches that allow us to characterize the expression of many CRISPRs in their natural environments, using community RNA-seq (metatranscriptomic) data. By exploiting public human gut metatranscriptomic data sets, we studied the expression of 56 repeat-sequence types of CRISPRs, revealing that most CRISPRs are transcribed in one direction (producing crRNAs). In rarer cases, including a type II system associated with Bacteroides fragilis, CRISPRs are transcribed in both directions. Type III CRISPR–Cas systems were found in the microbiomes, but metatranscriptomic reads were barely found for their CRISPRs. We observed individual-level variation of the crRNA transcription, and an even greater transcription of a CRISPR from the antisense strand than the crRNA strand in one sample. The orientations of CRISPR expression implicated by metatranscriptomic data are largely in agreement with prior predictions for CRISPRs, with exceptions. Our study shows the promise of exploiting community RNA-seq data for investigating the transcription of CRISPR–Cas systems. PMID:27190232

  19. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  20. Transcription of Inflammatory Genes: Long Noncoding RNA and Beyond

    PubMed Central

    Carpenter, Susan

    2015-01-01

    The innate immune system must coordinate elaborate signaling pathways to turn on expression of hundreds of genes to provide protection against pathogens and resolve acute inflammation. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional on and off switches in response to distinct external stimuli. Three classes of transcription factors act together with transcriptional coregulators and chromatin-modifying complexes to control these programs. In addition, newer studies implicate long noncoding RNA (lncRNA) as additional regulators of these responses. LncRNAs promote, fine-tune, and restrain the inflammatory program. In this study, we provide an overview of gene regulation and the emerging importance of lncRNAs in the immune system. PMID:25250698

  1. RNA binding specificity of Ebola virus transcription factor VP30.

    PubMed

    Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

    2016-09-01

    The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences. PMID:27315567

  2. HuR, a protein implicated in oncogene and growth factor mRNA decay, binds to the 3' ends of hepatitis C virus RNA of both polarities.

    PubMed

    Spångberg, K; Wiklund, L; Schwartz, S

    2000-09-01

    To identify cellular factors that interact with hepatitis C virus RNA, cellular extracts were subjected to UV cross-linking to radiolabeled RNAs corresponding to the hepatitis C virus 5' and 3' untranslated regions of positive and negative polarities. Our results demonstrate that the U-rich region of the hepatitis C virus 3' untranslated region of the positive RNA strand is a hot spot for cellular RNA binding proteins. Two of these proteins were identified as the ELAV-like HuR protein and hnRNP C. Interestingly, HuR and hnRNP C also interacted with the 3' end of the RNA representing the negative strand of the HCV genome. The binding of HuR and hnRNP C to the 3' ends of the HCV RNAs of both negative and positive polarities suggests that HuR and hnRNP C may be involved in the transcription of the HCV RNA genome. Alternatively, they act by protecting the HCV RNAs from premature degradation by binding to their 3' ends. However, we were unable to demonstrate an effect on HCV RNA stability by the HuR protein. These interactions may be necessary for the establishment of chronic active infections that may develop into cirrhosis or hepatocellular carcinoma. PMID:10964780

  3. The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    PubMed Central

    Linnstaedt, Sarah D.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Casey, John L.

    2006-01-01

    RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification. PMID:16790843

  4. A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription.

    PubMed Central

    Zhang, X; Lai, M M

    1996-01-01

    Coronavirus transcription is a discontinuous process, involving interactions between a trans-acting leader and the intergenic transcription initiation sequences. A 9-nucleotide (nt) sequence (UUUAUAAAC), which is located immediately downstream of the leader at the 5' terminus of the mouse hepatitis virus (MHV) genomic RNA, contains a sequence resembling the consensus intergenic sequence (UCUAAAC). It has been shown previously that the presence of the 9-nt sequence facilitates leader RNA switching and may enhance subgenomic mRNA transcription. It is unclear how the 9-nt sequence exerts these functions. In this study, we inserted the 9-nt sequence into a defective interfering (DI) RNA reporter system and demonstrated that mRNA transcription could be initiated from the 9-nt sequence almost as efficiently as from the intergenic sequence between genes 6 and 7. Sequence analysis of the mRNAs showed that the 9-nt sequence served as a site of fusion between the leaders and mRNA. The transcription initiation function of the 9-nt sequence could not be substituted by other 5'-terminal sequences. When the entire 5'-terminal sequence, including four copies of the UCUAA sequence plus the 9-nt sequence, was present, transcription could be initiated from any of the UCUAA copies or the 9-nt sequence, resulting in different copy numbers of the UCUAA sequence and the deletion of the 9-nt sequence in some mRNAs. All of these heterogeneous RNA species were also detected from the 5'-terminal region of the viral genomic-length RNA in MHV-infected cells. These results thus suggest tha the heterogeneity of the copy number of UCUAA sequences at the 5' end, the deletion of the 9-nt sequence in viral and DI RNAs, and the leader RNA switching are the results of transcriptional initiation from the 9-nt site. They also show that an mRNA species (mRNA 1) that lacks the 9-nt sequence can be synthesized during MHV infection. Therefore, MHV genomic RNA replication and mRNA 1 transcription may be

  5. Deciphering Transcriptional Dynamics In Vivo by Counting Nascent RNA Molecules

    PubMed Central

    Choubey, Sandeep; Kondev, Jane; Sanchez, Alvaro

    2015-01-01

    Abstract Deciphering how the regulatory DNA sequence of a gene dictates its expression in response to intra and extracellular cues is one of the leading challenges in modern genomics. The development of novel single-cell sequencing and imaging techniques, as well as a better exploitation of currently available single-molecule imaging techniques, provides an avenue to interrogate the process of transcription and its dynamics in cells by quantifying the number of RNA polymerases engaged in the transcription of a gene (or equivalently the number of nascent RNAs) at a given moment in time. In this paper, we propose that measurements of the cell-to-cell variability in the number of nascent RNAs provide a mostly unexplored method for deciphering mechanisms of transcription initiation in cells. We propose a simple kinetic model of transcription initiation and elongation from which we calculate nascent RNA copy-number fluctuations. To demonstrate the usefulness of this approach, we test our theory against published nascent RNA data for twelve constitutively expressed yeast genes. Rather than transcription being initiated through a single rate limiting step, as it had been previously proposed, our single-cell analysis reveals the presence of at least two rate limiting steps. Surprisingly, half of the genes analyzed have nearly identical rates of transcription initiation, suggesting a common mechanism. Our analytical framework can be used to extract quantitative information about dynamics of transcription from single-cell sequencing data, as well as from single-molecule imaging and electron micrographs of fixed cells, and provides the mathematical means to exploit the quantitative power of these technologies. PMID:26544860

  6. RNA exosome regulated long non-coding RNA transcription controls super-enhancer activity

    PubMed Central

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N.; Bradner, James E.; Rabadan, Raul; Basu, Uttiya

    2015-01-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem (ES) cells by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3’ regulatory region super-enhancer function. CRISPRCas9 mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3’regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers, by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. PMID:25957685

  7. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    PubMed

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. PMID:25957685

  8. Structural insights into transcription initiation by RNA polymerase II

    PubMed Central

    Grünberg, Sebastian; Hahn, Steven

    2013-01-01

    Transcriptional regulation is one of the most important steps in control of cell identity, growth, differentiation and development. Many signaling pathways controlling these processes ultimately target the core transcription machinery that, for protein coding genes, consists of RNA polymerase II (Pol II) and the general transcription factors (GTFs). New studies on the structure and mechanism of the core assembly and how it interfaces with promoter DNA and coactivator complexes have given tremendous insight into early steps in the initiation process, genome-wide binding, and mechanisms conserved for all nuclear and archaeal Pols. Here we review recent developments in dissecting the architecture of the Pol II core machinery with a focus on early and regulated steps in transcription initiation. PMID:24120742

  9. Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients.

    PubMed Central

    Stoll-Becker, S; Repp, R; Glebe, D; Schaefer, S; Kreuder, J; Kann, M; Lampert, F; Gerlich, W H

    1997-01-01

    Hepatitis B virus (HBV) has been reported to exist in peripheral blood mononuclear cells (PBMC), but it is not clear whether it replicates there. A precondition for replication should be the formation of covalently closed viral DNA and transcription of all essential viral mRNAs. The mRNAs of HBV form a nested box with common 3' ends. In order to detect even low levels of potential replication, we developed a quantitative reverse transcription-PCR method for detection of a smaller HBV mRNA species in the presence of the larger ones. All three highly viremic patients tested so far had mRNAs for the large and the small surface proteins and the X protein of the virus within PBMC but not in the virus from their sera. Furthermore, we detected by PCR covalently closed viral DNA in their PBMC. These data suggest that HBV may be not only taken up but also replicated by mononuclear blood cells and that these cells may be an extrahepatic site of viral persistence. X mRNA was detected in the largest amount. Possibly, X protein interferes with functions of the mononuclear cells during the immune response against the virus. PMID:9188611

  10. Transcription of ribosomal RNA: the role of antitermination of RNA polymerase

    NASA Astrophysics Data System (ADS)

    Klumpp, Stefan; Hwa, Terry

    2007-03-01

    The genes encoding ribosomal RNA are transcribed at high rates of 1-2 transcripts per second. These high transcription rates are crucial to maintain the large concentration of ribosomes necessary in fast growing bacteria. To understand how transcription is regulated under these conditions, we developed a model for the traffic of transcribing RNA polymerases (RNAP). Our simulations show that the transcription rate is limited by the elongation stage of transcription rather than by transcript initiation. The maximal transcription rate is severly impaired by RNAP pausing with pause durations in the second range which is ubiquitous under single-molecule conditions. We propose that ribosomal antitermination reduces pauses and thereby increases the transcription rate. This idea is in quantitative agreement with the observed increase of the elongation rate due to antitermination and predicts a two-fold increase of the transcription rate. Antitermination must be highly efficient, since incomplete antitermination with only a few percent of non-antiterminated, i.e. slow, RNAPs completely abolishes its effect. This result suggests that rho-dependent termination may selectively terminate slow RNAPs.

  11. Interaction of nucleolin with ribosomal RNA genes and its role in RNA polymerase I transcription

    PubMed Central

    Cong, Rong; Das, Sadhan; Ugrinova, Iva; Kumar, Sanjeev; Mongelard, Fabien; Wong, Jiemin; Bouvet, Philippe

    2012-01-01

    Nucleolin is a multi-functional nucleolar protein that is required for ribosomal RNA gene (rRNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (RNAPI) transcription is not well understood. Nucleolin depletion results in an increase in the heterochromatin mark H3K9me2 and a decrease in H4K12Ac and H3K4me3 euchromatin histone marks in rRNA genes. ChIP-seq experiments identified an enrichment of nucleolin in the ribosomal DNA (rDNA) coding and promoter region. Nucleolin is preferentially associated with unmethylated rRNA genes and its depletion leads to the accumulation of RNAPI at the beginning of the transcription unit and a decrease in UBF along the coding and promoter regions. Nucleolin is able to affect the binding of transcription termination factor-1 on the promoter-proximal terminator T0, thus inhibiting the recruitment of TIP5 and HDAC1 and the establishment of a repressive heterochromatin state. These results reveal the importance of nucleolin for the maintenance of the euchromatin state and transcription elongation of rDNA. PMID:22859736

  12. RNA polymerase II ternary transcription complexes generated in vitro.

    PubMed Central

    Ackerman, S; Bunick, D; Zandomeni, R; Weinmann, R

    1983-01-01

    Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation. Images PMID:6193489

  13. Bacterial RNA polymerase can retain σ70 throughout transcription.

    PubMed

    Harden, Timothy T; Wells, Christopher D; Friedman, Larry J; Landick, Robert; Hochschild, Ann; Kondev, Jane; Gelles, Jeff

    2016-01-19

    Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription. PMID:26733675

  14. Dynamics of transcription and mRNA export

    PubMed Central

    Darzacq, Xavier; Singer, Robert H; Shav-Tal, Yaron

    2016-01-01

    Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists of converting the genetic code into a dynamic messenger RNA that will specify a required cellular function following translocation to the cytoplasm and translation. We now possess an in-depth understanding of the mechanism and regulations of transcription. By contrast, an understanding of the dynamics of an individual gene's expression in real time is just beginning to emerge following recent technological developments. PMID:15901505

  15. Hepatitis C virus RNA functionally sequesters miR-122

    PubMed Central

    Luna, Joseph M.; Scheel, Troels K. H.; Danino, Tal; Shaw, Katharina S.; Mele, Aldo; Fak, John J.; Nishiuchi, Eiko; Takacs, Constantin N.; Catanese, Maria Teresa; de Jong, Ype P.; Jacobson, Ira M.; Rice, Charles M.; Darnell, Robert B.

    2015-01-01

    Summary Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5′UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV. PMID:25768906

  16. Sex-related differences in murine hepatic transcriptional and proteomic responses to TCDD.

    PubMed

    Prokopec, Stephenie D; Watson, John D; Lee, Jamie; Pohjanvirta, Raimo; Boutros, Paul C

    2015-04-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities in most mammals. In rodents alone, there is a huge divergence in the toxicological response across species, as well as among different strains within a species. But there are also significant differences between males and females animals of a single strain. These differences are inconsistent across model systems: the severity of toxicity is greater in female rats than males, while male mice and guinea pigs are more sensitive than females. Because the specific events that underlie this difference remain unclear, we characterized the hepatic transcriptional response of adult male and female C57BL/6 mice to 500μg/kg TCDD at multiple time-points. The transcriptional profile diverged significantly between the sexes. Female mice demonstrated a large number of altered transcripts as early as 6h following treatment, suggesting a large primary response. Conversely, male animals showed the greatest TCDD-mediated response 144h following exposure, potentially implicating significant secondary responses. Nr1i3 was statistically significantly induced at all time-points in the sensitive male animals. This mRNA encodes the constitutive androstane receptor (CAR), a transcription factor involved in the regulation of xenobiotic metabolism, lipid metabolism, cell cycle and apoptosis. Surprisingly though, changes at the protein level (aside from the positive control, CYP1A1) were modest, with only FMO3 showing clear induction, and no genes with sex-differences. Thus, while male and female mice show transcriptional differences in their response to TCDD, their association with TCDD-induced toxicities remains unclear. PMID:25703434

  17. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

    PubMed Central

    Zhou, Ping; Cai, Qing; Chen, You-Chun; Zhang, Mu-Sen; Guan, Jian; Li, Xiao-Juan

    1997-01-01

    AIM: To investigate the existence and clinical significance of hepatitis C virus (HCV) RNA in the serum and peripheral blood mononuclear cells (PBMC) of patients with hepatitis C. METHODS: HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 patients with chronic hepatitis C (CHC). RESULTS: The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC (P < 0.01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti-HCV positive patients with normal alanine aminotransferase (ALT) levels (P < 0.01). HCV RNA was negative in the serum of two patients, but could be detected in PBMC. In 12 patients, anti HCV was negative while HCV RNA was positive in serum. CONCLUSION: (1) detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti-HCV negative hepatitis C; (2) liver damage in patients with hepatitis C might be correlated with HCV-viremia; (3) infection of PBMC by HCV might play an important role in chronic liver damage in patients with HCV and in the chronicity of its clinical course; and (4) PBMC might be considered as a “reservoir” for HCV. PMID:27041960

  18. In vitro and ex vivo delivery of short hairpin RNAs for control of hepatitis C viral transcript expression.

    PubMed

    Lonze, Bonnie E; Holzer, Horatio T; Knabel, Matthew K; Locke, Jayme E; DiCamillo, Gregory A; Karhadkar, Sunil S; Montgomery, Robert A; Sun, Zhaoli; Warren, Daniel S; Cameron, Andrew M

    2012-04-01

    Recurrent hepatitis C virus (HCV) infection is the most common cause of graft loss and patient death after transplantation for HCV cirrhosis. Transplant surgeons have access to uninfected explanted livers before transplantation and an opportunity to deliver RNA interference-based protective gene therapy to uninfected grafts. Conserved HCV sequences were used to design short interfering RNAs and test their ability to knockdown HCV transcript expression in an in vitro model, both by transfection and when delivered via an adeno-associated viral vector. In a rodent model of liver transplantation, portal venous perfusion of explanted grafts with an adeno-associated viral vector before transplantation produced detectable short hairpin RNA transcript expression after transplantation. The ability to deliver anti-HCV short hairpin RNAs to uninfected livers before transplantation and subsequent exposure to HCV offers hope for the possibility of preventing the currently inevitable subsequent infection of liver grafts with HCV. PMID:22508787

  19. Distinct roles of transcription factors TFIIIB and TFIIIC in RNA polymerase III transcription reinitiation.

    PubMed

    Ferrari, Roberto; Rivetti, Claudio; Acker, Joël; Dieci, Giorgio

    2004-09-14

    Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable preinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short ( approximately 100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high reinitiation rate. Transcription reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes. PMID:15347814

  20. The short transcript of Leishmania RNA virus is generated by RNA cleavage.

    PubMed Central

    MacBeth, K J; Patterson, J L

    1995-01-01

    Leishmania RNA virus 1 produces a short viral RNA transcript corresponding to the 5' end of positive-sense single-stranded RNAs both in virally infected cells and in in vitro polymerase assays. We hypothesized that this short transcript was generated via cleavage of full-length positive-sense single-stranded RNA. A putative cleavage site was mapped by primer extension analysis to nucleotide 320 of the viral genome. To address the hypothesis that the short transcript is generated via cleavage at this site, two substrate RNAs that possessed viral sequence encompassing the putative cleavage site were created. When incubated with sucrose-purified viral particles, these substrate RNAs were site-specifically cleaved. The cleavage site of the in vitro-processed RNAs also mapped to viral nucleotide 320. The short-transcript-generating activity could be specifically abolished by proteinase K treatment of sucrose-purified viral particles and high concentrations of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], suggesting that the activity requires a proteinaceous factor and possibly intact viral particles. The cleavage activity is directly associated with short-transcript-generating activity, since only viral particle preparations which were capable of generating the short transcript in polymerase assays were also active in the cleavage assay. Furthermore, the short-transcript-generating activity is independent of the viral polymerase's transcriptase and replicase activities. We present a working model whereby cleavage of Leishmaniavirus RNA transcripts functions in the maintenance of a low-level persistent infection. PMID:7745692

  1. Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

    PubMed

    Fishburn, James; Galburt, Eric; Hahn, Steven

    2016-06-17

    Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction. PMID:27129284

  2. Identification of two spliced leader RNA transcripts from Perkinsus marinus.

    PubMed

    Hearne, Jennifer L; Pitula, Joseph S

    2011-01-01

    Spliced leader (SL) variants are present in a number of mRNAs from Perkinsus marinus. Three different SLs of 22 nucleotides (nt) in length were previously reported, with a consensus sequence of (DCCGUAGCCAUYUUGGCUCAAG). A truncated 21 nt SL, with an (A) at nt-1 and a (U) deletion at nt-13, has also been reported. Here, we report an additional 21 nt SL variant with (G) at nt-1. Using cDNA analysis, a full-length SL RNA transcript was identified for both 21 nt SLs (SL2[A] and SL2[G]). This transcript is 81 nt in length and contains a conserved transcription termination sequence present in closely related dinoflagellates. PMID:21435079

  3. Prospects for inhibiting the post-transcriptional regulation of gene expression in hepatitis B virus.

    PubMed

    Chen, Augustine; Panjaworayan T-Thienprasert, Nattanan; Brown, Chris M

    2014-07-01

    There is a continuing need for novel antivirals to treat hepatitis B virus (HBV) infection, as it remains a major health problem worldwide. Ideally new classes of antivirals would target multiple steps in the viral lifecycle. In this review, we consider the steps in which HBV RNAs are processed, exported from the nucleus and translated. These are often overlooked steps in the HBV life-cycle. HBV, like retroviruses, incorporates a number of unusual steps in these processes, which use a combination of viral and host cellular machinery. Some of these unusual steps deserve a closer scrutiny. They may provide alternative targets to existing antiviral therapies, which are associated with increasing drug resistance. The RNA post-transcriptional regulatory element identified 20 years ago promotes nucleocytoplasmic export of all unspliced HBV RNAs. There is evidence that inhibition of this step is part of the antiviral action of interferon. Similarly, the structured RNA epsilon element situated at the 5' end of the polycistronic HBV pregenomic RNA also performs key roles during HBV replication. The pregenomic RNA, which is the template for translation of both the viral core and polymerase proteins, is also encapsidated and used in replication. This complex process, regulated at the epsilon element, also presents an attractive antiviral target. These RNA elements that mediate and regulate gene expression are highly conserved and could be targeted using novel strategies employing RNAi, miRNAs or aptamers. Such approaches targeting these functionally constrained genomic regions should avoid escape mutations. Therefore understanding these regulatory elements, along with providing potential targets, may also facilitate the development of other new classes of antiviral drugs. PMID:25009369

  4. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  5. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction

    PubMed Central

    Fritz, Brian R.; Jewett, Michael C.

    2014-01-01

    In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3′ modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

  6. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells

    SciTech Connect

    Yap, Chui Sun; Sinha, Rohit Anthony; Ota, Sho; Katsuki, Masahito; Yen, Paul Michael

    2013-11-01

    Highlights: •Thyroid hormone induces miR-181d expression in human hepatic cells and mouse livers. •Thyroid hormone downregulates CDX2 and SOAT2 (or ACAT2) via miR-181d. •miR-181d reduces cholesterol output from human hepatic cells. -- Abstract: Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

  7. Branch-PCR Constructed Stable shRNA Transcription Nanoparticles Have Long-Lasting RNAi Effect.

    PubMed

    Liu, Jianbing; Wang, Runyu; Ma, Dejun; Li, Yanyan; Wei, Chao; Xi, Zhen

    2016-06-01

    RNA interference (RNAi) is a cellular process for gene silencing. Because of poor serum stability, transferring dsRNA directly into the target cells is a challenge. We report a facile and universal strategy to construct short hairpin RNA (shRNA) transcription nanoparticles with multiple shRNA transcription templates by PCR with flexible branched primers (branch-PCR). Compared with conventional linear shRNA transcription templates, these shRNA transcription nanoparticles show excellent stability against digestion by exonuclease III. Importantly, we found that our highly stable shRNA transcription nanoparticles can also be transcribed and thus induce efficient and long-lasting RNAi with picomolar activity in living mammalian cells. These chemically well-defined branch-PCR-generated stable shRNA transcription nanoparticles might facilitate RNAi delivery with a long-lasting RNAi effects. PMID:26972444

  8. Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator.

    PubMed Central

    Kato, N; Lan, K H; Ono-Nita, S K; Shiratori, Y; Omata, M

    1997-01-01

    The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis. PMID:9343247

  9. A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

    PubMed

    Nechaev, Sergei; Yuzenkova, Yulia; Niedziela-Majka, Anita; Heyduk, Tomasz; Severinov, Konstantin

    2002-06-28

    Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination. PMID:12079331

  10. Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

    PubMed Central

    Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

    2006-01-01

    The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

  11. Hepatitis C virus RNA: molecular switches mediated by long-range RNA–RNA interactions?

    PubMed Central

    Shetty, Sumangala; Stefanovic, Snezana; Mihailescu, Mihaela Rita

    2013-01-01

    Multiple conserved structural cis-acting regulatory elements have been recognized both in the coding and untranslated regions (UTRs) of the hepatitis C virus (HCV) genome. For example, the cis-element 5BSL3.2 in the HCV-coding region has been predicted to use both its apical and internal loops to interact with the X RNA in the 3′-UTR, with the IIId domain in the 5′-UTR and with the Alt sequence in the coding region. Additionally, the X RNA region uses a palindromic sequence that overlaps the sequence required for the interaction with 5BSL3.2, to dimerize with another HCV genome. The ability of the 5BSL3.2 and X RNA regions to engage in multi-interactions suggests the existence of one or more molecular RNA switches which may regulate different steps of the HCV life cycle. In this study, we used biophysical methods to characterize the essential interactions of these HCV cis-elements at the molecular level. Our results indicate that X RNA interacts with 5BSL3.2 and another X RNA molecule by adopting two different conformations and that 5BSL3.2 engages simultaneously in kissing interactions using its apical and internal loops. Based on these results, we propose a mode of action for possible molecular switches involving the HCV RNA. PMID:23275555

  12. Characteristics and significance of intergenic polyadenylated RNA transcription in Arabidopsis.

    PubMed

    Moghe, Gaurav D; Lehti-Shiu, Melissa D; Seddon, Alex E; Yin, Shan; Chen, Yani; Juntawong, Piyada; Brandizzi, Federica; Bailey-Serres, Julia; Shiu, Shin-Han

    2013-01-01

    The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these "translated" ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from "junk" DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes. PMID:23132786

  13. piRNA-guided slicing specifies transcripts for Zucchini dependent, phased piRNA biogenesis

    PubMed Central

    Brennecke, Julius

    2016-01-01

    In animal gonads PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound piRNAs. These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3′ directed and phased process that, in the Drosophila germline, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endo-nucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3′ and 5′ ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3′ end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism where Zucchini plays a central role in defining piRNA 5′ and 3′ ends. PMID:25977553

  14. RNA transcription modulates phase transition-driven nuclear body assembly

    PubMed Central

    Berry, Joel; Weber, Stephanie C.; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford P.

    2015-01-01

    Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid–liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of “extranucleolar droplets” (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism. PMID:26351690

  15. RNA transcription modulates phase transition-driven nuclear body assembly.

    PubMed

    Berry, Joel; Weber, Stephanie C; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford P

    2015-09-22

    Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid-liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of "extranucleolar droplets" (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism. PMID:26351690

  16. Rescue of Mtp siRNA-induced hepatic steatosis by DGAT2 siRNA silencing.

    PubMed

    Tep, Samnang; Mihaila, Radu; Freeman, Alexander; Pickering, Victoria; Huynh, Felicia; Huyhn, Felicia; Tadin-Strapps, Marija; Stracks, Allison; Hubbard, Brian; Caldwell, Jeremy; Flanagan, W Michael; Kuklin, Nelly A; Ason, Brandon

    2012-05-01

    Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response. PMID:22355095

  17. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  18. RNA editing by G-nucleotide insertion in mumps virus P-gene mRNA transcripts.

    PubMed Central

    Paterson, R G; Lamb, R A

    1990-01-01

    A guanine nucleotide insertion event has been shown to occur at a specific site within mumps virus P-gene mRNA transcripts. The region of the mRNA containing the site expected to be used for RNA editing and the complementary portion of the genomic RNA were cloned, and their nucleotide sequences were obtained. The genomic RNA was found to possess six C residues at the insertion site, whereas 63% of the P-gene-specific mRNA transcripts were found to have from two to five G residues inserted at this position in the RNA. An unedited mRNA was shown to encode the mumps virus cysteine-rich protein V, and mRNA transcripts containing two and four inserted G residues were translated to yield the mumps virus P and I proteins, respectively. Images PMID:2166809

  19. Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.

