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Sample records for hepatitis rna transcripts

  1. Hepatic rRNA transcription regulates high-fat-diet-induced obesity.

    PubMed

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Tokunaga, Shinji; Waku, Tsuyoshi; Han, Song-Iee; Iwasaki, Naoya; Mikogai, Aya; Yasuzawa-Tanaka, Kayoko; Kishimoto, Hiroyuki; Hiyoshi, Hiromi; Nakajima, Yuka; Araki, Toshiyuki; Kimura, Keiji; Yanagisawa, Junn; Murayama, Akiko

    2014-05-08

    Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Isolation of hepatitis C virus RNA from serum for reverse transcription-PCR.

    PubMed Central

    Nolte, F S; Thurmond, C; Mitchell, P S

    1994-01-01

    Standard multistep extraction and isolation of RNA for hepatitis C virus (HCV) reverse transcription (RT)-PCR are impractical for routine use in clinical laboratories. We compared three simple commercially available methods for RNA isolation (RNAzol B, TRISOLV, and ULTRASPEC; Biotecx Laboratories, Houston, Tex.) and a total nucleic acid isolation method (IsoQuick; MicroProbe Corp., Garden Grove, Calif.) for the recovery of HCV RNA from sera obtained from 12 viremic patients for RT-PCR. RNAzol B, TRISOLV, ULTRASPEC, and IsoQuick extraction methods detected 87.5, 79.2, 33.3, and 58.3% of the paired positive samples, respectively. The method used for isolation of RNA is an important concern when optimizing HCV RT-PCR. Images PMID:8150964

  3. Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay.

    PubMed Central

    Young, K K; Resnick, R M; Myers, T W

    1993-01-01

    Amplification of RNA by the polymerase chain reaction (PCR) is normally a two-step process requiring separate enzymes and buffer conditions. We describe a combined reverse transcription-PCR (RT-PCR) assay for hepatitis C virus (HCV) RNA amplification in which a single enzyme and buffer condition are used. In this assay, both the RT and PCR steps are carried out with the thermoactive DNA polymerase of Thermus thermophilus. A transcription vector containing HCV sequences has also been constructed to generate quantifiable HCV RNA templates that can be used to optimize reaction conditions and to assess the efficiency of amplification. Amplification from < or = 100 copies of RNA was detected reproducibly by gel electrophoresis. The assay sensitivity was increased to 10 RNA copies by hybridization to a probe. The patterns of viremia in three individuals infected with HCV were examined by amplification of HCV RNA from plasma samples collected serially over a period of 1 year. These results were correlated with the times of seroconversion and the onset of rise in levels of alanine aminotransferase in serum. In all three subjects, HCV RNA was detected prior to seroconversion and the initial rise in levels of alanine aminotransferase in serum. Upon seroconversion, HCV RNA fell to a level below the detection limit of the assay. This pattern of transient viremia appears to be characteristic of acute, resolving HCV infections. The combined RT-PCR assay is a sensitive method which circumvents the problems associated with PCR amplification of RNA. Using this assay, we demonstrated that three donors infected by the same index case all have similar patterns of viremia. Images PMID:8385151

  4. Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Wang, Qin-qin; Zhang, Jie; Hu, Jin-song; Chen, Hao-tai; Du, Li; Wu, Li-qin; Ding, Yao-zhong; Xiong, Sheng-he; Huang, Xin-cheng; Zhang, Yin-hong; Liu, Yong-sheng

    2011-10-01

    The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid diagnosis of hepatitis C virus (HCV) RNA was evaluated. This assay showed higher sensitivities than that of nested RT-PCR, with a detection limit of 600 IU mL(-1) , and no cross-reactivity was observed with hepatitis A virus, hepatitis B virus and hepatitis E virus. Furthermore, 106 stored sera from recently diagnosed cases were retrospectively investigated with real-time RT-PCR, the nested RT-PCR, in parallel with this new assay. The general detection rates of HCV RT-LAMP, real-time PCR and the nested RT-PCR for 106 stored sera samples were 95%, 96% and 88%, respectively. This study provides the first data on the usefulness of HCV RT-LAMP in the diagnosis of HCV RNA, especially in the early clinical diagnosis of acute HCV infection.

  5. 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

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

    PubMed

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

    2015-11-01

    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 HBV(G2335A) 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. 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 infected with other HBV

  7. RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

    PubMed

    Cossaboom, Caitlin M; Huang, Yao-Wei; Yugo, Danielle M; Kenney, Scott P; Piñeyro, Pablo; Matzinger, Shannon R; Heffron, C Lynn; Pierson, F William; Meng, Xiang-Jin

    2014-11-07

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

  8. Comparison of transcription mediated amplification (TMA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of hepatitis C virus RNA in liver tissue.

    PubMed

    Hofmann, Wolf Peter; Dries, Volker; Herrmann, Eva; Gärtner, Barbara; Zeuzem, Stefan; Sarrazin, Christoph

    2005-04-01

    Transcription mediated amplification (TMA) is known to be one of the most sensitive detection assays for hepatitis C virus (HCV) RNA in serum but has not yet been evaluated in liver tissue. It is unknown whether the higher sensitivity of TMA in comparison with polymerase chain reaction (PCR)-based assays is related to a higher efficiency of the extraction and/or amplification step. The sensitivity of a TMA-based assay (Versant HCV RNA Qualitative assay, Bayer Diagnostics) and a standard RT-PCR-based assay (Cobas Amplicor HCV 2.0, Roche Diagnostics) was compared in formalin-fixed paraffin-embedded liver biopsy specimens of patients with chronic hepatitis C. After deparaffinization of 7.5 microm liver sections HCV RNA was extracted by standard phenol/chloroform. HCV RNA dilution panels were transferred in parallel to cDNA synthesis and amplification steps of PCR and TMA. Furthermore, TMA amplification from stepwise diluted HCV sera was performed following RNA extraction by either microcentrifuge colums (QIAmp Viral RNA spin Kit, Qiagen, Hilden, Germany) or magnetic microparticles (VERSANT HCV RNA Qualitative assay). The total number of HCV RNA positive liver specimens detected by TMA was higher compared with those detected by RT-PCR (P=0.032). The total number of TMA positive serum samples was higher when HCV RNA was extracted using magnetic microparticles in comparison with multicentrifuge column extraction (P=0.019). Our results suggest that both the extraction and amplification step of the TMA-based assay contribute to the higher sensitivity compared with standard RT-PCR.

  9. A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

    PubMed

    Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

    2015-12-01

    Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

  10. Regulation of the microRNA processor DGCR8 by hepatitis B virus proteins via the transcription factor YY1.

    PubMed

    Shan, Xuefeng; Ren, Min; Chen, Ke; Huang, Ailong; Tang, Hua

    2015-03-01

    MicroRNAs (miRNAs) are a new class of well-conserved small noncoding RNAs that mediate posttranscriptional gene regulation. Hepatitis B virus (HBV) causes various liver diseases, including chronic hepatitis, liver cirrhosis and hepatocellular cancer. Recent data have indicated HBV alters miRNAs expression patterns, but the underlying mechanisms have not been fully established so far. Here, we provide a hypothesis that HBV alters the expressions of miRNAs by playing a role in the microRNA production process. In this study, we demonstrate that HBV downregulates miRNAs processor DGCR8 mRNA and protein expression in stable and transient HBV-expressing cells. HBV downregulates DGCR8 expression by inhibiting its promoter activity, and HBs and HBx may be involved in this process. Ectopic expression and knockdown of YY1 revealed that YY1 suppresses the activity of the DGCR8 promoter, while YY1 expression is significantly upregulated by HBV. In conclusion, our data show that HBV proteins repress DGCR8 promoter activity by upregulating the expression of transcription factor YY1. This provides a new insight into the mechanism of HBV-induced miRNA dysregulation.

  11. The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription.

    PubMed

    Tan, Zhenning; Pionek, Karolyn; Unchwaniwala, Nuruddin; Maguire, Megan L; Loeb, Daniel D; Zlotnick, Adam

    2015-03-01

    Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry

  12. The Interface between Hepatitis B Virus Capsid Proteins Affects Self-Assembly, Pregenomic RNA Packaging, and Reverse Transcription

    PubMed Central

    Tan, Zhenning; Pionek, Karolyn; Unchwaniwala, Nuruddin; Maguire, Megan L.

    2015-01-01

    ABSTRACT Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. IMPORTANCE The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the

  13. PRMT5 Restricts Hepatitis B Virus Replication via Epigenetic Repression of cccDNA Transcription and Interference with pgRNA Encapsidation.

    PubMed

    Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

    2017-02-25

    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscured. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In the cell culture-based models for HBV infection and the liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on cccDNAwas a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered H4R3me2s on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based hSWI/SNF chromatin remodeler, which resulted in the downregulation of the binding of RNA Pol II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independent of its methyltransferase activity. Further study revealed that PRMT5 interfered with pre-genomic RNA (pgRNA) encapsidation by preventing its interaction with viral polymerase protein through binding to the RT-RH region of polymerase which is crucial for the polymerase-pgRNA interaction.

  14. 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.

  15. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

    PubMed

    Wang, H D; Trivedi, A; Johnson, D L

    1997-12-01

    Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

  16. Messenger RNA transcripts

    Treesearch

    Dan Cullen

    2004-01-01

    In contrast to DNA, messenger RNA (mRNA) in complex substrata is rarely analyzed, in large part because labile RNA molecules are difficult to purify. Nucleic acid extractions from fungi that colonize soil are particularly difficult and plagued by humic substances that interfere with Taq polymerase (Tebbe and Vahjen 1993 and references therein). Magnetic capture...

  17. Novel mechanism for reverse transcription in hepatitis B viruses.

    PubMed Central

    Wang, G H; Seeger, C

    1993-01-01

    Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis. However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992). We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein. Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues. Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription. This mechanism is without precedent among all known retroid elements and among other viruses and bacteriophages that use protein as a primer for RNA or DNA synthesis. It could provide an effective target for antiviral therapy, which is required for the treatment of more than 300 million carriers of hepatitis B virus. Images PMID:7692081

  18. RNA Polymerase II Transcription: Structure and Mechanism

    PubMed Central

    Liu, Xin; Bushnell, David A.; Kornberg, Roger D.

    2014-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. PMID:23000482

  19. RNA polymerase II transcription: structure and mechanism.

    PubMed

    Liu, Xin; Bushnell, David A; Kornberg, Roger D

    2013-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  20. 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

  1. Transcription by RNA polymerases I and III

    PubMed Central

    Paule, Marvin R.; White, Robert J.

    2000-01-01

    The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesises the large rRNA, pol II synthesises mRNA and pol III synthesises tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences. PMID:10684922

  2. TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

    PubMed

    Troxler, Salome; Marek, Ana; Prokofieva, Irina; Bilic, Ivana; Hess, Michael

    2011-04-01

    Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

  3. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  4. 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.

  5. Archaeal RNA polymerase and transcription regulation

    PubMed Central

    Jun, Sung-Hoon; Reichlen, Matthew J.; Tajiri, Momoko; Murakami, Katsuhiko S.

    2010-01-01

    To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high resolution X-ray crystal structures together with structure-guided biochemical, biophysical and genetics studies are essential. The recently-solved X-ray crystal structures of archaeal RNA polymerase (RNAP) allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors, is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all three domains of life. PMID:21250781

  6. FoxO1 deacetylation regulates thyroid hormone-induced transcription of key hepatic gluconeogenic genes.

    PubMed

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

    2013-10-18

    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.

  7. The RNA polymerase I transcription machinery.

    PubMed

    Russell, Jackie; Zomerdijk, Joost C B M

    2006-01-01

    The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

  8. 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

  9. Regulatory sequences of duck hepatitis B virus C gene transcription.

    PubMed Central

    Schneider, R; Will, H

    1991-01-01

    The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements. Images PMID:1920612

  10. Transcription and recombination: when RNA meets DNA.

    PubMed

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    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. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  11. MicroRNA circuits for transcriptional logic.

    PubMed

    Leisner, Madeleine; Bleris, Leonidas; Lohmueller, Jason; Xie, Zhen; Benenson, Yaakov

    2012-01-01

    One of the longstanding challenges in synthetic biology is rational design of complex regulatory circuitry with multiple biological inputs, complex internal processing, and physiologically active outputs. We have previously proposed how to address this challenge in the case of transcription factor inputs. Here we describe the methods used to construct these synthetic circuits, capable of performing logic integration of transcription factor inputs using microRNA expression vectors and RNA interference (RNAi). The circuits operate in mammalian cells and they can serve as starting point for more complex synthetic information processing networks in these cells.

  12. 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

  13. Competitor template RNA for detection and quantitation of hepatitis A virus by PCR.

    PubMed

    Goswami, B B; Koch, W H; Cebula, T A

    1994-01-01

    PCR was used to introduce a 63-bp deletion into the putative RNA replicase coding sequence of hepatitis A virus. RNA was synthesized in vitro from the deletion mutant cloned into a transcription vector. Upon amplification by PCR, cDNA made from the competitor RNA generated an amplified fragment that could be easily distinguished from the product generated from wild-type hepatitis A virus genomic RNA by gel electrophoresis, when the same primers were used, without further manipulation. The competitor RNA was used as a positive control in PCR-based detection of very low copy numbers of hepatitis A virus genomic RNA in the presence of unrelated hard-shell clam RNA. When the competitor RNA was used for competitive PCR to quantitate wild-type RNA, the presence of one template at a 10-fold to 100-fold higher level almost completely inhibited product formation from the underrepresented template. The competitor RNA should be useful as a control for reverse transcription and PCRs to determine hepatitis A virus genome RNA when accidental contamination of test samples by a wild-type positive control template would compromise the results.

  14. Functional Integration of Transcriptional and RNA Processing Machineries

    PubMed Central

    Pandit, Shatakshi; Wang, Dong; Fu, Xiang-Dong

    2009-01-01

    Co-transcriptional RNA processing not only permits temporal RNA processing before the completion of transcription, but also allows sequential recognition of RNA processing signals on nascent transcripts threading out from the elongating RNAPII complex. Rapid progress in recent years has established multiple contacts that physically connect the transcription and RNA processing machineries, which centers on the C-terminal domain (CTD) of the largest subunit of RNAPII. While co-transcriptional RNA processing has been substantiated, the evidence for “reciprocal” coupling starts to emerge, which emphasizes functional integration of transcription and RNA processing machineries in a mutually beneficial manner for efficient and regulated gene expression. PMID:18436438

  15. 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

  16. 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.

  17. Suppression of rat hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fat.

    PubMed

    Blake, W L; Clarke, S D

    1990-12-01

    The objective of this research was to determine whether dietary polyunsaturated fatty acids suppress hepatic fatty acid synthase (FAS) mRNA levels by altering FAS gene transcription. Male Sprague-Dawley rats were meal-fed for 10 d a high glucose diet supplemented with 20% digestible energy as menhaden oil or tripalmitin. The transcription rate for FAS was determined by nuclear run-on analysis in hepatic nuclei isolated from rats 2 h postmeal. The values for transcription rates of FAS and S14 (a putative lipogenic protein) in rats fed menhaden oil were only 6 and 21%, respectively, of the rates in rats fed the tripalmitin diet (p less than 0.02). Gene transcription for beta-actin and phosphoenolpyruvate carboxykinase did not differ between treatments. The reduction in hepatic FAS mRNA levels caused by dietary polyunsaturated fats appears to be caused primarily by an inhibition of FAS transcription. The control of transcription by polyunsaturated fats appears not to be mediated by cAMP because the transcription rate for phosphoenolpyruvate carboxykinase (whose gene is very sensitive to cAMP stimulation) was unaffected by the source of dietary fat.

  18. RNA polymerase: the vehicle of transcription.

    PubMed

    Borukhov, Sergei; Nudler, Evgeny

    2008-03-01

    RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation for all three divisions of life: Eukaryota, Archaea and Bacteria. Recent progress in the structural and biochemical characterization of RNAP illuminates this enzyme as a flexible, multifunctional molecular machine. During each step of the transcription cycle, RNAP undergoes elaborate conformational changes. As many fundamental and previously mysterious aspects of how RNAP works begin to be understood, this enzyme reveals intriguing similarities to man-made engineered devices. These resemblances can be found in the mechanics of RNAP-DNA complex formation, in RNA chain initiation and in the elongation processes. Here we highlight recent advances in understanding RNAP function and regulation.

  19. 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

  20. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  1. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Processivity and Coupling in Messenger RNA Transcription

    PubMed Central

    Aitken, Stuart; Robert, Marie-Cécile; Alexander, Ross D.; Goryanin, Igor; Bertrand, Edouard; Beggs, Jean D.

    2010-01-01

    Background The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model. Methodology In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range. Conclusions We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low—as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise—a prediction of the on/off model that has no supporting evidence. PMID

  3. 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

  4. BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript.

    PubMed Central

    Martignetti, J A; Brosius, J

    1995-01-01

    Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product. By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription. The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element. The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced. Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed. These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated. PMID:7862155

  5. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  6. RNA-protein interactions at the 3' end of the hepatitis A virus RNA.

    PubMed Central

    Kusov, Y; Weitz, M; Dollenmeier, G; Gauss-Müller, V; Siegl, G

    1996-01-01

    The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3'-nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3'-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis. PMID:8627714

  7. DNA wrapping in transcription initiation by RNA polymerase II.

    PubMed

    Coulombe, B

    1999-01-01

    The DNA wrapping model of transcription stipulates that DNA bending and wrapping around RNA polymerase causes an unwinding of the DNA helix at the enzyme catalytic center that stimulates strand separation prior to initiation and during transcript elongation. Recent experiments with mammalian RNA polymerase II indicate the significance of DNA bending and wrapping in transcriptional mechanisms. These findings have important implications in our understanding of the role of the general transcription factors in transcriptional initiation and the mechanisms underlying transcriptional regulation.

  8. Are there any subgenomic forms of hepatitis C virus RNA in the liver?

    PubMed

    Quadri, R; Negro, F

    2001-01-01

    Hepatitis C virus has a single stranded positive RNA genoma. Although believed to replicate via semi-conservative transcription of a negative-stranded, genomic-length RNA intermediate, detailed steps of its replicative cycle are unknown. To quantify some of intrahepatic hepatitis C virus RNA forms, as inferred from comparison with replication of other members of the Flaviviridae family. Genomic and negative-stranded hepatitis C virus RNA were semi-quantitated by strand-specific reverse transcriptase-polymerase chain reaction at both their 5' and 3' ends in liver of 10 patients with recurrent hepatitis C after liver transplantation. Our data are consistent with the existence of hitherto unrecognized, very large amounts (up to approximately 10,000 fold the amount of the replication intermediate proper) of subgenomic hepatitis C virus RNAs of genomic polarity, starting in the 5' untranslated region, of unknown length. Similarly, subgenomic RNAs of negative polarity, starting in the 3' untranslated region, may also be produced, albeit to a less extent. We found no correlation between the amount of these forms and any clinical, histological or virological feature. However, the number of subgenomic RNA molecules of negative polarity tended to be inversely correlated with viraemia (r = 0.7, p = 0.058), suggesting their possible role in controlling rate of virion production. Hepatitis C virus replication results in transcription of huge amounts of subgenomic RNAs both of genomic and negative polarity, which may either regulate translation of excess structural antigens of hepatitis C virus, or play the role of defective RNAs interfering with viral replication. A revised model of hepatitis C virus RNA replication is proposed.