    PubMed Central

    Liao, C L; Lai, M M

    1994-01-01

    We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The

  20. A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

    PubMed

    Atianand, Maninjay K; Hu, Wenqian; Satpathy, Ansuman T; Shen, Ying; Ricci, Emiliano P; Alvarez-Dominguez, Juan R; Bhatta, Ankit; Schattgen, Stefan A; McGowan, Jason D; Blin, Juliana; Braun, Joerg E; Gandhi, Pallavi; Moore, Melissa J; Chang, Howard Y; Lodish, Harvey F; Caffrey, Daniel R; Fitzgerald, Katherine A

    2016-06-16

    Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system. PMID:27315481

  1. RNA-Skim: a rapid method for RNA-Seq quantification at transcript level

    PubMed Central

    Zhang, Zhaojun; Wang, Wei

    2014-01-01

    Motivation: RNA-Seq technique has been demonstrated as a revolutionary means for exploring transcriptome because it provides deep coverage and base pair-level resolution. RNA-Seq quantification is proven to be an efficient alternative to Microarray technique in gene expression study, and it is a critical component in RNA-Seq differential expression analysis. Most existing RNA-Seq quantification tools require the alignments of fragments to either a genome or a transcriptome, entailing a time-consuming and intricate alignment step. To improve the performance of RNA-Seq quantification, an alignment-free method, Sailfish, has been recently proposed to quantify transcript abundances using all k-mers in the transcriptome, demonstrating the feasibility of designing an efficient alignment-free method for transcriptome quantification. Even though Sailfish is substantially faster than alternative alignment-dependent methods such as Cufflinks, using all k-mers in the transcriptome quantification impedes the scalability of the method. Results: We propose a novel RNA-Seq quantification method, RNA-Skim, which partitions the transcriptome into disjoint transcript clusters based on sequence similarity, and introduces the notion of sig-mers, which are a special type of k-mers uniquely associated with each cluster. We demonstrate that the sig-mer counts within a cluster are sufficient for estimating transcript abundances with accuracy comparable with any state-of-the-art method. This enables RNA-Skim to perform transcript quantification on each cluster independently, reducing a complex optimization problem into smaller optimization tasks that can be run in parallel. As a result, RNA-Skim uses <4% of the k-mers and <10% of the CPU time required by Sailfish. It is able to finish transcriptome quantification in <10 min per sample by using just a single thread on a commodity computer, which represents >100 speedup over the state-of-the-art alignment-based methods, while delivering

  2. Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis.

    PubMed

    Mohn, Fabio; Handler, Dominik; Brennecke, Julius

    2015-05-15

    In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends. PMID:25977553

  3. Unusual properties of adenovirus E2E transcription by RNA polymerase III.

    PubMed

    Huang, Wenlin; Flint, S J

    2003-04-01

    In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T(1) protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual. PMID:12634361

  4. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

    PubMed Central

    Bouloy, Michele; Plotch, Stephen J.; Krug, Robert M.

    1978-01-01

    Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro. Images PMID:283399

  5. Ontogeny of Hepatic Energy Metabolism Genes in Mice as Revealed by RNA-Sequencing

    PubMed Central

    Renaud, Helen J.; Cui, Yue Julia; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2014-01-01

    The liver plays a central role in metabolic homeostasis by coordinating synthesis, storage, breakdown, and redistribution of nutrients. Hepatic energy metabolism is dynamically regulated throughout different life stages due to different demands for energy during growth and development. However, changes in gene expression patterns throughout ontogeny for factors important in hepatic energy metabolism are not well understood. We performed detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6J mice were collected at twelve ages, including perinatal and postnatal time points (n = 3/age). The mRNA was quantified by RNA-Sequencing, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental age compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5–Day 5 (perinatal-enriched), Day 10–Day 20 (pre-weaning-enriched), and Day 25–Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed three patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally-enriched; glycogenolysis was the only pathway enriched during pre-weaning ages; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/adulthood. This study reveals novel findings such as the divergent expression of the fatty acid β-oxidation enzymes Acyl-CoA oxidase 1 and Carnitine palmitoyltransferase 1a, indicating a switch from mitochondrial to peroxisomal β-oxidation after weaning; as well as the dynamic ontogeny of genes implicated in obesity such as Stearoyl-CoA desaturase 1 and Elongation of very long chain fatty acids-like 3

  6. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    PubMed

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

  7. Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention

    PubMed Central

    Ahmed-Belkacem, Abdelhakim; Guichou, Jean-François; Brillet, Rozenn; Ahnou, Nazim; Hernandez, Eva; Pallier, Coralie; Pawlotsky, Jean-Michel

    2014-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key target for antiviral intervention. The goal of this study was to identify the binding site and unravel the molecular mechanism by which natural flavonoids efficiently inhibit HCV RdRp. Screening identified the flavonol quercetagetin as the most potent inhibitor of HCV RdRp activity. Quercetagetin was found to inhibit RdRp through inhibition of RNA binding to the viral polymerase, a yet unknown antiviral mechanism. X-ray crystallographic structure analysis of the RdRp-quercetagetin complex identified quercetagetin's binding site at the entrance of the RNA template tunnel, confirming its original mode of action. This antiviral mechanism was associated with a high barrier to resistance in both site-directed mutagenesis and long-term selection experiments. In conclusion, we identified a new mechanism for non-nucleoside inhibition of HCV RdRp through inhibition of RNA binding to the enzyme, a mechanism associated with broad genotypic activity and a high barrier to resistance. Our results open the way to new antiviral approaches for HCV and other viruses that use an RdRp based on RNA binding inhibition, that could prove to be useful in human, animal or plant viral infections. PMID:25053847

  8. Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

    PubMed Central

    Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

    2007-01-01

    Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription. PMID:17356699

  9. Sex-related differences in murine hepatic transcriptional and proteomic responses to TCDD

    SciTech Connect

    Prokopec, Stephenie D.; Watson, John D.; Lee, Jamie; Pohjanvirta, Raimo; Boutros, Paul C.

    2015-04-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities in most mammals. In rodents alone, there is a huge divergence in the toxicological response across species, as well as among different strains within a species. But there are also significant differences between males and females animals of a single strain. These differences are inconsistent across model systems: the severity of toxicity is greater in female rats than males, while male mice and guinea pigs are more sensitive than females. Because the specific events that underlie this difference remain unclear, we characterized the hepatic transcriptional response of adult male and female C57BL/6 mice to 500 μg/kg TCDD at multiple time-points. The transcriptional profile diverged significantly between the sexes. Female mice demonstrated a large number of altered transcripts as early as 6 h following treatment, suggesting a large primary response. Conversely, male animals showed the greatest TCDD-mediated response 144 h following exposure, potentially implicating significant secondary responses. Nr1i3 was statistically significantly induced at all time-points in the sensitive male animals. This mRNA encodes the constitutive androstane receptor (CAR), a transcription factor involved in the regulation of xenobiotic metabolism, lipid metabolism, cell cycle and apoptosis. Surprisingly though, changes at the protein level (aside from the positive control, CYP1A1) were modest, with only FMO3 showing clear induction, and no genes with sex-differences. Thus, while male and female mice show transcriptional differences in their response to TCDD, their association with TCDD-induced toxicities remains unclear. - Highlights: • Differences exist between the toxicity phenotypes to TCDD in male and female mice. • TCDD-mediated transcriptomic differences were identified between the sexes. • Resistant female mice displayed a large, early-onset, transcriptomic response.

  10. Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions

    PubMed Central

    Stojic, Lovorka; Niemczyk, Malwina; Orjalo, Arturo; Ito, Yoko; Ruijter, Anna Elisabeth Maria; Uribe-Lewis, Santiago; Joseph, Nimesh; Weston, Stephen; Menon, Suraj; Odom, Duncan T.; Rinn, John; Gergely, Fanni; Murrell, Adele

    2016-01-01

    Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription. PMID:26832224

  11. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  12. A putative ATPase mediates RNA transcription and capping in a dsRNA virus

    PubMed Central

    Yu, Xuekui; Jiang, Jiansen; Sun, Jingchen; Zhou, Z Hong

    2015-01-01

    mRNA transcription in dsRNA viruses is a highly regulated process but the mechanism of this regulation is not known. Here, by nucleoside triphosphatase (NTPase) assay and comparisons of six high-resolution (2.9–3.1 Å) cryo-electron microscopy structures of cytoplasmic polyhedrosis virus with bound ligands, we show that the large sub-domain of the guanylyltransferase (GTase) domain of the turret protein (TP) also has an ATP-binding site and is likely an ATPase. S-adenosyl-L-methionine (SAM) acts as a signal and binds the methylase-2 domain of TP to induce conformational change of the viral capsid, which in turn activates the putative ATPase. ATP binding/hydrolysis leads to an enlarged capsid for efficient mRNA synthesis, an open GTase domain for His217-mediated guanylyl transfer, and an open methylase-1 domain for SAM binding and methyl transfer. Taken together, our data support a role of the putative ATPase in mediating the activation of mRNA transcription and capping within the confines of the virus. DOI: http://dx.doi.org/10.7554/eLife.07901.001 PMID:26240998

  13. Regulation of Transcription from Two ssrS Promoters in 6S RNA Biogenesis

    PubMed Central

    Lee, Ji Young; Park, Hongmarn; Bak, Geunu; Kim, Kwang-sun; Lee, Younghoon

    2013-01-01

    ssrS-encoded 6S RNA is an abundant noncoding RNA that binds σ70-RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is transcribed from two tandem promoters, ssrS P1 and ssrS P2. Regulation of transcription from two ssrS promoters in 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA. PMID:23864284

  14. RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

    PubMed Central

    Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar

    2014-01-01

    Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV. PMID:24505321

  15. Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product

    PubMed Central

    Braun, Katherine A.; Dombek, Kenneth M.

    2015-01-01

    In the yeast Saccharomyces cerevisiae, the switch from respiratory metabolism to fermentation causes rapid decay of transcripts encoding proteins uniquely required for aerobic metabolism. Snf1, the yeast ortholog of AMP-activated protein kinase, has been implicated in this process because inhibiting Snf1 mimics the addition of glucose. In this study, we show that the SNF1-dependent ADH2 promoter, or just the major transcription factor binding site, is sufficient to confer glucose-induced mRNA decay upon heterologous transcripts. SNF1-independent expression from the ADH2 promoter prevented glucose-induced mRNA decay without altering the start site of transcription. SNF1-dependent transcripts are enriched for the binding motif of the RNA binding protein Vts1, an important mediator of mRNA decay and mRNA repression whose expression is correlated with decreased abundance of SNF1-dependent transcripts during the yeast metabolic cycle. However, deletion of VTS1 did not slow the rate of glucose-induced mRNA decay. ADH2 mRNA rapidly dissociated from polysomes after glucose repletion, and sequences bound by RNA binding proteins were enriched in the transcripts from repressed cells. Inhibiting the protein kinase A pathway did not affect glucose-induced decay of ADH2 mRNA. Our results suggest that Snf1 may influence mRNA stability by altering the recruitment activity of the transcription factor Adr1. PMID:26667037

  16. Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

    PubMed Central

    Kopp, K.; Gasiorowski, J. Z.; Chen, D.; Gilmore, R.; Norton, J. T.; Wang, C.; Leary, D. J.; Chan, E.K.L.; Dean, D. A.

    2007-01-01

    Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription. PMID:17108330

  17. The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C.

    PubMed Central

    Shindo, M; Di Bisceglie, A M; Akatsuka, T; Fong, T L; Scaglione, L; Donets, M; Hoofnagle, J H; Feinstone, S M

    1994-01-01

    Negative strands of the hepatitis C virus (HCV) genome (a positive-stranded RNA virus) have been found in a nuclease-resistant form in the serum of patients with HCV infections. We determined whether a complete negative-strand copy is present in the serum, whether the negative strand is particle-associated, and finally, whether it is virion-associated and encapsidated like the positive (genomic) strand. Isopyknic sucrose and cesium chloride density ultracentrifugation followed by a strand-specific reverse transcription-polymerase chain reaction on the collected fractions was performed to determine whether both positive and negative strands were associated with similar particles. Both strands comigrated to approximately the same density (1.11-1.16 g/cm3) in sucrose. After treatment of the plasma with detergent (0.1% Nonidet P-40) to remove the viral envelope and centrifugation on cesium chloride gradients, the positive strands shifted to a density of 1.35 g/cm3, and the negative strands were not detected. By using antibodies specific for the HCV core or envelope glycoproteins E1 or E2 coated onto the wells of a microtiter plate, it was possible to specifically bind HCV or viral cores to the solid phase. Pelleted virus particles were resuspended in either PBS or PBS with 0.1% Nonidet P-40 to expose the core. These pellets were then incubated in antibody-coated microtiter wells. RNA extracted from the bound and unbound fractions was tested for HCV RNA. The anti-core antibody was able to bind positive strands but not negative strands only in detergent-treated samples. In the nondetergent-treated pellets, the anti-E1 and -E2 bound the positive strand, but only anti-E1 bound the negative strands. These findings indicate that while both strands of HCV RNA can be detected in serum, the positive strand is encapsidated within the enveloped core, and the negative strand appears to be in a membrane particle associated with the viral envelope protein E1 but does not appear to

  18. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II[OPEN

    PubMed Central

    Qu, Jie; Ji, Shaoyi; Wallace, Andrew J.; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-01-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  19. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II.

    PubMed

    Wang, Ying; Qu, Jie; Ji, Shaoyi; Wallace, Andrew J; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-05-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP PSTVd replication in the nucleoplasm generates (-)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (-)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  20. Intrinsic noise in post-transcriptional gene regulation by small non-coding RNA.

    PubMed

    Jia, Ya; Liu, Wangheng; Li, Anbang; Yang, Lijian; Zhan, Xuan

    2009-07-01

    Small non-coding RNA (sRNA) plays very important role in the post transcriptional regulation in various organisms. In complex regulatory networks, highly significant relative fluctuations in RNAs copy numbers can not be neglected due to very small copy number of individual RNA molecules. Here we consider two simple regulation schemes, where one is single target gene regulated by a sRNA and the other is two target mRNAs (mRNA(R) and mRNA(T)) regulated by one sRNA. The Fano factor (a measure of the relative size of the internal fluctuations) formulae of RNA molecules in the post transcriptional regulation are theoretically derived by using of the Langevin theory. For single target gene regulated by a sRNA, it is shown that the intrinsic noise of both mRNA and sRNA approaches the bare Poissonian limit in the regimen of both target RNA silencing and surviving. However, the strong anti-correlation between the fluctuations of two components result in a large intrinsic fluctuations in the level of RNA molecules in the regimen of crossover. For two target mRNAs regulated by one sRNA, in the regimen of crossover, it is found that, with the increasing of transcription rate of target mRNA(T), the maximal intrinsic fluctuation of RNA molecules is shifted from sRNA to target mRNA(R), and then to target mRNA(T). The intrinsic noise intensity of target mRNA(R) is determined by both the transcriptional rate of itself and that of sRNA, and independent of the transcriptional rate of the other target mRNA(T). PMID:19403234

  1. Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone.

    PubMed

    Melia, Tisha; Hao, Pengying; Yilmaz, Feyza; Waxman, David J

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the

  2. Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone

    PubMed Central

    Melia, Tisha; Hao, Pengying; Yilmaz, Feyza

    2015-01-01

    Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the

  3. A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus.

    PubMed

    Beckel-Mitchener, A; Summers, J

    1997-10-01

    We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net. PMID:9311882

  4. Transcription inactivation through local refolding of the RNA polymerase structure

    SciTech Connect

    Belogurov, Georgiy A.; Vassylyeva, Marina N.; Sevostyanova, Anastasiya; Appleman, James R.; Xiang, Alan X.; Lira, Ricardo; Webber, Stephen E.; Klyuyev, Sergiy; Nudler, Evgeny; Artsimovitch, Irina; Vassylyev, Dmitry G.

    2009-02-12

    Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx - a desmethyl derivative of myxopyronin B - complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the {beta}'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex - the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

  5. RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts.

    PubMed

    Xiol, Jordi; Spinelli, Pietro; Laussmann, Maike A; Homolka, David; Yang, Zhaolin; Cora, Elisa; Couté, Yohann; Conn, Simon; Kadlec, Jan; Sachidanandam, Ravi; Kaksonen, Marko; Cusack, Stephen; Ephrussi, Anne; Pillai, Ramesh S

    2014-06-19

    Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons. PMID:24910301

  6. Structural basis of initial RNA polymerase II transcription

    PubMed Central

    Cheung, Alan C M; Sainsbury, Sarah; Cramer, Patrick

    2011-01-01

    During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II–DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site. The ITC-mimicking structures show that two invariant lysine residues anchor the 3′-proximal phosphate of short RNAs. Short DNA–RNA hybrids adopt a tilted conformation that excludes the +1 template nt from the active site. NTP binding induces complete DNA translocation and the standard hybrid conformation. Conserved NTP contacts indicate a universal mechanism of NTP selection. The essential residue Q1078 in the closed trigger loop binds the NTP 2′-OH group, explaining how the trigger loop couples catalysis to NTP selection, suppressing dNTP binding and DNA synthesis. PMID:22056778

  7. DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

    PubMed Central

    Coulombe, Benoit; Burton, Zachary F.

    1999-01-01

    A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. PMID:10357858

  8. Microprocessor mediates transcriptional termination in long noncoding microRNA genes

    PubMed Central

    Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J.; Jopling, Catherine L.

    2015-01-01

    MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with co-transcriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. While most miRNA are located within introns of protein coding genes, a substantial minority of miRNA originate from long non coding (lnc) RNA where transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) pathway, but instead use Microprocessor cleavage to terminate transcription. This Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. PMID:25730776

  9. Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro.

    PubMed Central

    Khanna, N C; Lakhani, S; Tewari, K K

    1991-01-01

    We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast

  10. Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

    PubMed Central

    Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

    2000-01-01

    Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640

  11. Transcriptional regulation mechanism mediated by miRNA-DNA•DNA triplex structure stabilized by Argonaute.

    PubMed

    Toscano-Garibay, Julia D; Aquino-Jarquin, Guillermo

    2014-11-01

    Transcription regulation depends on interactions between repressor or activator proteins with promoter sequences, while post-transcriptional regulation typically relies on microRNA (miRNA) interaction with sequences in 5' and 3'-Untranslated regions (UTRs) of messenger RNA (mRNA). However, several pieces of evidence suggest that miRNA:Argonaute (AGO) complexes may also suppress transcription through RNA interference (RNAi) components and epigenetic mechanisms. However, recent observations suggest that miRNA-induced transcriptional silencing could be exerted by an unknown mechanism independent of chromatin modifiers. The RNA-DNA•DNA triplex structure has emerged as an important RNA tertiary motif in which successive non-canonical base pairs form between a DNA-DNA duplex and a third strand. Frequently, promoters have Purine (PU)-rich tracts, and some Triplex-forming oligonucleotides (TFOs) targeting these regulatory regions have been shown to inhibit transcription selectively. Here, we summarize observations suggesting that miRNAs exert regulation over promoter regions through miRNA-DNA•DNA triplex structure formation stabilized by AGO proteins which represents a plausible model of RNA-mediated Transcriptional gene silencing (TGS). PMID:25086339

  12. Accurate transcription of homologous 5S rRNA and tRNA genes and splicing of tRNA in vitro by soluble extracts of Neurospora.

    PubMed Central

    Tyler, B M; Giles, N H

    1984-01-01

    We have developed soluble extracts from Neurospora crassa capable of accurately and efficiently transcribing homologous 5S rRNA and tRNA genes. The extracts also appear to quantitatively end-process and splice the primary tRNA transcripts. Although the extracts could not transcribe a heterologous (yeast) 5S rRNA gene, they did transcribe a yeast tRNALeu gene and slowly process the transcripts. In addition, we have developed a novel strategy for rapidly sequencing uniformly labelled RNAs using base-specific ribonucleases. We have used this procedure to verify the identity of the in vitro transcripts and processing products. Images PMID:6235482

  13. Digital inventory of Arabidopsis transcripts revealed by 61 RNA sequencing samples.

    PubMed

    Sun, Xiaoyong; Yang, Qiuying; Deng, Zhiping; Ye, Xinfu

    2014-10-01

    Alternative splicing is an essential biological process to generate proteome diversity and phenotypic complexity. Recent improvements in RNA sequencing accuracy and computational algorithms have provided unprecedented opportunities to examine the expression levels of Arabidopsis (Arabidopsis thaliana) transcripts. In this article, we analyzed 61 RNA sequencing samples from 10 totally independent studies of Arabidopsis and calculated the transcript expression levels in different tissues, treatments, developmental stages, and varieties. These data provide a comprehensive profile of Arabidopsis transcripts with single-base resolution. We quantified the expression levels of 40,745 transcripts annotated in The Arabidopsis Information Resource 10, comprising 73% common transcripts, 15% rare transcripts, and 12% nondetectable transcripts. In addition, we investigated diverse common transcripts in detail, including ubiquitous transcripts, dominant/subordinate transcripts, and switch transcripts, in terms of their expression and transcript ratio. Interestingly, alternative splicing was the highly enriched function for the genes related to dominant/subordinate transcripts and switch transcripts. In addition, motif analysis revealed that TC motifs were enriched in dominant transcripts but not in subordinate transcripts. These motifs were found to have a strong relationship with transcription factor activity. Our results shed light on the complexity of alternative splicing and the diversity of the contributing factors. PMID:25118256

  14. Circulating microRNA-196a as a candidate diagnostic biomarker for chronic hepatitis C

    PubMed Central

    LIU, BO; XIANG, YING; ZHANG, HENG-SHU

    2015-01-01

    Previous studies have demonstrated the inhibitory effect of microRNA (miR)-196a on hepatitis C virus (HCV) expression in human hepatocytes. However, the clinical implications of aberrant miR-196a expression and the application of circulating miR-196a in the diagnosis and management of chronic hepatitis C (CHC) require further investigation. The present study aimed to examine the possibility of using serum miR-196a as a biomarker for CHC. The Affymetrix miRNA array platform was used for miRNA expression profiling in adenovirus (Ad)-HCV core-infected (HepG2-HCV) and Ad-enhanced green fluorescence protein (EGFP)-infected HepG2 cells (HepG2-control). miR-196a downregulation and levels were analyzed using stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the sera of 43 patients with CHC and 22 healthy controls. A total of six miRNAs were identified as significantly different (≥1.5 fold; P≤0.05) between the two groups. Of note, significant miR-196a downregulation was observed in HepG2-HCV as compared with HepG2-EGFP. Furthermore, as compared with that of the healthy control group, serum miR-196a was demonstrated to be significantly lower in patients with CHC. In addition, analysis of the receiver operating characteristic (ROC) curve for serum miR-196a revealed an area under the ROC curve of 0.849 (95% confidence interval, 0.756–0.941; P<0.001) with 81.8% sensitivity and 76.7% specificity in discriminating chronic HCV infection from healthy controls at a cut-off value of 6.115×10‒5, demonstrating significant diagnostic value for CHC. However, no correlation was identified between serum miR-196a and alanine aminotransferase, aspartate aminotransferase or HCV-RNA. In conclusion, the present study identified circulating miR-196a as a specific and noninvasive candidate biomarker for the diagnosis of CHC. PMID:25738504

  15. Circulating microRNA-196a as a candidate diagnostic biomarker for chronic hepatitis C.

    PubMed

    Liu, Bo; Xiang, Ying; Zhang, Heng-Shu

    2015-07-01

    Previous studies have demonstrated the inhibitory effect of microRNA (miR)-196a on hepatitis C virus (HCV) expression in human hepatocytes. However, the clinical implications of aberrant miR-196a expression and the application of circulating miR-196a in the diagnosis and management of chronic hepatitis C (CHC) require further investigation. The present study aimed to examine the possibility of using serum miR-196a as a biomarker for CHC. The Affymetrix miRNA array platform was used for miRNA expression profiling in adenovirus (Ad)-HCV core-infected (HepG2-HCV) and Ad-enhanced green fluorescence protein (EGFP)-infected HepG2 cells (HepG2-control). miR-196a downregulation and levels were analyzed using stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the sera of 43 patients with CHC and 22 healthy controls. A total of six miRNAs were identified as significantly different (≥ 1.5 fold; P ≤ 0.05) between the two groups. Of note, significant miR-196a downregulation was observed in HepG2-HCV as compared with HepG2‑EGFP. Furthermore, as compared with that of the healthy control group, serum miR-196a was demonstrated to be significantly lower in patients with CHC. In addition, analysis of the receiver operating characteristic (ROC) curve for serum miR-196a revealed an area under the ROC curve of 0.849 (95% confidence interval, 0.756-0.941; P<0.001) with 81.8% sensitivity and 76.7% specificity in discriminating chronic HCV infection from healthy controls at a cut-off value of 6.115 x 10(-5), demonstrating significant diagnostic value for CHC. However, no correlation was identified between serum miR-196a and alanine aminotransferase, aspartate aminotransferase or HCV-RNA. In conclusion, the present study identified circulating miR-196a as a specific and noninvasive candidate biomarker for the diagnosis of CHC. PMID:25738504

  16. A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription.

    PubMed

    Eggert, F; Kath-Schorr, S

    2016-06-01

    Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base is functionalized with a cyclopropene moiety and used in transcription reactions to produce site-specifically cyclopropene-modified RNA molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction with a selected tetrazine-fluorophore conjugate is demonstrated. PMID:27181840

  17. Effect of epinephrine and serotonin on hepatic poly(A)/sup +/ RNA synthesis

    SciTech Connect

    Roy, A.K.; Bhadra, R.; Datta, A.G.

    1985-06-17

    In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of /sup 14/C-orotic acid into Poly(A)/sup +/ RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments. 21 references, 1 figure, 3 tables.

  18. Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis.

    PubMed

    Zou, Yongkang; Gong, Ning; Cui, Ying; Wang, Xiaojuan; Cui, Anfang; Chen, Qi; Jiao, Tao; Dong, Xueyu; Yang, Hao; Zhang, Shutian; Fang, Fude; Chang, Yongsheng

    2015-12-18

    Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis. PMID:26504089

  19. Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator

    PubMed Central

    Severinov, Konstantin; Minakhin, Leonid; Sekine, Shun-ichi; Lopatina, Anna; Yokoyama, Shigeyuki

    2014-01-01

    Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue. PMID:25105059

  20. Preparation of Chromatin Templates to Study RNA Polymerase I Transcription In Vitro.

    PubMed

    Längst, Gernot

    2016-01-01

    Cellular DNA is packaged into chromatin, which is the substrate of all DNA-dependent processes in eukaryotes. The regulation of chromatin requires specialized enzyme activities to allow the access of sequence-specific binding proteins and RNA polymerases. In order to dissect chromatin-dependent features of transcription regulation in detail, in vitro systems to generate defined chromatin templates for transcription are required. I present a protocol that allows the assembly of nucleosomes on ribosomal RNA (rRNA) minigenes by salt gradient dialysis and subsequent sucrose gradient centrifugation. This procedure yields high nucleosome occupancy and high dynamic response in subsequent transcriptional analysis. It provides an invaluable tool to study rRNA gene transcription, as transcription on free DNA is clearly different from the more in vivo-like transcription on reconstituted chromatin templates. PMID:27576714

  1. Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters.

    PubMed

    Chen, Yun; Pai, Athma A; Herudek, Jan; Lubas, Michal; Meola, Nicola; Järvelin, Aino I; Andersson, Robin; Pelechano, Vicent; Steinmetz, Lars M; Jensen, Torben Heick; Sandelin, Albin

    2016-09-01

    Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes. PMID:27455346

  2. A yeast transcription system for the 5S rRNA gene.

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin. Images PMID:7145700

  3. New pseudodimeric aurones as palm pocket inhibitors of Hepatitis C virus RNA-dependent RNA polymerase.

    PubMed

    Meguellati, Amel; Ahmed-Belkacem, Abdelhakim; Nurisso, Alessandra; Yi, Wei; Brillet, Rozenn; Berqouch, Nawel; Chavoutier, Laura; Fortuné, Antoine; Pawlotsky, Jean-Michel; Boumendjel, Ahcène; Peuchmaur, Marine

    2016-06-10

    The NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme for Hepatitis C Virus (HCV) replication. In addition to the catalytic site, this enzyme is characterized by the presence of at least four allosteric pockets making it an interesting target for development of inhibitors as potential anti-HCV drugs. Based on a previous study showing the potential of the naturally occurring aurones as inhibitors of NS5B, we pursued our efforts to focus on pseudodimeric aurones that have never been investigated so far. Hence, 14 original compounds characterized by the presence of a spacer between the benzofuranone moieties were synthesized and investigated as HCV RdRp inhibitors by means of an in vitro assay. The most active inhibitor, pseudodimeric aurone 4, induced high inhibition activity (IC50 = 1.3 μM). Mutagenic and molecular modeling studies reveal that the binding site for the most active derivatives probably is the palm pocket I instead of the thumb pocket I as for the monomeric derivatives. PMID:27017550

  4. Long non-coding RNA expression profiles of hepatitis C virus-related dysplasia and hepatocellular carcinoma

    PubMed Central

    Li, Ming; Wu, Liangcai; Yang, Xiaobo; Wan, Xueshuai; Wang, Anqiang; Zhang, Michael Q.; Sang, Xinting; Zhao, Haitao

    2015-01-01

    Recently, long non-coding RNAs (lncRNAs) were found to be implicated in cancer progression. However, the contributions of lncRNAs to Hepatitis C virus-related hepatocellular carcinoma (HCC) remain largely unknown. Here, we characterized lncRNA expression in 73 tissue samples from several different developmental stages of HCV-related hepatocarcinogenesis by repurposing microarray data sets. We found that the expression of 7 lncRNAs in preneoplastic lesions and HCC was significantly different. Among these significantly differently expressed lncRNAs, the lncRNA LINC01419 transcripts were expressed at higher levels in early stage HCC compared to dysplasia and as compared with early stage HCC, lncRNA AK021443 level increase in advanced stage HCC while lncRNA AF070632 level decrease in advanced stage HCC. Using quantitative real-time reverse-transcription PCR, we validated that LINC01419 was significantly overexpressed in HBV-related and HCV-related HCC when compared with matched non-tumor liver tissues. Moreover, functional predictions suggested that LINC01419 and AK021443 regulate cell cycle genes, whereas AF070632 is associated with cofactor binding, oxidation-reduction and carboxylic acid catabolic process. These findings provide the first large-scale survey of lncRNAs associated with the development of hepatocarcinogenesis and may offer new diagnostic biomarkers and therapeutic targets for HCV-related HCC. PMID:26540467

  5. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

    PubMed Central

    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. PMID:25654332

  6. Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase.

    PubMed

    Parks, Adam R; Court, Carolyn; Lubkowska, Lucyna; Jin, Ding J; Kashlev, Mikhail; Court, Donald L

    2014-05-01

    Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5' end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination. PMID:24711367

  7. Bacteriophage λ N protein inhibits transcription slippage by Escherichia coli RNA polymerase

    PubMed Central

    Parks, Adam R.; Court, Carolyn; Lubkowska, Lucyna; Jin, Ding J.; Kashlev, Mikhail; Court, Donald L.

    2014-01-01

    Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination. PMID:24711367

  8. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  9. Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice.

    PubMed

    Dawson, Paul Anthony; Gardiner, Brooke; Grimmond, Sean; Markovich, Daniel

    2006-07-12

    Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout (Nas1-/-) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1-/- mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased (n = 51) or decreased (n = 41) in Nas1-/- mice when compared with Nas1+/+ mice. The most upregulated transcript levels in Nas1-/- mice were found for the sulfotransferase genes, Sult3a1 (approximately 500% increase) and Sult2a2 (100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes (70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated (> or = 30% increase) in Nas1-/- mice. In addition, Nas1-/- mice exhibited increased levels of hepatic lipid (approximately 16% increase), serum cholesterol (approximately 20% increase), and low-density lipoprotein (approximately 100% increase) and reduced hepatic glycogen (approximately 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1-/- mouse and provide new insights into the metabolic state of the liver in Nas1-/- mice. PMID:16621889

  10. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  11. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus

    PubMed Central

    Xu, Yili; Barauskas, Ona; Perry, Jason K.; Ahmadyar, Shekeba; Stepan, George; Yu, Helen; Babusis, Darius; Park, Yeojin; McCutcheon, Krista; Perron, Michel; Schultz, Brian E.; Sakowicz, Roman; Ray, Adrian S.

    2015-01-01

    Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing. PMID:26596942

  12. RNA-Sequencing Quantification of Hepatic Ontogeny and Tissue Distribution of mRNAs of Phase II Enzymes in Mice

    PubMed Central

    Gunewardena, Sumedha; Cui, Julia Y.; Yoo, Byunggil; Zhong, Xiao-bo; Klaassen, Curtis D.

    2013-01-01

    Phase II conjugating enzymes play key roles in the metabolism of xenobiotics. In the present study, RNA sequencing was used to elucidate hepatic ontogeny and tissue distribution of mRNA expression of all major known Phase II enzymes, including enzymes involved in glucuronidation, sulfation, glutathione conjugation, acetylation, methylation, and amino acid conjugation, as well as enzymes for the synthesis of Phase II cosubstrates, in male C57BL/6J mice. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. Many of these Phase II enzymes were expressed at much higher levels in adult livers than in perinatal livers, such as Ugt1a6b, -2a3, -2b1, -2b5, -2b36, -3a1, and -3a2; Gsta1, -m1, -p1, -p2, and -z1; mGst1; Nat8; Comt; Nnmt; Baat; Ugdh; and Gclc. In contrast, hepatic mRNA expression of a few Phase II enzymes decreased during postnatal liver development, such as mGst2, mGst3, Gclm, and Mat2a. Hepatic expression of certain Phase II enzymes peaked during the adolescent stage, such as Ugt1a1, Sult1a1, Sult1c2, Sult1d1, Sult2as, Sult5a1, Tpmt, Glyat, Ugp2, and Mat1a. In adult mice, the total transcripts for Phase II enzymes were comparable in liver, kidney, and small intestine; however, individual Phase II enzymes displayed marked tissue specificity among the three organs. In conclusion, this study unveils for the first time developmental changes in mRNA abundance of all major known Phase II enzymes in mouse liver, as well as their tissue-specific expression in key drug-metabolizing organs. The age- and tissue-specific expression of Phase II enzymes indicate that the detoxification of xenobiotics is highly regulated by age and cell type. PMID:23382457

  13. A Novel Peroxisome Proliferator Response Element Modulates Hepatic Low Density Lipoprotein Receptor Gene Transcription in Response to PPARδ Activation

    PubMed Central

    Shende, Vikram R.; Singh, Amar Bahadur; Liu, Jingwen

    2016-01-01

    The hepatic expression of LDLR gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative PPAR-response element (PPRE) sequence motif located at −768 to −752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin mediated transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression. PMID:26443862

  14. Probing the transcription mechanisms of reovirus cores with molecules that alter RNA duplex stability.

    PubMed

    Demidenko, Alexander A; Nibert, Max L

    2009-06-01

    The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps. PMID:19297468

  15. The Potential Role of Th9 Cell Related Cytokine and Transcription Factors in Patients with Hepatic Alveolar Echinococcosis

    PubMed Central

    Tuxun, Tuerhongjiang; Apaer, Shadike; Ma, Hai-Zhang; Zhang, Heng; Aierken, Amina; Lin, Ren-Yong; Wen, Hao

    2015-01-01

    Human alveolar echinococcosis (AE) is a lethal parasitic infectious disease which may lead to liver failure if left untreated. It is caused by the larval stage of the fox tapeworm Echinococcus multilocularis and usually develops a substantial infiltrative occupation in solid organs. During the infection, T helper subsets are known to play crucial role in crosstalk between the parasite and human host. Th9 cells, a new member of CD4+ T cell family which is characterized by its specific cytokine IL-9 and transcription factors PU.1 and IRF-4, have been known recently to have a critical role in allergic diseases, and cancers as well as the parasitic infection. To assess the potential role of Th9 cells during the infection, the mRNA levels of IL-9, PU.1, and IRF-4 both in peripheral blood mononuclear cells and in liver tissues were, respectively, detected by using real-time PCR. The plasma concentration levels of IL-9 were detected by using enzyme linked immunosorbent assay (ELISA). Th9 related cytokine IL-9 and transcription factors PU.1 and IRF-4 mRNA levels elevated both in PBMCs, and in hepatic lesion and paralesion tissues in AE patients. This may facilitate the infiltrative growth of the parasite and its persistence in human host. PMID:26509179

  16. The Potential Role of Th9 Cell Related Cytokine and Transcription Factors in Patients with Hepatic Alveolar Echinococcosis.

    PubMed

    Tuxun, Tuerhongjiang; Apaer, Shadike; Ma, Hai-Zhang; Zhang, Heng; Aierken, Amina; Lin, Ren-Yong; Wen, Hao

    2015-01-01

    Human alveolar echinococcosis (AE) is a lethal parasitic infectious disease which may lead to liver failure if left untreated. It is caused by the larval stage of the fox tapeworm Echinococcus multilocularis and usually develops a substantial infiltrative occupation in solid organs. During the infection, T helper subsets are known to play crucial role in crosstalk between the parasite and human host. Th9 cells, a new member of CD4(+) T cell family which is characterized by its specific cytokine IL-9 and transcription factors PU.1 and IRF-4, have been known recently to have a critical role in allergic diseases, and cancers as well as the parasitic infection. To assess the potential role of Th9 cells during the infection, the mRNA levels of IL-9, PU.1, and IRF-4 both in peripheral blood mononuclear cells and in liver tissues were, respectively, detected by using real-time PCR. The plasma concentration levels of IL-9 were detected by using enzyme linked immunosorbent assay (ELISA). Th9 related cytokine IL-9 and transcription factors PU.1 and IRF-4 mRNA levels elevated both in PBMCs, and in hepatic lesion and paralesion tissues in AE patients. This may facilitate the infiltrative growth of the parasite and its persistence in human host. PMID:26509179

  17. RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing

    PubMed Central

    Djupedal, Ingela; Portoso, Manuela; Spåhr, Henrik; Bonilla, Carolina; Gustafsson, Claes M.; Allshire, Robin C.; Ekwall, Karl

    2005-01-01

    Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing. PMID:16204182

  18. Transcription profile of boar spermatozoa as revealed by RNA-sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput RNA sequencing (RNA-Seq) overcomes the limitations of the current hybridization-based techniques to detect the actual pool of RNA transcripts in spermatozoa. The application of this technology in livestock can speed the discovery of potential predictors of male fertility. As a first ...

  19. Regulation of human hepatic hydroxysteroid sulfotransferase gene expression by the peroxisome proliferator-activated receptor alpha transcription factor.

    PubMed

    Fang, Hai-Lin; Strom, Stephen C; Cai, Hongbo; Falany, Charles N; Kocarek, Thomas A; Runge-Morris, Melissa

    2005-04-01

    Human hydroxysteroid sulfotransferase or (HUMAN)SULT2A1 catalyzes the sulfonation of procarcinogen xenobiotics, hydroxysteroids, and bile acids and plays a dynamic role in hepatic cholesterol homeostasis. The treatment of primary cultured human hepatocytes with a peroxisome proliferator-activated receptor alpha (PPARalpha)-activating concentration of ciprofibrate (10(-) (4) M) increased (HUMAN)SULT2A1 mRNA, immunoreactive protein, and enzymatic activity levels by approximately 2-fold. By contrast, expression of (RAT)SULT2A3, the rat counterpart to (HUMAN)SULT2A1, was induced by treatment of primary hepatocyte cultures with an activator of the pregnane X receptor, but not PPARalpha. In HepG2 cells, transient transfection analyses of luciferase reporter constructs containing upstream regions of the (HUMAN)SULT2A1 gene implicated a candidate peroxisome proliferator response element (PPRE) at nucleotides (nt) -5949 to -5929 relative to the transcription start site. Site-directed mutagenesis and electrophoretic mobility shift assay studies confirmed that this distal PPRE (dPPRE), a direct repeat nuclear receptor motif containing one intervening nt, represented a functional PPRE. Chromatin immunoprecipitation analysis indicated that the (HUMAN)SULT2A1 dPPRE was also a functional element in the context of the human genome. These data support a major role for the PPARalpha transcription factor in the regulation of hepatic (HUMAN)SULT2A1. Results also indicate that important species differences govern the transactivation of SULT2A gene transcription by nuclear receptors. PMID:15635043

  20. Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA

    PubMed Central

    Brown, Timothy A.; Tkachuk, Ariana N.; Clayton, David A.

    2015-01-01

    Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein. PMID:26545237

  1. The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

    PubMed

    Ojha, Sandeep; Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation. PMID:24200639

  2. Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome.

    PubMed

    Rozovski, Uri; Hazan-Halevy, Inbal; Calin, George; Harris, David; Li, Ping; Liu, Zhiming; Keating, Michael J; Estrov, Zeev

    2016-01-01

    While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells. PMID:27341356

  3. A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

    PubMed Central

    Einav, Shirit; Elazar, Menashe; Danieli, Tsafi; Glenn, Jeffrey S.

    2004-01-01

    Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies. PMID:15452248

  4. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    PubMed

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. PMID:25881991

  5. PTRF/Cavin-1 promotes efficient ribosomal RNA transcription in response to metabolic challenges.

    PubMed

    Liu, Libin; Pilch, Paul F

    2016-01-01

    Ribosomal RNA transcription mediated by RNA polymerase I represents the rate-limiting step in ribosome biogenesis. In eukaryotic cells, nutrients and growth factors regulate ribosomal RNA transcription through various key factors coupled to cell growth. We show here in mature adipocytes, ribosomal transcription can be acutely regulated in response to metabolic challenges. This acute response is mediated by PTRF (polymerase I transcription and release factor, also known as cavin-1), which has previously been shown to play a critical role in caveolae formation. The caveolae-independent rDNA transcriptional role of PTRF not only explains the lipodystrophy phenotype observed in PTRF deficient mice and humans, but also highlights its crucial physiological role in maintaining adipocyte allostasis. Multiple post-translational modifications of PTRF provide mechanistic bases for its regulation. The role of PTRF in ribosomal transcriptional efficiency is likely relevant to many additional physiological situations of cell growth and organismal metabolism. PMID:27528195

  6. RNA Structural Elements of Hepatitis C Virus Controlling Viral RNA Translation and the Implications for Viral Pathogenesis

    PubMed Central

    Piñeiro, David; Martinez-Salas, Encarnación

    2012-01-01

    Hepatitis C virus (HCV) genome multiplication requires the concerted action of the viral RNA, host factors and viral proteins. Recent studies have provided information about the requirement of specific viral RNA motifs that play an active role in the viral life cycle. RNA regulatory motifs controlling translation and replication of the viral RNA are mostly found at the 5' and 3' untranslated regions (UTRs). In particular, viral protein synthesis is under the control of the internal ribosome entry site (IRES) element, a complex RNA structure located at the 5'UTR that recruits the ribosomal subunits to the initiator codon. Accordingly, interfering with this RNA structural motif causes the abrogation of the viral cycle. In addition, RNA translation initiation is modulated by cellular factors, including miRNAs and RNA-binding proteins. Interestingly, a RNA structural motif located at the 3'end controls viral replication and establishes long-range RNA-RNA interactions with the 5'UTR, generating functional bridges between both ends on the viral genome. In this article, we review recent advances on virus-host interaction and translation control modulating viral gene expression in infected cells. PMID:23202462

  7. Alcohol Deranges Hepatic Lipid Metabolism via Altered Transcriptional Regulation.

    PubMed Central

    Crabb, David W.

    2004-01-01

    Alcohol has classically been thought to cause fatty liver by way of altered redox potential in the liver, which inhibits fatty acid oxidation. Additional effects appear to play a role both in impairing fat oxidation and stimulating lipogenesis. Alcohol reduces the DNA binding and transcription-activating properties of peroxisome proliferator-activated receptor alpha (PPARalpha), both in cultured cells and in mice fed alcohol. Treatment of alcohol-fed mice with a PPARalpha agonist reverses fatty liver despite continued alcohol consumption. Alcohol also activates sterol response element- binding protein 1 (SREBP-1), inducing a battery of lipogenic enzymes. This effect may be due in part to inhibition of AMP-dependent protein kinase. This understanding of alcohol effects provides new therapeutic targets to reverse alcoholic fatty liver. Images Fig. 4 Fig. 6 PMID:17060973

  8. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    NASA Astrophysics Data System (ADS)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  9. MicroRNA 152 regulates hepatic glycogenesis by targeting PTEN.

    PubMed

    Wang, Shuyue; Wang, Lilin; Dou, Lin; Guo, Jun; Fang, Weiwei; Li, Meng; Meng, Xiangyu; Man, Yong; Shen, Tao; Huang, Xiuqing; Li, Jian

    2016-05-01

    Hepatic insulin resistance, defined as a diminished ability of hepatocytes to respond to the action of insulin, plays an important role in the development of type 2 diabetes and metabolic syndrome. Aberrant expression of mmu-miR-152-3p (miR-152) is related to the pathogenesis of tumors such as hepatitis B virus related hepatocellular carcinoma. However, the role of miR-152 in hepatic insulin resistance remains unknown. In the present study, we identified the potential role of miR-152 in regulating hepatic glycogenesis. The expression of miR-152 and the level of glycogen were significantly downregulated in the liver of db/db mice and mice fed a high fat diet. In vivo and in vitro results suggest that inhibition of miR-152 expression induced impaired glycogenesis in hepatocytes. Interestingly, miR-152 expression, glycogen synthesis and protein kinase B/glycogen synthase kinase (AKT/GSK) pathway activation were significantly decreased in the liver of mice injected with 16 μg·mL(-1) interleukin 6 (IL-6) by pumps for 7 days and in NCTC 1469 cells treated with 10 ng·mL(-1) IL-6 for 24 h. Moreover, hepatic overexpression of miR-152 rescued IL-6-induced impaired glycogenesis. Finally, phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-152 to mediate hepatic glycogen synthesis. Our findings provide mechanistic insight into the effects of miR-152 on the regulation of the AKT/GSK pathway and the synthesis of glycogen in hepatocytes. Downregulated miR-152 induced impaired hepatic glycogenesis by targeting PTEN. PTEN participated in miR-152-mediated glycogenesis in hepatocytes via regulation of the AKT/GSK pathway. PMID:26996529

  10. Enhanced hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations

    PubMed Central

    Wang, Xinmei; Yu, Bo; Ren, Wei; Mo, Xiaokui; Zhou, Chenguang; He, Hongyan; Jia, HuLiang; Wang, Lu; Jacob, Samson T.; Lee, Robert J.; Ghoshal, Kalpana; Lee, L. James

    2015-01-01

    Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid; into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, OA containing LNPs delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, OA containing LNPs showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. OA containing LNPs are a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases. PMID:24121065

  11. Short Hairpin RNA Causes the Methylation of Transforming Growth Factor-β Receptor II Promoter and Silencing of the Target Gene in Rat Hepatic Stellate Cells

    PubMed Central

    Kim, Jin-Wook; Zhang, Yan-Hong; Zern, Mark A; Rossi, John J.; Wu, Jian

    2008-01-01

    Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-β receptor (TGFβRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFβRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFβRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters. PMID:17533113

  12. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana.

    PubMed

    Calderwood, Alexander; Kopriva, Stanislav; Morris, Richard J

    2016-03-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements. PMID:26952566

  13. Transcript Abundance Explains mRNA Mobility Data in Arabidopsis thaliana[OPEN

    PubMed Central

    Calderwood, Alexander

    2016-01-01

    Recently, a large population of mRNA was shown to be able to travel between plant organs via sieve elements as a putative long-distance signaling molecule. However, a mechanistic basis by which transcripts are selected for transport has not yet been identified. Here, we show that experimental mRNA mobility data in Arabidopsis can be explained by transcript abundance and half-life. This suggests that the majority of identified mobile transcripts can be accounted for by non-sequence-specific movement of mRNA from companion cells into sieve elements. PMID:26952566

  14. Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    PubMed Central

    Wang, Xingyu; Li, Can; Gao, Xiaomeng; Wang, Jing; Liang, Xingguo

    2015-01-01

    A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2′-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 103 times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume. PMID:25584899

  15. Mapping in vivo initiation sites of RNA transcription and determining their relative use.

    PubMed Central

    Kessler, M; Aloni, Y

    1984-01-01

    Runoff transcripts were generated on viral transcriptional complexes cleaved with restriction enzymes and incubated in vitro with [alpha-32P]UTP under pulse-chase conditions. As viral transcriptional complexes in vitro elongated the nascent RNA preinitiated in vivo, size analysis by gel electrophoresis of the runoff transcripts allowed identification of the in vivo initiation sites. Moreover, scanning the intensities of the runoff bands as they appeared in the autoradiogram of the gel allowed determination of the relative use of these sites. A model system in which the initiation sites of simian virus 40 late RNA were identified and their relative use determined is presented. Images PMID:6090704

  16. MiRNA-Based Regulation of Hemostatic Factors through Hepatic Nuclear Factor-4 Alpha

    PubMed Central

    Salloum-Asfar, Salam; Arroyo, Ana B.; Teruel-Montoya, Raúl; García-Barberá, Nuria; Roldán, Vanessa; Vicente, Vicente; Martínez, Constantino; González-Conejero, Rocío

    2016-01-01

    MiRNAs have been reported as CIS-acting elements of several hemostatic factors, however, their mechanism as TRANS-acting elements mediated by a transcription factor is little known and could have important effects. HNF4α has a direct and important role in the regulation of multiple hepatic coagulation genes. Previous in vitro studies have demonstrated that miR-24-3p and miR-34a-5p regulate HNF4A expression. Here we aimed to investigate the molecular mechanisms of miR-24 and miR-34a on coagulation through HNF4A. Transfections with miR-24 and miR-34a in HepG2 cells decreased not only HNF4A but also F10, F12, SERPINC1, PROS1, PROC, and PROZ transcripts levels. Positive and significant correlations were observed between levels of HNF4A and several hemostatic factors (F5, F8, F9, F11, F12, SERPINC1, PROC, and PROS1) in human liver samples (N = 104). However, miR-24 and miR-34a levels of the low (10th) and high (90th) percentiles of those liver samples were inversely correlated with HNF4A and almost all hemostatic factors expression levels. These outcomes suggest that miR-24 and miR-34a might be two indirect elements of regulation of several hemostatic factors. Additionally, variations in miRNA expression profiles could justify, at least in part, changes in HNF4A expression levels and its downstream targets of coagulation. PMID:27135744

  17. CK2 phosphorylation of Bdp1 executes cell cycle-specific RNA polymerase III transcription repression.

    PubMed

    Hu, Ping; Samudre, Kalpana; Wu, Si; Sun, Yuling; Hernandez, Nouria

    2004-10-01

    RNA polymerase III (pol III) transcription from the human U6 snRNA promoter can be reconstituted with the recombinant factors SNAPc and Brf2-TFIIIB combined with purified pol III. In this system, CK2 treatment of the pol III complex is required for transcription, whereas treatment of Brf2-TFIIIB is inhibitory. Here we show that CK2 inhibits Brf2-TFIIIB by specifically phosphorylating its Bdp1 component. Bdp1 is phosphorylated by CK2 during mitosis, and this is accompanied by Bdp1 dissociation from the U6 promoter and from chromatin in general and by transcription repression. Remarkably, whereas inhibition of CK2 in mitotic extracts restores pol III transcription, inhibition of CK2 in active S phase extracts debilitates transcription. Thus, CK2 is directed to phosphorylate different targets within the basal pol III transcription machinery at different times during the cell cycle, with opposite transcriptional effects. PMID:15469824

  18. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    SciTech Connect

    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.

  19. Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription

    PubMed Central

    Petushkov, Ivan; Pupov, Danil; Bass, Irina; Kulbachinskiy, Andrey

    2015-01-01

    During transcription, the catalytic core of RNA polymerase (RNAP) must interact with the DNA template with low-sequence specificity to ensure efficient enzyme translocation and RNA extension. Unexpectedly, recent structural studies of bacterial promoter complexes revealed specific interactions between the nontemplate DNA strand at the downstream edge of the transcription bubble (CRE, core recognition element) and a protein pocket formed by core RNAP (CRE pocket). We investigated the roles of these interactions in transcription by analyzing point amino acid substitutions and deletions in Escherichia coli RNAP. The mutations affected multiple steps of transcription, including promoter recognition, RNA elongation and termination. In particular, we showed that interactions of the CRE pocket with a nontemplate guanine immediately downstream of the active center stimulate RNA-hairpin-dependent transcription pausing but not other types of pausing. Thus, conformational changes of the elongation complex induced by nascent RNA can modulate CRE effects on transcription. The results highlight the roles of specific core RNAP–DNA interactions at different steps of RNA synthesis and suggest their importance for transcription regulation in various organisms. PMID:25990734

  20. miRNA studies in in vitro and in vivo activated hepatic stellate cells

    PubMed Central

    Maubach, Gunter; Lim, Michelle Chin Chia; Chen, Jinmiao; Yang, Henry; Zhuo, Lang

    2011-01-01

    AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation. METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated. RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type I transcription. CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA. PMID:21734783

  1. Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

    PubMed

    Abernathy, Emma; Gilbertson, Sarah; Alla, Ravi; Glaunsinger, Britt

    2015-08-12

    Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5'-3' mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression. PMID:26211836

  2. Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization

    PubMed Central

    Wu, Tse-Hsiang; Kuo, Yuan-Yeh; Lee, Hsiao-Hui; Kuo, Jean-Cheng; Ou, Meng-Hsin; Chang, Zee-Fen

    2016-01-01

    It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress. PMID:27350000

  3. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

    PubMed Central

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Rao, Kummara Madhusudana; Park, Soo Yong; Chung, Ildoo; Ha, Chang-Sik; Kim, Sang-Woo; Yun, Yang H.; Jung, Youngmi

    2016-01-01

    Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis. PMID:27001906

  4. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression.