  9. 7SK small nuclear RNA, a multifunctional transcriptional regulatory RNA with gene-specific features.

    PubMed

    Egloff, Sylvain; Studniarek, Cécilia; Kiss, Tamás

    2017-08-18

    The 7SK small nuclear RNA is a multifunctional transcriptional regulatory RNA that controls the nuclear activity of the positive transcription elongation factor b (P-TEFb), specifically targets P-TEFb to the promoter regions of selected protein-coding genes and promotes transcription of RNA polymerase II-specific spliceosomal small nuclear RNA genes.

  10. The RNA polymerase II CTD coordinates transcription and RNA processing

    PubMed Central

    Hsin, Jing-Ping; Manley, James L.

    2012-01-01

    The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1–Ser2–Pro3–Thr4–Ser5–Pro6–Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity. PMID:23028141

  11. 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

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

    PubMed

    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.

  13. Transcription factor networks regulating hepatic fatty acid metabolism.

    PubMed

    Karagianni, Panagiota; Talianidis, Iannis

    2015-01-01

    Tight regulation of lipid levels is critical for cellular and organismal homeostasis, not only in terms of energy utilization and storage, but also to prevent potential toxicity. The liver utilizes a set of hepatic transcription factors to regulate the expression of genes implicated in all aspects of lipid metabolism including catabolism, transport, and synthesis. In this article, we will review the main transcriptional mechanisms regulating the expression of genes involved in hepatic lipid metabolism. The principal regulatory pathways are composed of simple modules of transcription factor crosstalks, which correspond to building blocks of more complex regulatory networks. These transcriptional networks contribute to the regulation of proper lipid homeostasis in parallel to posttranslational mechanisms and end product-mediated modulation of lipid metabolizing enzymes. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  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. Surveillance study of hepatitis A virus RNA on fig and date samples.

    PubMed

    Boxman, Ingeborg L A; te Loeke, Nathalie A J M; Klunder, Kyara; Hägele, Geke; Jansen, Claudia C C

    2012-02-01

    A total of 91 fig and 185 date samples were analyzed by reverse transcription (RT) real-time PCR for the presence of hepatitis A virus (HAV) RNA. Two batches of dates tested positive, and the HAV RNA detected was genotyped as IA. These findings warrant further development of methods applicable to food which is consumed untreated and is exported from countries in which HAV is endemic.

  16. Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol.

    PubMed

    Bhan, Arunoday; Hussain, Imran; Ansari, Khairul I; Kasiri, Sahba; Bashyal, Aarti; Mandal, Subhrangsu S

    2013-10-09

    HOTAIR (HOX antisense intergenic RNA) is a long noncoding RNA (lncRNA) that is transcribed from the antisense strand of homeobox C gene locus in chromosome 12. HOTAIR coordinates with chromatin-modifying enzymes and regulates gene silencing. It is overexpressed in various carcinomas including breast cancer. Herein, we demonstrated that HOTAIR is crucial for cell growth and viability and its knockdown induced apoptosis in breast cancer cells. We also demonstrated that HOTAIR is transcriptionally induced by estradiol (E2). Its promoter contains multiple functional estrogen response elements (EREs). Estrogen receptors (ERs) along with various ER coregulators such as histone methylases MLL1 (mixed lineage leukemia 1) and MLL3 and CREB-binding protein/p300 bind to the promoter of HOTAIR in an E2-dependent manner. Level of histone H3 lysine-4 trimethylation, histone acetylation, and RNA polymerase II recruitment is enriched at the HOTAIR promoter in the presence of E2. Knockdown of ERs and MLLs downregulated the E2-induced HOTAIR expression. Thus, similar to protein-coding gene transcription, E2-induced transcription of antisense transcript HOTAIR is coordinated via ERs and ER coregulators, and this mechanism of HOTAIR overexpression potentially contributes towards breast cancer progression. Published by Elsevier Ltd.

  17. RNA Polymerase II Collision Interrupts Convergent Transcription

    PubMed Central

    Hobson, David J.; Wei, Wu; Steinmetz, Lars M.; Svejstrup, Jesper Q.

    2012-01-01

    Summary Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision. PMID:23041286

  18. Optical tweezers studies of transcription by eukaryotic RNA polymerases.

    PubMed

    Lisica, Ana; Grill, Stephan W

    2017-02-21

    Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

  19. RNA-RNA and RNA-protein interactions in coronavirus replication and transcription

    PubMed Central

    Sola, Isabel; Mateos-Gomez, Pedro A; Almazan, Fernando; Zuñiga, Sonia

    2011-01-01

    Coronavirus (CoV) RNA synthesis includes the replication of the viral genome, and the transcription of sgRNAs by a discontinuous mechanism. Both processes are regulated by RNA sequences such as the 5′ and 3′ untranslated regions (UTRs), and the transcription regulating sequences (TRSs) of the leader (TRS-L) and those preceding each gene (TRS-Bs). These distant RNA regulatory sequences interact with each other directly and probably through protein-RNA and protein-protein interactions involving viral and cellular proteins.1 By analogy to other plus-stranded RNA viruses, such as polioviruses, in which translation and replication switch involves a cellular factor (PCBP) and a viral protein (3CD),2 it is conceivable that in CoVs the switch between replication and transcription is also associated with the binding of proteins that are specifically recruited by the replication or transcription complexes. Complexes between RNA motifs such as TRS-L and the TRS-Bs located along the CoV genome are probably formed previously to the transcription start, and most likely promote template-switch of the nascent minus RNA to the TRS-L region.3 Many cellular proteins interacting with regulatory CoV RNA sequences4 are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA-binding proteins, involved in mRNA processing and transport, which shuttle between the nucleus and the cytoplasm. In the context of CoV RNA synthesis, these cellular ribonucleoproteins might also participate in RNA-protein complexes to bring into physical proximity TRS-L and distant TRS-B, as proposed for CoV discontinuous transcription.5–7 In this review, we summarize RNA-RNA and RNA-protein interactions that represent modest examples of complex quaternary RNA-protein structures required for the fine-tuning of virus replication. Design of chemically defined replication and transcription systems will help to clarify the nature and activity of these structures. PMID:21378501

  20. Coupled Evolution of Transcription and mRNA Degradation

    PubMed Central

    Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

    2011-01-01

    mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

  1. Dietary protein-related changes in hepatic transcription correspond to modifications in hepatic protein expression in growing pigs.

    PubMed

    Junghans, Peter; Kaehne, Thilo; Beyer, Manfred; Metges, Cornelia C; Schwerin, Manfred

    2004-01-01

    In a previous investigation we showed by expression profiling based on transcription analysis using differential display RT-PCR (DDRT-PCR) and real-time RT-PCR that a soy protein diet (SPI) significantly changes the hepatic transcription pattern compared with a casein diet (CAS). The present study was conducted to determine whether the transcriptional modulation is translated into protein expression. The hepatic mRNA abundance of four genes (EP24.16, LC3, NPAP60L, RFC2) that showed diet-related expression in previous DDRT-PCR experiments was analyzed by real-time RT-PCR. Two pigs that showed the most prominent SPI-related changes of transcription and two casein-fed pigs were selected and their hepatic protein pattern was studied comparatively by two-dimensional gel electrophoresis and peptide mass fingerprinting. The two-dimensional protein gel electrophoresis revealed a predominant SPI-associated upregulation of protein expression that corresponded to the results of the mRNA study. Of 380 diet-related protein spots displayed, 215 appeared exclusively or enlarged in the two SPI pigs; 10 of 39 diet-related expressed protein spots extracted could be identified by peptide mass fingerprinting and database search. Compared with the transcriptomics approach, the proteomics approach led in part to the identification of the same diet-associated expressed molecules (plasminogen, trypsin, phospholipase A2, glutathione-S-transferase alpha, retinal binding protein) or at least molecules belonging to the same metabolic pathways (protein and amino acid metabolism, oxidative stress response, lipid metabolism). The present results at the proteome level confirm SPI-related increased oxidative stress response and significant effects on protein biosynthesis already observed at the transcriptome level.

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

    PubMed

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

    2011-01-01

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

  3. 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.

  4. 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

  5. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Preclinical evaluation of AMPLICOR hepatitis C virus test for detection of hepatitis C virus RNA.

    PubMed Central

    Nolte, F S; Thurmond, C; Fried, M W

    1995-01-01

    We compared a single-enzyme, combined reverse transcription-PCR (RT-PCR; AMPLICOR HCV Test; Roche Molecular Systems, Branchburg, N.J.) with an independent, two-enzyme, standard RT-PCR (SRT-PCR) assay for the detection of hepatitis C virus (HCV) RNA in serum and plasma. Test samples included a proficiency testing panel consisting of 10 undiluted plasma samples, three separate dilution series, and sera from 99 patients with chronic liver disease. The quantity of HCV RNA in each patient serum sample was determined by a branched DNA (bDNA) signal amplification assay (Quantiplex HCV-RNA assay; Chiron, Emeryville, Calif.). There was complete concordance between the results of the RT-PCR assays with the 10 undiluted plasma samples used for proficiency testing (3 positive and 7 negative samples). However, the analytical sensitivity of SRT-PCR was 4- to 10-fold greater than that of the AMPLICOR test in the dilution series. HCV RNA was detected in 44, 45, and 40 of the patient serum samples, by SRT-PCR, the AMPLICOR test, and the bDNA assay, respectively. There was 97% agreement between the results of the RT-PCR assays, with only three discrepancies. Review of the patients' medical records resolved all three discrepancies in favor of the AMPLICOR results (two false-negative SRT-PCR results and one false-positive SRT-PCR result). The quantity of HCV RNA in sera from five (11%) patients with viremia detected by AMPLICOR was below the bDNA assay cutoff (< 3.5 x 10(5) RNA equivalents per ml).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7665645

  7. Downregulation of rRNA transcription triggers cell differentiation.

    PubMed

    Hayashi, Yuki; Kuroda, Takao; Kishimoto, Hiroyuki; Wang, Changshan; Iwama, Atsushi; Kimura, Keiji

    2014-01-01

    Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

  8. Transcriptional regulation of mammalian miRNA genes

    PubMed Central

    Schanen, Brian C.; Li, Xiaoman

    2010-01-01

    MicroRNAs (miRNAs) are members of a growing family of non-coding transcripts, 21-23 nucleotides long, which regulate a diverse collection of biological processes and various diseases by RNA-mediated gene-silencing mechanisms. While currently many studies focus on defining the regulatory functions of miRNAs, few are directed towards how miRNA genes are themselves transcriptionally regulated. Recent studies of miRNA transcription have elucidated RNA polymerase II as the major polymerase of miRNAs, however, little is known of the structural features of miRNA promoters, especially those of mammalian miRNAs. Here, we review the current literature regarding features conserved among miRNA promoters useful for their detection and the current novel methodologies available to enable researchers to advance our understanding of the transcriptional regulation of miRNA genes. PMID:20977933

  9. Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

    PubMed

    Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

    2009-06-01

    Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

  10. FACT facilitates chromatin transcription by RNA polymerases I and III

    PubMed Central

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I; Panova, Tatiana B; Andersen, Jens S; Owen-Hughes, Tom A; Russell, Jackie; Lee, Sheng-Chung; Zomerdijk, Joost C B M

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes. PMID:19214185

  11. Single molecule studies of RNA polymerase II transcription in vitro

    PubMed Central

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

    2014-01-01

    Eukaryotic mRNA transcription by RNA polymerase II (RNAP II) is the first step in gene expression and a key determinant of cellular regulation. Elucidating the mechanism by which RNAP II synthesizes RNA is therefore vital to determining how genes are controlled under diverse biological conditions. Significant advances in understanding RNAP II transcription have been achieved using classical biochemical and structural techniques; however, aspects of the transcription mechanism cannot be assessed using these approaches. The application of single-molecule techniques to study RNAP II transcription has provided new insight only obtainable by studying molecules in this complex system one at a time. PMID:25764112

  12. Single molecule studies of RNA polymerase II transcription in vitro.

    PubMed

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

    2014-01-01

    Eukaryotic mRNA transcription by RNA polymerase II (RNAP II) is the first step in gene expression and a key determinant of cellular regulation. Elucidating the mechanism by which RNAP II synthesizes RNA is therefore vital to determining how genes are controlled under diverse biological conditions. Significant advances in understanding RNAP II transcription have been achieved using classical biochemical and structural techniques; however, aspects of the transcription mechanism cannot be assessed using these approaches. The application of single-molecule techniques to study RNAP II transcription has provided new insight only obtainable by studying molecules in this complex system one at a time.

  13. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Structural basis of transcription initiation by RNA polymerase II.

    PubMed

    Sainsbury, Sarah; Bernecky, Carrie; Cramer, Patrick

    2015-03-01

    Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained. Although mechanistic details await elucidation, available data outline how Pol II cooperates with the general transcription factors to bind to and open promoter DNA, and how Pol II directs RNA synthesis and escapes from the promoter.

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

    PubMed

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

    2014-01-01

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

  16. 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. Copyright © 2015, American Association for the Advancement of Science.

  17. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  18. RNA polymerase II transcription on the fast lane.

    PubMed

    Marcello, Alessandro

    2012-01-01

    Transcription by RNA polymerase II is the process that copies DNA into RNA leading to the expression of a specific gene. Averaged estimates of polymerase elongation rates in mammalian cells have been shown to vary between 1 and 4 kilobases per minute. However, recent advances in live cell imaging allowed direct measurements of RNA biogenesis from a single gene exceeded 50 kb·min(-1) . This unexpected finding opens novel and intriguing perspectives on the control of metazoan transcription.

  19. Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme*

    PubMed Central

    Basu, Ritwika S.; Warner, Brittany A.; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S.

    2014-01-01

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5′-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. PMID:24973216

  20. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme.

    PubMed

    Basu, Ritwika S; Warner, Brittany A; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S

    2014-08-29

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. DNA display of folded RNA libraries enabling RNA-SELEX without reverse transcription.

    PubMed

    MacPherson, I S; Temme, J S; Krauss, I J

    2017-03-02

    A method for the physical attachment of folded RNA libraries to their encoding DNA is presented as a way to circumvent the reverse transcription step during systematic evolution of RNA ligands by exponential enrichment (RNA-SELEX). A DNA library is modified with one isodC base to stall T7 polymerase and a 5' "capture strand" which anneals to the nascent RNA transcript. This method is validated in a selection of RNA aptamers against human α-thrombin with dissociation constants in the low nanomolar range. This method will be useful in the discovery of RNA aptamers and ribozymes containing base modifications that make them resistant to accurate reverse transcription.

  2. 2-Selenouridine triphosphate synthesis and Se-RNA transcription.

    PubMed

    Sun, Huiyan; Jiang, Sibo; Caton-Williams, Julianne; Liu, Hehua; Huang, Zhen

    2013-09-01

    2-Selenouridine ((Se)U) is one of the naturally occurring modifications of Se-tRNAs ((Se)U-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the (Se)U-RNA via 2-selenouridine triphosphate ((Se)UTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized (Se)UTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of (Se)U-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed (Se)U-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of (Se)U-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.

  3. Transcript-RNA-templated DNA recombination and repair.

    PubMed

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  4. In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    PubMed Central

    Jones, Scott A.; Boregowda, Rajeev; Spratt, Thomas E.

    2012-01-01

    Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming. PMID:22379076

  5. Regulation of mammalian ribosomal gene transcription by RNA polymerase I.

    PubMed

    Grummt, I

    1999-01-01

    All cells, from prokaryotes to vertebrates, synthesize vast amounts of ribosomal RNA to produce the several million new ribosomes per generation that are required to maintain the protein synthetic capacity of the daughter cells. Ribosomal gene (rDNA) transcription is governed by RNA polymerase I (Pol I) assisted by a dedicated set of transcription factors that mediate the specificity of transcription and are the targets of the pleiotrophic pathways the cell uses to adapt rRNA synthesis to cell growth. In the past few years we have begun to understand the specific functions of individual factors involved in rDNA transcription and to elucidate on a molecular level how transcriptional regulation is achieved. This article reviews our present knowledge of the molecular mechanism of rDNA transcriptional regulation.

  6. HDAC5 Inhibits Hepatic Lipogenic Genes Expression by Attenuating the Transcriptional Activity of Liver X Receptor.

    PubMed

    Jia, Hai-Yan; Li, Quan-Zhong; Lv, Li-Fang

    2016-01-01

    Liver X receptor (LXR), a member of the nuclear receptor superfamily, is known to induce the expression of SREBP-1c and ChREBP, two master regulators of hepatic lipogenesis. Histone deacyetylases (HDACs) have been shown to play critical roles in glucose and lipids metabolism. However, the exact role of HDAC5 in lipogenesis remains elusive. mRNA and protein levels of HDAC5 were analyzed by quantitative real-time PCR and Western blots in high-fat-diet-induced and leptin receptor deficiency-induced obese mice. HDAC5 was overexpressed or depleted in HepG2 cells, followed by analysis of cellular triglycerides contents. Quantitative real-time PCR was used to detect the expression levels of lipogenic genes. Luciferase reporter assay was used to determine the regulation of HDAC on the transcriptional activity of LXR. Co-immunoprecipitation experiment was used to determine the interaction between HDAC5 and LXR. We found that mRNA and protein expression levels of hepatic HDAC5 were reduced in high-fat-diet-induced and leptin receptor deficiency-induced obese mice. In vitro studies further demonstrated that knockdown of HDAC5 promoted cellular triglycerides accumulation, accompanied with up-regulation of lipogenic genes. At the molecular level, HDAC5 was shown to interact with LXR, thereby attenuating its transcriptional activity. Overall, our data suggest that hepatic HDAC5 is an important regulator of lipogenesis. © 2016 The Author(s) Published by S. Karger AG, Basel.

  7. CLOCK Regulates Circadian Rhythms of Hepatic Glycogen Synthesis through Transcriptional Activation of Gys2*

    PubMed Central

    Doi, Ryosuke; Oishi, Katsutaka; Ishida, Norio

    2010-01-01

    Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2. PMID:20430893

  8. The transcription cofactor CRTC1 protects from aberrant hepatic lipid accumulation

    PubMed Central

    Kim, Hwijin

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a rapidly emerging global health-problem. NAFLD encompasses a range of conditions associated with hepatic steatosis, aberrant accumulation of fat in hepatocytes. Although obesity and metabolic syndrome are considered to have a strong association with NAFLD, genetic factors that predispose liver to NAFLD and molecular mechanisms by which excess hepatic lipid develops remain largely unknown. We report that the transcription cofactor CRTC1 confers broad spectrum protection against hepatic steatosis development. CRTC1 directly interferes with the expression of genes regulated by lipogenic transcription factors, most prominently liver x receptor α (LXRα). Accordingly, Crtc1 deficient mice develop spontaneous hepatic steatosis in young age. As a cyclic AMP effector, CRTC1 mediates anti-steatotic effects of calorie restriction (CR). Notably, CRTC1 also mediates anti-lipogenic effects of bile acid signaling, whereas it is negatively regulated by miR-34a, a pathogenic microRNA upregulated in a broad spectrum of NAFLD. These patterns of gene function and regulation of CRTC1 are distinct from other CR-responsive proteins, highlighting critical protective roles that CRTC1 selectively plays against NAFLD development, which in turn provides novel opportunities for selectively targeting beneficial therapeutic effects of CR. PMID:27869139

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

    PubMed

    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

    2010-12-01

    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.

  10. Versatile RNA-sensing transcriptional regulators for engineering genetic networks.

    PubMed

    Lucks, Julius B; Qi, Lei; Mutalik, Vivek K; Wang, Denise; Arkin, Adam P

    2011-05-24

    The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.

  11. Versatile RNA-sensing transcriptional regulators for engineering genetic networks

    PubMed Central

    Lucks, Julius B.; Qi, Lei; Mutalik, Vivek K.; Wang, Denise; Arkin, Adam P.

    2011-01-01

    The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules. PMID:21555549

  12. Natural antisense and noncoding RNA transcripts as potential drug targets.

    PubMed

    Wahlestedt, Claes

    2006-06-01

    Information on the complexity of mammalian RNA transcription has increased greatly in the past few years. Notably, thousands of sense transcripts (conventional protein-coding genes) have antisense transcript partners, most of which are noncoding. Interestingly, a number of antisense transcripts regulate the expression of their sense partners, either in a discordant (antisense knockdown results in sense-transcript elevation) or concordant (antisense knockdown results in concomitant sense-transcript reduction) manner. Two new pharmacological strategies based on the knockdown of antisense RNA transcripts by siRNA (or another RNA targeting principle) are proposed in this review. In the case of discordant regulation, knockdown of antisense transcript elevates the expression of the conventional (sense) gene, thereby conceivably mimicking agonist-activator action. In the case of concordant regulation, knockdown of antisense transcript, or concomitant knockdown of antisense and sense transcripts, results in an additive or even synergistic reduction of the conventional gene expression. Although both strategies have been demonstrated to be valid in cell culture, it remains to be seen whether they provide advantages in other contexts.

  13. Nascent RNA sequencing reveals distinct features in plant transcription

    PubMed Central

    Hetzel, Jonathan; Duttke, Sascha H.; Benner, Christopher; Chory, Joanne

    2016-01-01

    Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole. PMID:27729530

  14. Bovine somatotropin increases hepatic phosphoenolpyruvate carboxykinase mRNA in lactating dairy cows.

    PubMed

    Velez, J C; Donkin, S S

    2004-05-01

    Somatotropin (ST) increases milk production and through coordinated changes in hepatic glucose synthesis and amino acid metabolism in dairy cows. The objective of this study was to determine the effects of ST on hepatic mRNA expression for phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC), enzymes that are critical to the synthesis of glucose in liver and hepatic mRNA expression for carbamylphosphate synthetase I (CPS-I), argininosuccinate synthetase (AS), and ornithine transcarbamylase (OTC), critical enzymes of the urea cycle. Eighteen cows were randomly allocated to 2 treatment groups and received either recombinant bovine ST (Posilac; Monsanto, St. Louis, MO) or saline injections at 14-d intervals during a 42-d period. Expression of mRNA was determined using Northern blot analysis. Nuclei, isolated from liver biopsy samples, were used to determine effects of ST on transcription rate of PEPCK. Milk production was increased with ST (37.3 vs. 35.1+/-0.6 kg/ d). Plasma NEFA was increased with ST (299 vs. 156+/-34 microM). There were no differences in the expression of CPS-I, AS, and OTC mRNA with ST. Expression of PEPCK and IGF-I mRNA were increased with ST but PC mRNA was unchanged. The data indicate increased PEPCK mRNA in cows given ST and indicates a greater capacity for gluconeogenesis from gluconeogenic precursors that form oxaloacetate. The effects of ST to elevate PEPCK mRNA expression require chronic administration and involve increased transcription of the PEPCK gene.

  15. A Specific Hepatic Transfer RNA for Phosphoserine*

    PubMed Central

    Mäenpää, Pekka H.; Bernfield, Merton R.

    1970-01-01

    Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

  16. 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

  17. Understanding the Molecular Basis of RNA Polymerase II Transcription.

    PubMed

    Zhang, Su; Wang, Dong

    2013-06-01

    Synthetic nucleic acid analogues have profoundly advanced our knowledge of DNA and RNA, as well as the complex biological processes that involve nucleic acids. As a pivotal enzyme, eukaryotic RNA polymerase II (Pol II) is responsible for transcribing DNA into messenger RNA, which serves as a template to direct protein synthesis. Chemically modified nucleic acid analogues have greatly facilitated the structural elucidation of RNA Pol II elongation complex and understanding the key chemical interactions governing RNA Pol II transcriptional fidelity. This review addresses major progress in RNA polymerase II mechanistic studies using modified nucleic acid analogues in recent years.

  18. Transcript RNA supports precise repair of its own DNA gene.

    PubMed

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  19. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells

    PubMed Central

    Sigova, Alla A.; Mullen, Alan C.; Molinie, Benoit; Gupta, Sumeet; Orlando, David A.; Guenther, Matthew G.; Almada, Albert E.; Lin, Charles; Sharp, Phillip A.; Giallourakis, Cosmas C.; Young, Richard A.

    2013-01-01

    Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. PMID:23382218

  20. Mechanism of genome transcription in segmented dsRNA viruses.

    PubMed

    Lawton, J A; Estes, M K; Prasad, B V

    2000-01-01

    Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for

  1. Recent highlights of RNA-polymerase-II-mediated transcription.

    PubMed

    Sims, Robert J; Mandal, Subhrangsu S; Reinberg, Danny

    2004-06-01

    Considerable advances into the basis of RNA-polymerase-II-mediated transcriptional regulation have recently emerged. Biochemical, genetic and structural studies have contributed to novel insights into transcription, as well as the functional significance of covalent histone modifications. New details regarding transcription elongation through chromatin have further defined the mechanism behind this action, and identified how chromatin structure may be maintained after RNAP II traverses a nucleosome. ATP-dependent chromatin remodeling complexes, along with histone chaperone complexes, were recently discovered to facilitate histone exchange. In addition, it has become increasingly clear that transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm.

  2. 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.

  3. 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

  4. Nuclear stability and transcriptional directionality separate functionally distinct RNA species.

    PubMed

    Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind; Core, Leighton J; Bornholdt, Jette; Boyd, Mette; Heick Jensen, Torben; Sandelin, Albin

    2014-11-12

    Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

  5. Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription.

    PubMed

    Han, Sangwoo; Kim, Hyejin; Lee, Jong Bum

    2017-08-30

    Since the discovery of RNA interference (RNAi), small interfering RNA (siRNA) has been powerful tools for gene downregulation in biomedical applications. Despite the outstanding efficacy of siRNA, the development of a therapeutic delivery system remains a challenge owing to the instability of RNA. In this study, we describe a new method for the design of siRNA-generating nanosponges by using complementary rolling circle transcription (cRCT), a technique that requires two complementary circular DNA. The sequences of one of the circular DNA are designed to have complete complementarity to the target mRNA resulting in double stranded RNA (dsRNA) that can be digested to siRNA by cellular Dicer activity. This siRNA design, called 'library siRNA', could be universally applied to fabricate RNA nanosponges targeting any known mRNA sequence.

  6. Promoter RNA links transcriptional regulation of inflammatory pathway genes

    PubMed Central

    Matsui, Masayuki; Chu, Yongjun; Zhang, Huiying; Gagnon, Keith T.; Shaikh, Sarfraz; Kuchimanchi, Satya; Manoharan, Muthiah; Corey, David R.; Janowski, Bethany A.

    2013-01-01

    Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping. PMID:23999091

  7. Enhanced amplification of hepatitis C virus (HCV) cDNA by PCR: detection of HCV RNA in archival sera.

    PubMed Central

    Beardsley, A M; Gowans, E J; Burrell, C J; Marmion, B P

    1996-01-01

    A reverse transcription-PCR assay which successfully amplified hepatitis C virus RNA from poorly stored archival sera was optimized. Maximum sensitivity was achieved with Moloney murine leukemia virus RNase H- reverse transcriptase and by a single round of PCR amplification of a short (112-bp) fragment of the 5' untranslated region of the viral genome. PMID:8735126

  8. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  9. Disengaging polymerase: Terminating RNA polymerase II transcription in budding yeast☆

    PubMed Central

    Mischo, Hannah E.; Proudfoot, Nick J.

    2013-01-01

    Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3′ end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation. PMID:23085255

  10. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II

    PubMed Central

    Serebryannyy, Leonid A.; Parilla, Megan; Cruz, Christina M.; Laster, Kyle; Kudryashov, Dmitri; Kosak, Steven T.

    2016-01-01

    ABSTRACT Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin–RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II. PMID:27505898

  11. Transcription rate of RNA polymerase under rotary torque

    NASA Astrophysics Data System (ADS)

    Kiryu, H.

    2004-04-01

    We investigated the transcription rates of RNA polymerases that were subjected to rotational drag. By combining chemical kinetics with mechanical equations, we derived formulas for the transcription rate in the case where the torque was caused by the hydrodynamic drag to DNA rotation.

  12. 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.

  13. 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

  14. Resetting the transcription factor network reverses terminal chronic hepatic failure

    PubMed Central

    Nishikawa, Taichiro; Bell, Aaron; Brooks, Jenna M.; Setoyama, Kentaro; Melis, Marta; Han, Bing; Fukumitsu, Ken; Handa, Kan; Tian, Jianmin; Kaestner, Klaus H.; Vodovotz, Yoram; Locker, Joseph; Soto-Gutierrez, Alejandro; Fox, Ira J.

    2015-01-01

    The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement. PMID:25774505

  15. DNA-RNA transcription as an impact of viscosity

    NASA Astrophysics Data System (ADS)

    Zdravković, Slobodan; Satarić, Miljko V.; Hadžievski, Ljupčo

    2010-12-01

    The impact of viscosity on DNA dynamics is studied both analytically and numerically. It is assumed that the viscosity exists at the segments where DNA molecule is surrounded by RNA polymerase. We demonstrate that the frictional forces destroy the modulation of the incoming solitonic wave. We show that viscosity, crucial for demodulation, is essential for DNA-RNA transcription.

  16. The transcriptional factor Osterix directly interacts with RNA helicase A.

    PubMed

    Amorim, Bruna Rabelo; Okamura, Hirohiko; Yoshida, Kaya; Qiu, Lihong; Morimoto, Hiroyuki; Haneji, Tatsuji

    2007-04-06

    Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation.

  17. Glucocorticoids promote hepatic cholestasis in mice by inhibiting the transcriptional activity of the farnesoid X receptor.

    PubMed

    Lu, Yan; Zhang, Zhijian; Xiong, Xuelian; Wang, Xiaolin; Li, Jin; Shi, Guojun; Yang, Jian; Zhang, Xianfeng; Zhang, Huijie; Hong, Jie; Xia, Xuefeng; Ning, Guang; Li, Xiaoying

    2012-12-01

    Glucocorticoids have potent anti-inflammatory effects, but also can cause insulin resistance, osteoporosis, and muscle wasting, preventing their long-term use. Glucocorticoids also have been associated with the development of hepatic cholestasis and gallstone disease, but little is known about their pathogenic mechanisms. We analyzed levels of bile acids (BAs) and glucocorticoids in serum samples from patients with Cushing disease and obese individuals (body mass index, >30). C57BL/6 mice were injected with dexamethasone and db/db obese mice were injected with glucocorticoid receptor (GR) antagonists or small hairpin RNAs. We analyzed farnesoid X receptor (FXR) signaling in HepG2 cells and cells from mice using immunoprecipitation, luciferase reporter, and glutathione-s-transferase and chromatin immunoprecipitation assays. We analyzed BA metabolism in FXR-/- mice and mice with reduced levels of the transcription factor C-terminal binding protein (CtBP). Serum levels of BAs were higher in patients with Cushing disease or obesity than in individuals with normal levels of glucocorticoids. Administration of dexamethasone promoted cholestasis and overproduction of BAs in C57BL/6 mice, but not in FXR-/- mice. GR antagonists, or injection of an adenoviral small hairpin RNA against GR, reduced features of hepatic cholestasis in db/db mice. The GR interacted with FXR to reduce its transcriptional activity by recruiting CtBP co-repressor complexes. Mice with reduced levels of CtBP were resistant to induction of hepatic cholestasis by dexamethasone. Glucocorticoids promote hepatic cholestasis in mice by recruiting CtBP co-repressor complexes to FXR and thereby blocking the transcriptional activity. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  18. 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.

  19. 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.

  20. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss

    PubMed Central

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong; Baek, Songjoon; Nielsen, Ronni; Mandrup, Susanne; Hager, Gordon L.; Chung, Jay H.; Grøntved, Lars

    2017-01-01

    Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome. PMID:28071704

  1. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss.

    PubMed

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong; Baek, Songjoon; Nielsen, Ronni; Mandrup, Susanne; Hager, Gordon L; Chung, Jay H; Grøntved, Lars

    2017-01-10

    Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

  2. Structural basis of transcription by bacterial and eukaryotic RNA polymerases.

    PubMed

    Sekine, Shun-ichi; Tagami, Shunsuke; Yokoyama, Shigeyuki

    2012-02-01

    DNA-dependent RNA polymerase (RNAP) is responsible for cellular gene transcription. Although crystallographic studies on prokaryotic and eukaryotic RNAPs have elucidated the basic RNAP architectures, the structural details of many essential events during transcription initiation, elongation, and termination are still largely unknown. Recent crystallographic studies on a bacterial RNAP and yeast RNAP II have revealed different RNAP structural states from that of the normal transcribing complex, as well as the basis of transcription factor functions, advancing our understanding of transcription. These studies have highlighted unexpected similarities in many fundamental aspects of transcription mechanisms between the bacterial and eukaryotic transcription machineries. Remarkable differences also exist between the bacterial and eukaryotic transcription systems, suggesting directions for future studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

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

    PubMed Central

    Proudfoot, Nick J.

    2016-01-01

    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

  5. 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

  6. Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

    PubMed

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

    2015-04-01

    MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which 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 lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

  7. 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.

  8. 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

  9. Nuclear networking fashions pre-messenger RNA and primary microRNA transcripts for function

    PubMed Central

    Pawlicki, Jan M.; Steitz, Joan A.

    2010-01-01

    The expression of protein-coding genes is enhanced by the exquisite coupling of transcription by RNA polymerase II with pre-messenger RNA processing reactions, such as 5′-end capping, splicing and 3′-end formation. Integration between cotranscriptional processing events extends beyond the nucleus, as proteins that bind cotranscriptionally can affect the localization, translation and degradation of the mature messenger RNA. MicroRNAs are RNA polymerase II transcripts with crucial roles in the regulation of gene expression. Recent data demonstrate that processing of primary microRNA transcripts might be yet another cotranscriptional event that is woven into this elaborate nuclear network. This review discusses the extensive molecular interactions that couple the earliest steps in gene expression and therefore influence the final fate and function of the mature messenger RNA or microRNA produced. PMID:20004579

  10. Zinc-mediated RNA fragmentation allows robust transcript reassembly upon whole transcriptome RNA-Seq.

    PubMed

    Wery, Maxime; Descrimes, Marc; Thermes, Claude; Gautheret, Daniel; Morillon, Antonin

    2013-09-01

    Whole transcriptome RNA-Seq has emerged as a powerful tool in transcriptomics, enabling genome-wide quantitative analysis of gene expression and qualitative identification of novel coding or non-coding RNA species through transcriptome reassembly. Common protocols for preparation of RNA-Seq libraries include an RNA fragmentation step for which several RNA sizing techniques are commercially available. To date, there is no global information about their putative bias on transcriptome analysis. Here we compared the effects of RNase III- and zinc-mediated RNA fragmentation on transcript expression measurement and transcriptome reassembly in the budding yeast Saccharomyces cerevisiae. We observed that RNA cleavage by RNase III is heterogeneous along transcripts with a striking decrease of autocorrelation between adjacent nucleotides along the transcriptome. This had little impact on mRNA expression measurement, but specific classes of transcripts such as abundant non-coding RNAs were underrepresented in the libraries constructed using RNase III. Furthermore, zinc-mediated fragmentation allows proper reassembly of more transcripts, with more precise 5' and 3' ends. Together, our results show that transcriptome reassembly from RNA-Seq data is very sensitive to the RNA fragmentation technique, and that zinc-mediated fragmentation provides more robust and accurate transcript identification than cleavage by RNase III. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. 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

  12. Global RNA association with the transcriptionally active chromosome of chloroplasts.

    PubMed

    Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

    2017-09-08

    Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

  13. Post-transcriptional gene regulation by long noncoding RNA

    PubMed Central

    Yoon, Je-Hyun; Abdelmohsen, Kotb; Gorospe, Myriam

    2012-01-01

    Eukaryotic cells transcribe a vast number of noncoding RNA species. Among them, long noncoding RNAs (lncRNAs) have been widely implicated in the regulation of gene transcription. However, examples of post-transcriptional gene regulation by lncRNAs are emerging. For example, through extended base-pairing, lncRNAs can stabilize or promote the translation of target mRNAs, while partial base-pairing facilitates mRNA decay or inhibits target mRNA translation. In the absence of complementarity, lncRNAs can suppress pre-mRNA splicing and translation by acting as decoys of RNA-binding proteins or microRNAs, and can compete for microRNA-mediated inhibition leading to increased expression of the mRNA. Through these regulatory mechanisms, lncRNAs can elicit differentiation, proliferation, and cytoprotective programs, underscoring the rising recognition of lncRNA roles in human disease. In this review, we summarize the mechanism of post-transcriptional gene regulation by lncRNAs. PMID:23178169

  14. RNA Sequencing and Bioinformatics Analysis Implicate the Regulatory Role of a Long Noncoding RNA-mRNA Network in Hepatic Stellate Cell Activation.