    PubMed

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Rao, Kummara Madhusudana; Park, Soo Yong; Chung, Ildoo; Ha, Chang-Sik; Kim, Sang-Woo; Yun, Yang H; Jung, Youngmi

    2016-01-01

    Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis. PMID:27001906

  5. Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

    PubMed Central

    Canella, Donatella; Praz, Viviane; Reina, Jaime H.; Cousin, Pascal; Hernandez, Nouria

    2010-01-01

    Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units. PMID:20413673

  6. Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

    PubMed Central

    Young, L S; Rivier, D H; Sprague, K U

    1991-01-01

    We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes. Images PMID:1996100

  7. Alterations in the hepatic transcriptional landscape after RNAi mediated ApoB silencing in cynomolgus monkeys.

    PubMed

    Hamza, M Sabry; Kumar, Chanchal; Chia, Ser Mien; Anandalakshmi, Vidhya; Boo, Nicole; Strapps, Walter; Robinson, Michael; Caguyong, Michelle; Bartz, Steven; Tadin-Strapps, Marija; van Gool, Alain; Shih, Shian-Jiun

    2015-10-01

    The greater genomic conservation between humans and non-human primates (NHP) enables target validation studies for developing of therapeutic strategies for human diseases. Together with predicting activity and potential adverse clinical signs, the inclusion of NHP testing bequeaths to efficacy models for dose titration and pharmacodynamic effects. We have used lipid nanoparticle encapsulated siRNA to silence ApoB in the liver and assessed the phenotypic effects on serum lipids with various levels of hepatic ApoB mRNA knockdown in healthy lean cynomolgus monkeys. ApoB siRNA dosed animals demonstrated significant reductions of hepatic ApoB mRNA and serum APOB protein, with a substantial lowering of plasma lipid levels without obvious signs of toxicity. Microarray based assessment of ApoB siRNA mediated effects revealed a number of differentially expressed genes which mapped onto biological pathways and processes related to lipid and cholesterol metabolism. Furthermore, we identified potential targets and cellular effects that could be studied for therapeutic benchmarking of APOB mediated effects. The network of ApoB regulated genes should be of significance for the understanding and development of novel hypercholesterolemia therapies. PMID:26275376

  8. A Comparative Study of RNA Polymerase II Transcription Machinery in Yeasts

    NASA Astrophysics Data System (ADS)

    Sharma, Nimisha; Mehta, Surbhi

    The control of gene expression, predominantly at the level of transcription, plays a fundamental role in biological processes determining the phenotypic changes in cells and organisms. The eukaryotes have evolved a complex and sophisticated transcription machinery to transcribe DNA into RNA. RNA polymerase II enzyme lies at the centre of the transcription apparatus that comprises nearly 60 polypeptides and is responsible for the expression and regulation of proteinencoding genes. Much of our present understanding and knowledge of the RNA polymerase II transcription apparatus in eukaryotes has been derived from studies in Saccharomyces cerevisiae. More recently, Schizosaccharomyces pombe has emerged as a better model system to study transcription because the transcription mechanism in this yeast is closer to that in higher eukaryotes. Also, studies on components of the basal transcription machinery have revealed a number of properties that are common with other eukaryotes, but have also highlighted some features unique to S. pombe. In fact, the fungal transcription associated protein families show greater species specificity and only 15% of these proteins contain homologues shared between both S. cerevisiae and S. pombe. In this chapter, we compare the RNA polymerase II transcription apparatus in different yeasts.

  9. RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation.

    PubMed Central

    Hanrahan, C J; Palladino, M J; Ganetzky, B; Reenan, R A

    2000-01-01

    Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na(+) channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na(+) channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs. PMID:10880477

  10. Transcriptional properties and splicing of the flamenco piRNA cluster.

    PubMed

    Goriaux, Coline; Desset, Sophie; Renaud, Yoan; Vaury, Chantal; Brasset, Emilie

    2014-04-01

    In Drosophila, the piRNA cluster, flamenco, produces most of the piRNAs (PIWI-interacting RNAs) that silence transposable elements in the somatic follicle cells during oogenesis. These piRNAs are thought to be processed from a long single-stranded precursor transcript. Here, we demonstrate that flamenco transcription is initiated from an RNA polymerase II promoter containing an initiator motif (Inr) and downstream promoter element (DPE) and requires the transcription factor, Cubitus interruptus. We show that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed to piRNAs. Our data reveal dynamic processing steps giving rise to piRNA cluster precursors. PMID:24562610

  11. Controlled Transcription of Exogenous mRNA in Platelets Using Protocells.

    PubMed

    Chan, Vivienne; Novakowski, Stefanie K; Law, Simon; Klein-Bosgoed, Christa; Kastrup, Christian J

    2015-11-01

    Transcribing exogenous RNA in eukaryotic cells requires delivering DNA to their nuclei and changing their genome. Nuclear delivery is often inefficient, limiting the potential scope of gene therapy and synthetic biology. These challenges may be overcome by techniques that allow for extranucleate transcription within eukaryotic cells. Protocells have been developed that enable transcription inside of liposomes; however, it has not yet been demonstrated whether this technology can be extended for use within eukaryotic cells. Here we show RNA-synthesizing nanoliposomes allow transcription of exogenous RNA inside anucleate cells. To accomplish this, components of transcription were encapsulated into liposomes and delivered to platelets. These liposomes were capable of light-induced transcription in platelets, providing proof-of-concept that protocell technology can be adapted for use within mammalian cells. PMID:26368852

  12. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  13. Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock.

    PubMed

    Dang, Fabin; Sun, Xiujie; Ma, Xiang; Wu, Rong; Zhang, Deyi; Chen, Yaqiong; Xu, Qian; Wu, Yuting; Liu, Yi

    2016-01-01

    Although food availability is a potent synchronizer of the peripheral circadian clock in mammals, the underlying mechanisms are unclear. Here, we show that hepatic Bmal1, a core transcription activator of the molecular clock, is post-transcriptionally regulated by signals from insulin, an important hormone that is temporally controlled by feeding. Insulin promotes postprandial Akt-mediated Ser42-phosphorylation of Bmal1 to induce its dissociation from DNA, interaction with 14-3-3 protein and subsequently nuclear exclusion, which results in the suppression of Bmal1 transcriptional activity. Inverted feeding cycles not only shift the phase of daily insulin oscillation, but also elevate the amplitude due to food overconsumption. This enhanced and reversed insulin signalling initiates the reset of clock gene rhythms by altering Bmal1 nuclear accumulation in mouse liver. These results reveal the molecular mechanism of insulin signalling in regulating peripheral circadian rhythms. PMID:27576939

  14. RNA processing of nitrogenase transcripts in the cyanobacterium Anabaena variabilis.

    PubMed

    Ungerer, Justin L; Pratte, Brenda S; Thiel, Teresa

    2010-07-01

    Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, with the nifB1 promoter contributing to most of the expression. Similarly, while the region upstream of vnfH, containing the putative transcription start site, did not drive expression of lacZ, the region that included the promoter for the upstream gene, ava4055, did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5'RACE (5' rapid amplification of cDNA ends) revealed that these 5' ends resulted from processing of larger transcripts rather than by de novo transcription initiation. The 5' positions of both the vnfH and nifH1 transcripts lie at the base of a stem-loop structure that may serve to stabilize the nifHDK1 and vnfH specific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis. PMID:20435734

  15. Noncoding RNA transcription targets AID to divergently transcribed loci in B cells

    PubMed Central

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Chao, Jaime; Rabadan, Raul; Economides, Aris N.; Basu, Uttiya

    2015-01-01

    The vast majority of the mammalian genome has the potential to expressnoncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3′–5′ exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome1. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID)2,3. The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells4,5. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity. PMID:25119026

  16. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    SciTech Connect

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  17. Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays

    PubMed Central

    Honkela, Antti; Peltonen, Jaakko; Topa, Hande; Charapitsa, Iryna; Matarese, Filomena; Grote, Korbinian; Stunnenberg, Hendrik G.; Reid, George; Lawrence, Neil D.; Rattray, Magnus

    2015-01-01

    Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes. PMID:26438844

  18. Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays.

    PubMed

    Honkela, Antti; Peltonen, Jaakko; Topa, Hande; Charapitsa, Iryna; Matarese, Filomena; Grote, Korbinian; Stunnenberg, Hendrik G; Reid, George; Lawrence, Neil D; Rattray, Magnus

    2015-10-20

    Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes. PMID:26438844

  19. lincRNA-p21 inhibits hepatic stellate cell activation and liver fibrogenesis via p21.

    PubMed

    Zheng, Jianjian; Dong, Peihong; Mao, Yuqing; Chen, Shaolong; Wu, Xiaoli; Li, Guojun; Lu, Zhongqiu; Yu, Fujun

    2015-12-01

    Long non-coding RNAs are involved in various biological processes and diseases. The biological role of long intergenic non-coding RNA-p21 (lincRNA-p21) in liver fibrosis remains unknown before this study. In this study, we observed marked reduction of lincRNA-p21 expression in mice liver fibrosis models and human cirrhotic liver. Over-expression of lincRNA-p21 suppressed activation of hepatic stellate cells (HSCs) in vitro. Lentivirus-mediated lincRNA-p21 transfer into mice decreased the severity of liver fibrosis in vivo. Additionally, lincRNA-p21 reversed the activation of HSCs to their quiescent phenotype. The mRNA levels of lincRNA-p21 and p21 were positively correlated. Our results show that over-expression of lincRNA-p21 promotes up-regulation of p21 at both the mRNA and protein levels. Furthermore, lincRNA-p21 inhibited cell-cycle progression and proliferation of primary HSCs through enhancement of p21 expression. Compared with healthy subjects, serum lincRNA-p21 levels were significantly lower in patients with liver cirrhosis, especially those with decompensation. These findings collectively indicate that lincRNA-p21 is a mediator of HSC activation, supporting its utility as a novel therapeutic target for liver fibrosis. PMID:26433205

  20. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

    NASA Astrophysics Data System (ADS)

    Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

    2009-06-01

    Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

  1. CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

    PubMed Central

    Yoshinari, S; Nagy, P D; Simon, A E; Dreher, T W

    2000-01-01

    It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible. PMID:10836791

  2. Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

    PubMed Central

    Schwalbe, Martin; Ohlenschläger, Oliver; Marchanka, Aliaksandr; Ramachandran, Ramadurai; Häfner, Sabine; Heise, Tilman; Görlach, Matthias

    2008-01-01

    Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα. PMID:18263618

  3. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    NASA Astrophysics Data System (ADS)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  4. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.

    PubMed

    Gnatt, A L; Cramer, P; Fu, J; Bushnell, D A; Kornberg, R D

    2001-06-01

    The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation. PMID:11313499

  5. Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA.

    PubMed Central

    Volkmann, S; Jendis, J; Frauendorf, A; Moelling, K

    1995-01-01

    Reverse transcription of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). A highly conserved polypurine tract (PPT) on the viral RNA serves as primer for plus-strand DNA synthesis and is a possible target for triple-helix formation. Triple-helix formation during reverse transcription involves either single-stranded RNA or an RNA.DNA hybrid. The effect of triple-helix formation on reverse transcription has been analyzed here in vitro using a three-strand-system consisting of an RNA.DNA hybrid and triplex-forming oligonucleotides (TFOs) consisting either of DNA or RNA. Three strand triple-helices inhibit RNase H cleavage of the PPT-RNA.DNA hybrid and initiation of plus-strand DNA synthesis in vitro. Triple-helix formation on a single-stranded RNA target has also been tested in a two-strand-system with TFOs comprising Watson-Crick and Hoogsteen base-pairing sequences, both targeted to the PPT-RNA, on a single strand connected by a linker (T)4. TFOs prevent RNase H cleavage of the PPT-RNA and initiation of plus-strand DNA synthesis in vitro. In cell culture experiments one TFO is an efficient inhibitor of retrovirus replication, leading to a block of p24 synthesis and inhibition of syncytia formation in newly infected cells. Images PMID:7537875

  6. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model. PMID:8882638

  7. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III.

    PubMed Central

    Gottlieb, E; Steitz, J A

    1989-01-01

    We have tested the hypothesis that the mammalian La protein, which appears to be required for accurate and efficient RNA polymerase III transcription, is a transcription termination factor. Our data suggest that 3' foreshortened transcripts generated in La's absence are components of a novel transcription intermediate containing a paused polymerase. These transcripts are produced by fractionated transcription complexes, are synthesized with kinetics different from full-length transcripts, and are chasable to completion from the stalled transcription complexes. Together, these findings argue that termination by RNA polymerase III requires auxilliary factor(s) and implicate La as such a factor. Since La appears to facilitate transcript completion and release and also binds the resulting RNA product, it may be a regulator of RNA polymerase III transcription. Images PMID:2470590

  8. Every little piece counts: the many faces of tRNA transcripts.

    PubMed

    Lalaouna, David; Carrier, Marie-Claude; Massé, Eric

    2015-01-01

    For over half a century, tRNAs have been exclusively known as decoders of genomic information. However, recent reports evidenced that tRNA transcripts are also bearers of functional RNAs, which are able to execute various tasks through an array of mechanisms. Here, we succinctly review the diversity and functions of RNAs deriving from tRNA loci. PMID:26595434

  9. Probing the structure of Nun transcription arrest factor bound to RNA polymerase.

    PubMed

    Mustaev, Arkady; Vitiello, Christal L; Gottesman, Max E

    2016-08-01

    The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5' and the 3' ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3' end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5' end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors. PMID:27436904

  10. EGR1 regulates hepatic clock gene amplitude by activating Per1 transcription

    PubMed Central

    Tao, Weiwei; Wu, Jing; Zhang, Qian; Lai, Shan-Shan; Jiang, Shan; Jiang, Chen; Xu, Ying; Xue, Bin; Du, Jie; Li, Chao-Jun

    2015-01-01

    The mammalian clock system is composed of a master clock and peripheral clocks. At the molecular level, the rhythm-generating mechanism is controlled by a molecular clock composed of positive and negative feedback loops. However, the underlying mechanisms for molecular clock regulation that affect circadian clock function remain unclear. Here, we show that Egr1 (early growth response 1), an early growth response gene, is expressed in mouse liver in a circadian manner. Consistently, Egr1 is transactivated by the CLOCK/BMAL1 heterodimer through a conserved E-box response element. In hepatocytes, EGR1 regulates the transcription of several core clock genes, including Bmal1, Per1, Per2, Rev-erbα and Rev-erbβ, and the rhythm amplitude of their expression is dependent on EGR1’s transcriptional function. Further mechanistic studies indicated that EGR1 binds to the proximal region of the Per1 promoter to activate its transcription directly. When the peripheral clock is altered by light or feeding behavior transposition in Egr1-deficient mice, the expression phase of hepatic clock genes shifts normally, but the amplitude is also altered. Our data reveal a critical role for EGR1 in the regulation of hepatic clock circuitry, which may contribute to the rhythm stability of peripheral clock oscillators. PMID:26471974

  11. Post-Transcriptional Regulation of RNA Polymerase II Levels in Caenorhabditis Elegans

    PubMed Central

    Dalley, B. K.; Rogalski, T. M.; Tullis, G. E.; Riddle, D. L.; Golomb, M.

    1993-01-01

    To investigate the regulation of RNA polymerase II levels in Caenorhabditis elegans, we have constructed nematode strains having one, two, or three copies of ama-1, the gene for the largest subunit of RNA polymerase II. Steady-state levels of RNA polymerase II polypeptides and solubilized enzyme activity are invariant with gene dosage, indicating regulatory compensation. However, steady-state levels of ama-1 mRNA are directly proportional to gene dosage. These results imply that RNA polymerase II levels in C. elegans are regulated post-transcriptionally. PMID:8436272

  12. Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

    PubMed Central

    Pollard, V W; Hajduk, S L

    1991-01-01

    The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function. Images PMID:1825348

  13. RNA-Seq Reveals Different mRNA Abundance of Transporters and Their Alternative Transcript Isoforms During Liver Development

    PubMed Central

    Cui, Julia Yue; Gunewardena, Sumedha S.; Yoo, Byunggil; Liu, Jie; Renaud, Helen J.; Lu, Hong; Zhong, Xiao-bo; Klaassen, Curtis D.

    2012-01-01

    During development, the maturation of liver transporters is essential for chemical elimination in newborns and children. One cannot compare the real abundance of transcripts by conventional messenger RNA (mRNA) profiling methods; in comparison, RNA-Seq provides a “true quantification” of transcript counts and an unbiased detection of novel transcripts. The purpose of this study was to compare the mRNA abundance of liver transporters and seek their novel transcripts during liver development. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. The transcriptome was determined by RNA-Seq, with transcript abundance estimated by Cufflinks. Among 498 known transporters, the ontogeny of 62 known critical xenobiotic transporters was examined in detail. The cumulative mRNAs of the uptake transporters increased more than the efflux transporters in livers after birth. A heatmap revealed three ontogenic patterns of these transporters, namely perinatal (reaching maximal expression before birth), adolescent (about 20 days), and adult enriched (about 60 days of age). Before birth, equilibrative nucleoside transporter 1 was the transporter with highest expression in liver (29%), followed by breast cancer resistance protein (Bcrp) (26%). Within 1 day after birth, the mRNAs of these two transporters decreased markedly, and Ntcp became the transporter with highest expression (52%). In adult liver, the transporters with highest expression were organic cation transporter 1 and Ntcp (23% and 22%, respectively). Three isoforms of Bcrp with alternate leading exons were identified (E1a, E1b, and E1c), with E1b being the major isoform. In conclusion, this study reveals the mRNA abundance of transporters in liver and demonstrates that the expression of liver transporters is both age and isoform specific. PMID:22454430

  14. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells

    PubMed Central

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A.; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-01-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression. PMID

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

    PubMed Central

    Zenkin, Nikolay; Yuzenkova, Yulia

    2015-01-01

    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. PMID:26120903

  16. Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome.

    PubMed

    Kim, Kyumin; Heo, Dong-Hyuk; Kim, Iktae; Suh, Jeong-Yong; Kim, Minkyu

    2016-06-17

    The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease. PMID:27076633

  17. Intragenic transcription of a noncoding RNA modulates expression of ASP3 in budding yeast

    PubMed Central

    Huang, Yu-Ching; Chen, Hung-Ta; Teng, Shu-Chun

    2010-01-01

    Inter- and intragenic noncoding transcription is widespread in eukaryotic genomes; however, the purpose of these types of transcription is still poorly understood. Here, we show that intragenic sense-oriented transcription within the budding yeast ASP3 coding region regulates a constitutively and immediately accessible promoter for the transcription of full-length ASP3. Expression of this short intragenic transcript is independent of GATA transcription factors, which are essential for the activation of full-length ASP3, and independent of RNA polymerase II (RNAPII). Furthermore, we found that an intragenic control element is required for the expression of this noncoding RNA (ncRNA). Continuous expression of the short ncRNA maintains a high level of trimethylation of histone H3 at lysine 4 (H3K4me3) at the ASP3 promoter and makes this region more accessible for RNAPII to transcribe the full-length ASP3. Our results show for the first time that intragenic noncoding transcription promotes gene expression. PMID:20817754

  18. Transcription of eucaryotic tRNA1met and 5SRNA genes by RNA polymerase III is blocked by base mismatches in the intragenic control regions.

    PubMed Central

    Sullivan, M A; Folk, W R

    1987-01-01

    We have constructed duplex DNAs containing single G-T or A-C mismatches in the X. laevis tRNA1met gene. Mismatches within regions of this gene which are bound by transcription factor TFIIIC prevent transcription by RNA polymerase III. Homoduplexes with G-C----A-T mutations at some of the same sites, however, are transcribed efficiently in oocytes. Mismatches outside of the tRNA1met gene have no effect upon transcription. A survey of several point mutants in the Syrian hamster 5SRNA gene indicates that mismatches outside the internal control region somewhat reduce transcription, but a mismatch within the internal control region blocks transcription. Thus, the presence of mismatched bases in the region of DNA which interacts with RNA polymerase III transcription factors blocks transcription, perhaps by interfering with DNA renaturation following transit of the RNA polymerase. Images PMID:3645544

  19. Direct assessment of transcription fidelity by high-resolution RNA sequencing

    PubMed Central

    Imashimizu, Masahiko; Oshima, Taku; Lubkowska, Lucyna; Kashlev, Mikhail

    2013-01-01

    Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10−4–10−5 per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ∼103 nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity. PMID:23925128

  20. Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

    PubMed Central

    Huang, Lingyan; Behlke, Mark A.; Tsourkas, Andrew

    2014-01-01

    The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3’-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts. PMID:25146531

  1. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain

    PubMed Central

    Cho, Eun-Jung; Takagi, Toshimitsu; Moore, Christine R.; Buratowski, Stephen

    1997-01-01

    Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD–kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD. PMID:9407025

  2. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  3. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    PubMed

    Rijal, Keshab; Maraia, Richard J

    2016-08-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

  4. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.

    2016-01-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

  5. Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

    SciTech Connect

    Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

    2010-10-22

    Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

  6. TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation.

    PubMed Central

    Bohjanen, P R; Liu, Y; Garcia-Blanco, M A

    1997-01-01

    The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes. PMID:9358155

  7. Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo

    PubMed Central

    Lin, Tzung-Yi; Chou, Chi-Fu; Chung, Hsin-Yu; Chiang, Chia-Yin; Li, Chung-Hao; Wu, Jen-Leih; Lin, Han-Jia; Pai, Tun-Wen; Hu, Chin-Hwa; Tzou, Wen-Shyong

    2014-01-01

    The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment. PMID:25000307

  8. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  9. MITIE: Simultaneous RNA-Seq-based transcript identification and quantification in multiple samples

    PubMed Central

    Behr, Jonas; Kahles, André; Zhong, Yi; Sreedharan, Vipin T.; Drewe, Philipp; Rätsch, Gunnar

    2013-01-01

    Motivation: High-throughput sequencing of mRNA (RNA-Seq) has led to tremendous improvements in the detection of expressed genes and reconstruction of RNA transcripts. However, the extensive dynamic range of gene expression, technical limitations and biases, as well as the observed complexity of the transcriptional landscape, pose profound computational challenges for transcriptome reconstruction. Results: We present the novel framework MITIE (Mixed Integer Transcript IdEntification) for simultaneous transcript reconstruction and quantification. We define a likelihood function based on the negative binomial distribution, use a regularization approach to select a few transcripts collectively explaining the observed read data and show how to find the optimal solution using Mixed Integer Programming. MITIE can (i) take advantage of known transcripts, (ii) reconstruct and quantify transcripts simultaneously in multiple samples, and (iii) resolve the location of multi-mapping reads. It is designed for genome- and assembly-based transcriptome reconstruction. We present an extensive study based on realistic simulated RNA-Seq data. When compared with state-of-the-art approaches, MITIE proves to be significantly more sensitive and overall more accurate. Moreover, MITIE yields substantial performance gains when used with multiple samples. We applied our system to 38 Drosophila melanogaster modENCODE RNA-Seq libraries and estimated the sensitivity of reconstructing omitted transcript annotations and the specificity with respect to annotated transcripts. Our results corroborate that a well-motivated objective paired with appropriate optimization techniques lead to significant improvements over the state-of-the-art in transcriptome reconstruction. Availability: MITIE is implemented in C++ and is available from http://bioweb.me/mitie under the GPL license. Contact: Jonas_Behr@web.de and raetsch@cbio.mskcc.org Supplementary information: Supplementary data are available at

  10. A cell-based screening system for influenza A viral RNA transcription/replication inhibitors

    PubMed Central

    Ozawa, Makoto; Shimojima, Masayuki; Goto, Hideo; Watanabe, Shinji; Hatta, Yasuko; Kiso, Maki; Furuta, Yousuke; Horimoto, Taisuke; Peters, Noel R.; Hoffmann, F. Michael; Kawaoka, Yoshihiro

    2013-01-01

    Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens. PMID:23346363

  11. Repression of host RNA polymerase II transcription by herpes simplex virus type 1.

    PubMed Central

    Spencer, C A; Dahmus, M E; Rice, S A

    1997-01-01

    Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II. PMID:9032335

  12. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    PubMed Central

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

    2013-01-01

    The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

  13. Metastasis-suppressor transcript destabilization through TARBP2 binding of mRNA hairpins.

    PubMed

    Goodarzi, Hani; Zhang, Steven; Buss, Colin G; Fish, Lisa; Tavazoie, Saeed; Tavazoie, Sohail F

    2014-09-11

    Aberrant regulation of RNA stability has an important role in many disease states. Deregulated post-transcriptional modulation, such as that governed by microRNAs targeting linear sequence elements in messenger RNAs, has been implicated in the progression of many cancer types. A defining feature of RNA is its ability to fold into structures. However, the roles of structural mRNA elements in cancer progression remain unexplored. Here we performed an unbiased search for post-transcriptional modulators of mRNA stability in breast cancer by conducting whole-genome transcript stability measurements in poorly and highly metastatic isogenic human breast cancer lines. Using a computational framework that searches RNA sequence and structure space, we discovered a family of GC-rich structural cis-regulatory RNA elements, termed sRSEs for structural RNA stability elements, which are significantly overrepresented in transcripts displaying reduced stability in highly metastatic cells. By integrating computational and biochemical approaches, we identified TARBP2, a double-stranded RNA-binding protein implicated in microRNA processing, as the trans factor that binds the sRSE family and similar structural elements--collectively termed TARBP2-binding structural elements (TBSEs)--in transcripts. TARBP2 is overexpressed in metastatic cells and metastatic human breast tumours and destabilizes transcripts containing TBSEs. Endogenous TARBP2 promotes metastatic cell invasion and colonization by destabilizing amyloid precursor protein (APP) and ZNF395 transcripts, two genes previously associated with Alzheimer's and Huntington's disease, respectively. We reveal these genes to be novel metastasis suppressor genes in breast cancer. The cleavage product of APP, extracellular amyloid-α peptide, directly suppresses invasion while ZNF395 transcriptionally represses a pro-metastatic gene expression program. The expression levels of TARBP2, APP and ZNF395 in human breast carcinomas support

  14. An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

    PubMed

    Crawford, David W; Blakeley, Brett D; Chen, Po-Han; Sherpa, Chringma; Le Grice, Stuart F J; Laird-Offringa, Ite A; McNaughton, Brian R

    2016-08-19

    Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA. PMID:27253715

  15. Hepatitis

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Hepatitis KidsHealth > For Kids > Hepatitis Print A A A ... an important digestive liquid called bile . What Is Hepatitis? Hepatitis is an inflammation (say: in-fluh-MAY- ...

  16. Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    PubMed Central

    Reynolds, David; Hofmeister, Brigitte T.; Cliffe, Laura; Alabady, Magdy; Siegel, T. Nicolai; Schmitz, Robert J.; Sabatini, Robert

    2016-01-01

    Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG) transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination. PMID:26796527

  17. B2 RNA and 7SK RNA, RNA polymerase III transcripts, have a cap-like structure at their 5' end.

    PubMed Central

    Shumyatsky, G P; Tillib, S V; Kramerov, D A

    1990-01-01

    We found that hydrolysates of poly(A)+ RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xppp5'G, where x has a non-nucleotide structure. About half of all B2 RNAs contained this group at the 5' end. Previously, Epstein et al. (1) detected a similar structure at the 5' end of small nuclear U6 RNA. Later, Singh and Reddy (2) showed methyl to be the blocking group in the component x of U6 RNA. Besides B2 RNA, we found 5' ends containing methyl groups in 7SK RNA. Images PMID:1700854

  18. RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

    PubMed

    Goic, Bertsy; Vodovar, Nicolas; Mondotte, Juan A; Monot, Clément; Frangeul, Lionel; Blanc, Hervé; Gausson, Valérie; Vera-Otarola, Jorge; Cristofari, Gael; Saleh, Maria-Carla

    2013-04-01

    How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence. PMID:23435119

  19. RNA polymerase III dependence of the human L1 promoter and possible participation of the RNA polymerase II factor YY1 in the RNA polymerase III transcription system.