    PubMed

    Guo, Can-Jie; Xiao, Xiao; Sheng, Li; Chen, Lili; Zhong, Wei; Li, Hai; Hua, Jing; Ma, Xiong

    2017-08-11

    To analyze the long noncoding (lncRNA)-mRNA expression network and potential roles in rat hepatic stellate cells (HSCs) during activation. LncRNA expression was analyzed in quiescent and culture-activated HSCs by RNA sequencing, and differentially expressed lncRNAs verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were subjected to bioinformatics analysis. In vivo analyses of differential lncRNA-mRNA expression were performed on a rat model of liver fibrosis. We identified upregulation of 12 lncRNAs and 155 mRNAs and downregulation of 12 lncRNAs and 374 mRNAs in activated HSCs. Additionally, we identified the differential expression of upregulated lncRNAs (NONRATT012636.2, NONRATT016788.2, and NONRATT021402.2) and downregulated lncRNAs (NONRATT007863.2, NONRATT019720.2, and NONRATT024061.2) in activated HSCs relative to levels observed in quiescent HSCs, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that changes in lncRNAs associated with HSC activation revealed 11 significantly enriched pathways according to their predicted targets. Moreover, based on the predicted co-expression network, the relative dynamic levels of NONRATT013819.2 and lysyl oxidase (Lox) were compared during HSC activation both in vitro and in vivo. Our results confirmed the upregulation of lncRNA NONRATT013819.2 and Lox mRNA associated with the extracellular matrix (ECM)-related signaling pathway in HSCs and fibrotic livers. Our results detailing a dysregulated lncRNA-mRNA network might provide new treatment strategies for hepatic fibrosis based on findings indicating potentially critical roles for NONRATT013819.2 and Lox in ECM remodeling during HSC activation. © 2017 The Author(s). Published by S. Karger AG, Basel.

  15. 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.

  16. Structural insights into transcription initiation by yeast RNA polymerase I.

    PubMed

    Sadian, Yashar; Tafur, Lucas; Kosinski, Jan; Jakobi, Arjen J; Wetzel, Rene; Buczak, Katarzyna; Hagen, Wim Jh; Beck, Martin; Sachse, Carsten; Müller, Christoph W

    2017-09-15

    In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  17. 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

  18. The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts

    PubMed Central

    Shimada, Yukiko; Mohn, Fabio; Bühler, Marc

    2016-01-01

    Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5′ end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA–DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms. PMID:27941123

  19. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  20. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    PubMed

    Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

    2013-01-01

    Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the

  1. Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.

    PubMed

    Sasano, Yu; Kariya, Takahiro; Usugi, Shogo; Sugiyama, Minetaka; Harashima, Satoshi

    2017-12-01

    As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400-700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

  2. Breastmilk hepatitis A virus RNA in nursing mothers with acute hepatitis A virus infection.

    PubMed

    Daudi, Nili; Shouval, Daniel; Stein-Zamir, Chen; Ackerman, Zvi

    2012-08-01

    Breastmilk specimens from three women with acute hepatitis A virus (HAV) infection were studied. Anti-HAV immunoglobulin M and immunoglobulin G antibodies were detected in serum and breastmilk specimens of the three women. The three women also had serum HAV RNA. However, HAV RNA was detected only in two of the three breastmilk specimens. It is interesting that none of the three infants contracted clinical HAV infection. Furthermore, mothers with HAV infection should not be encouraged to discontinue breastfeeding.

  3. 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.

  4. CDK9-dependent RNA polymerase II pausing controls transcription initiation.

    PubMed

    Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

    2017-10-10

    Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

  5. 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

  6. Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

    PubMed

    Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

    2017-04-01

    Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD(+)-dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Repairing RNA Transcripts that Mediate Breast Cancer Susceptibility

    DTIC Science & Technology

    2005-08-01

    is actually the yield of TES product plus the yield of cryptic is in contrast to hammerhead and hairpin ribozymes , which products. This increases the...therapeutics. To this end, we have developed a novel biomolecule (a ribozyme ) that can specifically excise regions from RNA transcripts. In this work, we...designed a ribozyme that excises an insertion mutation that is linked to breast cancer predisposition from a short mimic of the p53 transcript in a

  8. Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

    PubMed

    Song, Wenjiao; Filonov, Grigory S; Kim, Hyaeyeong; Hirsch, Markus; Li, Xing; Moon, Jared D; Jaffrey, Samie R

    2017-09-25

    Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

  9. 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.

  10. Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript.

    PubMed Central

    Okamoto, K; Freundlich, M

    1986-01-01

    Expression of the crp gene is negatively autoregulated by the complex of cyclic AMP and its receptor protein (cAMP-CRP). We find a second promoter in this region that is strongly activated in vitro and in vivo by cAMP-CRP. Transcription from this promoter is initiated 3 nucleotides upstream and on the opposite strand from the start of crp mRNA. The addition of the purified 5' segment of the divergent RNA specifically inhibits crp transcription in vitro. cAMP-CRP does not block crp expression if the new promoter is altered so that divergent RNA cannot be made. The initial nucleotides of the divergent RNA are complementary to 10 of the first 11 nucleotides of the crp mRNA. Since the next 11 nucleotides of crp mRNA are A + U-rich, and RNA hybrid between the divergent RNA and the 5' end of crp mRNA could produce a structure similar to a rho-independent terminator, leading to inhibition of crp transcription. Images PMID:2425359

  11. 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. © 2016 Ye and Zhang; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.

    PubMed Central

    Quincey, R V; Arnold, R E

    1984-01-01

    A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes. The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx. 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207. Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA. Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping. These transcripts were initiated at the site used by the host rRNA genes, were approx. 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing. Images Fig. 4. Fig. 5. Fig. 6. PMID:6097222

  13. Giardia lamblia RNA polymerase II: amanitin-resistant transcription.

    PubMed

    Seshadri, Vishwas; McArthur, Andrew G; Sogin, Mitchell L; Adam, Rodney D

    2003-07-25

    Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G. lamblia demonstrate important differences from these processes in higher eukaryotes. The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to alpha-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 microg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template. To better understand transcription in G. lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpb1 from G. lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms. These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G. lamblia.

  14. 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

  15. Searching for nuclear export elements in hepatitis D virus RNA.

    PubMed

    Freitas, Natália; Cunha, Celso

    2013-08-12

    To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.

  16. 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.

  17. A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

    PubMed Central

    Feng, Hui; Beck, Jürgen; Nassal, Michael; Hu, Kang-hong

    2011-01-01

    Background The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. Methodology/Principal Findings Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. Conclusions/Significance This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B. PMID:22125633

  18. Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

    PubMed

    Brannan, Kris; Bentley, David L

    2012-01-01

    The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

  19. Gastrointestinal hormone mRNA expression in human colonic adenocarcinomas, hepatic metastases and cell lines

    PubMed Central

    Monges, G; Biagini, P; Cantaloube, J F; De Micco, P; Parriaux, D; Seitz, J F; Delpero, J R; Hassoun, J

    1996-01-01

    Aims—(1) To investigate the expression of the four main hormones of the digestive tract by performing reverse transcription polymerase chain reaction (RT-PCR) on a series of samples, comprising tumoral and healthy colonic tissues, hepatic metastases and colonic cell line samples; and (2) to study the patterns of labelling obtained with serological and morphological markers. Methods—After extraction and reverse transcription, gastrin, somatostatin, cholecystokinin (CCK) and transforming growth factor α (TGFα) mRNAs were detected by PCR and nested PCR using specific primers. The corresponding proteins were detected by immunohistochemistry. Results—The cell lines expressed all four mRNAs. Gastrin mRNA was present in most tumoral and metastatic samples, while the somatostatin transcript was detected in all samples and was frequently overexpressed in the normal colon. TGFα mRNA was expressed systematically in tumours of the right and transverse colon, but not in those located in the left colon; the expression of CCK mRNA was systematically absent in the left colon. Conclusions—The data presented here shed some light on the transcriptional events involved in the production of the various hormones present in the gastrointestinal tract, in both healthy and tumoral tissues. The various mRNAs expressed in cell lines are therefore not systematically expressed in the human pathology. Images PMID:16696065

  20. 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

  1. L_RNA_scaffolder: scaffolding genomes with transcripts

    PubMed Central

    2013-01-01

    Background Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments. Results We describe L_RNA_scaffolder, a novel genome scaffolding method that uses long transcriptome reads to order, orient and combine genomic fragments into larger sequences. To demonstrate the accuracy of the method, the zebrafish genome was scaffolded. With expanded human transcriptome data, the N50 of human genome was doubled and L_RNA_scaffolder out-performed most scaffolding results by existing scaffolders which employ mate-pair libraries. In these two examples, the transcript coverage was almost complete, especially for long transcripts. We applied L_RNA_scaffolder to the highly polymorphic pearl oyster draft genome and the gene model length significantly increased. Conclusions The simplicity and high-throughput of RNA-seq data makes this approach suitable for genome scaffolding. L_RNA_scaffolder is available at http://www.fishbrowser.org/software/L_RNA_scaffolder. PMID:24010822

  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. TFIIB-related Factors in RNA Polymerase I Transcription

    PubMed Central

    Knutson, Bruce A.; Hahn, Steven

    2012-01-01

    Eukaryotic RNA polymerases (Pol) I, II, III and archaeal Pol use a related set of general transcription factors to recognize promoter sequences, recruit Pol to promoters and to function at key points in the transcription initiation mechanism. The TFIIB-like general transcription factors (GTFs) function during several important and conserved steps in the initiation pathway for Pol II, III, and archaeal Pol. Until recently, the mechanism of Pol I initiation seemed unique, since it appeared to lack a GTF paralogous to the TFIIB-like proteins. The surprising recent discovery of TFIIB-related Pol I general factors in yeast and humans highlights the evolutionary conservation of transcription initiation mechanisms for all eukaryotic and archaeal Pols. These findings reveal new roles for the function of the Pol I GTFs and insight into the function of TFIIB-related factors. Models for Pol I transcription initiation are reexamined in light of these recent findings. PMID:22960599

  4. Cooperation between translating ribosomes and RNA polymerase in transcription elongation.

    PubMed

    Proshkin, Sergey; Rahmouni, A Rachid; Mironov, Alexander; Nudler, Evgeny

    2010-04-23

    During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that translates the newly synthesized transcript. Our in vivo measurements show that the overall elongation rate of transcription is tightly controlled by the rate of translation. Acceleration and deceleration of a ribosome result in corresponding changes in the speed of RNAP. Moreover, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. The stimulating effect of a ribosome on RNAP is achieved by preventing its spontaneous backtracking, which enhances the pace and also facilitates readthrough of roadblocks in vivo. Such a cooperative mechanism ensures that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions.

  5. Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

    PubMed

    Tongaonkar, Prasad; French, Sarah L; Oakes, Melanie L; Vu, Loan; Schneider, David A; Beyer, Ann L; Nomura, Masayasu

    2005-07-19

    Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

  6. When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

    PubMed

    Pipathsouk, Anne; Belotserkovskii, Boris P; Hanawalt, Philip C

    2017-02-01

    Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Alterations in hepatic miRNA expression during negative energy balance in postpartum dairy cattle.

    PubMed

    Fatima, Attia; Waters, Sinead; O'Boyle, Padraig; Seoighe, Cathal; Morris, Dermot G

    2014-01-15

    Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the 'samr' statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor

  8. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  9. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. In vitro transcription of a cloned mouse ribosomal RNA gene.

    PubMed Central

    Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

    1981-01-01

    An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

  11. 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

  12. 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

  13. 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.

  14. SAGA Is a General Cofactor for RNA Polymerase II Transcription.

    PubMed

    Baptista, Tiago; Grünberg, Sebastian; Minoungou, Nadège; Koster, Maria J E; Timmers, H T Marc; Hahn, Steve; Devys, Didier; Tora, László

    2017-10-05

    Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Up-regulation of intercellular adhesion molecule 1 transcription by hepatitis B virus X protein.

    PubMed Central

    Hu, K Q; Yu, C H; Vierling, J M

    1992-01-01

    Intercellular adhesion molecule 1 (ICAM-1), a counter-receptor for lymphocyte function-associated antigen 1 on T cells, is critically important to a wide variety of adhesion-dependent leukocyte functions, including antigen presentation and target cell lysis. ICAM-1 expression by hepatocytes is increased in areas of inflammation and necrosis during chronic hepatitis B. Whether induction of ICAM-1 is due to the effect of inflammatory cytokines or involves a direct effect of the hepatitis B virus (HBV) remains unknown. In the present study, transfection of the HBV genome into human hepatoma cell lines resulted in enhanced expression of ICAM-1 protein and RNA in the absence of inflammation. Results of subgenomic transfections indicated that the HBV X protein (pX) induced ICAM-1 expression. Nuclear run-on assays showed that pX induced the ICAM-1 gene by increasing its rate of transcription. Although both pX and interferon gamma induced transcription of ICAM-1, addition of interferon gamma to cells expressing pX did not show an additive or synergistic effect. These results indicate that pX can directly regulate expression of ICAM-1 and may participate in the immunopathogenesis of HBV infection. Images PMID:1360668

  16. 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

  17. 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. 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

  18. Transcription-mediated amplification (TMA) for the assessment of viremia in hemodialysis patients with hepatitis C.

    PubMed

    Bastos, Dauana O; Perez, Renata M; Silva, Ivonete Souza; Lemos, Lara Barros; Simonetti, José Pascoal; Medina-Pestana, José Osmar; Silva, Antonio Eduardo B; Ferraz, Maria Lucia

    2012-04-01

    The diagnosis of hepatitis C virus (HCV) infection in hemodialysis patients is difficult particularly due to the presence of intermittent viremia. The aims of this study were: (a) to determine the prevalence of intermittent viremia in hemodialysis patients with anti-HCV antibodies who tested negative for HCV RNA by PCR at the first evaluation and (b) to evaluate the contribution of the transcription-mediated amplification method (TMA) to the diagnosis of viremia in the PCR-negative samples. One hundred and six patients with anti-HCV antibodies and an initial negative result for HCV RNA by PCR were included. An additional sample was collected for a second HCV RNA test by PCR after a minimum interval of 3 months and a positive result characterized intermittent viremia. HCV RNA was investigated by TMA in the PCR-negative sample of patients with intermittent viremia, and in the most recent sample from patients with PCR-negative results in both determinations. Intermittent viremia was observed in 60/106 (57%) patients (57% men; age: 45 ± 10 years). Fifty-one of the 60 negative samples from patients with intermittent viremia and 29/46 double-negative patients were tested by TMA. This assay detected viremia in 20/51 (39%) samples of intermittent viremia and in 2/29 (7%) of double-negative samples. The results showed that intermittent viremia is frequent in hemodialysis patients who tested negative for HCV RNA by PCR. Therefore, a second HCV RNA test is necessary for all HCV RNA-negative patients. The TMA assay appears to be the best first screening test for viremia in this population. Copyright © 2012 Wiley Periodicals, Inc.

  19. RNA Polymerase III Accurately Initiates Transcription from RNA Polymerase II Promoters in Vitro*

    PubMed Central

    Duttke, Sascha H. C.

    2014-01-01

    In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the downstream promoter element (DPE) or the motif ten element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature. PMID:24917680

  20. Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed.

    PubMed Central

    Zhang, X; Lai, M M

    1995-01-01

    Previous studies suggested that coronavirus RNA transcription involves interaction between leader RNA and the intergenic (IG) sequences, probably via protein-RNA interactions (X. M. Zhang, C.-L. Liao, and M. M. C. Lai, J. Virol., 68:4738-4746, 1994; X. M. Zhang and M. M. C. Lai, J. Virol., 68:6626-6633, 1994). To determine whether cellular proteins are involved in this process, we performed UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG (promoter) sequence between genes 6 and 7 (IG7) and the 5' untranslational region of mouse hepatitis virus genomic RNA. We demonstrated that three different cellular proteins (p70, p48, and p35/38) bound to the promoter sequence of the template RNA. Deletion analyses of the template RNA mapped the binding site of p35/38 at the consensus transcription initiation signal. In contrast, the binding of p70 and p48 was less specific. p35/38 is the same protein as the one previously identified to bind to the complementary strand of the leader RNA; its binding affinity to the leader was approximately 15 times stronger than that to IG7. Site-directed mutagenesis of the IG sequence revealed that mutations in the consensus sequence of IG7 (UCUAAUCUAAAC to UCGAAAC and GCUAAAG), which resulted in reduced subgenomic mRNA transcription, also caused correspondingly reduced levels of p35/38 binding. These results demonstrated that the extent of protein binding to the IG sequences correlated with the amounts of subgenomic mRNAs transcribed from the IG site. These studies suggest that these RNA-binding proteins are involved in coronavirus RNA transcription and may represent transcription factors. PMID:7853499

  1. Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

    PubMed

    Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

    2014-10-01

    MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs.

  2. The RNA polymerase flow model of gene transcription.

    PubMed

    Edri, Shlomit; Gazit, Eran; Cohen, Eyal; Tuller, Tamir

    2014-02-01

    Gene expression is a fundamental cellular process by which proteins are synthesized based on the information coded in the genes. The two major steps of this process are the transcription of the DNA segment corresponding to a gene to mRNA molecules and the translation of the mRNA molecules to proteins by the ribosome. Thus, understanding, modeling and engineering the different stages of this process have both important biotechnological applications and contributions to basic life science. In previous studies we have introduced the Homogenous Ribosome Flow Model (HRFM) and demonstrated its advantages in analyses of the translation process. In this study we introduce the RNA Polymerase Flow Model (RPFM), a non trivial extension of the HRFM, which also includes a backward flow and can be used for modeling transcription and maybe other similar processes. We compare the HRFM and the RPFM in the three regimes of the transcription process: rate limiting initiation, rate limiting elongation and rate limiting termination via a simulative and analytical analysis. In addition, based on experimental data, we show that RPFM is a better choice for modeling transcription process.

  3. Molecular basis for coordinating transcription termination with noncoding RNA degradation.

    PubMed

    Tudek, Agnieszka; Porrua, Odil; Kabzinski, Tomasz; Lidschreiber, Michael; Kubicek, Karel; Fortova, Andrea; Lacroute, François; Vanacova, Stepanka; Cramer, Patrick; Stefl, Richard; Libri, Domenico

    2014-08-07

    The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Molecular Basis for Coordinating Transcription Termination with Noncoding RNA Degradation

    PubMed Central

    Tudek, Agnieszka; Porrua, Odil; Kabzinski, Tomasz; Lidschreiber, Michael; Kubicek, Karel; Fortova, Andrea; Lacroute, François; Vanacova, Stepanka; Cramer, Patrick; Stefl, Richard; Libri, Domenico

    2014-01-01

    Summary The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex. PMID:25066235

  5. Mechanism of histone survival during transcription by RNA polymerase II.

    PubMed

    Kulaeva, Olga I; Studitsky, Vasily M

    2010-01-01

    This work is related to and stems from our recent NSMB paper, "Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II" (December 2009). Synopsis. Recent genomic studies from many laboratories have suggested that nucleosomes are not displaced from moderately transcribed genes. Furthermore, histones H3/H4 carrying the primary epigenetic marks are not displaced or exchanged (in contrast to H2A/H2B histones) during moderate transcription by RNA polymerase II (Pol II) in vivo. These exciting observations suggest that the large molecule of Pol II passes through chromatin structure without even transient displacement of H3/H4 histones. The most recent analysis of the RNA polymerase II (Pol II)-type mechanism of chromatin remodeling in vitro (described in our NSMB 2009 paper) suggests that nucleosome survival is tightly coupled with formation of a novel intermediate: a very small intranucleosomal DNA loop (Ø-loop) containing transcribing Pol II. In the submitted manuscript we critically evaluate one of the key predictions of this model: the lack of even transient displacement of histones H3/H4 during Pol II transcription in vitro. The data suggest that, indeed, histones H3/H4 are not displaced during Pol II transcription in vitro. These studies are directly connected with the observation in vivo on the lack of exchange of histones H3/H4 during Pol II transcription.

  6. Rapid detection of duck hepatitis virus type-1 by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Song, Cuiping; Wan, Hongquan; Yu, Shengqing; Han, Xiangan; Qiu, Xusheng; Hu, Qinghai; Tan, Lei; Ding, Chan

    2012-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of duck hepatitis virus type-1 (DHV-1) was established. Using primers specific to the highly conserved 3D gene of DHV-1, the developed RT-LAMP assay detected the viral RNA of DHV-1 extracted from both allantoic fluid and liver samples of infected ducks. The assay is as sensitive as RT-PCR, and shows no cross-reaction with other common avian viral and bacterial pathogens. In addition to detection via ethidium bromide staining following gel electrophoresis, naked-eye observation after staining with SYBR Green I dye can be used to detect RT-LAMP products; this enables field application of this assay. The findings demonstrate that RT-LAMP can serve as a helpful tool for the detection and surveillance of DHV-1 in the poultry industry.

  7. 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.

  8. 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.

  9. RNA-directed DNA methylation induces transcriptional activation in plants

    PubMed Central

    Shibuya, Kenichi; Fukushima, Setsuko; Takatsuji, Hiroshi

    2009-01-01

    A class-C floral homeotic gene of Petunia, pMADS3, is specifically expressed in the stamen and carpels of developing flowers. We had previously reported the ect-pMADS3 phenomenon in which introduction of a part of the pMADS3 genomic sequence, including intron 2, induces ectopic expression of endogenous pMADS3. Unlike transcriptional or posttranscriptional gene silencing triggered by the introduction of homologous sequences, this observation is unique in that the gene expression is up-regulated. In this study, we demonstrated that the ect-pMADS3 phenomenon is due to transcriptional activation based on RNA-directed DNA methylation (RdDM) occurring in a particular CG in a putative cis-element in pMADS3 intron 2. The CG methylation was maintained over generations, along with pMADS3 ectopic expression, even in the absence of RNA triggers. These results demonstrate a previously undescribed transcriptional regulatory mechanism that could lead to the generation of a transcriptionally active epiallele, thereby contributing to plant evolution. Our results also reveal a putative negative cis-element for organ-specific transcriptional regulation of class-C floral homeotic genes, which could be difficult to identify by other approaches. PMID:19164525

  10. Post-transcriptional gene regulation by mRNA modifications

    PubMed Central

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan

    2016-01-01

    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  11. RNA-eXpress annotates novel transcript features in RNA-seq data.

    PubMed

    Forster, Samuel C; Finkel, Alexander M; Gould, Jodee A; Hertzog, Paul J

    2013-03-15

    Next-generation sequencing is rapidly becoming the approach of choice for transcriptional analysis experiments. Substantial advances have been achieved in computational approaches to support these technologies. These approaches typically rely on existing transcript annotations, introducing a bias towards known genes, require specific experimental design and computational resources, or focus only on identification of splice variants (ignoring other biologically relevant transcribed features contained within the data that may be important for downstream analysis). Biologically relevant transcribed features also include large and small non-coding RNA, new transcription start sites, alternative promoters, RNA editing and processing of coding transcripts. Also, many existing solutions lack accessible interfaces required for wide scale adoption. We present a user-friendly, rapid and computation-efficient feature annotation framework (RNA-eXpress) that enables identification of transcripts and other genomic and transcriptional features independently of current annotations. RNA-eXpress accepts mapped reads in the standard binary alignment (BAM) format and produces a study-specific feature annotation in GTF format, comparison statistics, sequence extraction and feature counts. The framework is designed to be easily accessible while allowing advanced users to integrate new feature-identification algorithms through simple class extension, thus facilitating expansion to novel feature types or identification of study-specific feature types.

  12. Control of rRNA transcription in Escherichia coli.

    PubMed Central

    Condon, C; Squires, C; Squires, C L

    1995-01-01

    The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response. PMID:8531889

  13. Novel layers of RNA polymerase III control affecting tRNA gene transcription in eukaryotes

    PubMed Central

    Leśniewska, Ewa

    2017-01-01

    RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency. PMID:28228471

  14. Novel layers of RNA polymerase III control affecting tRNA gene transcription in eukaryotes.

    PubMed

    Leśniewska, Ewa; Boguta, Magdalena

    2017-02-01

    RNA polymerase III (Pol III) transcribes a limited set of short genes in eukaryotes producing abundant small RNAs, mostly tRNA. The originally defined yeast Pol III transcriptome appears to be expanding owing to the application of new methods. Also, several factors required for assembly and nuclear import of Pol III complex have been identified recently. Models of Pol III based on cryo-electron microscopy reconstructions of distinct Pol III conformations reveal unique features distinguishing Pol III from other polymerases. Novel concepts concerning Pol III functioning involve recruitment of general Pol III-specific transcription factors and distinctive mechanisms of transcription initiation, elongation and termination. Despite the short length of Pol III transcription units, mapping of transcriptionally active Pol III with nucleotide resolution has revealed strikingly uneven polymerase distribution along all genes. This may be related, at least in part, to the transcription factors bound at the internal promoter regions. Pol III uses also a specific negative regulator, Maf1, which binds to polymerase under stress conditions; however, a subset of Pol III genes is not controlled by Maf1. Among other RNA polymerases, Pol III machinery represents unique features related to a short transcript length and high transcription efficiency.

  15. Transcription by RNA polymerase III: insights into mechanism and regulation

    PubMed Central

    Turowski, Tomasz W.; Tollervey, David

    2016-01-01

    The highly abundant, small stable RNAs that are synthesized by RNA polymerase III (RNAPIII) have key functional roles, particularly in the protein synthesis apparatus. Their expression is metabolically demanding, and is therefore coupled to changing demands for protein synthesis during cell growth and division. Here, we review the regulatory mechanisms that control the levels of RNAPIII transcripts and discuss their potential physiological relevance. Recent analyses have revealed differential regulation of tRNA expression at all steps on its biogenesis, with significant deregulation of mature tRNAs in cancer cells. PMID:27911719

  16. Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

    PubMed Central

    Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

    1992-01-01

    Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID

  17. Hepatic HNF1 transcription factors control the induction of PCSK9 mediated by rosuvastatin in normolipidemic hamsters.

    PubMed

    Dong, Bin; Singh, Amar Bahadur; Shende, Vikram Ravindra; Liu, Jingwen

    2017-03-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) impedes low‑density lipoprotein (LDL) receptor (LDLR)-mediated LDL-cholesterol uptake and has hence emerged as a critical regulator of serum cholesterol levels and a new therapeutic target for the treatment of hypercholesterolemia. Statins have been shown to elevate circulating PCSK9 levels by stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL‑cholesterol reduction. The transcription of PCSK9 is partially controlled by the hepatocyte nuclear factor 1 (HNF1) binding site embedded in the proximal region of its promoter. In this study, we utilized adenoviral shRNA delivery vectors to generate liver-specific knockdown of HNF1α (Ad‑shHNF1α) or HNF1β (Ad‑shHNF1β) in hamsters to examine the impact of reduced hepatic expression of HNF1 transcription factors on statin‑induced elevation of PCSK9 expression and serum cholesterol levels. We showed that the administration of rosuvastatin (RSV) to normolipidemic hamsters significantly augmented hepatic PCSK9 expression and serum PCSK9 levels. In addition, RSV treatment increased hepatic HNF1α protein levels without a clear effect on HNF1α mRNA expression. Injection of Ad-shHNF1α or Ad‑shHNF1β into hamsters both blunted RSV‑induced elevation of PCSK9 serum concentration and hepatic mRNA and protein levels, which led to significant increases in liver LDLR protein abundance. Furthermore, hepatic depletion of HNF1 factors lowered circulating total cholesterol and non‑high density lipoprotein cholesterol levels in RSV‑treated hamsters. Our study demonstrates that both HNF1α and HNF1β are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver-specific knockdown of either HNF1α or HNF1β could antagonize the RSV‑induced elevation of serum PCSK9 and reduce circulating cholesterol levels.

  18. Hepatic HNF1 transcription factors control the induction of PCSK9 mediated by rosuvastatin in normolipidemic hamsters.

    PubMed

    Dong, Bin; Singh, Amar Bahadur; Shende, Vikram Ravindra; Liu, Jingwen

    2017-02-06

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) impedes low‑density lipoprotein (LDL) receptor (LDLR)-mediated LDL-cholesterol uptake and has hence emerged as a critical regulator of serum cholesterol levels and a new therapeutic target for the treatment of hypercholesterolemia. Statins have been shown to elevate circulating PCSK9 levels by stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL‑cholesterol reduction. The transcription of PCSK9 is partially controlled by the hepatocyte nuclear factor 1 (HNF1) binding site embedded in the proximal region of its promoter. In this study, we utilized adenoviral shRNA delivery vectors to generate liver-specific knockdown of HNF1α (Ad‑shHNF1α) or HNF1β (Ad‑shHNF1β) in hamsters to examine the impact of reduced hepatic expression of HNF1 transcription factors on statin‑induced elevation of PCSK9 expression and serum cholesterol levels. We showed that the administration of rosuvastatin (RSV) to normolipidemic hamsters significantly augmented hepatic PCSK9 expression and serum PCSK9 levels. In addition, RSV treatment increased hepatic HNF1α protein levels without a clear effect on HNF1α mRNA expression. Injection of Ad-shHNF1α or Ad‑shHNF1β into hamsters both blunted RSV‑induced elevation of PCSK9 serum concentration and hepatic mRNA and protein levels, which led to significant increases in liver LDLR protein abundance. Furthermore, hepatic depletion of HNF1 factors lowered circulating total cholesterol and non‑high density lipoprotein cholesterol levels in RSV‑treated hamsters. Our study demonstrates that both HNF1α and HNF1β are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver-specific knockdown of either HNF1α or HNF1β could antagonize the RSV‑induced elevation of serum PCSK9 and reduce circulating cholesterol levels.

  19. The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I.

    PubMed

    Zhang, Yinfeng; French, Sarah L; Beyer, Ann L; Schneider, David A

    2016-02-05

    Although ribosomal RNA represents the majority of cellular RNA, and ribosome synthesis is closely connected to cell growth and proliferation rates, a complete understanding of the factors that influence transcription of ribosomal DNA is lacking. Here, we show that the THO complex positively affects transcription by RNA polymerase I (Pol I). We found that THO physically associates with the rDNA repeat and interacts genetically with Pol I transcription initiation factors. Pol I transcription in hpr1 or tho2 null mutants is dramatically reduced to less than 20% of the WT level. Pol I occupancy of the coding region of the rDNA in THO mutants is decreased to ~50% of WT level. Furthermore, although the percentage of active rDNA repeats remains unaffected in the mutant cells, the overall rDNA copy number increases ~2-fold compared with WT. Together, these data show that perturbation of THO function impairs transcription initiation and elongation by Pol I, identifying a new cellular target for the conserved THO complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Laser-mediated, site-specific inactivation of RNA transcripts

    PubMed Central

    Grate, Dilara; Wilson, Charles

    1999-01-01

    The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction. PMID:10339553

  1. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

    PubMed Central

    Nishimura, Yoshiki; Kikis, Elise A.; Zimmer, Sara L.; Komine, Yutaka; Stern, David B.

    2004-01-01

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5

  2. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    DTIC Science & Technology

    2016-05-26

    only allow entirely new investigations into the replication of these viruses , but also how this method can be applied to any virus with a known...resulted in the development of a variety of useful assays for virus detection. For hemorrhagic fever viruses with segmented genomes such as Crimean Congo...Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses

  3. Transcription termination by the eukaryotic RNA polymerase III

    PubMed Central

    Arimbasseri, Aneeshkumar G.; Rijal, Keshab; Maraia, Richard J.

    2012-01-01

    RNA polymerase (pol) III transcribes a multitude of tRNA and 5S rRNA genes as well as other small RNA genes distributed through the genome. By being sequence-specific, precise and efficient, transcription termination by pol III not only defines the 3′ end of the nascent RNA which directs subsequent association with the stabilizing La protein, it also prevents transcription into downstream DNA and promotes efficient recycling. Each of the RNA polymerases appears to have evolved unique mechanisms to initiate the process of termination in response to different types of termination signals. However, in eukaryotes much less is known about the final stage of termination, destabilization of the elongation complex with release of the RNA and DNA from the polymerase active center. By comparison to pols I & II, pol III exhibits the most direct coupling of the initial and final stages of termination, both of which occur at a short oligo(dT) tract on the non-template strand (dA on the template) of the DNA. While pol III termination is autonomous involving the core subunits C2 and probably C1, it also involves subunits C11, C37 and C53, which act on the pol III catalytic center and exhibit homology to the pol II elongation factor TFIIS, and TFIIFα/β respectively. Here we compile knowledge of pol III termination and associate mutations that affect this process with structural elements of the polymerase that illustrate the importance of C53/37 both at its docking site on the pol III lobe and in the active center. The models suggest that some of these features may apply to the other eukaryotic pols. PMID:23099421

  4. RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction.

    PubMed

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

    2017-05-18

    Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 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.

  6. Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA.

    PubMed

    Upadhyay, Alok; Dixit, Updesh; Manvar, Dinesh; Chaturvedi, Nootan; Pandey, Virendra N

    2013-06-01

    Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.

  7. Correlation between hepatitis B virus protein and microRNA processor Drosha in cells expressing HBV.

    PubMed

    Ren, Min; Qin, Dongdong; Li, Kai; Qu, Jialin; Wang, Liying; Wang, Zengchan; Huang, Ailong; Tang, Hua

    2012-06-01

    Drosha regulates the biogenesis of microRNAs (miRNAs) and plays an essential role in the regulation of gene expression. Infection with hepatitis B virus (HBV) causes chronic hepatitis and liver cirrhosis. It is also a major risk factor for hepatocellular carcinoma. Emerging evidence suggests that HBV alters miRNA expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. We therefore examined how HBV affected the production of miRNAs. We found that Drosha mRNA and protein expression were downregulated in cells expressing the HBV genome. This was associated with a reduction in the activity of the Drosha gene promoter. Gene silencing of HBx by RNA interference significantly restored the expression of Drosha. In conclusion, our data show that HBV could downregulate Drosha expression by inhibiting promoter activity, and the transcription factors SP1 and AP-2α may be involved in this process. This provides a new understanding of the mechanism of HBV-induced miRNAs dysregulation.

  8. Prevalence of hepatitis A viral RNA and antibodies among Chinese blood donors.

    PubMed

    Sun, P; Su, N; Lin, F Z; Ma, L; Wang, H J; Rong, X; Dai, Y D; Li, J; Jian, Z W; Tang, L H; Xiao, W; Li, C Q

    2015-12-09

    Like other developing countries, China was reported to have a relatively high seroprevalence of anti-hepatitis A antibodies (anti-HAV). However, no studies have evaluated the prevalence of anti-HAV and HAV RNA among voluntary blood donors with or without elevated serum alanine transaminase (ALT) levels. Anti-HAV antibodies were detected using an enzyme-linked immunosorbent assay, and reverse transcription quantitative polymerase chain reaction was carried out for detection of HAV RNA. In the current study, we analyzed a total of 450 serum samples with elevated ALT levels (≥40 U/L) and 278 serum samples with non-elevated ALT levels. Seroprevalence rates of anti-HAV were 51.6% in donors with elevated ALT and 41.4% in donors with non-elevated ALT; however, none of the samples was positive for HAV RNA. The results of our study showed lower seroprevalence rates of anti-HAV in blood donors (irrespective of ALT levels) than those in published data on Chinese populations. Although donors with elevated ALT had statistically higher prevalence rates of anti- HAV than did those with non-elevated ALT, none of the serum samples had detectable levels of the active virus. In conclusion, our results demonstrate that the transmission of hepatitis A by blood transfusion will occur rarely.

  9. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds

    PubMed Central

    Zalatan, Jesse G.; Lee, Michael E.; Almeida, Ricardo; Gilbert, Luke A.; Whitehead, Evan H.; La Russa, Marie; Tsai, Jordan C.; Weissman, Jonathan S.; Dueber, John E.; Qi, Lei S.; Lim, Wendell A.

    2014-01-01

    Summary Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multi-gene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications including rewiring cell fates or engineering metabolic pathways. PMID:25533786

  10. The RNA Epistructurome: Uncovering RNA Function by Studying Structure and Post-Transcriptional Modifications.

    PubMed

    Incarnato, Danny; Oliviero, Salvatore

    2017-04-01

    A large fraction of higher metazoan genomes transcribe RNA molecules whose functions extend far beyond carrying instructions for protein synthesis. Although RNA is apparently a simple molecule, the ways in which it performs many of its functions have remained highly elusive for decades. As learned from studying ribosomal and transfer RNAs, two of the key features influencing the function of RNA are its structure and post-transcriptional modifications. A deep understanding of RNA function therefore requires rapid and straightforward approaches to study the complex and intricate landscape of RNA structures and modifications. In this review we summarize and discuss the most recent methods and findings in the field of RNA biology, with an eye toward new frontiers and open questions.

  11. 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.

  12. 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

  13. Transcription, reverse transcription, and analysis of RNA containing artificial genetic components.

    PubMed

    Leal, Nicole A; Kim, Hyo-Joong; Hoshika, Shuichi; Kim, Myong-Jung; Carrigan, Matthew A; Benner, Steven A

    2015-04-17

    Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.