    PubMed Central

    Kurose, K; Hata, K; Hattori, M; Sakaki, Y

    1995-01-01

    From the general views of the eukaryotic transcription systems, L1 (or L1-like) retrotransposons that encode some proteins are unusual. L1, unlike other protein-coding elements, is transcribed through an internal promoter. And the L1 internal promoter, unlike other internal promoters, is thought to be RNA polymerase II (pol II) dependent, because the L1 transcript has a large size (approximately 6 kb), protein coding capacity and a 3' terminal polyadenylation signal followed by a poly(A) tail, and also because transcription from the promoter of Drosophila L1-like element jockey was highly sensitive to alpha-amanitin. However, our in vitro transcription study reveals that transcription from the human L1 promoter is highly sensitive to tagetitoxin, a selective inhibitor of RNA polymerase III (pol III), but insensitive to 1 micrograms/ml of alpha-amanitin, indicating that the human L1 promoter is pol III-dependent. The pol III dependence is further supported by our observation that L1 and pol III-dependent tRNA gene promoters share a common nuclear factor YY1. There is evidence that YY1 is also a pol II transcription factor. We thus propose that YY1 is a possible member of the pol III transcription system. Images PMID:7479000

  20. Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

    PubMed Central

    Song, Alice Tung Wan; de Mello, Evandro Sobroza; Alves, Venâncio Avancini Ferreira; Cavalheiro, Norma de Paula; Melo, Carlos Eduardo; Bonazzi, Patricia Rodrigues; Tengan, Fatima Mitiko; Freire, Maristela Pinheiro; Barone, Antonio Alci; D'Albuquerque, Luiz Augusto Carneiro; Abdala, Edson

    2015-01-01

    Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection. PMID:25742264

  1. Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

    PubMed Central

    Park, E A; Mynatt, R L; Cook, G A; Kashfi, K

    1995-01-01

    The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene. Images Figure 2 Figure 4 PMID:7575418

  2. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

    PubMed

    Cramer, P; Bushnell, D A; Kornberg, R D

    2001-06-01

    Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain. PMID:11313498

  3. The little elongation complex functions at initiation and elongation phases of snRNA gene transcription.

    PubMed

    Hu, Deqing; Smith, Edwin R; Garruss, Alexander S; Mohaghegh, Nima; Varberg, Joseph M; Lin, Chengqi; Jackson, Jessica; Gao, Xin; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Eissenberg, Joel C; Shilatifard, Ali

    2013-08-22

    The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL. PMID:23932780

  4. The Little Elongation Complex functions at initiation and elongation phases of snRNA gene transcription

    PubMed Central

    Hu, Deqing; Smith, Edwin R.; Garruss, Alexander S.; Mohaghegh, Nima; Varberg, Joseph M.; Lin, Chengqi; Jackson, Jessica; Gao, Xin; Saraf, Anita; Florens, Laurence; Washburn, Michael P.; Eissenberg, Joel C.; Shilatifard, Ali

    2014-01-01

    SUMMARY The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA Polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the Little Elongation Complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL. PMID:23932780

  5. MYC Regulation of Cell Growth through Control of Transcription by RNA Polymerases I and III

    PubMed Central

    Campbell, Kirsteen J.; White, Robert J.

    2014-01-01

    MYC’s tumorigenic potential involves increased ribosome biogenesis and translational capacity, which supply the cell with protein required for enhanced cell growth and subsequent cell division. In addition to activation of protein-encoding genes transcribed by RNA polymerase II, MYC must stimulate transcription by RNA polymerase I and RNA polymerase III to meet this synthetic demand. In the past decade our knowledge of the mechanisms and importance of MYC regulation of RNA polymerases I and III has flourished. Here we discuss MYC’s influence on transcription by these “odd” RNA polymerases and the physiological impact of this regulation is evaluated with relevance to cancer development and treatment. PMID:24789877

  6. Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex

    PubMed Central

    Mekler, Vladimir; Minakhin, Leonid; Borukhov, Sergei; Mustaev, Arkady; Severinov, Konstantin

    2014-01-01

    Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT-rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis. PMID:25311862

  7. Possible formation of mitochondrial-RNA containing chimeric or trimeric RNA implies a post-transcriptional and post-splicing mechanism for RNA fusion.

    PubMed

    Yang, Wei; Wu, Jian-min; Bi, An-ding; Ou-Yang, Yong-chang; Shen, Hai-hong; Chirn, Gung-wei; Zhou, Jian-hua; Weiss, Emily; Holman, Emily Pauline; Liao, D Joshua

    2013-01-01

    Human cells are known to express many chimeric RNAs, i.e. RNAs containing two genes' sequences. Wondering whether there also is trimeric RNA, i.e. an RNA containing three genes' sequences, we wrote simple computer code to screen human expression sequence tags (ESTs) deposited in different public databases, and obtained hundreds of putative trimeric ESTs. We then used NCBI Blast and UCSC Blat browsers to further analyze their sequences, and identified 61 trimeric and two tetrameric ESTs (one EST containing four different sequences). We also identified 57 chimeric, trimeric or teterameric ESTs that contained both mitochondrial (mt) RNA and nuclear RNA (nRNA), i.e. were mtRNA-nRNA fusions. In some trimeric ESTs, the downstream partner was fused to the poly-A tail of the upstream partner, which, together with the mtRNA-nRNA fusions, suggests a possible new mechanism for RNA fusion that occurs after both transcription and splicing have been terminated, and possibly outside the nucleus, in contrast to the two current hypothetical mechanisms, trans-splicing and transcriptional-slippage, that occur in the nucleus. The mt-sequences in the mtRNA-nRNA fusions had pseudogenes in the nucleus but, surprisingly, localized mainly in chromosomes 1 and 5. In some mtRNA-nRNA fusions, as well as in some ESTs that were derived only from mtRNA, the mt-sequences might be cis- or trans-spliced. Actually, we cloned a new cis-spliced mtRNA, coined as 16SrRNA-s. Hence, mtDNA may not always be intron-less. Fusion of three or more RNAs to one, fusion of nRNA to mtRNA, and cis- or trans-splicing of mtRNA should all enlarge the cellular RNA repertoire, in turn enlarging the cellular functions. Therefore, future experimental verification of the existence of these novel classes of fusion RNAs and spliced mtRNAs in human cells should significantly advance our understanding of biology and medicine. PMID:24204722

  8. E. coli 6S RNA: a universal transcriptional regulator within the centre of growth adaptation.

    PubMed

    Geissen, René; Steuten, Benedikt; Polen, Tino; Wagner, Rolf

    2010-01-01

    Bacterial 6S RNA has been shown to bind with high affinity to σ(70)-containing RNA polymerase, suppressing σ(70)-dependent transcription during stationary phase, when 6S RNA concentrations are highest. We recently reported a genome-wide transcriptional comparison of wild-type and 6S RNA deficient E. coli strains. Contrary to the expected σ(70)- and stationary phase-specific regulatory effect of 6S RNA it turned out that mRNA levels derived from many alternative sigma factors, including σ(38) or σ(32), were affected during exponential and stationary growth. Among the most noticeably down-regulated genes at stationary growth are ribosomal proteins and factors involved in translation. In addition, a striking number of mRNA levels coding for enzymes involved in the purine metabolism, for transporters and stress regulators are altered both during log- and stationary phase. During the study we discovered a link between 6S RNA and the general stress alarmone ppGpp, which has a higher basal level in cells deficient in 6S RNA. This finding points to a functional interrelation of 6S RNA and the global network of stress and growth adaptation. PMID:20930516

  9. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Liu, Zhong; Zhao, Rui; Giles, Keith E.

    2016-01-01

    It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells. PMID:27299313

  10. Mutational analysis of the transcription start site of the yeast tRNA(Leu3) gene.

    PubMed Central

    Fruscoloni, P; Zamboni, M; Panetta, G; De Paolis, A; Tocchini-Valentini, G P

    1995-01-01

    In addition to the well-known internal promoter elements of tRNA genes, 5' flanking sequences can also influence the efficiency of transcription by Saccharomyces cerevisiae extracts in vitro. A consensus sequence of yeast tRNA genes in the vicinity of the transcriptional start site can be derived. To determine whether the activity of this region can be attributed to particular sequence features we studied in vitro mutants of the start site region. We found that the start site can be shifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purine motif (with transcription initiating at the purine) and a small T:A base pair block upstream are important for efficient transcription in vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNA(Leu3) gene does play a role in determining transcription efficiency and fixing the precise site of initiation by RNA polymerase III. Images PMID:7659514

  11. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    PubMed Central

    Ducey, Thomas F.; Jackson, Lydgia; Orvis, Joshua; Dyer, David W.

    2016-01-01

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in Escherichia coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5′ start of the transcript, using a single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping. PMID:19162160

  12. Transcription of the tRNA-tufB operon of Escherichia coli: activation, termination and antitermination.

    PubMed Central

    van Delft, J H; Mariñon, B; Schmidt, D S; Bosch, L

    1987-01-01

    Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping. TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions. Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB transcription. This demonstrates the presence of a region, upstream of the tRNA-tufB promoter, that enhances the expression of the operon. DNA fragments bearing this upstream activator region do not display an abnormal electrophoretic mobility, as has been observed for the rrnB P1 upstream activator. Deletions starting in the first tRNA gene and directing towards tufB reveal at least two sites that influence tufB transcription. One signals transcription termination in the intergenic region between thrT and tufB. The other may be involved in antitermination. Possible mechanisms underlying antitermination and termination are considered in the light of the nucleotide sequence. Images PMID:3317280

  13. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

    PubMed

    Weitzmann, C J; Cunningham, P R; Ofengand, J

    1990-06-25

    The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. PMID:2194163

  14. Genome-guided transcript assembly from integrative analysis of RNA sequence data

    PubMed Central

    Boley, Nathan; Stoiber, Marcus H.; Booth, Benjamin W.; Wan, Kenneth H.; Hoskins, Roger A.; Bickel, Peter J.; Celniker, Susan E.; Brown, James B.

    2014-01-01

    The identification of full length transcripts entirely from short-read RNA sequencing data (RNA-seq) remains a challenge in genome annotation pipelines. Here we describe an automated pipeline for genome annotation that integrates RNA-seq and gene-boundary data sets, which we call generalized RNA integration tool, or GRIT. By applying GRIT to Drosophila melanogaster short-read RNA-seq, cap analysis of gene expression (CAGE) and poly(A)-site-seq data collected for the modENCODE project, we recover the vast majority of previously annotated transcripts and double the total number of transcripts cataloged. We find that 20% of protein coding genes encode multiple protein-localization signals, and that, in 20 day old adult fly heads, genes with multiple poly-adenylation sites are more common than genes with alternate splicing or alternate promoters. When compared to the most widely used transcript assembly tools, GRIT recovers a larger fraction of annotated transcripts at higher precision. GRIT will enable the automated generation of high-quality genome annotations without necessitating extensive manual annotation. PMID:24633242

  15. An RNA motif advances transcription by preventing Rho-dependent termination

    PubMed Central

    Sevostyanova, Anastasia; Groisman, Eduardo A.

    2015-01-01

    The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity. PMID:26630006

  16. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3.

    PubMed Central

    Wang, X W; Forrester, K; Yeh, H; Feitelson, M A; Gu, J R; Harris, C C

    1994-01-01

    Chronic active hepatitis caused by infection with hepatitis B virus, a DNA virus, is a major risk factor for human hepatocellular carcinoma. Since the oncogenicity of several DNA viruses is dependent on the interaction of their viral oncoproteins with cellular tumor-suppressor gene products, we investigated the interaction between hepatitis B virus X protein (HBX) and human wild-type p53 protein. HBX complexes with the wild-type p53 protein and inhibits its sequence-specific DNA binding in vitro. HBX expression also inhibits p53-mediated transcriptional activation in vivo and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Therefore, HBX may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma. Images PMID:8134379

  17. HNF6 and Rev-erbα integrate hepatic lipid metabolism by overlapping and distinct transcriptional mechanisms.

    PubMed

    Zhang, Yuxiang; Fang, Bin; Damle, Manashree; Guan, Dongyin; Li, Zhenghui; Kim, Yong Hoon; Gannon, Maureen; Lazar, Mitchell A

    2016-07-15

    Hepatocyte nuclear factor 6 (HNF6) is required for liver development, but its role in adult liver metabolism is not known. Here we show that deletion of HNF6 in livers of adult C57Bl/6 mice leads to hepatic steatosis in mice fed normal laboratory chow. Although HNF6 is known mainly as a transcriptional activator, hepatic loss of HNF6 up-regulated many lipogenic genes bound directly by HNF6. Many of these genes are targets of the circadian nuclear receptor Rev-erbα, and binding of Rev-erbα at these sites was lost when HNF6 was ablated in the liver. While HNF6 and Rev-erbα coordinately regulate hepatic lipid metabolism, each factor also affects additional gene sets independently. These findings highlight a novel mechanism of transcriptional repression by HNF6 and demonstrate how overlapping and distinct mechanisms of transcription factor function contribute to the integrated physiology of the liver. PMID:27445394

  18. A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor.

    PubMed Central

    White, R J; Jackson, S P; Rigby, P W

    1992-01-01

    The major class of vertebrate genes transcribed by RNA polymerase (EC 2.7.7.6) III, which includes 5S rRNA genes, tRNA genes, and the adenovirus VA genes, is characterized by split internal promoters and no absolute dependence upon specific upstream sequences. Fractionation experiments have shown that transcription of such genes requires two general RNA polymerase III-specific factors, TFIIIB and TFIIIC. We now demonstrate that a third general factor is also employed by these genes. This is the TATA-box-binding protein originally identified as being a component of the general RNA polymerase II transcription factor TFIID. This protein is involved in the transcription by RNA polymerase III of every template tested, even though the promoters of VA and most vertebrate tRNA and 5S rRNA genes do not contain recognizable TATA elements. Images PMID:1542692

  19. saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription

    PubMed Central

    Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

    2016-01-01

    Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process. PMID:26902284

  20. RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1

    PubMed Central

    Erkizan, Hayriye Verda; Schneider, Jeffrey A.; Sajwan, Kamal; Graham, Garrett T.; Griffin, Brittany; Chasovskikh, Sergey; Youbi, Sarah E.; Kallarakal, Abraham; Chruszcz, Maksymilian; Padmanabhan, Radhakrishnan; Casey, John L.; Üren, Aykut; Toretsky, Jeffrey A.

    2015-01-01

    RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy. PMID:25564528

  1. The identification and characterization of novel transcripts from RNA-seq data.

    PubMed

    Weirick, Tyler; Militello, Giuseppe; Müller, Raphael; John, David; Dimmeler, Stefanie; Uchida, Shizuka

    2016-07-01

    Owing greatly to the advancement of next-generation sequencing (NGS), the amount of NGS data is increasing rapidly. Although there are many NGS applications, one of the most commonly used techniques 'RNA sequencing (RNA-seq)' is rapidly replacing microarray-based techniques in laboratories around the world. As more and more of such techniques are standardized, allowing technicians to perform these experiments with minimal hands-on time and reduced experimental/operator-dependent biases, the bottleneck of such techniques is clearly visible; that is, data analysis. Further complicating the matter, increasing evidence suggests most of the genome is transcribed into RNA; however, the majority of these RNAs are not translated into proteins. These RNAs that do not become proteins are called 'noncoding RNAs (ncRNAs)'. Although some time has passed since the discovery of ncRNAs, their annotations remain poor, making analysis of RNA-seq data challenging. Here, we examine the current limitations of RNA-seq analysis using case studies focused on the detection of novel transcripts and examination of their characteristics. Finally, we validate the presence of novel transcripts using biological experiments, showing novel transcripts can be accurately identified when a series of filters is applied. In conclusion, novel transcripts that are identified from RNA-seq must be examined carefully before proceeding to biological experiments. PMID:26283677

  2. An HIV-Encoded Antisense Long Noncoding RNA Epigenetically Regulates Viral Transcription

    PubMed Central

    Saayman, Sheena; Ackley, Amanda; Turner, Anne-Marie W; Famiglietti, Marylinda; Bosque, Alberto; Clemson, Matthew; Planelles, Vicente; Morris, Kevin V

    2014-01-01

    The abundance of long noncoding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus-encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this human immunodeficiency virus-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single-stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5′ long-term repeats (LTR) and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating human immunodeficiency virus gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter. PMID:24576854

  3. Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

    PubMed

    Tullo, A; Tanzariello, F; D'Erchia, A M; Nardelli, M; Papeo, P A; Sbisà, E; Saccone, C

    1994-10-31

    We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature. PMID:7957896

  4. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

    PubMed Central

    Reyes, Juan M; Chitwood, James L; Ross, Pablo J

    2014-01-01

    Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P<0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3’-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack there of (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation. PMID:25560149

  5. TAGLN2, a novel regulator involved in Hepatitis B virus transcription and replication.

    PubMed

    Yu, Youjia; He, Zhiliang; Cao, Yong; Tang, Hong; Huang, Feijun

    2016-09-01

    Hepatitis B virus (HBV) infection is one of the major health problems in the world. Transgelin-2 (TAGLN2) expression has been revealed to be significantly altered in previous studies concerning HBV-host interaction. The present study investigated TAGLN2 expression patterns in HBV related hepatocellular carcinoma (HCC) tissues and its role in HBV transcription and replication. We collected 59 HBV related HCC tissue samples, their adjacent non-tumoral tissues and 16 normal livers to make the tissue microarray. TAGLN2 protein was detected by immunohistochemistry and the transcriptional levels of TAGLN2, HBc, HBs and HBx were detected by qRT-PCR. Then we investigated the function of TAGLN2 on HBV transcription and replication in vitro by ectopic expressing or knocking down TAGLN2 in HepG2 and HepG2.2.15 cell lines. We further studied the effect of HBx on TAGLN2 expression with a Tet-on HBx expressing cell line. TAGLN2 protein expression was lower in normal livers and HBV-HCC tissues comparing to adjacent non-tumoral tissues. The transcriptional levels of TAGLN2 in HBV-HCC tissues and their adjacent tissues were positively related to that of HBc, HBs and HBx (P < 0.05). Ectopic expression of TAGLN2 in vitro could enhance HBV transcription and replication while suppressing TAGLN2 had the contrary effect. TAGLN2 could be induced by HBx in a dose-dependent manner. Our data demonstrated that TAGLN2 might be an HBx induced positive host factor involved in HBV transcription and replication and HBx related liver fibrosis and tumorigenesis. PMID:27402267

  6. Getting up to speed with transcription elongation by RNA polymerase II

    PubMed Central

    Jonkers, Iris; Lis, John T.

    2016-01-01

    Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II. PMID:25693130

  7. Active transcription and essential role of RNA polymerase II at the centromere during mitosis

    PubMed Central

    Chan, F. Lyn; Marshall, Owen J.; Saffery, Richard; Won Kim, Bo; Earle, Elizabeth; Choo, K. H. Andy; Wong, Lee H.

    2012-01-01

    Transcription of the centromeric regions has been reported to occur in G1 and S phase in different species. Here, we investigate whether transcription also occurs and plays a functional role at the mammalian centromere during mitosis. We show the presence of actively transcribing RNA polymerase II (RNAPII) and its associated transcription factors, coupled with the production of centromere satellite transcripts at the mitotic kinetochore. Specific inhibition of RNAPII activity during mitosis leads to a decrease in centromeric α-satellite transcription and a concomitant increase in anaphase-lagging cells, with the lagging chromosomes showing reduced centromere protein C binding. These findings demonstrate an essential role of RNAPII in the transcription of α-satellite DNA, binding of centromere protein C, and the proper functioning of the mitotic kinetochore. PMID:22308327

  8. Cell cycle-dependent regulation of RNA polymerase II basal transcription activity.

    PubMed Central

    Yonaha, M; Chibazakura, T; Kitajima, S; Yasukochi, Y

    1995-01-01

    Regulation of transcription by RNA polymerase II (pol II) in eukaryotic cells requires both basal and regulatory transcription factors. In this report we have investigated in vitro pol II basal transcription activity during the cell cycle by using nuclear extracts from synchronized HeLa cells. It is shown that pol II basal transcription activity is low in the S and G2 phases and high in early G1 phase and TFIID is the rate limiting component of pol II basal transcription activity during the cell cycle. Further analyses reveal that TFIID exists as a less active form in the S and G2 phases and nuclear extracts from S and G2 phase cells contain a heat-sensitive repressor(s) of TATA box binding protein (TBP). These results suggest that pol II basal transcription activity is regulated by a qualitative change in the TFIID complex, which could involve repression of TBP, during the cell cycle. Images PMID:7479063

  9. Natural Antisense Transcripts and Long Non-Coding RNA in Neurospora crassa

    PubMed Central

    Arthanari, Yamini; Heintzen, Christian; Griffiths-Jones, Sam; Crosthwaite, Susan K.

    2014-01-01

    The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs. PMID:24621812

  10. A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei

    PubMed Central

    Schimanski, Bernd; Brandenburg, Jens; Nguyen, Tu Ngoc; Caimano, Melissa Jo; Günzl, Arthur

    2006-01-01

    The lack of general class II transcription factors was a hallmark of the genomic sequences of the human parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. However, the recent identification of TFIIA as part of a protein complex essential for RNA polymerase II-mediated transcription of SLRNA genes, which encode the trans splicing-specific spliced leader RNA, suggests that trypanosomatids assemble a highly divergent set of these factors at the SLRNA promoter. Here we report the identification of a trypanosomatid TFIIB-like (TFIIBlike) protein which has limited overall sequence homology to eukaryotic TFIIB and archaeal TFB but harbors conserved residues within the N-terminal zinc ribbon domain, the B finger and cyclin repeat I. In accordance with the function of TFIIB, T.brucei TFIIBlike is encoded by an essential gene, localizes to the nucleus, specifically binds to the SLRNA promoter, interacts with RNA polymerase II, and is absolutely required for SLRNA transcription. PMID:16554554

  11. Detection of messenger RNA from the isoleucine--valine operons of Salmonella typhimurium by heterologous DNA-RNA hybridization: involvement of transfer RNA in transcriptional repression.

    PubMed

    Childs, G; Sonnenberg, F; Freundlich, M

    1977-03-01

    A hybridization assay using Escherichia coli K-12 DNA isolated from the specialized transducing bacteriophage gammaCI857St68h80 dilv was used to examine the rate of synthesis of the messenger RNA's (mRNA) derived from the isoleucine-valine (ilv) gene cluster of Salmonella typhimurium. In all cases examined, changes in ilv enzyme levels could be correlated with changes in the rate of synthesis of ilv mRNA. Several well characterized regulatory mutants of S. typhimurium had rates of synthesis of ilv mRNA 3 to 8-fold higher than the repressed wild-type strain. The increased rates of ilv mRNA synthesis found in a hisT strain as well as in isoleucyl-and leucyl-tRNA SYNTHETASE MUTANTS, STRONGLY SUGGESTS A ROLE FOR BRANCHED-CHAIN AMINOACYL-TRNA's in transcriptional control. PMID:327261

  12. Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

    PubMed Central

    Sikora, Dorota; Zhang, Dajiang; Bojic, Teodora; Beeharry, Yasnee; Tanara, Ali; Pelchat, Martin

    2013-01-01

    The hepatitis delta virus (HDV) is a small (∼1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used. PMID:23349975

  13. Ccr4-Not Regulates RNA Polymerase I Transcription and Couples Nutrient Signaling to the Control of Ribosomal RNA Biogenesis

    PubMed Central

    Laribee, R. Nicholas; Hosni-Ahmed, Amira; Workman, Jason J.; Chen, Hongfeng

    2015-01-01

    Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis. PMID:25815716

  14. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    PubMed Central

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  15. ChiTaRS 2.1--an improved database of the chimeric transcripts and RNA-seq data with novel sense-antisense chimeric RNA transcripts.

    PubMed

    Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Vucenovic, Dunja; Maestre, Lorena; Valencia, Alfonso

    2015-01-01

    Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS 2.1 database of chimeric transcripts and RNA-Seq data (http://chitars.bioinfo.cnio.es/) is the second version of the ChiTaRS database and includes improvements in content and functionality. Chimeras from eight organisms have been collated including novel sense-antisense (SAS) chimeras resulting from the slippage of the sense and anti-sense intragenic regions. The new database version collects more than 29,000 chimeric transcripts and indicates the expression and tissue specificity for 333 entries confirmed by RNA-seq reads mapping the chimeric junction sites. User interface allows for rapid and easy analysis of evolutionary conservation of fusions, literature references and experimental data supporting fusions in different organisms. More than 1428 cancer breakpoints have been automatically collected from public databases and manually verified to identify their correct cross-references, genomic sequences and junction sites. As a result, the ChiTaRS 2.1 collection of chimeras from eight organisms and human cancer breakpoints extends our understanding of the evolution of chimeric transcripts in eukaryotes as well as their functional role in carcinogenic processes. PMID:25414346

  16. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA

    PubMed Central

    Zuccaro, Antonio; Guarracino, Mario Rosario

    2015-01-01

    RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation’s enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein—protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely

  17. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA.

    PubMed

    Tripathi, Kumar Parijat; Evangelista, Daniela; Zuccaro, Antonio; Guarracino, Mario Rosario

    2015-01-01

    RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation's enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein-protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely

  18. RNA Editing of Androgen Receptor Gene Transcripts in Prostate Cancer Cells*S⃞

    PubMed Central

    Martinez, Harryl D.; Jasavala, Rohini J.; Hinkson, Izumi; Fitzgerald, Latricia D.; Trimmer, James S.; Kung, Hsing-Jien; Wright, Michael E.