  14. Fast transcription rates of RNA polymerase II in human cells

    PubMed Central

    Maiuri, Paolo; Knezevich, Anna; De Marco, Alex; Mazza, Davide; Kula, Anna; McNally, Jim G; Marcello, Alessandro

    2011-01-01

    Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1-derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells. PMID:22015688

  15. Structural Basis of RNA Polymerase I Transcription Initiation.

    PubMed

    Engel, Christoph; Gubbey, Tobias; Neyer, Simon; Sainsbury, Sarah; Oberthuer, Christiane; Baejen, Carlo; Bernecky, Carrie; Cramer, Patrick

    2017-03-23

    Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

  16. 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

  17. Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription.

    PubMed

    Sutcliffe, J E; Brown, T R; Allison, S J; Scott, P H; White, R J

    2000-12-01

    The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

  18. Selective repression of SINE transcription by RNA polymerase III.

    PubMed

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J; Cairns, Bradley R; White, Robert J

    2015-01-01

    A million copies of the Alu short interspersed nuclear element (SINE) are scattered throughout the human genome, providing ∼11% of our total DNA. SINEs spread by retrotransposition, using a transcript generated by RNA polymerase (pol) III from an internal promoter. Levels of these pol III-dependent Alu transcripts are far lower than might be expected from the abundance of the template. This was believed to reflect transcriptional suppression through DNA methylation, denying pol III access to most SINEs through chromatin-mediated effects. Contrary to expectations, our recent study found no evidence that methylation of SINE DNA reduces its occupancy or expression by pol III. However, histone H3 associated with SINEs is prominently methylated on lysine 9, a mark that correlates with transcriptional silencing. The SUV39 methyltransferases that deposit this mark can be found at many SINEs. Furthermore, a selective inhibitor of SUV39 stimulates pol III recruitment to these loci, as well as SINE expression. These data suggest that methylation of histone H3 rather than DNA may mediate repression of SINE transcription by pol III, at least under the conditions we studied.

  19. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

    PubMed

    Takada, Hiraku; Shimada, Tomohiro; Dey, Debashish; Quyyum, M Zuhaib; Nakano, Masahiro; Ishiguro, Akira; Yoshida, Hideji; Yamamoto, Kaneyoshi; Sen, Ranjan; Ishihama, Akira

    2016-01-01

    Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed

  20. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli

    PubMed Central

    Takada, Hiraku; Shimada, Tomohiro; Dey, Debashish; Quyyum, M. Zuhaib; Nakano, Masahiro; Ishiguro, Akira; Yoshida, Hideji; Yamamoto, Kaneyoshi; Sen, Ranjan

    2016-01-01

    Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3’ proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed

  1. 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.

  2. The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

    PubMed

    Wittmann, Sina; Renner, Max; Watts, Beth R; Adams, Oliver; Huseyin, Miles; Baejen, Carlo; El Omari, Kamel; Kilchert, Cornelia; Heo, Dong-Hyuk; Kecman, Tea; Cramer, Patrick; Grimes, Jonathan M; Vasiljeva, Lidia

    2017-04-03

    Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.

  3. 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

  4. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase.

    PubMed

    Clark, Daniel N; Jones, Scott A; Hu, Jianming

    2017-01-01

    The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.

  5. In silico methods for co-transcriptional RNA secondary structure prediction and for investigating alternative RNA structure expression.

    PubMed

    Meyer, Irmtraud M

    2017-05-01

    RNA transcripts are the primary products of active genes in any living organism, including many viruses. Their cellular destiny not only depends on primary sequence signals, but can also be determined by RNA structure. Recent experimental evidence shows that many transcripts can be assigned more than a single functional RNA structure throughout their cellular life and that structure formation happens co-transcriptionally, i.e. as the transcript is synthesised in the cell. Moreover, functional RNA structures are not limited to non-coding transcripts, but can also feature in coding transcripts. The picture that now emerges is that RNA structures constitute an additional layer of information that can be encoded in any RNA transcript (and on top of other layers of information such as protein-context) in order to exert a wide range of functional roles. Moreover, different encoded RNA structures can be expressed at different stages of a transcript's life in order to alter the transcript's behaviour depending on its actual cellular context. Similar to the concept of alternative splicing for protein-coding genes, where a single transcript can yield different proteins depending on cellular context, it is thus appropriate to propose the notion of alternative RNA structure expression for any given transcript. This review introduces several computational strategies that my group developed to detect different aspects of RNA structure expression in vivo. Two aspects are of particular interest to us: (1) RNA secondary structure features that emerge during co-transcriptional folding and (2) functional RNA structure features that are expressed at different times of a transcript's life and potentially mutually exclusive. Copyright © 2017. Published by Elsevier Inc.

  6. De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

    PubMed Central

    Luo, Guangxiang; Hamatake, Robert K.; Mathis, Danielle M.; Racela, Jason; Rigat, Karen L.; Lemm, Julie; Colonno, Richard J.

    2000-01-01

    Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo. PMID:10623748

  7. 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.

  8. 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

  9. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

    PubMed Central

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2016-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

  10. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications.

    PubMed

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2015-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress toward understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation.

  11. Role of non-coding RNA transcription around gene regulatory elements in transcription factor recruitment

    PubMed Central

    Ohta, Kunihiro

    2017-01-01

    ABSTRACT Eukaryotic cells produce a variety of non-coding RNAs (ncRNAs), many of which have been shown to play pivotal roles in biological processes such as differentiation, maintenance of pluripotency of stem cells, and cellular response to various stresses. Genome-wide analyses have revealed that many ncRNAs are transcribed around regulatory DNA elements located proximal or distal to gene promoters, but their biological functions are largely unknown. Recently, it has been demonstrated in yeast and mouse that ncRNA transcription around gene promoters and enhancers facilitates DNA binding of transcription factors to their target sites. These results suggest universal roles of promoter/enhancer-associated ncRNAs in the recruitment of transcription factors to their binding sites. PMID:27763805

  12. RNA polymerase active center: the molecular engine of transcription.

    PubMed

    Nudler, Evgeny

    2009-01-01

    RNA polymerase (RNAP) is a complex molecular machine that governs gene expression and its regulation in all cellular organisms. To accomplish its function of accurately producing a full-length RNA copy of a gene, RNAP performs a plethora of chemical reactions and undergoes multiple conformational changes in response to cellular conditions. At the heart of this machine is the active center, the engine, which is composed of distinct fixed and moving parts that serve as the ultimate acceptor of regulatory signals and as the target of inhibitory drugs. Recent advances in the structural and biochemical characterization of RNAP explain the active center at the atomic level and enable new approaches to understanding the entire transcription mechanism, its exceptional fidelity and control.

  13. 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.

  14. Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

    PubMed

    Arndt, Karen M; Reines, Daniel

    2015-01-01

    The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.

  15. Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II

    PubMed Central

    Arndt, Karen M.; Reines, Daniel

    2016-01-01

    The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1–Nab3–Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes. PMID:25747400

  16. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis.

    PubMed

    Oh, Kyoung-Jin; Han, Hye-Sook; Kim, Min-Jung; Koo, Seung-Hoi

    2013-12-01

    Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed.

  18. The mRNA assembly line: transcription and processing machines in the same factory.

    PubMed

    Bentley, David

    2002-06-01

    Processing of RNA precursors to their mature form often occurs co-transcriptionally. Consequently, the ternary complex of DNA template, RNA polymerase and nascent RNA chain is the physiological substrate for factors that modify the nascent RNA by capping, splicing and cleavage/polyadenylation. mRNA production is thought to occur within a "factory" that contains the RNA polymerase II transcription machine and the processing machines. Newly discovered protein-protein contacts between RNA polymerase and factors that process mRNA precursors are beginning to illuminate how the "mRNA factory" works.

  19. Conformational changes accompany activation of reovirus RNA-dependent RNA transcription

    PubMed Central

    Mendez, Israel I.; Weiner, Scott G.; She, Yi-Min; Yeager, Mark; Coombs, Kevin M.

    2009-01-01

    Many critical biologic processes involve dynamic interactions between proteins and nucleic acids. Such dynamic processes are often difficult to delineate by conventional static methods. For example, while a variety of nucleic acid polymerase structures have been determined at atomic resolution, the details of how some multi-protein transcriptase complexes actively produce mRNA, as well as conformational changes associated with activation of such complexes, remain poorly understood. The mammalian reovirus innermost capsid (core) manifests all enzymatic activities necessary to produce mRNA from each of the 10 encased double-stranded RNA genes. We used rapid freezing and electron cryo-microscopy to trap and visualize transcriptionally active reovirus core particles and compared them to inactive core images. Rod-like density centered within actively transcribing core spike channels was attributed to exiting nascent mRNA. Comparative radial density plots of active and inactive core particles identified several structural changes in both internal and external regions of the icosahedral core capsid. Inactive and transcriptionally active cores were partially digested with trypsin and identities of initial tryptic peptides determined by mass spectrometry. Differentially-digested peptides, which also suggest transcription-associated conformational changes, were placed within the known 3-dimensional structures of major core proteins. PMID:18321727

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

    PubMed

    Zhang, Zhaojun; Wang, Wei

    2014-06-15

    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. 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 comparable or higher

  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. Copyright © 2015, American Association for the Advancement of Science.

  3. Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III Transcription

    PubMed Central

    Sutcliffe, Josephine E.; Brown, Timothy R. P.; Allison, Simon J.; Scott, Pamela H.; White, Robert J.

    2000-01-01

    The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression. PMID:11094071

  4. Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

    PubMed Central

    Wang, H W; Chen, P J; Lee, C Z; Wu, H L; Chen, D S

    1994-01-01

    Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging. Images PMID:8083975

  5. 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

  6. Quantification of co-transcriptional splicing from RNA-Seq data.

    PubMed

    Herzel, Lydia; Neugebauer, Karla M

    2015-09-01

    During gene expression, protein-coding transcripts are shaped by multiple processing events: 5' end capping, pre-mRNA splicing, RNA editing, and 3' end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3' end cleavage or post-transcriptionally. Pre-mRNA splicing - the removal of introns and ligation of exons - can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.

  7. Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing

    PubMed Central

    Arribere, Joshua A.; Gilbert, Wendy V.

    2013-01-01

    Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m7G-capped mRNA 5′ ends with high-throughput sequencing. TL-seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Surprisingly, TL-seq identified transcription initiation sites within 6% of protein-coding regions, and these sites were concentrated near the 5′ ends of ORFs. Furthermore, ribosome density analysis showed these truncated mRNAs are translated. Translation-associated TL-seq (TATL-seq), which combines TL-seq with polysome fractionation, enabled annotation of TLs, and simultaneously assayed their function in translation. Using TATL-seq to address relationships between TL features and translation of the downstream ORF, we observed that upstream AUGs (uAUGs), and no other upstream codons, were associated with poor translation and nonsense-mediated mRNA decay (NMD). We also identified hundreds of genes with very short TLs, and demonstrated that short TLs were associated with poor translation initiation at the annotated start codon and increased initiation at downstream AUGs. This frequently resulted in out-of-frame translation and subsequent termination at premature termination codons, culminating in NMD of the transcript. Unlike previous approaches, our technique enabled observation of alternative TL variants for hundreds of genes and revealed significant differences in translation in genes with distinct TL isoforms. TL-seq and TATL-seq are useful tools for annotation and functional characterization of TLs, and can be applied to any eukaryotic system to investigate TL-mediated regulation of gene expression. PMID:23580730

  8. Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome.

    PubMed

    Chong, Chun Kong; Cheng, Ching Yan Serene; Tsoi, Sin Yi Jasmine; Huang, Fung-Yu; Liu, Fen; Seto, Wai-Kay; Lai, Ching-Lung; Yuen, Man-Fung; Wong, Danny Ka-Ho

    2017-08-01

    The hepatitis B core protein (HBc) has been suggested to interact with covalently closed circular DNA (cccDNA) and regulate hepatitis B virus (HBV) transcription. However, direct evidence is lacking. We aimed to identify the specific HBc region(s) responsible for transcription regulation and its interaction with cccDNA. Seventeen mutants with mutations at the four arginine-rich clusters of the HBc carboxyl-terminal domain (CTD) were created. The effect of HBc mutations on the levels of HBV DNA, RNA, and hepatitis B surface antigen (HBsAg) were measured. The association of cccDNA with mutant HBc and histone acetyltransferases (HATs) was assessed by chromatin immunoprecipitation (ChIP). Compared with wild-type HBc, HBc mutants with mutations in clusters III and IV resulted in a significant reduction in HBV RNA levels (all P < 0.05). HBc arginine clusters III and IV mutants also had a significantly lower levels of intracellular HBV DNA (<5% of wild-type; P < 0.001) and HBsAg (<10% of wild-type; P < 0.0001). cccDNA-ChIP assay demonstrated that HBc clusters III and IV mutants had a smaller degree of association with cccDNA (P < 0.001). In the HBc mutants, the association between HATs with cccDNA were reduced. In conclusion, HBc-CTD arginine residues at clusters III and IV play an important role in the regulation of HBV transcription as well as subsequent replication steps, likely through the reduced interaction of HBc with cccDNA and reduced acetylation of cccDNA-bound histones. These findings may provide clues to the identification of novel therapeutic targets against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Vitamin C modulates cadmium-induced hepatic antioxidants' gene transcripts and toxicopathic changes in Nile tilapia, Oreochromis niloticus.

    PubMed

    El-Sayed, Yasser S; El-Gazzar, Ahmed M; El-Nahas, Abeer F; Ashry, Khaled M

    2016-01-01

    Cadmium (Cd) is one of the naturally occurring heavy metals having adverse effects, while vitamin C (L-ascorbic acid) is an essential micronutrient for fish, which can attenuate tissue damage owing to its chain-breaking antioxidant and free radical scavenger properties. The adult Nile tilapia fish were exposed to Cd at 5 mg/l with and without vitamin C (500 mg/kg diet) for 45 days in addition to negative and positive controls fed with the basal diet and basal diet supplemented with vitamin C, respectively. Hepatic relative mRNA expression of genes involved in antioxidant function, metallothionein (MT), glutathione S-transferase (GST-α1), and glutathione peroxidase (GPx1), was assessed using real-time reverse transcription polymerase chain reaction (RT-PCR). Hepatic architecture was also histopathologically examined. Tilapia exposed to Cd exhibited upregulated antioxidants' gene transcript levels, GST-⍺1, GPx1, and MT by 6.10-, 4.60-, and 4.29-fold, respectively. Histopathologically, Cd caused severe hepatic changes of multifocal hepatocellular and pancreatic acinar necrosis, and lytic hepatocytes infiltrated with eosinophilic granular cells. Co-treatment of Cd-exposed fish with vitamin C overexpressed antioxidant enzyme-related genes, GST-⍺1 (16.26-fold) and GPx1 (18.68-fold), and maintained the expression of MT gene close to control (1.07-fold), averting the toxicopathic lesions induced by Cd. These results suggested that vitamin C has the potential to protect Nile tilapia from Cd hepatotoxicity via sustaining hepatic antioxidants' genes transcripts and normal histoarchitecture.

  10. Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation.

    PubMed

    Lahiry, Ashwin; Stimple, Samuel D; Wood, David W; Lease, Richard A

    2017-01-24

    Multitargeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multitargeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes. Accordingly, we began with the well-characterized E. coli native DsrA sRNA. The merits of using DsrA include its well-characterized separation of function into two independently folded stem-loop domains, wherein alterations at one stem do not necessarily abolish activity at the other stem. Expression of the sRNA and each reporter mRNA was independently controlled by small inducer molecules, allowing precise quantification of the regulatory effects of each sRNA:mRNA interaction in vivo with a microtiter plate assay. Using this system, we semirationally designed DsrA variants screened in E. coli for their ability to regulate key mRNA leader sequences from the Clostridium acetobutylicum n-butanol synthesis pathway. To coordinate intervention at two points in a metabolic pathway, we created bifunctional sRNA prototypes by combining sequences from two singly retargeted DsrA variants. This approach constitutes a platform for designing sRNAs to specifically target arbitrary mRNA transcript sequences, and thus provides a generalizable tool for retargeting and characterizing multitarget sRNAs for metabolic engineering.

  11. 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.

  12. 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

  13. Ontogeny of hepatic energy metabolism genes in mice as revealed by RNA-sequencing.

    PubMed

    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. These

  14. New therapeutic opportunities for Hepatitis C based on small RNA

    PubMed Central

    Pan, Qiu-Wei; Henry, Scot D; Scholte, Bob J; Tilanus, Hugo W; Janssen, Harry LA; van der Laan, Luc JW

    2007-01-01

    Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease, including cirrhosis and liver cancer and is therefore, the most common indication for liver transplantation. Conventional antiviral drugs such as pegylated interferon-alpha, taken in combination with ribavirin, represent a milestone in the therapy of this disease. However, due to different viral and host factors, clinical success can be achieved only in approximately half of patients, making urgent the requirement of exploiting alternative approaches for HCV therapy. Fortunately, recent advances in the understanding of HCV viral replication and host cell interactions have opened new possibilities for therapeutic intervention. The most recent technologies, such as small interference RNA mediated gene-silencing, anti-sense oligonucleotides (ASO), or viral vector based gene delivery systems, have paved the way to develop novel therapeutic modalities for HCV. In this review, we outline the application of these technologies in the context of HCV therapy. In particular, we will focus on the newly defined role of cellular microRNA (miR-122) in viral replication and discuss its potential for HCV molecular therapy. PMID:17724797

  15. MicroRNA-34a Promotes Hepatic Stellate Cell Activation via Targeting ACSL1

    PubMed Central

    Yan, Gangli; Li, Binbin; Xin, Xuan; Xu, Midie; Ji, Guoqing; Yu, Hongyu

    2015-01-01

    Background The incidence of liver fibrosis remains high due to the lack of effective therapies. Our previous work found that microRNA (miR)-34a expression was increased, while acy1-CoA synthetase long-chain family member1 (ACSL1) was decreased, in a dimethylnitrosamine (DNS)-induced hepatic fibrosis rat model. We hypothesized that miR-34a may play a role in the process of hepatic fibrosis by targeting ACSL1. Material/Methods From days 2 to 14, cultured primary hepatic stellate cells (HSCs) underwent cell morphology, immunocytochemical staining, and quantitative reverse transcription PCR (RT-qPCR) for alpha smooth muscle actin (α-SMA), desmin, rno-miR-34a, and ACSL1 expression. Wild-type and mutant luciferase reporter plasmids were constructed according to the predicted miR-34a binding site on the 3′-untranslated region (UTR) of the ACSL1 mRNA and then transfected into HEK293 cells. rno-miR-34a was silenced in HSCs to confirm that rno-miR-34a negatively regulates ACSL1 expression. mRNA and protein expression of α-SMA, type I collagen, and desmin were assayed in miR-34a-silenced HSCs. Results HSCs were deemed quiescent during the first 3 days and activated after 10 days. rno-miR-34a expression increased, and ACSL1 expression decreased, from day 2 to 7 to 14. rno-miR-34a was shown to specifically bind to the 3′-UTR of ACSL1. miR-34a-silenced HSCs showed higher ACSL1and lower α-SMA, type I collagen, and desmin expression than that of matching negative controls and non-transfected cells. Conclusions miR-34a appears to play an important role in the process of liver fibrosis by targeting ACSL1 and may show promise as a therapeutic molecular target for hepatic fibrosis. PMID:26437572

  16. Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines.

    PubMed Central

    Raney, A K; Milich, D R; McLachlan, A

    1991-01-01

    A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene. PMID:1651407

  17. 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

  18. Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II.

    PubMed

    Cline, Susan D; Riggins, James N; Tornaletti, Silvia; Marnett, Lawrence J; Hanawalt, Philip C

    2004-05-11

    Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

  19. 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.

  20. Chromatin Remodeling around Nucleosome-Free Regions Leads to Repression of Noncoding RNA Transcription

    PubMed Central

    Yadon, Adam N.; Van de Mark, Daniel; Basom, Ryan; Delrow, Jeffrey; Whitehouse, Iestyn; Tsukiyama, Toshio

    2010-01-01

    Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of transcription initiation for mRNA and noncoding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest that the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5′ and 3′ ends of genes, within open reading frames (ORFs), and far from ORFs. The ATP-dependent chromatin-remodeling enzyme Isw2 was found enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus, by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we established, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting that one biological consequence of repression of ncRNA, by Isw2 or by the exosome, is prevention of transcriptional interference of mRNA. PMID:20805356

  1. 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.

  2. 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

  3. Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes.

    PubMed

    Cook, Linda; Sullivan, KaWing; Krantz, Elizabeth M; Bagabag, Arthur; Jerome, Keith R

    2006-11-01

    A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

  4. TATA elements direct bi-directional transcription by RNA polymerases II and III.

    PubMed Central

    Huang, W; Wong, J M; Bateman, E

    1996-01-01

    Eukaryotic promoter elements specify the direction and efficiency of transcription, as well as the type of RNA polymerase to be used. One such element, the TATA box, is thought to participate in determining the direction of transcription and can function within promoters for RNA polymerase II or III, depending on the sequence context. In this report the ability of four different TATA boxes to support transcription in vitro was determined. It was found that TATA elements are not directional. However, they support transcription by RNA polymerases II and III. An upstream activating sequence was found to stimulate downstream transcription by RNA polymerase II and to inhibit upstream transcription by RNA polymerases II and III. Thus a promoter necessarily consists of a TATA element and upstream sequences in order to specify the direction of transcription and the type of polymerase to be used. PMID:8604352

  5. Species-specificity of rRNA gene transcription in plants manifested as a switch in RNA polymerase specificity.

    PubMed Central

    Doelling, J H; Pikaard, C S

    1996-01-01

    Rapid evolution of ribosomal RNA (rRNA) gene promoters often prevents their recognition in a foreign species. Unlike animal systems, we show that foreign plant rRNA gene promoters are recognized in an alien species, but tend to program transcription by a different polymerase. In plants, RNA polymerase I transcripts initiate at a TATATA element (+1 is underlined) important for promoter strength and start-site selection. However, transcripts initiate from +32 following transfection of a tomato promoter into Arabidopsis. The rRNA gene promoter of a more closely related species, Brassica oleracea, programs both +1 and +29 transcription. A point mutation at +2 improving the identity between the Brassica and Arabidopsis promoters increases +1 transcription, indicating a role for the initiator element in species-specificity. Brassica +29 transcripts can be translated to express a luciferase reporter gene, implicating RNA polymerase II. TATA mutations that disrupt TATA-binding protein (TBP) interactions inhibit +29 transcription and luciferase expression. Co-expressed TBP proteins bearing compensatory mutations restore +29 transcription and luciferase activity, suggesting a direct TBP-TATA interaction. Importantly, +1 transcription is unaffected by the TATA mutations, suggesting that in the context of pol I recognition, the TATA-containing initiator element serves a function other than TBP binding. PMID:8972859

  6. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

    PubMed Central

    Schneider, David Alan

    2016-01-01

    The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come. PMID:22113301

  7. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.

    PubMed

    Sydow, Jasmin F; Brueckner, Florian; Cheung, Alan C M; Damsma, Gerke E; Dengl, Stefan; Lehmann, Elisabeth; Vassylyev, Dmitry; Cramer, Patrick

    2009-06-26

    We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.

  8. Integrated analysis of microRNA and mRNA expression profiles in HBx-expressing hepatic cells

    PubMed Central

    Chen, Ruo-Chan; Wang, Juan; Kuang, Xu-Yuan; Peng, Fang; Fu, Yong-Ming; Huang, Yan; Li, Ning; Fan, Xue-Gong

    2017-01-01

    AIM To identify the miRNA-mRNA regulatory network in hepatitis B virus X (HBx)-expressing hepatic cells. METHODS A stable HBx-expressing human liver cell line L02 was established. The mRNA and miRNA expression profiles of L02/HBx and L02/pcDNA liver cells were identified by RNA-sequencing analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to investigate the function of candidate biomarkers, and the relationship between miRNA and mRNA was studied by network analysis. RESULTS Compared with L02/pcDNA cells, 742 unregulated genes and 501 downregulated genes were determined as differentially expressed in L02/HBx cells. Gene ontology analysis suggested that the differentially expressed genes were relevant to different biological processes. Concurrently, 22 differential miRNAs were also determined in L02/HBx cells. Furthermore, integrated analysis of miRNA and mRNA expression profiles identified a core miRNA-mRNA regulatory network that is correlated with the carcinogenic role of HBx. CONCLUSION Collectively, the miRNA-mRNA network-based analysis could be useful to elucidate the potential role of HBx in liver cell malignant transformation and shed light on the underlying molecular mechanism and novel therapy targets for hepatocellular carcinoma. PMID:28348484

  9. Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

    PubMed

    Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

    2014-07-01

    Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants.

  10. The plant RNA polymerase II elongation complex: a hub coordinating transcript elongation and mRNA processing.

    PubMed

    Grasser, Marion; Grasser, Klaus D

    2017-09-08

    Characterisation of the Arabidopsis RNA polymerase II (RNAPII) elongation complex revealed an assembly of a conserved set of transcript elongation factors associated with chromatin remodellers, histone modifiers as well as with various pre-mRNA splicing and polyadenylation factors. Therefore, transcribing RNAPII streamlines the processes of mRNA synthesis and processing in plants.

  11. Multihormonal induction of hepatic α2u-globulin mRNA as measured by hybridization to complementary DNA

    PubMed Central

    Kurtz, David T.; Feigelson, Philip

    1977-01-01

    A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u-globulin, which binds to the nascent α2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis. PMID:73184

  12. 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

  13. 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.

  14. Clearance of HCV RNA following acute hepatitis A superinfection.

    PubMed

    Cacopardo, B; Nunnari, G; Nigro, L

    2009-05-01

    A transient reduction of hepatitis C virus replication during the course of acute hepatitis A virus infection has already been reported in the literature. The present study reports the case study of a subject with chronic hepatitis due to hepatitis C virus who went on to develop an acute hepatitis A. From the early onset of acute disease, hepatitis C virus ribonucleic acid became undetectable. Following recovery from acute hepatitis, alanine amino-transferase levels became persistently normal and liver biopsy revealed a reduction in the Knodell histological activity index score. Hepatitis C virus ribonucleic acid clearance was maintained up to 4 years after the onset of acute hepatitis A. During the course of the acute disease, a sharp increase in interferon gamma levels was detected in serum and in the supernatant of both unstimulated and phytoemagglutinin/lipopolysaccharide-stimulated peripheral blood mononuclear cells. Interferon gamma levels were still high 3 months later. We hypothesize that acute hepatitis A virus superinfection during the course of chronic hepatitis C may lead to hepatitis C virus ribonucleic acid clearance through an immunological mechanism related to interferon gamma production.

  15. Multicenter Quality Control for the Detection of Hepatitis C Virus RNA in Seminal Plasma Specimens

    PubMed Central

    Bourlet, Thomas; Levy, Rachel; Laporte, Silvy; Blachier, Stéphane; Bocket, Laurence; Cassuto, Guy; Chollet, Lionel; Leruez-Ville, Marianne; Maertens, Anne; Mousnier, Fabienne; Pasquier, Christophe; Payan, Christopher; Pellegrin, Bertrand; Schvoerer, Evelyne; Zavadzki, Patricia; Chouteau, Jacques; Duverlie, Gilles; Izopet, Jacques; Lunel-Fabiani, Françoise; Pawlotsky, Jean-Michel; Profizi, Nerina; Rouzioux, Christine; Stoll-Keller, Françoise; Thibault, Vincent; Wattré, Pierre; Pozzetto, Bruno

    2003-01-01

    The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20,000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction. PMID:12574284

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

    PubMed

    Braun, Katherine A; Dombek, Kenneth M; Young, Elton T

    2015-12-14

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. 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

  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. Structural basis of initial RNA polymerase II transcription.

    PubMed

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

    2011-11-04

    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.

  20. Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana.

    PubMed

    Fulnecek, Jaroslav; Kovarik, Ales

    2007-11-01

    In plants, 5S rRNA genes (5S rDNA) encoding 120-nt structural RNA molecules of ribosomes are organized in tandem arrays comprising thousands of units. Failure to correctly terminate transcription would generate longer inaccurately processed transcripts interfering with ribosome biogenesis. Hence multiple termination signals occur immediately after the 5S rRNA coding sequence. To obtain information about the efficiency of termination of 5S rDNA transcription in plants we analyzed 5S rRNA pools in three Nicotiana species, N. sylvestris, N. tomentosiformis and N. tabacum. In addition to highly abundant 120-nt 5S rRNA transcripts, we also detected RNA species composed of a genic region and variable lengths of intergenic sequences. These genic-intergenic RNA molecules occur at a frequency severalfold lower than the mature 120-nt transcripts, and are posttranscriptionally modified by polyadenylation at their 3' end in contrast to 120-nt transcripts. An absence of 5S small RNAs (smRNA) argue against a dominant role for the smRNA biosynthesis pathway in the degradation of aberrant 5S rRNA in Nicotiana. This work is the first description of polyadenylated 5S rRNA species in higher eukaryotes originating from a read-through transcription into the intergenic spacer. We propose that polyadenylation may function in a "quality control" pathway ensuring that only correctly processed molecules enter the ribosome biogenesis.

  1. Identification and characterization of multiple TRIM proteins that inhibit hepatitis B virus transcription.

    PubMed

    Zhang, Shijian; Guo, Ju-Tao; Wu, Jim Z; Yang, Guang

    2013-01-01

    Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.

  2. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

    PubMed Central

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-01-01

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. PMID:25588787

  3. Mapping Transcription Regulatory Networks with ChIP-seq and RNA-seq.

    PubMed

    Wade, Joseph T

    2015-01-01

    Bacterial genomes encode numerous transcription factors, DNA-binding proteins that regulate transcription initiation. Identifying the regulatory targets of transcription factors is a major challenge of systems biology. Here I describe the use of two genome-scale approaches, ChIP-seq and RNA-seq, that are used to map transcription factor regulons. ChIP-seq maps the association of transcription factors with DNA, and RNA-seq determines changes in RNA levels associated with transcription factor perturbation. I discuss the strengths and weaknesses of these and related approaches, and I describe how ChIP-seq and RNA-seq can be combined to map individual transcription factor regulons and entire regulatory networks.

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

    PubMed Central

    Beckel-Mitchener, A; Summers, J

    1997-01-01

    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

  5. 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

  6. 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

  7. 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

  8. Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

    NASA Astrophysics Data System (ADS)

    Fujita, Keisuke; Iwaki, Mitsuhiro; Yanagida, Toshio

    2016-12-01

    Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

  9. Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

    PubMed Central

    Fujita, Keisuke; Iwaki, Mitsuhiro; Yanagida, Toshio

    2016-01-01

    Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting. PMID:27924870

  10. Identification and characterization of lncRNA mediated transcriptional dysregulation dictates lncRNA roles in glioblastoma

    PubMed Central

    Zhao, Zheng; Zhang, Jinwen; Lu, Jianping; Xu, Juan; Li, Xia

    2016-01-01

    Long non-coding RNAs (lncRNAs) modulate gene expression, and lncRNA misregulation is associated with cancer. However, precise functional roles in biological and disease processes have been described for only a few lncRNAs. Identification of genome-wide lncRNA-mediated transcriptional dysregulations may improve cancer treatments. In the present study, we used a computational framework that combined lncRNA and gene expression profiles with transcription factor (TF)-target relationships to comprehensively identify dysregulatory lncRNA-TF-gene triplets. In glioblastoma (GBM), we found that most lncRNAs affect multiple targets and primarily affect TF activity in trans. Six different classes of lncRNA-mediated transcriptional dysregulations were identified, with most lncRNAs either enhancing or attenuating target gene expression. Functional analysis of lncRNAs via their dysregulated targets implicated lncRNA modulators in some hallmarks of cancer, providing a new way to predict lncRNA function. Finally, we identified several lncRNA-TF-gene triplets (including HOTAIR-MXI1-CD58/PRKCE and HOTAIR-ATF5-NCAM1) that are associated with glioblastoma prognosis. The integration of lncRNA modulators into transcriptional regulatory networks will further enhance our understanding of lncRNA functions in cancer. PMID:26943771

  11. Conditional knockdown of target gene expression by tetracycline regulated transcription of double strand RNA.

    PubMed

    Hou, Xubin; Omi, Minoru; Harada, Hidekiyo; Ishii, Shunsuke; Takahashi, Yoshiko; Nakamura, Harukazu

    2011-01-01

    In vivo electroporation has served as an effective tool for the study of developmental biology. Here we report tetracycline inducible gene knockdown by electroporation. Our system consists of genome integration of a cassette encoding long double strand RNA (dsRNA) of a gene of interest by electroporation, transcription of which is assured by RNA polymerase II, and induction of transcription of dsRNA by tetracyclin. Long dsRNA decapped by ribozyme in the cassette and without poly A tail is processed into siRNA within nuclei. We could successfully induce knockdown of En2 and Coactosin by Dox administration.

  12. Transcriptional Elongation Control of Hepatitis B Virus Covalently Closed Circular DNA Transcription by Super Elongation Complex and BRD4.

    PubMed

    Francisco, Joel Celio; Dai, Qian; Luo, Zhuojuan; Wang, Yan; Chong, Roxanne Hui-Heng; Tan, Yee Joo; Xie, Wei; Lee, Guan-Huei; Lin, Chengqi

    2017-10-01

    Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. HBV reactivation during or after chemotherapy is a potentially fatal complication for cancer patients with chronic HBV infection. Transcription of HBV is a critical intermediate step of the HBV life cycle. However, factors controlling HBV transcription remain largely unknown. Here, we found that different P-TEFb complexes are involved in the transcription of the HBV viral genome. Both BRD4 and the super elongation complex (SEC) bind to the HBV genome. The treatment of bromodomain inhibitor JQ1 stimulates HBV transcription and increases the occupancy of BRD4 on the HBV genome, suggesting the bromodomain-independent recruitment of BRD4 to the HBV genome. JQ1 also leads to the increased binding of SEC to the HBV genome, and SEC is required for JQ1-induced HBV transcription. These findings reveal a novel mechanism by which the HBV genome hijacks the host P-TEFb-containing complexes to promote its own transcription. Our findings also point out an important clinical implication, that is, the potential risk of HBV reactivation during therapy with a BRD4 inhibitor, such as JQ1 or its analogues, which are a potential treatment for acute myeloid leukemia. Copyright © 2017 American Society for Microbiology.

  13. Ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus

    SciTech Connect

    Branch, A.D.; Benenfeld, B.J.; Baroudy, B.M.; Wells, F.V.; Gerin, J.L.; Robertson, H.D.

    1989-02-03

    The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.

  14. Structural Organization of Pregenomic RNA and the Carboxy-Terminal Domain of the Capsid Protein of Hepatitis B Virus

    PubMed Central

    Wang, Joseph C.-Y.; Dhason, Mary S.; Zlotnick, Adam

    2012-01-01

    The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of in vitro assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone. PMID:23028319

  15. JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

    PubMed Central

    Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

    2017-01-01

    The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule

  16. An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.

    PubMed

    Chen, Jieliang; Zhang, Wen; Lin, Junyu; Wang, Fan; Wu, Min; Chen, Cuncun; Zheng, Ye; Peng, Xiuhua; Li, Jianhua; Yuan, Zhenghong

    2014-02-01

    The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which are present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg, and pgRNA, resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.

  17. Ribonuclease T1 generates circular RNA molecules from viroid-specific RNA transcripts by cleavage and intramolecular ligation.

    PubMed Central

    Tsagris, M; Tabler, M; Sänger, H L

    1991-01-01

    A 406 nucleotide long potato spindle tuber viroid (PSTVd)-specific linear RNA transcript was synthesized in vitro and subjected to limited digestion with ribonuclease (RNase) T1. Under certain conditions this guanosine-specific endoribonuclease proved to be capable of processing the longer-than-unit-length, precursor-like viroid RNA transcript by cleaving out a linear 358 nucleotide long product and ligating that to a circular RNA molecule. The new finding that RNase T1 acts as an RNA processing enzyme and, in particular, as an RNA 'circulase' can be explained by the unique structural preconditions inherent in the viroid-specific substrate and by the well characterized two-step cleavage mechanism of the enzyme. These in vitro potentials of RNase T1 suggest that also in vivo procaryotic and eucaryotic RNases with a similar reaction mechanism might not only be involved in RNA degradation and trimming, but also in processing, ligation and recombination of RNA. Images PMID:1709278

  18. 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

  19. Suppression of long chain acyl-CoA synthetase 3 decreases hepatic de novo fatty acid synthesis through decreased transcriptional activity.

    PubMed

    Bu, So Young; Mashek, Mara T; Mashek, Douglas G

    2009-10-30

    Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-gamma, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-alpha and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-(14)C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis.

  20. 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.

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

    PubMed Central

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

    2016-01-01

    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 find 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 due 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, provides a framework with which evolution shapes transcriptomes. PMID:27455346

  2. The RNA polymerase trigger loop functions in all three phases of the transcription cycle

    PubMed Central

    Fouqueau, Thomas; Zeller, Mirijam E.; Cheung, Alan C.; Cramer, Patrick; Thomm, Michael

    2013-01-01

    The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites. PMID:23737452

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

    PubMed

    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.