    2008-01-01

    Reactivation of the androgen receptor (AR) signaling pathway represents a critical step in the growth and survival of androgen-independent (AI) prostate cancer (CaP). In this study we show the DU145 and PC3 AI human CaP cell lines respond to androgens and require AR expression for optimal proliferation in vitro. Interestingly, AR gene transcripts in DU145 and PC3 cells harbored a large number of single base pair nucleotide transitions that resulted in missense mutations in selected AR codons. The most notable lesion detected in AR gene transcripts included the oncogenic codon 877T→A gain-of-function mutation. Surprisingly, AR gene transcript nucleotide transitions were not genome-encoded substitutions, but instead the mutations co-localized to putative A-to-I, U-to-C, C-to-U, and G-to-A RNA editing sites, suggesting the lesions were mediated through RNA editing mechanisms. Higher levels of mRNA encoding the A-to-I RNA editing enzymes ADAR1 and ADARB1 were observed in DU145 and PC3 cells relative to the androgen-responsive LNCaP and 22Rv1 human CaP cell lines, which correlated with higher levels of AR gene transcript A-to-I editing detected in DU145 and PC3 cells. Our results suggest that AR gene transcripts are targeted by different RNA editing enzymes in DU145 and PC3 cells. Thus RNA editing of AR gene transcripts may contribute to the etiology of hormone-refractory phenotypes in advanced stage AI CaP. PMID:18708348

  19. Hepatitis C virus RNA assays: a comparison of SuperQuant and Monitor.

    PubMed

    Hadziyannis, E; Hadziyannis, A; Yen-Lieberman, B; Kiwi, M L; Hodnick, S; Spanou, F; Starkey, C; Younossi, Z M

    2001-07-01

    Hepatitis C RNA testing has been used extensively to assess the efficacy of antiviral therapy and has increasingly become an integral part of clinical management of patients with chronic hepatitis C. A variety of commercially available hepatitis C virus (HCV) RNA tests are used to detect HCV RNA qualitatively or quantitatively. These commercial tests have fundamental differences that are reflected on the values they generate. We compared two widely used assays, HCV SuperQuant (SQ) and Amplicor HCV Monitor (M1 and M2), in sera of patients with chronic hepatitis C. A total of 506 sera from 79 patients were tested with both assays. The data were logarithmically transformed and analyzed by linear regression and measurement of agreement. Two hundred thirty-eight sera had HCV RNA values within the dynamic range of both assays. The correlation between the assays was fair, with a correlation coefficient (r) of 0.699. Overall, SQ generated higher values than M1 with a mean difference of 0.558 log (SD = 0.624). One hundred ninety-four (38%) and 121 (24%) of the sera were below the dynamic range of M1 and SQ, respectively. Seventy-three sera, undetectable by M1, were positive by SQ. The Amplicor HCV Monitor 2.0 (M2) was performed in 66 sera. All were positive by SQ and M2, but only 38 were within the dynamic range of M1. The correlations between these tests were good (r = 0.68-0.78), but the agreement was rather poor. In conclusion, this study confirms that both SQ and M2 are more sensitive than M1. Additionally, our results show rather poor agreements between these assays. The recent attempts in standardizing the reporting of these assays should make their results more easily interchangeable. PMID:11418790

  20. Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein

    PubMed Central

    Wang, Horng-Dar; Trivedi, Alpa; Johnson, Deborah L.

    1998-01-01

    The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838–6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the

  1. Quality control of transcription start site selection by nonsense-mediated-mRNA decay.

    PubMed

    Malabat, Christophe; Feuerbach, Frank; Ma, Laurence; Saveanu, Cosmin; Jacquier, Alain

    2015-01-01

    Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA quality-control pathway targeting transcripts such as messenger RNAs harboring premature stop-codons or short upstream open reading frame (uORFs). Our transcription start sites (TSSs) analysis of Saccharomyces cerevisiae cells deficient for RNA degradation pathways revealed that about half of the pervasive transcripts are degraded by NMD, which provides a fail-safe mechanism to remove spurious transcripts that escaped degradation in the nucleus. Moreover, we found that the low specificity of RNA polymerase II TSSs selection generates, for 47% of the expressed genes, NMD-sensitive transcript isoforms carrying uORFs or starting downstream of the ATG START codon. Despite the low abundance of this last category of isoforms, their presence seems to constrain genomic sequences, as suggested by the significant bias against in-frame ATGs specifically found at the beginning of the corresponding genes and reflected by a depletion of methionines in the N-terminus of the encoded proteins. PMID:25905671

  2. Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms.

    PubMed

    Bushnell, David A; Westover, Kenneth D; Davis, Ralph E; Kornberg, Roger D

    2004-02-13

    The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process. PMID:14963322

  3. 7SK small nuclear RNA directly affects HMGA1 function in transcription regulation

    PubMed Central

    Eilebrecht, Sebastian; Brysbaert, Guillaume; Wegert, Thomas; Urlaub, Henning; Benecke, Bernd-Joachim; Benecke, Arndt

    2011-01-01

    Non-coding (nc) RNAs are increasingly recognized to play important regulatory roles in eukaryotic gene expression. The highly abundant and essential 7SK ncRNA has been shown to negatively regulate RNA Polymerase II transcription by inactivating the positive transcription elongation factor b (P-TEFb) in cellular and Tat-dependent HIV transcription. Here, we identify a more general, P-TEFb-independent role of 7SK RNA in directly affecting the function of the architectural transcription factor and chromatin regulator HMGA1. An important regulatory role of 7SK RNA in HMGA1-dependent cell differentiation and proliferation regulation is uncovered with the identification of over 1500 7SK-responsive HMGA1 target genes. Elevated HMGA1 expression is observed in nearly every type of cancer making the use of a 7SK substructure in the inhibition of HMGA1 activity, as pioneered here, potentially useful in therapy. The 7SK-HMGA1 interaction not only adds an essential facet to the comprehension of transcriptional plasticity at the coupling of initiation and elongation, but also might provide a molecular link between HIV reprogramming of cellular gene expression-associated oncogenesis. PMID:21087998

  4. Structure of a Neurospora RNA polymerase I promoter defined by transcription in vitro with homologous extracts.

    PubMed Central

    Tyler, B M; Giles, N H

    1985-01-01

    A Neurospora in vitro transcription system has been developed which specifically and efficiently initiates transcription of a cloned Neurospora crassa ribosomal RNA gene by RNA polymerase I. The initiation site of transcription (both in vitro and in vivo) appears to be located about 850 bp from the 5' end of mature 17S rRNA. However, the primary rRNA transcripts are normally cleaved very rapidly at a site 120-125 nt from the 5' end in vitro and in vivo. The nucleotide sequence surrounding the initiation site has been determined. The region from -16 to +9 exhibits partial homology to the corresponding sequences from a wide variety of organisms including yeast, but the most striking similarity is to the initiation region from Dictyostelium discoideum which displays 73% homology to the Neurospora sequence from -23 to +47. The Neurospora sequences from -96 to +97 have been shown to be sufficient for transcription. This region contains two sequences displaying 8/9 bp matches to elements of the 5S rDNA promoter. Images PMID:2989792

  5. Using Synthetic Mouse Spike-In Transcripts to Evaluate RNA-Seq Analysis Tools

    PubMed Central

    Leshkowitz, Dena; Feldmesser, Ester; Friedlander, Gilgi; Jona, Ghil; Ainbinder, Elena; Parmet, Yisrael; Horn-Saban, Shirley

    2016-01-01

    One of the key applications of next-generation sequencing (NGS) technologies is RNA-Seq for transcriptome genome-wide analysis. Although multiple studies have evaluated and benchmarked RNA-Seq tools dedicated to gene level analysis, few studies have assessed their effectiveness on the transcript-isoform level. Alternative splicing is a naturally occurring phenomenon in eukaryotes, significantly increasing the biodiversity of proteins that can be encoded by the genome. The aim of this study was to assess and compare the ability of the bioinformatics approaches and tools to assemble, quantify and detect differentially expressed transcripts using RNA-Seq data, in a controlled experiment. To this end, in vitro synthesized mouse spike-in control transcripts were added to the total RNA of differentiating mouse embryonic bodies, and their expression patterns were measured. This novel approach was used to assess the accuracy of the tools, as established by comparing the observed results versus the results expected of the mouse controlled spiked-in transcripts. We found that detection of differential expression at the gene level is adequate, yet on the transcript-isoform level, all tools tested lacked accuracy and precision. PMID:27100792

  6. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome.

    PubMed

    Van Driessche, Benoit; Rodari, Anthony; Delacourt, Nadège; Fauquenoy, Sylvain; Vanhulle, Caroline; Burny, Arsène; Rohr, Olivier; Van Lint, Carine

    2016-01-01

    Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3'LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5'LTR promoter. PMID:27545598

  7. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome

    PubMed Central

    Van Driessche, Benoit; Rodari, Anthony; Delacourt, Nadège; Fauquenoy, Sylvain; Vanhulle, Caroline; Burny, Arsène; Rohr, Olivier; Van Lint, Carine

    2016-01-01

    Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3′LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5′LTR promoter. PMID:27545598

  8. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  9. RNA sequence and transcriptional properties of the 3' end of the Newcastle disease virus genome

    SciTech Connect

    Kurilla, M.G.; Stone, H.O.; Keene, J.D.

    1985-09-01

    The 3' end of the genomic RNA of Newcastle disease virus (NDV) has been sequenced and the leader RNA defined. Using hybridization to a 3'-end-labeled genome, leader RNA species from in vitro transcription reactions and from infected cell extracts were found to be 47 and 53 nucleotides long. In addition, the start site of the 3'-proximal mRNA was determined by sequence analysis of in vitro (beta-32P)GTP-labeled transcription products. The genomic sequence extending beyond the leader region demonstrated an open reading frame for at least 42 amino acids and probably represents the amino terminus of the nucleocapsid protein (NP). The terminal 8 nucleotides of the NDV genome were identical to those of measles virus and Sendai virus while the sequence of the distal half of the leader region was more similar to that of vesicular stomatitis virus. These data argue for strong evolutionary relatedness between the paramyxovirus and rhabdovirus groups.

  10. Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay

    PubMed Central

    Hausburg, Melissa A; Doles, Jason D; Clement, Sandra L; Cadwallader, Adam B; Hall, Monica N; Blackshear, Perry J; Lykke-Andersen, Jens; Olwin, Bradley B

    2015-01-01

    Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3′ untranslated region. Upon satellite cell activation, p38α/β MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis. DOI: http://dx.doi.org/10.7554/eLife.03390.001 PMID:25815583

  11. T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates.

    PubMed Central

    Pleiss, J A; Derrick, M L; Uhlenbeck, O C

    1998-01-01

    The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5' end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5' end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5' ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5' end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5' end heterogeneity in combination with the 3' end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification. PMID:9769105

  12. Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay.

    PubMed

    Hausburg, Melissa A; Doles, Jason D; Clement, Sandra L; Cadwallader, Adam B; Hall, Monica N; Blackshear, Perry J; Lykke-Andersen, Jens; Olwin, Bradley B

    2015-01-01

    Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/β MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis. PMID:25815583

  13. Close association of RNA polymerase II and many transcription factors with Pol III genes.

    PubMed

    Raha, Debasish; Wang, Zhong; Moqtaderi, Zarmik; Wu, Linfeng; Zhong, Guoneng; Gerstein, Mark; Struhl, Kevin; Snyder, Michael

    2010-02-23

    Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using alpha-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression. PMID:20139302

  14. Different functional modes of p300 in activation of RNA polymerase III transcription from chromatin templates.

    PubMed

    Mertens, Claudia; Roeder, Robert G

    2008-09-01

    Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains. PMID:18644873

  15. Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates▿

    PubMed Central

    Mertens, Claudia; Roeder, Robert G.

    2008-01-01

    Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains. PMID:18644873

  16. Upstream Binding of Idling RNA Polymerase Modulates Transcription Initiation from a Nearby Promoter*

    PubMed Central

    Gerganova, Veneta; Maurer, Sebastian; Stoliar, Liubov; Japaridze, Aleksandre; Dietler, Giovanni; Nasser, William; Kutateladze, Tamara; Travers, Andrew; Muskhelishvili, Georgi

    2015-01-01

    The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter. PMID:25648898

  17. Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP

    PubMed Central

    Fan, Xiaochun; Shi, Hua; Lis, John T.

    2005-01-01

    The TATA-binding protein (TBP) is a general factor that is involved in transcription by all three types of nuclear RNA polymerase. To delineate the roles played by the DNA-binding surface of TBP in these transcription reactions, we used a set of RNA aptamers directed against TBP and examined their ability to perturb transcription in vitro by the different RNA polymerases. Distinct responses to the TBP aptamers were observed for transcription by different types of polymerase at either the initiation, reinitiation or both stages of the transcription cycle. We further probed the TBP interactions in the TFIIIB•DNA complex to elucidate the mechanism for the different sensitivity of Pol III dependent transcription before and after preinitiation complex (PIC) formation. Lastly, the aptamers were employed to measure the time required for Pol III PIC formation in vitro. This approach can be generalized to define the involvement of a particular region on the surface of a protein at particular stages in a biological process. PMID:15701755

  18. The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

    PubMed Central

    Reuter, L. Maximilian; Meinel, Dominik M.; Sträßer, Katja

    2015-01-01

    RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters. PMID:26220998

  19. PTRF/Cavin-1 promotes efficient ribosomal RNA transcription in response to metabolic challenges

    PubMed Central

    Liu, Libin; Pilch, Paul F

    2016-01-01

    Ribosomal RNA transcription mediated by RNA polymerase I represents the rate-limiting step in ribosome biogenesis. In eukaryotic cells, nutrients and growth factors regulate ribosomal RNA transcription through various key factors coupled to cell growth. We show here in mature adipocytes, ribosomal transcription can be acutely regulated in response to metabolic challenges. This acute response is mediated by PTRF (polymerase I transcription and release factor, also known as cavin-1), which has previously been shown to play a critical role in caveolae formation. The caveolae–independent rDNA transcriptional role of PTRF not only explains the lipodystrophy phenotype observed in PTRF deficient mice and humans, but also highlights its crucial physiological role in maintaining adipocyte allostasis. Multiple post-translational modifications of PTRF provide mechanistic bases for its regulation. The role of PTRF in ribosomal transcriptional efficiency is likely relevant to many additional physiological situations of cell growth and organismal metabolism. DOI: http://dx.doi.org/10.7554/eLife.17508.001 PMID:27528195

  20. Zinc metallothionein (MT) induction by parenteral iron and endotoxin: A temporal analysis of hepatic MT mRNA changes

    SciTech Connect

    McCormick, C.C. )

    1991-03-15

    The present study was undertaken to compare the temporal characteristics of iron-induced hepatic MT mRNA accumulation to that effected by endotoxin. Young chicks were given (ip) either endotoxin, ferrous gluconate or an equivalent volume of saline. At various times following injections, liver was obtained from 5 chicks per treatment for total RNA extraction. Equal amounts of total hepatic RNA from each chick were pooled and 10 {mu}g separated by denaturing agarose gel electrophoresis. Hepatic MT mRNA and albumin mRNA were analyzed by Northern blot analysis using synthetic oligonucleotides. The results indicated little temporal difference in the accumulation of hepatic MT mRNA as affected by either endotoxin or iron. In both treatments, MT mRNA was minimally affected at 3 hours post-injection. Maximum accumulation was achieved during a 6 h period from 6 to 12 hours post-injection. At 24 hours, MT mRNA was considerably higher in liver of endotoxin-injected chicks when compared to that of iron-injection chicks. Albumin expression appeared not to be substantially affected by either treatment. The results suggest that the induction of hepatic MT by iron injection is not substantially different than that observed following endotoxin administration. It would be speculative to suggest that the processes by which MT is induced under these conditions are also similar.

  1. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    SciTech Connect

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA

  2. Detecting miRNA by producing RNA: a sensitive assay that combines rolling-circle DNA polymerization and rolling circle transcription.

    PubMed

    Li, Xuemei; Zheng, Fuwei; Ren, Rui

    2015-08-01

    Target miRNA was detected by producing RNA: rolling circle polymerization (RCP) and rolling circle transcription (RCT) were interlinked to provide dual amplification, producing multiplied malachite green (MG) aptamers, and a signal was generated by the SERS (surface-enhanced Raman scattering) quantification of the MG molecules that were bound to the transcripts. PMID:26120604

  3. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. PMID:24291148

  4. RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.

    PubMed Central

    Kolodziej, P; Young, R A

    1989-01-01

    To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III. Images PMID:2685562

  5. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    SciTech Connect

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J.

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  6. Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

    PubMed Central

    Arimbasseri, Aneeshkumar G; Rijal, Keshab; Maraia, Richard J

    2013-01-01

    In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development. PMID:25764110

  7. Transcription termination and RNA processing in the 3'-end spacer of mouse ribosomal RNA genes.

    PubMed Central

    Miwa, T; Kominami, R; Yoshikura, H; Sudo, K; Muramatsu, M

    1987-01-01

    The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically. Images PMID:3031586

  8. Mediator and TREX-2: Emerging links between transcription initiation and mRNA export

    PubMed Central

    Schubert, Tobias; Köhler, Alwin

    2016-01-01

    ABSTRACT Nuclear pore proteins interact dynamically with chromatin to regulate gene activities. A key question is how nucleoporin interactions mechanistically alter a gene's intranuclear position and transcriptional output. We reported recently on a direct interaction between the nuclear pore-associated TREX-2 complex and promoter-bound Mediator. This highlights how nuclear-pore associated adaptors gain regulatory access to the core transcription machinery. In this Extra View, we discuss an additional implication that arises from our work and the recent literature: how promoter elements may regulate mRNA metabolism beyond transcription initiation. PMID:27028218

  9. Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

    PubMed Central

    Agbottah, Emmanuel T; Traviss, Christine; McArdle, James; Karki, Sambhav; St Laurent, Georges C; Kumar, Ajit

    2007-01-01

    Background Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression. Results Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene. Conclusion Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1. PMID:17565699

  10. Identification of Endogenous Controls for Analyzing Serum Exosomal miRNA in Patients with Hepatitis B or Hepatocellular Carcinoma

    PubMed Central

    Li, Yi; Zhang, Liqun; Liu, Fei; Xiang, Guiming; Jiang, Dongneng; Pu, Xiaoyun

    2015-01-01

    Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for diagnosing cancer. The canonical technique for measuring miRNA transcript levels is reverse transcription quantitative polymerase chain reaction (RT-qPCR). One prerequisite for validating RT-qPCR data is proper normalization with respect to stably expressed endogenous reference genes. However, genes that meet all of the criteria of a control gene for exosomal miRNAs have not yet been identified. To find out the control gene for exosomal miRNAs, we evaluated the expression stability of 11 well-known reference genes in circulating exosomes. In this study, we found that the combination of miR-221, miR-191, let-7a, miR-181a, and miR-26a can be an optimal gene reference set for normalizing the expression of liver-specific miRNAs. This combination enhanced the robustness of the relative quantification analyses. These findings highlight the importance of validating reference genes before quantifying target miRNAs. Furthermore, our findings will improve studies that monitor hepatitis progression and will aid in the discovery of noninvasive biomarkers to diagnose early stage HCC. PMID:25814782

  11. MicroRNA-27b Enhances the Hepatic Regenerative Properties of Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Chen, Kuang-Den; Huang, Kuang-Tzu; Lin, Chih-Che; Weng, Wei-Teng; Hsu, Li-Wen; Goto, Shigeru; Nakano, Toshiaki; Lai, Chia-Yun; Kung, Chao-Pin; Chiu, King-Wah; Wang, Chih-Chi; Cheng, Yu-Fan; Ma, Yen-Ying; Chen, Chao-Long

    2016-01-01

    Adipose-derived mesenchymal stem cells (ASCs) are readily available multipotent mesenchymal progenitor cells and have become an attractive therapeutic tool for regenerative medicine. We herein investigated the mechanistic role of how miR-27b modulated regenerative capacities of ASCs. Intravenous administration of miR-27b-transfected ASCs (ASCs-miR-27b) was conducted after 70% partial hepatectomy (PH). After PH, rats injected with ASCs-miR-27b had decreased inflammatory cytokines and increased hepatocyte growth factor and other related growth factors. We showed that the nature of ASCs-miR-27b to inhibit hepatic stellate cell activation was dependent upon peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in vitro. Moreover, expression of miR-27b in ASCs induced heme oxygenase-1 (HO-1), resulting in increased production of ATP, protective cytokines/growth factors, and genes involved in mitochondrial biogenesis in a PGC-1α-dependent manner. RNA sequencing (RNA-Seq) analysis revealed drastic transcriptional changes in livers treated with ASCs-miR-27b after PH. The differentially expressed genes classified into “regeneration,” “fibrosis,” and “mitochondrial biogenesis” clusters were mainly mitochondrial. The potential biological context reflecting the effects of PGC-1α by ASCs-miR-27b treatment was also observed by the subnetwork analysis with HO-1 and PGC-1α being the top-ranked regulatory genes. We demonstrate autologous ASCs-miR-27b enhances liver regeneration and, importantly, preserves hepatic function through paracrine actions which offers a viable therapeutic option to facilitate rapid recovery after liver injury. PMID:26836372

  12. Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation.

    PubMed

    Klauser, Benedikt; Saragliadis, Athanasios; Ausländer, Simon; Wieland, Markus; Berthold, Michael R; Hartig, Jörg S

    2012-09-01

    In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors. PMID:22777205

  13. Investigation of RNA Polymerase I Transcription under Force-Free Condition by Single Molecule Technique

    NASA Astrophysics Data System (ADS)

    Ucuncuoglu, Suleyman; Schneider, David A.; Dunlap, David; Finzi, Laura

    2014-03-01

    RNA Polymerase I (Pol I) conducts more than 60% of all the transcriptional activity in cells and also is responsible for synthesizing the RNA structure of the ribosome in eukaryotic cells. It is evident in many studies that Pol I transcription is affected by tumor suppressors and oncogenes which makes Pol I as a target for the anticancer therapeutics. The mechanistic pathways and kinetics of the Pol I transcription needs to be understood more precisely. Even though previous bulk studies measured the kinetics of the Pol I transcription, the results may hinder the intermediate states such as processivity and pausing during elongation. Here we used the single molecule approach to show that Pol I pauses more than Pol II during elongation step by using a novel single molecule instrument, multiplexed tethered particle motion microscopy (TPM). Our in-house developed TPM equipment is able to concurrently observe hundreds of single molecules. TPM technique has a major advantage to observe pausing under force-free condition unlike other single molecule techniques such as magnetic tweezers and optical tweezers. We also report that the processivity of Pol I is very low where only one out of fifteen transcription event reached the run-off site. We anticipate that our single molecule assays paved the way for observing more sophisticated aspects of Pol I transcription and it's relation with initiation and transcriptional factors.

  14. A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing

    PubMed Central

    Berdygulova, Zhanna; Esyunina, Daria; Miropolskaya, Nataliya; Mukhamedyarov, Damir; Kuznedelov, Konstantin; Nickels, Bryce E.; Severinov, Konstantin; Kulbachinskiy, Andrey; Minakhin, Leonid

    2012-01-01

    Gp39, a small protein encoded by Thermus thermophilus phage P23–45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity. PMID:22238378

  15. Review: Genomic era analyses of RNA secondary structure and RNA-binding proteins reveal their significance to post-transcriptional regulation in plants

    PubMed Central

    Silverman, Ian M.; Li, Fan; Gregory, Brian D.

    2014-01-01

    The eukaryotic transcriptome is regulated both transcriptionally and post-transcriptionally. Transcriptional control was the major focus of early research efforts, while more recently post-transcriptional mechanisms have gained recognition for their significant regulatory importance. At the heart of post-transcriptional regulatory pathways are cis- and trans-acting features and factors including RNA secondary structure as well as RNA-binding proteins and their recognition sites on target RNAs. Recent advances in genomic methodologies have significantly improved our understanding of both RNA secondary structure and RNA-binding proteins and their regulatory effects within the eukaryotic transcriptome. In this review, we focus specifically on the collection of these regulatory moieties in plant transcriptomes. We describe the approaches for studying RNA secondary structure and RNA-protein interaction sites, with an emphasis on recent methodological advances that produce transcriptome-wide datasets. We discuss how these methods that include genome-wide RNA secondary structure determination and RNA-protein interaction site mapping are significantly improving our understanding of the functions of these two elements in post-transcriptional regulation. Finally, we delineate the need for additional genome-wide studies of RNA secondary structure and RNA-protein interactions in plants. PMID:23498863

  16. Rule-based integration of RNA-Seq analyses tools for identification of novel transcripts.

    PubMed

    Inamdar, Harshal; Datta, Avik; Manjari, K Sunitha; Joshi, Rajendra

    2014-10-01

    Recent evidences suggest that a substantial amount of genome is transcribed more than that was anticipated, giving rise to a large number of unknown or novel transcripts. Identification of novel transcripts can provide key insights into understanding important cellular functions as well as molecular mechanisms underlying complex diseases like cancer. RNA-Seq has emerged as a powerful tool to detect novel transcripts, which previous profiling techniques failed to identify. A number of tools are available for enabling identification of novel transcripts at different levels. Read mappers such as TopHat, MapSplice, and SOAPsplice predict novel junctions, which are the indicators of novel transcripts. Cufflinks assembles novel transcripts based on alignment information and Oases performs de novo construction of transcripts. A common limitation of all these tools is prediction of sizable number of spurious or false positive (FP) novel transcripts. An approach that integrates information from all above sources and simultaneously scrutinizes FPs to correctly identify authentic novel transcripts of high confidence is proposed. PMID:25245144

  17. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  18. LNCipedia: a database for annotated human lncRNA transcript sequences and structures.

    PubMed

    Volders, Pieter-Jan; Helsens, Kenny; Wang, Xiaowei; Menten, Björn; Martens, Lennart; Gevaert, Kris; Vandesompele, Jo; Mestdagh, Pieter

    2013-01-01

    Here, we present LNCipedia (http://www.lncipedia.org), a novel database for human long non-coding RNA (lncRNA) transcripts and genes. LncRNAs constitute a large and diverse class of non-coding RNA genes. Although several lncRNAs have been functionally annotated, the majority remains to be characterized. Different high-throughput methods to identify new lncRNAs (including RNA sequencing and annotation of chromatin-state maps) have been applied in various studies resulting in multiple unrelated lncRNA data sets. LNCipedia offers 21 488 annotated human lncRNA transcripts obtained from different sources. In addition to basic transcript information and gene structure, several statistics are determined for each entry in the database, such as secondary structure information, protein coding potential and microRNA binding sites. Our analyses suggest that, much like microRNAs, many lncRNAs have a significant secondary structure, in-line with their presumed association with proteins or protein complexes. Available literature on specific lncRNAs is linked, and users or authors can submit articles through a web interface. Protein coding potential is assessed by two different prediction algorithms: Coding Potential Calculator and HMMER. In addition, a novel strategy has been integrated for detecting potentially coding lncRNAs by automatically re-analysing the large body of publicly available mass spectrometry data in the PRIDE database. LNCipedia is publicly available and allows users to query and download lncRNA sequences and structures based on different search criteria. The database may serve as a resource to initiate small- and large-scale lncRNA studies. As an example, the LNCipedia content was used to develop a custom microarray for expression profiling of all available lncRNAs. PMID:23042674

  19. The expression of IL-2, IL-4 and interferon-gamma (IFN-gamma) mRNA using liver biopsies at different phases of acute exacerbation of chronic hepatitis B.

    PubMed Central

    Fukuda, R; Ishimura, N; Nguyen, T X; Chowdhury, A; Ishihara, S; Kohge, N; Akagi, S; Watanabe, M; Fukumoto, S

    1995-01-01

    To investigate the hypothesis that Th1 phenotype cytokines are associated with the increasing activity of hepatitis and Th2 phenotype cytokines with decreasing activity in the liver of chronic viral hepatitis, expressions of the mRNA of the cytokines IL-2, IFN-gamma and IL-4 in the liver of 23 patients with chronic hepatitis B were investigated by reverse transcription polymerase chain reaction. Patients were divided into three groups according to the phase of acute exacerbation of hepatitis as increasing (n = 9), decreasing (n = 8), and stable phase (n = 6). Both IL-2 and IFN-gamma mRNA were preferentially expressed in increasing phase than in decreasing phase (P < 0.01, P < 0.05, respectively) and associated with the high serum alanine aminotransferase (ALT) level. On the other hand, IL-4 mRNA was detected in decreasing phase with significant frequency compared with increasing phase (P < 0.05). However, expression of IL-4 mRNA was not associated with serum ALT level. Our results suggest that Th1 phenotype cytokines up-regulate and Th2 phenotype cytokines down-regulate the liver inflammation of chronic viral hepatitis. PMID:7774054

  20. Host cellular microRNA involvement in the control of hepatitis B virus gene expression and replication

    PubMed Central

    Mizuguchi, Yoshiaki; Takizawa, Toshihiro; Uchida, Eiji

    2015-01-01

    A large number of studies have demonstrated that the synergistic collaboration of a number of microRNAs (miRNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. miRNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several miRNAs are involved in the hepatitis B virus (HBV) life cycle and infectivity, in addition to HBV-associated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). In turn, HBV can modulate the expression of several cellular miRNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular miRNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among miRNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease. PMID:25866606

  1. Transcriptional Regulation of the Group IIA Secretory Phospholipase A2 Gene by C/EBPδ in Rat liver and its Relationship to Hepatic Gluconeogenesis during Sepsis

    PubMed Central

    Yang, Rei-Cheng; Hsu, Chin; Lee, Tzu-Ying; Kuo, Kung-Kai; Wu, Shou-Mei; Chen, Yen-Hsu; Ho, Mei-Ling; Yao, Xing-Hai; Liu, Chia-Hsiung; Liu, Maw-Shung

    2014-01-01

    Background The present study was undertaken to test hypothesis that altered transcription of secretory Phospholipase A2 (sPLA2) gene in rat liver is regulated by CCAAT/enhancer binding protein δ (C/EBPδ), and to assess its relationship to hepatic gluconeogenesis during the progression of sepsis. Methods Sepsis was induced by Cecal Ligation and Puncture (CLP). Experiments were divided into three groups, control, early sepsis (9 h after CLP), and late sepsis (18 h after CLP). Results DNA mobility and super shift assays reveal that C/EBP complexes in the liver consisted of at least three isoforms: C/EBPα, C/EBPβ, and C/EBPδ; and various C/EBP isoforms were capable of interacting with each other. Hepatocyte transfection experiments demonstrate that under normal conditions, binding of C/EBPδ to sPLA2 gene enhanced sPLA2 promoter activity and the binding resulted in an increase in hepatic gluconeogenesis. Under pathological conditions such as sepsis, binding of C/EBPδ to sPLA2 promoter increased during early and late phases of sepsis, and the increases in C/EBPδ binding correlated with increases in sPLA2 mRNA abundance and sPLA2 protein levels. Under otherwise the identical experimental conditions, hepatic gluconeogenesis was reduced during early and late phases of sepsis and the sepsis-induced reductions in liver gluconeogenesis were aggravated by binding of C/EBPδ to sPLA2 gene. Conclusions These results link C/EBPδ binding to altered sPLA2 promoter, and to hepatic gluconeogenesis under normal and pathological conditions. It is suggested that C/EBPδ-sPLA2- hepatic gluconeogenesis may function as a signalling axis affecting glucose homeostasis during the progression of sepsis. PMID:25035816

  2. RNA transcript sequencing reveals inorganic sulfur compound oxidation pathways in the acidophile Acidithiobacillus ferrivorans.

    PubMed

    Christel, Stephan; Fridlund, Jimmy; Buetti-Dinh, Antoine; Buck, Moritz; Watkin, Elizabeth L; Dopson, Mark

    2016-04-01

    Acidithiobacillus ferrivorans is an acidophile implicated in low-temperature biomining for the recovery of metals from sulfide minerals. Acidithiobacillus ferrivorans obtains its energy from the oxidation of inorganic sulfur compounds, and genes encoding several alternative pathways have been identified. Next-generation sequencing of At. ferrivorans RNA transcripts identified the genes coding for metabolic and electron transport proteins for energy conservation from tetrathionate as electron donor. RNA transcripts suggested that tetrathionate was hydrolyzed by the tetH1 gene product to form thiosulfate, elemental sulfur and sulfate. Despite two of the genes being truncated, RNA transcripts for the SoxXYZAB complex had higher levels than for thiosulfate quinone oxidoreductase (doxDAgenes). However, a lack of heme-binding sites in soxX suggested that DoxDA was responsible for thiosulfate metabolism. Higher RNA transcript counts also suggested that elemental sulfur was metabolized by heterodisulfide reductase (hdrgenes) rather than sulfur oxygenase reductase (sor). The sulfite produced as a product of heterodisulfide reductase was suggested to be oxidized by a pathway involving the sat gene product or abiotically react with elemental sulfur to form thiosulfate. Finally, several electron transport complexes were involved in energy conservation. This study has elucidated the previously unknown At. ferrivorans tetrathionate metabolic pathway that is important in biomining. PMID:26956550

  3. Regulation of transcriptional pausing through the secondary channel of RNA polymerase.

    PubMed

    Esyunina, Daria; Agapov, Aleksei; Kulbachinskiy, Andrey

    2016-08-01

    Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg(2+) or Mn(2+)) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP. PMID:27432968

  4. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  5. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  6. PTEN represses RNA polymerase III-dependent transcription by targeting the TFIIIB complex.

    PubMed

    Woiwode, Annette; Johnson, Sandra A S; Zhong, Shuping; Zhang, Cheng; Roeder, Robert G; Teichmann, Martin; Johnson, Deborah L

    2008-06-01

    PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling. PMID:18391023

  7. HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

    PubMed Central

    McNamara, Ryan P; Forst, Christian V; D'Orso, Iván

    2015-01-01

    HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular ‘TAR-like’ motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but ‘multitasking’ protein to simultaneously control viral and cellular transcription. DOI: http://dx.doi.org/10.7554/eLife.08955.001 PMID:26488441

  8. The ratcheted and ratchetable structural states of RNA polymerase underlie multiple transcriptional functions.

    PubMed

    Sekine, Shun-ichi; Murayama, Yuko; Svetlov, Vladimir; Nudler, Evgeny; Yokoyama, Shigeyuki

    2015-02-01

    DNA-dependent RNA polymerase (RNAP) accomplishes multiple tasks during transcription by assuming different structural forms. Reportedly, the "tight" form performs nucleotide addition to nascent RNA, while the "ratcheted" form is adopted for transcription inhibition. In this study, we performed Cys-pair crosslinking (CPX) analyses of various transcription complexes of a bacterial RNAP and crystallographic analyses of its backtracked and Gre-factor-bound states to clarify which of the two forms is adopted. The ratcheted form was revealed to support GreA-dependent transcript cleavage, long backtracking, hairpin-dependent pausing, and termination. In contrast, the tight form correlated with nucleotide addition, mismatch-dependent pausing, one-nucleotide backtracking, and factor-independent transcript cleavage. RNAP in the paused/backtracked state, but not the nucleotide-addition state, readily transitions to the ratcheted form ("ratchetable"), indicating that the tight form represents two distinct regulatory states. The 3' end and the hairpin structure of the nascent RNA promote the ratchetable nature by modulating the trigger-loop conformation. PMID:25601758

  9. Transcription initiation factor DksA has diverse effects on RNA chain elongation

    PubMed Central

    Furman, Ran; Sevostyanova, Anastasiya; Artsimovitch, Irina

    2012-01-01

    Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex. PMID:22210857

  10. Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

    PubMed Central

    Sittka, Alexandra; Lucchini, Sacha; Papenfort, Kai; Sharma, Cynthia M.; Rolle, Katarzyna; Binnewies, Tim T.; Hinton, Jay C. D.; Vogel, Jörg

    2008-01-01

    Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria. PMID:18725932

  11. LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages

    PubMed Central

    Casero, David; Sandoval, Salemiz; Seet, Christopher S.; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M.

    2015-01-01

    To elucidate the transcriptional landscape that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitors spanning the earliest stages of B and T lymphoid specification. Over 3000 novel long non-coding RNA genes (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage-specific and more lineage-specific than protein coding patterns. Protein-coding genes co-expressed with neighboring lncRNA genes were enriched for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships between the earliest progenitors in the human bone marrow and thymus. PMID:26502406

  12. Characterization of Herpes Simplex Virus 2 Primary MicroRNA Transcript Regulation

    PubMed Central

    Bosch-Marce, Marta; Patel, Amita; Margolis, Todd P.

    2015-01-01

    ABSTRACT In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. IMPORTANCE The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5′ ends and evaluation of

  13. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment.

    PubMed

    Kwasniewska, Jolanta; Jaskowiak, Joanna

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  14. A dynamic model for PC4 coactivator function in RNA polymerase II transcription

    PubMed Central

    Malik, Sohail; Guermah, Mohamed; Roeder, Robert G.

    1998-01-01

    Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state. PMID:9482861

  15. Crystal structure of a transcribing RNA Polymerase II complex reveals a complete transcription bubble

    PubMed Central

    Barnes, Christopher O.; Calero, Monica; Malik, Indranil; Graham, Brian W.; Spahr, Henrik; Lin, Guowu; Cohen, Aina; Brown, Ian S.; Zhang, Qiangmin; Pullara, Filippo; Trakselis, Michael A.; Kaplan, Craig D.; Calero, Guillermo

    2015-01-01

    Summary Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation. PMID:26186291

  16. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment

    PubMed Central

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  17. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.

    PubMed

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-02-13

    The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape. PMID:14963331

  18. Transcriptional Bursting Explains the Noise–Versus–Mean Relationship in mRNA and Protein Levels

    PubMed Central

    Dar, Roy D.; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean. PMID:27467384

  19. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

    PubMed

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A; Shore, Paul

    2012-01-31

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  20. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    DOE PAGESBeta

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  1. Production and Processing of siRNA Precursor Transcripts from the Highly Repetitive Maize Genome

    PubMed Central

    Hale, Christopher J.; Erhard, Karl F.; Lisch, Damon; Hollick, Jay B.

    2009-01-01

    Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA–directed DNA methylation (RdDM) factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR) retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA–dependent RNA polymerase, RDR2 (MOP1). Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV) function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II–based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species. PMID:19680464

  2. Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

    PubMed Central

    Ho, Tho H.; Dang, Kien X.; Lintula, Susanna; Hotakainen, Kristina; Feng, Lin; Olkkonen, Vesa M.; Verschuren, Emmy W.; Tenkanen, Tuomas; Haglund, Caj; Kolho, Kaija-Leena; Stenman, Ulf-Hakan; Stenman, Jakob

    2015-01-01

    Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression. PMID:25378315

  3. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGESBeta

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  4. Novel mechanism of transcriptional repression of the human ATP binding cassette transporter A1 gene in hepatic cells by the winged helix/forkhead box transcription factor A2.

    PubMed

    Thymiakou, Efstathia; Kardassis, Dimitris

    2014-06-01

    ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXRα/RXRα. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene. PMID:24807696

  5. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    PubMed Central

    Van Assche, Elke; Van Puyvelde, Sandra; Vanderleyden, Jos; Steenackers, Hans P.

    2015-01-01

    Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed toward the role of small RNAs (sRNAs) in bacterial post-transcriptional regulation. However, sRNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins (RBPs) play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RBPs, which include (i) adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii) modulating the accessibility of the ribosome binding site of mRNAs, (iii) recruiting and assisting in the interaction of mRNAs with other molecules and (iv) regulating transcription terminator/antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future. PMID:25784899

  6. Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

    PubMed Central

    Pedone, F; Filetici, P; Ballario, P

    1982-01-01

    Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription. Images PMID:6292834

  7. World Health Organization International Standard to Harmonize Assays for Detection of Hepatitis E Virus RNA

    PubMed Central

    Blümel, Johannes; Mizusawa, Saeko; Matsubayashi, Keiji; Sakata, Hidekatsu; Okada, Yoshiaki; Nübling, C. Micha; Hanschmann, Kay-Martin O.

    2013-01-01

    Nucleic acid amplification technique–based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter. PMID:23647659

  8. Interference of hepatitis C virus RNA replication by short interfering RNAs

    NASA Astrophysics Data System (ADS)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  9. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA.

    PubMed

    Ascher, David B; Wielens, Jerome; Nero, Tracy L; Doughty, Larissa; Morton, Craig J; Parker, Michael W

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  10. Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA

    PubMed Central

    Ascher, David B.; Wielens, Jerome; Nero, Tracy L.; Doughty, Larissa; Morton, Craig J.; Parker, Michael W.

    2014-01-01

    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA. PMID:24755925

  11. Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II.

    PubMed

    Larson, Matthew H; Zhou, Jing; Kaplan, Craig D; Palangat, Murali; Kornberg, Roger D; Landick, Robert; Block, Steven M

    2012-04-24

    During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate. PMID:22493230

  12. Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

    PubMed Central

    Larson, Matthew H.; Zhou, Jing; Kaplan, Craig D.; Palangat, Murali; Kornberg, Roger D.; Landick, Robert; Block, Steven M.

    2012-01-01

    During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate. PMID:22493230

  13. Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma

    PubMed Central

    Grosso, Ana R; Leite, Ana P; Carvalho, Sílvia; Matos, Mafalda R; Martins, Filipa B; Vítor, Alexandra C; Desterro, Joana MP; Carmo-Fonseca, Maria; de Almeida, Sérgio F

    2015-01-01

    Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer. DOI: http://dx.doi.org/10.7554/eLife.09214.001 PMID:26575290

  14. Role of the intergenic dinucleotide in vesicular stomatitis virus RNA transcription.

    PubMed Central

    Barr, J N; Whelan, S P; Wertz, G W

    1997-01-01

    To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in modulation of polymerase activity, we analyzed the RNA synthesis activities of bicistrionic genomic analogs that contained either the authentic N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide intergenic sequences, or no intergenic sequence at all. Quantitative measurements of the amounts of upstream, downstream, and readthrough mRNAs that were transcribed by these mutant templates showed that the behavior of the viral polymerase was profoundly affected by the nucleotide sequence that it encountered as it traversed the gene junction, although the polymerase was able to accommodate a remarkable degree of sequence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic sequence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation of downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analogs with altered intergenic sequences directed abundant synthesis of a readthrough transcript without correspondingly high levels of downstream mRNA, an observation inconsistent with the shunting model of VSV transcription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findings of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript. PMID:9032308

  15. A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

    PubMed

    Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W; Oegema, Karen; Desai, Arshad

    2016-04-01

    Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

  16. Rational design of chemical genetic probes of RNA function and lead therapeutics targeting repeating transcripts

    PubMed Central

    Disney, Matthew D.

    2013-01-01

    RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA. PMID:23939337

  17. Rational design of chemical genetic probes of RNA function and lead therapeutics targeting repeating transcripts.

    PubMed

    Disney, Matthew D

    2013-12-01

    RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA. PMID:23939337

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

    ERIC Educational Resources Information Center

    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…

  19. Transforming growth factor (TGF)-beta stimulates hepatic jun-B and fos-B proto-oncogenes and decreases albumin mRNA.

    PubMed Central

    Beauchamp, R D; Sheng, H M; Ishizuka, J; Townsend, C M; Thompson, J C

    1992-01-01

    Transforming growth factor-beta (TGF-beta) modulates some components of the acute phase response in hepatic cells. The mechanisms for these actions of TGF-beta are largely unknown. The authors recently found that the decrease in albumin mRNA after TGF-beta 1 treatment required de novo RNA and protein synthesis, suggesting that TGF-beta acts through induction of another gene. The purpose of the current study was to determine whether TGF-beta 1 could regulate the expression of both the jun and fos genes that encode transcriptional regulatory proteins that constitute the AP-1 complex, and to determine whether expression of these genes may be coordinated with the decrease in albumin mRNA. Northern blot hybridization was used to determine levels of specific mRNAs. Transforming growth factor-beta 1 increased the levels of both jun-B and fos-B mRNA by 60 minutes after treatment of mouse hepatoma (BWTG3) cells. When TGF-beta 1 was removed from the media after 4 hours, there was a sustained effect of increased jun-B and decreased albumin mRNA (greater than 48 hours), and the subsequent decrease in jun-B levels coincided with the increase in albumin mRNA. The tumor-promoting phorbol ester (phorbol 12-myristate 13-acetate [PMA]), known to induce jun and fos gene expression, caused increases in jun-B and fos-B that preceded the decrease in albumin mRNA levels at 24 hours. These observations are consistent with our hypothesis that jun-B and fos-B induction may participate in downregulation of albumin synthesis as well as other hepatic responses to TGF-beta. Images FIG. 1. FIG. 2. FIG. 4. FIG. 5. FIG. 6. PMID:1417179

  20. DcpS is a transcript-specific modulator of RNA in mammalian cells

    PubMed Central

    Zhou, Mi; Bail, Sophie; Plasterer, Heather L.; Rusche, James

    2015-01-01

    The scavenger decapping enzyme DcpS is a multifunctional protein initially identified by its property to hydrolyze the resulting cap structure following 3′ end mRNA decay. In Saccharomyces cerevisiae, the DcpS homolog Dcs1 is an obligate cofactor for the 5′-3′ exoribonuclease Xrn1 while the Caenorhabditis elegans homolog Dcs-1, facilitates Xrn1 mediated microRNA turnover. In both cases, this function is independent of the decapping activity. Whether DcpS and its decapping activity can affect mRNA steady state or stability in mammalian cells remains unknown. We sought to determine DcpS target genes in mammalian cells using a cell-permeable DcpS inhibitor compound, RG3039 initially developed for therapeutic treatment of spinal muscular atrophy. Global mRNA levels were examined following DcpS decapping inhibition with RG3039. The steady-state levels of 222 RNAs were altered upon RG3039 treatment. Of a subset selected for validation, two transcripts that appear to be long noncoding RNAs HS370762 and BC011766, were dependent on DcpS and its scavenger decapping catalytic activity and referred to as DcpS-responsive noncoding transcripts (DRNT) 1 and 2, respectively. Interestingly, only the increase in DRNT1 transcript was accompanied with an increase of its RNA stability and this increase was dependent on both DcpS and Xrn1. Importantly, unlike in yeast where the DcpS homolog is an obligate cofactor for Xrn1, stability of additional Xrn1 dependent RNAs were not altered by a reduction in DcpS levels. Collectively, our data demonstrate that DcpS in conjunction with Xrn1 has the potential to regulate RNA stability in a transcript-selective manner in mammalian cells. PMID:26001796

  1. Does the linear Sry transcript function as a ceRNA for miR-138? The sense of antisense

    PubMed Central

    Granados-Riveron, Javier Tadeo; Aquino-Jarquin, Guillermo

    2014-01-01

    Recently, the sex determining region Y ( Sry) and the cerebellar degeneration-related protein 1 ( CDR1as) RNA transcripts have been described to function as a new class of post-transcriptional regulatory RNAs that behave as circular endogenous RNA sponges for the micro RNAs (miRNAs) miR-138 and miR-7, respectively. A special feature of the Sry gene is its ability to generate linear and circular transcripts, both transcribed in the sense orientation. Here we remark that both sense (e.g. Sry RNA) and antisense (e.g. CDR1as) transcripts could circularize and behave as miRNAs sponges, and importantly, that also protein-coding segments of mRNAs could also assume this role. Thus, it is reasonable to think that the linear Sry sense transcript could additionally act as a miRNA sponge, or as an endogenous competing RNA for miR-138. PMID:25580223

  2. Transcriptional Activation of the Interleukin-2 Promoter by Hepatitis C Virus Core Protein

    PubMed Central

    Bergqvist, Anders; Rice, Charles M.

    2001-01-01

    Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56lck, and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections. PMID:11134290

  3. Novel Mechanism of Blood Pressure Regulation By Foxo1-Mediated Transcriptional Control of Hepatic Angiotensinogen

    PubMed Central

    Qi, Yajuan; Zhang, Kebin; Wu, Yuxin; Xu, Zihui; Yong, QC; Kumar, Rajesh; Baker, Kenneth M.; Zhu, Qinglei; Chen, Shouwen; Guo, Shaodong

    2014-01-01

    The renin-angiotensin system (RAS) is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving three components: angiotensinogen (Agt), renin, and angiotensin-converting enzyme (ACE), which generate angiotensin II (Ang II) as a biologically active product. Agt is largely produced in the liver, acting as a major determinant of the circulating RAS, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of Agt is regulated is not completely understood. Here we hypothesize that Agt is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice (L-F1KO mice), which exhibited a reduction in plasma Agt and Ang II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased Agt expression, while hepatocytes lacking Foxo1 demonstrated a reduction of Agt gene expression and partially impaired insulin inhibition on Agt gene expression. Furthermore, mouse Agt prompter analysis demonstrated that the Agt promoter region contains a functional Foxo1 binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic Agt gene expression and controls plasma Agt and Ang II levels, modulating blood pressure control in mice. PMID:25069665

  4. Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR

    PubMed Central

    Ko, Gwangpyo; Cromeans, Theresa L.; Sobsey, Mark D.

    2003-01-01

    We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

  5. Conserved RNA secondary structures and long-range interactions in hepatitis C viruses.

    PubMed

    Fricke, Markus; Dünnes, Nadia; Zayas, Margarita; Bartenschlager, Ralf; Niepmann, Michael; Marz, Manja

    2015-07-01

    Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in nonstructural protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5' untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3' UTR. We propose an alternative structure in the 5' UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3' end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5' and 3' UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication. PMID:25964384

  6. Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5

    PubMed Central

    Kassube, Susanne A.; Jinek, Martin; Fang, Jie; Tsutakawa, Susan; Nogales, Eva

    2013-01-01

    RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein–coding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo–electron microscopy structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read–through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II–TFIIS interaction. PMID:23748380

  7. Hepatitis C virus RNA codes for proteins and replicates: does it also trigger the interferon response?

    PubMed

    Branch, A D

    2000-01-01

    Hepatitis C virus (HCV) is a positive sense virus with a genomic RNA molecule roughly 9,600 nucleotides in length. The single-stranded genomic RNA has a nontranslated region (NTR) at each end and a long open reading frame (coding region) in between. The 5'NTR and portions of the 3'NTR are the most conserved parts of HCV RNA. These conserved regions contain signals for replication and translation. Much of the 5'NTR is folded into a structure that binds ribosomes. This structure, an internal ribosome entry site, promotes the initiation of protein synthesis and is critical for HCV gene expression. The ribosome binding site may extend into the coding region; its exact boundaries are not known. The open reading frame encodes the HCV polyprotein, which is slightly more than 3,000 amino acids in length. The 3'NTR plays a key role in HCV replication and may also influence the rate of HCV protein synthesis. During replication, the genomic RNA is copied by virally encoded enzymes into a complementary antigenomic RNA, which itself is a template for the synthesis of progeny RNAs. At steady state, genomic strands outnumber antigenomic strands about 10 to 1. HCV RNA replication is thought to take place in the cytoplasm and is an error-prone process. It generates a mixed population of RNA sequences (quasispecies), including mutants that may be more fit than the parental type, less fit, or equally fit (but distinct). Natural selection acts upon the progeny RNAs, causing the population to change and drift. Over time, mutation, selection, and population bottlenecks led to the evolution of varied genotypes. The HCV replication complex is a potential source of double-stranded RNA, a powerful inducer of interferon. Thus, HCV-specific double-stranded RNA may trigger the first steps of innate immunity; however, for unknown reasons, the immune system often fails to clear the infection. The plasticity of the HCV genome and the low level of HCV gene expression may counterbalance any

  8. DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts

    PubMed Central

    Paraskevopoulou, Maria D.; Vlachos, Ioannis S.; Karagkouni, Dimitra; Georgakilas, Georgios; Kanellos, Ilias; Vergoulis, Thanasis; Zagganas, Konstantinos; Tsanakas, Panayiotis; Floros, Evangelos; Dalamagas, Theodore; Hatzigeorgiou, Artemis G.

    2016-01-01

    microRNAs (miRNAs) are short non-coding RNAs (ncRNAs) that act as post-transcriptional regulators of coding gene expression. Long non-coding RNAs (lncRNAs) have been recently reported to interact with miRNAs. The sponge-like function of lncRNAs introduces an extra layer of complexity in the miRNA interactome. DIANA-LncBase v1 provided a database of experimentally supported and in silico predicted miRNA Recognition Elements (MREs) on lncRNAs. The second version of LncBase (www.microrna.gr/LncBase) presents an extensive collection of miRNA:lncRNA interactions. The significantly enhanced database includes more than 70 000 low and high-throughput, (in)direct miRNA:lncRNA experimentally supported interactions, derived from manually curated publications and the analysis of 153 AGO CLIP-Seq libraries. The new experimental module presents a 14-fold increase compared to the previous release. LncBase v2 hosts in silico predicted miRNA targets on lncRNAs, identified with the DIANA-microT algorithm. The relevant module provides millions of predicted miRNA binding sites, accompanied with detailed metadata and MRE conservation metrics. LncBase v2 caters information regarding cell type specific miRNA:lncRNA regulation and enables users to easily identify interactions in 66 different cell types, spanning 36 tissues for human and mouse. Database entries are also supported by accurate lncRNA expression information, derived from the analysis of more than 6 billion RNA-Seq reads. PMID:26612864

  9. DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts.

    PubMed

    Paraskevopoulou, Maria D; Vlachos, Ioannis S; Karagkouni, Dimitra; Georgakilas, Georgios; Kanellos, Ilias; Vergoulis, Thanasis; Zagganas, Konstantinos; Tsanakas, Panayiotis; Floros, Evangelos; Dalamagas, Theodore; Hatzigeorgiou, Artemis G

    2016-01-01

    microRNAs (miRNAs) are short non-coding RNAs (ncRNAs) that act as post-transcriptional regulators of coding gene expression. Long non-coding RNAs (lncRNAs) have been recently reported to interact with miRNAs. The sponge-like function of lncRNAs introduces an extra layer of complexity in the miRNA interactome. DIANA-LncBase v1 provided a database of experimentally supported and in silico predicted miRNA Recognition Elements (MREs) on lncRNAs. The second version of LncBase (www.microrna.gr/LncBase) presents an extensive collection of miRNA:lncRNA interactions. The significantly enhanced database includes more than 70 000 low and high-throughput, (in)direct miRNA:lncRNA experimentally supported interactions, derived from manually curated publications and the analysis of 153 AGO CLIP-Seq libraries. The new experimental module presents a 14-fold increase compared to the previous release. LncBase v2 hosts in silico predicted miRNA targets on lncRNAs, identified with the DIANA-microT algorithm. The relevant module provides millions of predicted miRNA binding sites, accompanied with detailed metadata and MRE conservation metrics. LncBase v2 caters information regarding cell type specific miRNA:lncRNA regulation and enables users to easily identify interactions in 66 different cell types, spanning 36 tissues for human and mouse. Database entries are also supported by accurate lncRNA expression information, derived from the analysis of more than 6 billion RNA-Seq reads. PMID:26612864

  10. The hepatitis C virus core protein can modulate RNA-dependent RNA synthesis by the 2a polymerase

    PubMed Central

    Wen, Y.; Cheng Kao, C.

    2014-01-01

    RNA replication enzymes are multi-subunit protein complexes whose activity can be modulated by other viral and cellular factors. For genotype 1b Hepatitis C virus (HCV), the RNA-dependent RNA polymerase (RdRp) subunit of the replicase, NS5B, has been reported to interact with the HCV Core protein to decrease RNA synthesis (Kang et al., 2009). Here we used a cell-based assay for RNA synthesis to examine the Core–NS5B interaction of genotype 2a HCV. Unlike the 1b NS5B, the activity of the 2a NS5B was stimulated by the Core protein. Using the bimolecular fluorescence complementation assay, the 2a Core co-localized with 2a NS5B when they were transiently expressed in cells. The two proteins can form a coimmunoprecipitable complex. Deletion analysis showed that the N-terminal 75 residues of 2a Core were required to contact 2a NS5B to modulate its activity. The C-terminal transmembrane helix of 2a NS5B also contributes to the interaction with the 2a Core. To determine the basis for the differential effects of the Core–RdRp interaction, we found that the 2a RdRp activity was enhanced by both the 1b Core and 2a Core. However, the 1b NS5B activity was slightly inhibited by either Core protein. The replication of the 2a JFH-1 replicon was increased by co-expressed 2a Core while the genotype 1b Con1 replicon was not significantly affected by the corresponding Core. Mutations in 2a NS5B that affected the closed RdRp structure were found to be less responsive to 2a Core. Finally, we determined that RNA synthesis by the RdRps from genotypes 2a, 3a and 4a HCV were increased by the Core proteins from HCV of genotypes 1–4. These results reveal another difference between RNA syntheses by the different genotype RdRps and add additional examples of a viral structural protein regulating viral RNA synthesis. PMID:24874198

  11. Transcription factors YY1, Sp1 and Sp3 modulate dystrophin Dp71 gene expression in hepatic cells.

    PubMed

    Peñuelas-Urquides, Katia; Becerril-Esquivel, Carolina; Mendoza-de-León, Laura C; Silva-Ramírez, Beatriz; Dávila-Velderrain, José; Cisneros, Bulmaro; de León, Mario Bermúdez

    2016-07-01

    Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic β-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5'-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter. PMID:27143785

  12. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  13. Genome-wide analysis of FOXO3 mediated transcription regulation through RNA polymerase II profiling.

    PubMed

    Eijkelenboom, Astrid; Mokry, Michal; de Wit, Elzo; Smits, Lydia M; Polderman, Paulien E; van Triest, Miranda H; van Boxtel, Ruben; Schulze, Almut; de Laat, Wouter; Cuppen, Edwin; Burgering, Boudewijn M T

    2013-01-01

    Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO-regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. PMID:23340844

  14. BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

    PubMed

    Borck, Guntram; Hög, Friederike; Dentici, Maria Lisa; Tan, Perciliz L; Sowada, Nadine; Medeira, Ana; Gueneau, Lucie; Thiele, Holger; Kousi, Maria; Lepri, Francesca; Wenzeck, Larissa; Blumenthal, Ian; Radicioni, Antonio; Schwarzenberg, Tito Livio; Mandriani, Barbara; Fischetto, Rita; Morris-Rosendahl, Deborah J; Altmüller, Janine; Reymond, Alexandre; Nürnberg, Peter; Merla, Giuseppe; Dallapiccola, Bruno; Katsanis, Nicholas; Cramer, Patrick; Kubisch, Christian

    2015-02-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development. PMID:25561519

  15. BRF1 mutations alter RNA polymerase III–dependent transcription and cause neurodevelopmental anomalies

    PubMed Central

    Hög, Friederike; Dentici, Maria Lisa; Tan, Perciliz L.; Sowada, Nadine; Medeira, Ana; Gueneau, Lucie; Thiele, Holger; Kousi, Maria; Lepri, Francesca; Wenzeck, Larissa; Blumenthal, Ian; Radicioni, Antonio; Schwarzenberg, Tito Livio; Mandriani, Barbara; Fischetto, Rita; Morris-Rosendahl, Deborah J.; Altmüller, Janine; Reymond, Alexandre; Nürnberg, Peter; Merla, Giuseppe; Dallapiccola, Bruno; Katsanis, Nicholas; Cramer, Patrick; Kubisch, Christian

    2015-01-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III–related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development. PMID:25561519

  16. Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII.

    PubMed Central

    Szentirmay, M N; Sawadogo, M

    1993-01-01

    Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes. Images PMID:8223477

  17. Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

    PubMed Central

    Polosa, Paola Loguercio; Deceglie, Stefania; Falkenberg, Maria; Roberti, Marina; Di Ponzio, Barbara; Gadaleta, Maria Nicola; Cantatore, Palmiro

    2007-01-01

    Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms. PMID:17392338

  18. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  19. Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

    PubMed Central

    Mok, M; Maderious, A; Chen-Kiang, S

    1984-01-01

    We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication. Images PMID:6209554

  20. Real-time observation of the initiation of RNA polymerase II transcription.

    PubMed

    Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

    2015-09-10

    Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning. PMID:26331540

  1. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    PubMed Central

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  2. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

    PubMed

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  3. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    NASA Astrophysics Data System (ADS)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  4. The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent

    PubMed Central

    Hauenschild, Ralf; Tserovski, Lyudmil; Schmid, Katharina; Thüring, Kathrin; Winz, Marie-Luise; Sharma, Sunny; Entian, Karl-Dieter; Wacheul, Ludivine; Lafontaine, Denis L. J.; Anderson, James; Alfonzo, Juan; Hildebrandt, Andreas; Jäschke, Andres; Motorin, Yuri; Helm, Mark

    2015-01-01

    The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3′ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA. PMID:26365242

  5. Post-transcriptional regulation in corticogenesis: how RNA-binding proteins help build the brain

    PubMed Central

    Pilaz, Louis-Jan; Silver, Debra L.

    2015-01-01

    The cerebral cortex, the brain structure responsible for our higher cognitive functions, is built during embryonic development in a process called corticogenesis. During corticogenesis, neural stem cells generate distinct populations of progenitors and excitatory neurons. These new neurons migrate radially in the cortex, eventually forming neuronal layers and establishing synaptic connections with other neurons both within and outside the cortex. Perturbations to corticogenesis can result in severe neurodevelopmental disorders, thus emphasizing the need to better understand molecular regulation of brain development. Recent studies in both model organisms and humans have collectively highlighted roles for post-transcriptional regulation in virtually all steps of corticogenesis. Genomic approaches have revealed global RNA changes associated with spatial and temporal regulation of cortical development. Additionally, genetic studies have uncovered RNA-binding proteins (RBPs) critical for cell proliferation, differentiation, and migration within the developing neocortex. Many of these same RBPs play causal roles in neurodevelopmental pathologies. In the developing neocortex, RBPs influence diverse steps of mRNA metabolism, including splicing, stability, translation, and localization. With the advent of new technologies, researchers have begun to uncover key transcripts regulated by these RBPs. Given the complexity of the developing mammalian cortex, a major challenge for the future will be to understand how dynamic RNA regulation occurs within heterogeneous cell populations, across space and time. In sum, post-transcriptional regulation has emerged as a critical mechanism for driving corticogenesis and exciting direction of future research. PMID:26088328

  6. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  7. Isolation of the protein and RNA content of active sites of transcription from mammalian cells.

    PubMed

    Melnik, Svitlana; Caudron-Herger, Maïwen; Brant, Lilija; Carr, Ian M; Rippe, Karsten; Cook, Peter R; Papantonis, Argyris

    2016-03-01

    Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I-mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription. PMID:26914315

  8. Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

    PubMed Central

    Shashni, Rajesh; Qayyum, M. Zuhaib; Vishalini, V.; Dey, Debashish; Sen, Ranjan

    2014-01-01

    The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions. PMID:25081210

  9. Spatio-Temporal Dynamics of Yeast Mitochondrial Biogenesis: Transcriptional and Post-Transcriptional mRNA Oscillatory Modules

    PubMed Central

    Lelandais, Gaëlle; Saint-Georges, Yann; Geneix, Colette; Al-Shikhley, Liza; Dujardin, Geneviève; Jacq, Claude

    2009-01-01

    Examples of metabolic rhythms have recently emerged from studies of budding yeast. High density microarray analyses have produced a remarkably detailed picture of cycling gene expression that could be clustered according to metabolic functions. We developed a model-based approach for the decomposition of expression to analyze these data and to identify functional modules which, expressed sequentially and periodically, contribute to the complex and intricate mitochondrial architecture. This approach revealed that mitochondrial spatio-temporal modules are expressed during periodic spikes and specific cellular localizations, which cover the entire oscillatory period. For instance, assembly factors (32 genes) and translation regulators (47 genes) are expressed earlier than the components of the amino-acid synthesis pathways (31 genes). In addition, we could correlate the expression modules identified with particular post-transcriptional properties. Thus, mRNAs of modules expressed “early” are mostly translated in the vicinity of mitochondria under the control of the Puf3p mRNA-binding protein. This last spatio-temporal module concerns mostly mRNAs coding for basic elements of mitochondrial construction: assembly and regulatory factors. Prediction that unknown genes from this module code for important elements of mitochondrial biogenesis is supported by experimental evidence. More generally, these observations underscore the importance of post-transcriptional processes in mitochondrial biogenesis, highlighting close connections between nuclear transcription and cytoplasmic site-specific translation. PMID:19521515

  10. Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.

    PubMed

    Fels, A; Hu, K; Riesner, D

    2001-11-15

    Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, (105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (-)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties. PMID:11713308

  11. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    SciTech Connect

    Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

    1987-05-01

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific TSP-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect.

  12. Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

    PubMed Central

    Holoch, Daniel; Moazed, Danesh

    2015-01-01

    Argonautes and their small-RNA cofactors form the core effectors of ancient and diverse gene-silencing mechanisms whose roles include regulation of gene expression and defense against foreign genetic elements. Although Argonautes generally act within multisubunit complexes, what governs their assembly into these machineries is not well defined. Here, we show that loading of small RNAs onto Argonaute is a checkpoint for Argonaute’s association with conserved GW-protein components of silencing complexes. We demonstrate that the Argonaute small interfering RNA chaperone (ARC) complex mediates loading of small RNAs onto Ago1 in Schizosaccharomyces pombe and that deletion of its subunits, or mutations in Ago1 that prevent small-RNA loading, abolish the assembly of the GW protein–containing RNA-induced transcriptional silencing (RITS) complex. Our studies uncover a mechanism that ensures that Argonaute loading precedes RITS assembly and thereby averts the formation of inert and potentially deleterious complexes. PMID:25730778

  13. Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

    PubMed Central

    Hardin, P E; Hall, J C; Rosbash, M

    1992-01-01

    The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator. Images PMID:1465387

  14. Preferential use of RNA leader sequences during influenza A transcription initiation in vivo.

    PubMed

    Geerts-Dimitriadou, Christina; Goldbach, Rob; Kormelink, Richard

    2011-01-01

    In vitro transcription initiation studies revealed a preference of influenza A virus for capped RNA leader sequences with base complementarity to the viral RNA template. Here, these results were verified during an influenza infection in MDCK cells. Alfalfa mosaic virus RNA3 leader sequences mutated in their base complementarity to the viral template, or the nucleotides 5' of potential base-pairing residues, were tested for their use either singly or in competition. These analyses revealed that influenza transcriptase is able to use leaders from an exogenous mRNA source with a preference for leaders harboring base complementarity to the 3'-ultimate residues of the viral template, as previously observed during in vitro studies. Internal priming at the 3'-penultimate residue, as well as "prime-and-realign" was observed. The finding that multiple base-pairing promotes cap donor selection in vivo, and the earlier observed competitiveness of such molecules in vitro, offers new possibilities for antiviral drug design. PMID:21030059

  15. E sub 1 BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription

    SciTech Connect

    Ji Zhang; Huifeng Niu; Jacob, S.T. )

    1991-10-01

    The authors previously described the purification and characterization of E{sub 1}BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E{sub 1}BF was selectively phosphorylated. The labeled phosphate could be removed from the E{sub 1}BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E{sub 1}BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with ({gamma}-{sup 32}P)ATP resulted in the selective labeling o the 79-kDa band. The E{sub 1}BF-associated protein kinase did not phosphorylate casein or histone H1. These data demonstrate that (1) polymerase I promoter-binding factor E{sub 1}BF contains an intrinsic substrate-specific protein kinase and (2) E{sub 1}BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.

  16. Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

    PubMed

    Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

    2014-01-29

    We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression. PMID:24392825

  17. Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU

    SciTech Connect

    Iwamoto, Fumiko; Stadler, Michael; Chalupnikova, Katerina; Oakeley, Edward; Nagamine, Yoshikuni

    2008-04-01

    RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

  18. Catching RNA Polymerase in the act of Binding: Intermediates in Transcription Illuminated by Synchrotron Footprinting

    SciTech Connect

    Brenowitz,M.; Erie, D.; Chance, M.

    2005-01-01

    The article by Sclavi et al. in this issue of PNAS addresses 'initiation, ' the first step in transcription. Gene transcription is catalyzed in cells by large multisubunit proteins called RNA polymerases (RNAP). The eubacteria holoenzyme of RNAP is composed of five core subunits ({alpha}, {alpha}2, {beta}, {beta}', and {omega}) that contain the amino acid residues required for the enzyme's catalytic activity. A sixth subunit ({sigma}) guides RNAP to specific sequences on the genomic DNA (promoters) that mark the beginning of a gene or group of genes.

  19. Global Analysis of mRNA Half-Lives and de novo Transcription in a Dinoflagellate, Karenia brevis

    PubMed Central

    Morey, Jeanine S.; Van Dolah, Frances M.

    2013-01-01

    Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the roles of differential mRNA stability and de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of increases in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to

  20. Similar hepatitis C virus RNA kinetics in HIV/hepatitis C virus monoinfected genotype 2 or 3 matched controls during hepatitis C virus combination therapy.

    PubMed

    Karlström, Olle; Sönnerborg, Anders; Weiland, Ola

    2008-04-23

    We prospectively studied early hepatitis C virus kinetics and sustained virological response rates in HIV/HCV coinfected (n = 13) and HCV monoinfected matched controls (n = 26) with HCV genotype 2/3 treated with pegylated interferon (peg-IFN) alpha-2a 135 microg/week plus ribavirin 11 mg/kg daily during 24 weeks. No significant difference in HCV-RNA decay was seen at any time point during the initial 12 weeks of therapy. Sustained virological response was achieved in 9/13 (69%) versus 20/26 (77%) patients (intent-to-treat), respectively. The lower-than-standard peg-IFN dose offered high compliance and reasonable sustained virological response rates. PMID:18427210

  1. MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

    PubMed Central

    Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

    2016-01-01

    MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

  2. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    SciTech Connect

    Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  3. Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR.

    PubMed

    Bhan, Arunoday; Mandal, Subhrangsu S

    2016-01-01

    HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs. PMID:26585152

  4. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    NASA Astrophysics Data System (ADS)

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-07-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

  5. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    PubMed Central

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  6. Structure of the initiation-competent RNA polymerase I and its implication for transcription.

    PubMed

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  7. RNA Surveillance: Molecular Approaches in Transcript Quality Control and their Implications in Clinical Diseases

    PubMed Central

    Moraes, Karen CM

    2010-01-01

    Production of mature mRNAs that encode functional proteins involves highly complex pathways of synthesis, processing and surveillance. At numerous steps during the maturation process, the mRNA transcript undergoes scrutiny by cellular quality control machinery. This extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possibly deleterious proteins. Recent advances in elucidating the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and have revealed that a variety of human diseases are caused by disturbances in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss and gain of function are relevant, and a considerable number of different diseases exemplify the importance of the mechanistic function of RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, highlighting the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of therapeutic strategies. PMID:19829759

  8. Quercetin induces hepatic γ-glutamyl hydrolase expression in rats by suppressing hepatic microRNA rno-miR-125b-3p.

    PubMed

    Wein, Silvia Anette; Laviano, Alessandro; Wolffram, Siegfried

    2015-12-01

    Exogenous factors such as food components including the flavonoid quercetin are suspected to influence micro RNA (miRNA) concentrations and thus possibly target enzymes involved in xenobiotic metabolism. This study therefore investigates the influence of orally administered quercetin on hepatic miRNA and the identification of enzyme target mRNAs relevant in drug metabolism. Male Wistar rats (n=16) were fed either a diet without (C) or with (Q) the addition of 100-ppm quercetin for 7 weeks and subsequently euthanized at the end of the dark phase. To avoid strong effects of food deprivation on hepatic metabolism, food was not removed until 5 h prior to the procedure. Liver was immediately dissected and snap-frozen in liquid nitrogen. Concentrations of 352 hepatic miRNA were measured in pool samples of each dietary group (n=8) using the RT(2) miRNA PCR Array System. Differential expression of miRNAs was assumed with fold changes ≥3. Target genes of differentially expressed miRNAs were identified using the database TargetScan. Because rno-miR-125b-3p showed the most prominent fold-change (-9) we further analyzed the expression of its top predicted target gene gamma-glutamyl hydrolase (GGH) by quantitative real-time PCR using hypoxanthine phosphoribosyltransferase 1 (hprt1) as endogenous control. Compared to controls, 23 miRNAs were differentially expressed in rats fed quercetin. A ninefold reduction in hepatic miRNA rno-miR-125b-3p was paralleled by significant induction of GGH mRNA in liver of quercetin fed rats. Because increased GGH expressions were repeatedly associated with resistance to methotrexate, concomitant intake with quercetin should be monitored carefully. PMID:26432773

  9. Long Non-coding RNA Growth Arrest-specific Transcript 5 (GAS5) Inhibits Liver Fibrogenesis through a Mechanism of Competing Endogenous RNA.

    PubMed

    Yu, Fujun; Zheng, Jianjian; Mao, Yuqing; Dong, Peihong; Lu, Zhongqiu; Li, Guojun; Guo, Chuanyong; Liu, Zhanju; Fan, Xiaoming

    2015-11-20

    Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis. PMID:26446789

  10. Effects of blueberry on hepatic fibrosis and transcription factor Nrf2 in rats

    PubMed Central

    Wang, Yu-Ping; Cheng, Ming-Liang; Zhang, Bao-Fang; Mu, Mao; Wu, Jun

    2010-01-01

    AIM: To investigate the effects of blueberry on hepatic fibrosis and NF-E2-related factor 2 (Nrf2) transcription factor in rats. METHODS: Forty-five male Sprague-Dawley rats were randomly divided into control group (A); CCl4-induced hepatic fibrosis group (B); blueberry prevention group (C); Dan-shao-hua-xian capsule (DSHX) prevention group (D); and blueberry + DSHX prevention group (E). Liver fibrosis was induced in rats by subcutaneous injection of CCl4 and a high-lipid/low-protein diet for 8 wk (except the control group). The level of hyaluronic acid (HA) and alanine aminotransferase (ALT) in serum was examined. The activity of superoxide dismutase (SOD), glutathione-S-transferase (GST) and malondialdehyde (MDA) in liver homogenates was determined. The degree of hepatic fibrosis was evaluated by hematoxylin and eosin and Masson staining. Expression of Nrf2 and NADPH quinone oxidoreductase 1 (Nqo1) was detected by real-time reversed transcribed-polymerase chain reaction, immunohistochemical techniques, and western blotting. RESULTS: Compared with group B, liver indices, levels of serum HA and ALT of groups C, D and E were reduced (liver indices: 0.038 ± 0.008, 0.036 ± 0.007, 0.036 ± 0.005 vs 0.054 ± 0.009, P < 0.05; HA: 502.33 ± 110.57 ng/mL, 524.25 ± 255.42 ng/mL, 499.25 ± 198.10 ng/mL vs 828.50 ± 237.83 ng/mL, P < 0.05; ALT: 149.44 ± 16.51 U/L, 136.88 ± 10.07 U/L, 127.38 ± 11.03 U/L vs 203.25 ± 31.62 U/L, P < 0.05), and SOD level was significantly higher, but MDA level was lower, in liver homogenates (SOD: 1.36 ± 0.09 U/mg, 1.42 ± 0.13 U/mg, 1.50 ± 0.15 U/mg vs 1.08 ± 0.19 U/mg, P < 0.05; MDA: 0.294 ± 0.026 nmol/mg, 0.285 ± 0.025 nmol/mg, 0.284 ± 0.028 nmol/mg vs 0.335 ± 0.056 nmol/mg, P < 0.05). Meanwhile, the stage of hepatic fibrosis was significantly weakened (P < 0.05). Compared with group A, the activity of GST liver homogenates and expression levels of Nrf2 and Nqo1 in group B were elevated (P < 0.05). The expression level of Nrf2 and

  11. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  12. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

    PubMed

    Ahn, Jeong H; Rechsteiner, Andreas; Strome, Susan; Kelly, William G

    2016-08-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  13. Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing.

    PubMed

    Rigo, Frank; Hua, Yimin; Chun, Seung J; Prakash, Thazha P; Krainer, Adrian R; Bennett, C Frank

    2012-06-01

    We describe a new technology for recruiting specific proteins to RNA through selective recognition of heteroduplexes formed with chemically modified antisense oligonucleotides (ASOs). Typically, ASOs function by hybridizing to their RNA targets and blocking the binding of single-stranded RNA-binding proteins. Unexpectedly, we found that ASOs with 2'-deoxy-2'-fluoro (2'-F) nucleotides, but not with other 2' chemical modifications, have an additional property: they form heteroduplexes with RNA that are specifically recognized by the interleukin enhancer-binding factor 2 and 3 complex (ILF2/3). 2'-F ASO-directed recruitment of ILF2/3 to RNA can be harnessed to control gene expression by modulating alternative splicing of target transcripts. ILF2/3 recruitment to precursor mRNA near an exon results in omission of the exon from the mature mRNA, both in cell culture and in mice. We discuss the possibility of using chemically engineered ASOs that recruit specific proteins to modulate gene expression for therapeutic intervention. PMID:22504300

  14. Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA.

    PubMed

    Chen, Guangnan; Yanofsky, Charles

    2003-07-11

    The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon. Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp). Here we show that translation of AT also is regulated by uncharged tRNATrp. A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene. Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production. Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression. PMID:12855807

  15. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

  16. The emerging role of RNA in the regulation of gene transcription in human cells

    PubMed Central

    Morris, Kevin V.

    2011-01-01

    Recent evidence suggests that particular species of non-coding RNAs can modulate gene transcription in human cells. While such observations were in the past relegated to imprinted genes, it is now becoming apparent that several different genes in differentiated cells may be under some form of RNA based regulatory control. Studies carried out to date have begun to discern the mechanism of action whereby non-coding RNAs modulate gene transcription by the targeted recruitment of epigenetic silencing complexes to homology containing loci in the genome. The results of these studies will be considered in detail as well as the implications that a vast array of non-coding RNA based regulatory networks may be operative in human cells. PMID:21333746

  17. The regulation of mammalian mRNA transcription by long non-coding RNAs: Recent discoveries and current concepts

    PubMed Central

    Kugel, Jennifer F.; Goodrich, James A.

    2013-01-01

    Summary Transcription by RNA polymerase II (Pol II) is a tightly controlled process critical to normal cellular metabolism. Understanding how transcriptional regulation is orchestrated has mainly involved identifying and characterizing proteins that function as transcription factors. During the past decade, however, an increasing number of long non-coding RNAs (lncRNAs) have been identified as transcriptional regulators. This revelation has spurred new discoveries, novel techniques, and paradigm shifts, which together are redefining our understanding of transcriptional control and broadening our view of RNA function. Here we summarize recent discoveries concerning the role of lncRNAs as regulators of mammalian mRNA transcription, with a focus on key concepts that are guiding current research in the field. PMID:23414324

  18. Real-Time Observation of the Initiation of RNA Polymerase II Transcription

    PubMed Central

    Fazal, Furqan M.; Meng, Cong A.; Murakami, Kenji; Kornberg, Roger D.; Block, Steven M.

    2015-01-01

    Biochemical and structural studies have shown that the initiation of RNA polymerase II (pol II) transcription proceeds in the following stages: assembly of pol II with general transcription factors (GTFs) and promoter DNA in a “closed” preinitiation complex (PIC)1,2; unwinding about 15 bp of the promoter DNA to form an “open” complex3,4; scanning downstream to a transcription start site; synthesis of a short transcript, believed to be about 10 nucleotides; and promoter escape. We have assembled a 32-protein, 1.5 megadalton PIC5 derived from Saccharomyces cerevisiae and observed subsequent initiation processes in real time with optical tweezers6. Contrary to expectation, scanning driven by transcription factor IIH (TFIIH)7-12 entailed the rapid opening of an extended bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning. PMID:26331540

  19. Differential roles of phosphorylation in the formation of transcriptional active RNA polymerase I

    PubMed Central

    Fath, Stephan; Milkereit, Philipp; Peyroche, Gerald; Riva, Michel; Carles, Christophe; Tschochner, Herbert

    2001-01-01

    Regulation of rDNA transcription depends on the formation and dissociation of a functional complex between RNA polymerase I (pol I) and transcription initiation factor Rrn3p. We analyzed whether phosphorylation is involved in this molecular switch. Rrn3p is a phosphoprotein that is predominantly phosphorylated in vivo when it is not bound to pol I. In vitro, Rrn3p is able both to associate with pol I and to enter the transcription cycle in its nonphosphorylated form. By contrast, phosphorylation of pol I is required to form a stable pol I-Rrn3p complex for efficient transcription initiation. Furthermore, association of pol I with Rrn3p correlates with a change in the phosphorylation state of pol I in vivo. We suggest that phosphorylation at specific sites of pol I is a prerequisite for proper transcription initiation and that phosphorylation/dephosphorylation of pol I is one possibility to modulate cellular rDNA transcription activity. PMID:11717393

  20. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription

    PubMed Central

    Sammons, Morgan A.; Zhu, Jiajun; Berger, Shelley L.

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  1. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription.

    PubMed

    Sammons, Morgan A; Zhu, Jiajun; Berger, Shelley L

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  2. A common site on TBP for transcription by RNA polymerases II and III.

    PubMed

    Schröder, Oliver; Bryant, Gene O; Geiduschek, E Peter; Berk, Arnold J; Kassavetis, George A

    2003-10-01

    The TATA-binding protein (TBP) is involved in all nuclear transcription. We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III. TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB. A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB-DNA complexes reliant on only the TFIIB-related part of Brf1. Mutations impairing complex formation and transcription were identified and mapped on the surface of TBP. The most severe effects were observed for mutations in the C-terminal stirrup of TBP, which is the principal site of interaction between TBP and TFIIB. Structural modeling of the Brf1-TBP complex and comparison with its TFIIB-TBP analog further rationalizes the close resemblance of the TBP interaction with the N-proximal part of Brf1 and TFIIB, and establishes the conserved usage of a TBP surface in pol II and pol III transcription for a conserved function in the initiation of transcription. PMID:14517249

  3. RNA-sequencing reveals oligodendrocyte and neuronal transcripts in microglia relevant to central nervous system disease

    PubMed Central

    Walker, Jason; Wylie, Todd; Magrini, Vincent; Apicelli, Anthony J.; Griffith, Malachi; Griffith, Obi L.; Kohsaka, Shinichi; Wu, Gregory F.; Brody, David L.; Mardis, Elaine R.; Gutmann, David H.

    2014-01-01

    Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures (FACS) or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6-week old mice to compare the transcriptomes of FACS-sorted versus laser-captured (LCM) microglia. While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, ~50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis (EAE), increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury (TBI). Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations. PMID:25258010

  4. Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

    PubMed Central

    Ikegami, Tetsuro; Peters, C. J.; Makino, Shinji

    2005-01-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RN