  4. 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

  5. Landscape of post-transcriptional gene regulation during hepatitis C virus infection

    PubMed Central

    Schwerk, Johannes; Jarret, Abigail P.; Joslyn, Rochelle C.; Savan, Ram

    2015-01-01

    Post-transcriptional regulation of gene expression plays a pivotal role in various gene regulatory networks including, but not limited to metabolism, embryogenesis and immune responses. Different mechanisms of post-transcriptional regulation, which can act individually, synergistically, or even in an antagonistic manner have been described. Hepatitis C virus (HCV) is notorious for subverting host immune responses and indeed exploits several components of the host’s post-transcriptional regulatory machinery for its own benefit. At the same time, HCV replication is post-transcriptionally targeted by host cell components to blunt viral propagation. This review discusses the interplay of post-transcriptional mechanisms that affect host immune responses in the setting of HCV infection and highlights the sophisticated mechanisms both host and virus have evolved in the race for superiority. PMID:25890065

  6. 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.

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

    PubMed

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

    2015-08-12

    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.

  8. A novel conformation of RNA polymerase sheds light on the mechanism of transcription

    PubMed Central

    Tagami, Shunsuke; Sekine, Shun-ichi

    2011-01-01

    Transcription is a complicated, multistep process requiring stringent control. Its accuracy may be achieved in part by the conformational changes of RNA polymerase (RNAP). Here, we discuss the functional relevance of the recently reported conformational changes of RNAP, which may affect transcription control, RNAP translocation and transcription termination. PMID:21922057

  9. A new way to start: nanoRNA-mediated priming of transcription initiation.

    PubMed

    Nickels, Bryce E

    2012-01-01

    A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.

  10. Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

    PubMed Central

    Sharrock, R A; Gourse, R L; Nomura, M

    1985-01-01

    The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage lambda. By analyzing pulse-labeled RNA with an RNA.DNA filter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoter-proximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage lambda system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears that E. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons. PMID:3161080

  11. Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo

    PubMed Central

    Hoch, Philipp G.; Schlereth, Julia; Lechner, Marcus; Hartmann, Roland K.

    2016-01-01

    The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA. PMID:26873600

  12. Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo.

    PubMed

    Hoch, Philipp G; Schlereth, Julia; Lechner, Marcus; Hartmann, Roland K

    2016-04-01

    The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA.

  13. Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription.

    PubMed

    Surratt, C K; Milan, S C; Chamberlin, M J

    1991-09-15

    Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.

  14. 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.

  15. Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing

    PubMed Central

    Mayer, Andreas; Churchman, L. Stirling

    2017-01-01

    Many features of gene transcription in human cells remain unclear, mainly due to a lack of quantitative approaches to follow genome transcription with nucleotide precision in vivo. Here we present a robust genome-wide approach to study RNA polymerase (Pol) II-mediated transcription in human cells at single-nucleotide resolution by native elongating transcript sequencing (NET-seq). Elongating RNA polymerase and the associated nascent RNA is prepared by cell fractionation, avoiding immunoprecipitation or RNA labeling. The 3′-ends of nascent RNAs are captured through barcode linker ligation and converted into a DNA sequencing library. The identity and abundance of the 3′-ends are determined by high-throughput sequencing, revealing the exact genomic locations of Pol II. Human NET-seq can be applied to study the full spectrum of Pol II transcriptional activities, including the production of unstable RNAs and transcriptional pausing. Using the protocol described here, a NET-seq library can be obtained from human cells in 5 days. PMID:27010758

  16. Role of Hepatic-Specific Transcription Factors and Polycomb Repressive Complex 2 during Induction of Fibroblasts to Hepatic Fate

    PubMed Central

    Wee, Ping; Yaqubi, Moein

    2016-01-01

    Direct reprogramming using defined sets of transcription factors (TFs) is a recent strategy for generating induced hepatocytes (iHeps) from fibroblasts for use in regenerative medicine and drug development. Comprehensive studies detailing the regulatory role of TFs during this reprogramming process could help increase its efficiency. This study aimed to find the TFs with the greatest influences on the generation of iHeps from fibroblasts, and to further understand their roles in the regulation of the gene expression program. Here, we used systems biology approaches to analyze high quality expression data sets in combination with TF-binding sites data and protein-protein interactions data during the direct reprogramming of fibroblasts to iHeps. Our results revealed two main patterns for differentially expressed genes (DEGs): up-regulated genes were categorized as hepatic-specific pattern, and down-regulated genes were categorized as mesoderm- and fibroblast-specific pattern. Interestingly, hepatic-specific genes co-expressed and were regulated by hepatic-specific TFs, specifically Hnf4a and Foxa2. Conversely, the mesoderm- and fibroblast-specific pattern was mainly silenced by polycomb repressive complex 2 (PRC2) members, including Suz12, Mtf2, Ezh2, and Jarid2. Independent analysis of both the gene and core regulatory network of DE-TFs showed significant roles for Hnf4a, Foxa2, and PRC2 members in the regulation of the gene expression program and in biological processes during the direct conversion process. Altogether, using systems biology approaches, we clarified the role of Hnf4a and Foxa2 as hepatic-specific TFs, and for the first time, introduced the PRC2 complex as the main regulator that favors the direct reprogramming process in cooperation with hepatic-specific factors. PMID:27902735

  17. A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells

    PubMed Central

    Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V.

    2013-01-01

    PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs. PMID:23435381

  18. Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 micron DNA.

    PubMed Central

    Ballario, P; Buongiorno-Nardelli, M; Carnevali, F; Di Mauro, E; Pedone, F

    1981-01-01

    The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 micron sequences (BTYP 1, BTYH 2 and BTYH 3). Conditions for selective transcription of the 2 micron DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains. The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis. All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported. Images PMID:7029462

  19. The structure of RNA-free Rho termination factor indicates a dynamic mechanism of transcript capture.

    PubMed

    Canals, Albert; Usón, Isabel; Coll, Miquel

    2010-07-02

    The Rho factor is a ring-shaped ATP-dependent helicase that mediates transcription termination in most prokaryotic cells by disengaging the transcription elongation complex formed by the RNA polymerase, DNA, and the nascent RNA transcript. The crystal structures of key intermediates along the kinetic pathway of RNA binding to Rho unveiled an unprecedented mode of helicase loading and provided a model for the ATP turnover coupled to coordinated strand movement. Here we report the structure of the early RNA-free state of Rho, which had eluded crystallization for many years but now completes the series. The structure allows the characterization of the apo-form Rho from Thermotoga maritima to 2.3 A resolution, reveals an RNA-recruiting site that becomes hidden after occupancy of the adjacent specific primary RNA-binding site, and suggests an enriched model for mRNA capture that is consistent with previous data.

  20. MORPHEUS' MOLECULE1 is required to prevent aberrant RNA transcriptional read-through in Arabidopsis.

    PubMed

    Zhou, Yue; Zhang, Jun; Lin, Huixin; Guo, Guangqin; Guo, Yan

    2010-11-01

    Several pathways function to remove aberrant mRNA in eukaryotic cells; however, the exact mechanisms underlying the restriction of aberrant mRNA transcription are poorly understood. In this study, we found that MORPHEUS' MOLECULE1 (MOM1) is a key component of this regulatory machinery. The Arabidopsis (Arabidopsis thaliana) mom1-44 mutation was identified by luciferase imaging in transgenic plants harboring a cauliflower mosaic virus 35S promoter-LUCIFERASE transgene lacking the 3'-untranslated region. In the mom1-44 mutant, transcriptional read-though occurred in genes with an aberrant RNA structure. Analysis of an RNA-dependent RNA polymerase2 mom1 double mutant revealed that the RNA-directed DNA methylation pathway is not involved in this regulatory process. Moreover, the prevention of aberrant mRNA transcriptional read-through by MOM1 is gene locus and transgene copy number independent.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  2. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus.

    PubMed

    Miller, Eric

    2016-01-01

    Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5' and 3' untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts.

  3. Genome-wide annotation of microRNA primary transcript structures reveals novel regulatory mechanisms

    PubMed Central

    Chang, Tsung-Cheng; Pertea, Mihaela; Lee, Sungyul; Salzberg, Steven L.; Mendell, Joshua T.

    2015-01-01

    Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation. PMID:26290535

  4. Beneficial effect of berberine on hepatic insulin resistance in diabetic hamsters possibly involves in SREBPs, LXRα and PPARα transcriptional programs.

    PubMed

    Liu, Xuhan; Li, Guosheng; Zhu, Hua; Huang, Lan; Liu, Yali; Ma, Chunmei; Qin, Chuan

    2010-01-01

    The "lipotoxicity" hypothesis holds that fat-induced hepatic insulin resistance (FIHIR) may play a major role in the pathogenesis of type 2 diabetes. Berberine has been reported to have antidiabetic properties. However, the molecular mechanisms for this action are not fully clarified. Therefore, we will investigate the gene expression alterations involved in the therapeutic effect of berberine on FIHIR in diabetic hamsters and possible mechanisms. In this study, type 2 diabetic hamsters were induced by high-fat diet with streptozotocin injection. After 9 weeks of berberine-treatment, the gene expression alterations involved in the therapeutic molecular mechanisms of berberine on FIHIR will be studied by microarray technology and real time RT-PCR. Our study demonstrates berberine significantly improved fat-induced insulin resistance and diabetic phenotype in type 2 diabetic hamsters. The alterations of certain metabolism related genes and their main regulators: Liver X receptor (LXR) α, Peroxisome proliferator-activated receptor (PPAR) α and Sterol regulatory element-binding protein (SREBPs) are observed in the liver of treated and untreated diabetic hamsters. Compared with diabetic hamsters, the increased mRNA levels of LXRα and PPARα and the decreased mRNA levels of SREBPs are observed in berberine-treated diabetic hamster. The statistical significance of the expression of hepatic LXRα, SREBPs and PPARα and their certain target genes is found between treated and untreated diabetic hamsters. These results suggest that altered hepatic SREBPs, LXRα and PPARα transcriptional programs possibly involve in the therapeutic mechanisms of berberine on FIHIR in type 2 diabetic hamsters.

  5. A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes

    PubMed Central

    Bradrick, Shelton S.; Nagyal, Simardeep; Novatt, Hilary

    2013-01-01

    Micro(mi)RNAs are 21- to 23-nt RNAs that regulate multiple biological processes. In association with Argonaute (Ago) proteins and other factors that form the RNA-induced silencing complex (RISC), miRNAs typically bind mRNA 3′ untranslated regions (UTRs) and repress protein production through antagonizing translation and transcript stability. For a given mRNA–miRNA interaction, cis-acting RNA elements and trans-acting RNA-binding proteins (RBPs) may influence mRNA fate. This is particularly true of the hepatitis C virus (HCV) genome which interacts with miR-122, an abundant liver miRNA. miR-122 binding to HCV RNA considerably stimulates virus replication in cultured cells and primates, but the mechanism(s) and associated host factors required for enhancement of HCV replication have not been fully elucidated. We recapitulated miR-122–HCV RNA interactions in a cell-free translation system derived from cells that express miR-122. Specifically, lysates produced from HEK-293 cells that inducibly transcribe and process pri-miR-122 were characterized alongside those from isogenic cells lacking miR-122 expression. We observed a stimulatory effect of miR-122 on HCV reporter mRNAs in a manner that depended on expression of miR-122 and intact target sites within the HCV 5′ UTR. We took advantage of this system to affinity-purify miR-122-HCV RNP complexes. Similar to functional assays, we found that association of immobilized HCV internal ribosome entry site (IRES) RNA with endogenous Ago2 requires both miR-122 expression and intact miR-122 target sites in cis. This combined approach may be generalizable to affinity purification of miRNP complexes for selected target mRNAs, allowing identification of miRNP components and RBPs that may contribute to regulation. PMID:23793894

  6. Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

    PubMed

    Weng, Leiyun; Tian, Xiao; Gao, Yayi; Watashi, Koichi; Shimotohno, Kunitada; Wakita, Takaji; Kohara, Michinori; Toyoda, Tetsuya

    2012-12-01

    Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase.

    PubMed

    Weng, Leiyun; Kohara, Michinori; Wakita, Takaji; Shimotohno, Kunitada; Toyoda, Tetsuya

    2012-04-01

    Recently, we found that sphingomyelin bound and activated hepatitis C virus (HCV) 1b RNA polymerase (RdRp), thereby recruiting the HCV replication complex into lipid raft structures. Detergents are commonly used for resolving lipids and purifying proteins, including HCV RdRp. Here, we tested the effect of detergents on HCV RdRp activity in vitro and found that non-ionic (Triton X-100, NP-40, Tween 20, Tween 80, and Brij 35) and twitterionic (CHAPS) detergents activated HCV 1b RdRps by 8-16.6 folds, but did not affect 1a or 2a RdRps. The maximum effect of these detergents was observed at around their critical micelle concentrations. On the other hand, ionic detergents (SDS and DOC) completely inactivated polymerase activity at 0.01%. In the presence of Triton X-100, HCV 1b RdRp did not form oligomers, but recruited more template RNA and increased the speed of polymerization. Comparison of polymerase and RNA-binding activity between JFH1 RdRp and Triton X-100-activated 1b RdRp indicated that monomer RdRp showed high activity because JFH1 RdRp was a monomer in physiological conditions of transcription. Besides, 502H plays a key role on oligomerization of 1b RdRp, while 2a RdRps which have the amino acid S at position 502 are monomers. This oligomer formed by 502H was disrupted both by high salt and Triton X-100. On the contrary, HCV 1b RdRp completely lost fidelity in the presence of 0.02% Triton X-100, which suggests that caution should be exercised while using Triton X-100 in anti-HCV RdRp drug screening tests.

  8. 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

  9. A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

    PubMed

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

    2015-12-15

    The hepatic expression of low-density lipoprotein (LDL) receptor (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 peroxisome proliferator-activated receptor (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 (RSV)-mediated transactivation. EMSA and ChIP assay 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.

  10. Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

    PubMed Central

    Mishima, Y; Mitsuma, T; Ogata, K

    1985-01-01

    An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

  11. The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling.

    PubMed

    Shandilya, Jayasha; Roberts, Stefan G E

    2012-05-01

    The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.

  12. 5'-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events.

    PubMed

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

    2000-04-01

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

  13. Transcription and Maturation of mRNA in Dinoflagellates

    PubMed Central

    Roy, Sougata; Morse, David

    2013-01-01

    Dinoflagellates are of great importance to the marine ecosystem, yet scant details of how gene expression is regulated at the transcriptional level are available. Transcription is of interest in the context of the chromatin structure in the dinoflagellates as it shows many differences from more typical eukaryotic cells. Here we canvas recent transcriptome profiles to identify the molecular building blocks available for the construction of the transcriptional machinery and contrast these with those used by other systems. Dinoflagellates display a clear paucity of specific transcription factors, although surprisingly, the rest of the basic transcriptional machinery is not markedly different from what is found in the close relatives to the dinoflagellates. PMID:27694765

  14. Hepatic lipid composition and stearoyl-coenzyme A desaturase 1 mRNA expression can be estimated from plasma VLDL fatty acid ratios.

    PubMed

    Peter, Andreas; Cegan, Alexander; Wagner, Silvia; Lehmann, Rainer; Stefan, Norbert; Königsrainer, Alfred; Königsrainer, Ingmar; Häring, Hans-Ulrich; Schleicher, Erwin

    2009-12-01

    Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the limiting step of monounsaturated fatty acid synthesis in humans and is an important player in triglyceride generation. SCD1 has been repeatedly implicated in the pathogenesis of metabolic and inflammatory diseases. Therefore it is of great importance to determine SCD1 activity in human samples. In this study we aimed to evaluate a hepatic SCD1 activity index derived from plasma VLDL triglyceride composition as a tool to estimate hepatic SCD1 expression in humans. Additionally, we further evaluated commonly used fatty acid ratios [elongase, de novo lipogenesis, and Delta5 and Delta6 desaturase] in plasma VLDL and hepatic lipid fractions. Liver biopsies and plasma samples were simultaneously collected from 15 individuals. Plasma VLDL was obtained by ultracentrifugation. Hepatic and plasma VLDL lipids were fractionated by thin-layer chromatography, and the fatty acid composition of each fraction was analyzed by gas chromatography. Hepatic SCD1 expression was determined by real-time PCR. Hepatic SCD1 mRNA expression was associated with the product/precursor ratios (16:1/16:0 and 18:1/18:0) of hepatic lipid fractions. The 16:1/16:0 ratio in hepatic and VLDL triglycerides as well as the 18:1/18:0 ratio in plasma VLDL were closely associated with hepatic SCD1 expression. The hepatic de novo lipogenesis index from triglycerides was associated with expression of lipogenic genes [fatty acid synthase (FASN), acetyl-Coenzyme A carboxylase alpha (ACACA), and sterol regulatory element binding transcription factor 1 (SREBP-1)] and is closely reflected by the de novo lipogenesis index in VLDL triglycerides. We demonstrated for the first time that hepatic SCD1 expression can be estimated noninvasively from routine blood samples by measuring the SCD1 activity index in fasting plasma VLDL.

  15. Degraded RNA transcript stable regions (StaRs) as targets for enhanced forensic RNA body fluid identification.

    PubMed

    Lin, Meng-Han; Albani, Patricia P; Fleming, Rachel

    2016-01-01

    The detection of messenger RNA (mRNA) using reverse transcriptase PCR (RT-PCR) is becoming common practice for forensic body fluid identification. However, the degraded and scarce nature of RNA from forensic samples mean that mRNA transcripts are not consistently detected or remain undetected in practice. Conventional primer design for RT-PCR (and quantitative RT-PCR) includes targeting primers to span exon-exon boundaries or by having the primers on two separate exons, and satisfying common primer thermodynamic criteria. We have found that the conventional placement of primers is not always optimal for obtaining reproducible results from degraded samples. Using massively parallel sequencing data from degraded body fluids, we designed primers to amplify transcript regions of high read coverage, hence, higher stability, and compared these with primers designed using conventional methodology. Our findings are that primers designed for transcript regions of higher read coverage resulted in vastly improved detection of mRNA transcripts that were not previously detected or were not consistently detected in the same samples using conventional primers. We developed a new concept whereby primers targeted to transcript stable regions (StaRs) are able to consistently and specifically amplify a wide range of RNA biomarkers in various body fluids of varying degradation levels. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

    PubMed

    Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

    2017-02-22

    Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

  17. Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts

    PubMed Central

    Gildehaus, Nina; Neußer, Thomas; Wurm, Reinhild; Wagner, Rolf

    2007-01-01

    Escherichia coli 6S RNA represents a non-coding RNA (ncRNA), which, based on the conserved secondary structure and previous functional studies, had been suggested to interfere with transcription. Selective inhibition of sigma-70 holoenzymes, preferentially at extended −10 promoters, but not stationary-phase-specific transcription was described, suggesting a direct role of 6S RNA in the transition from exponential to stationary phase. To elucidate the underlying mechanism, we have analysed 6S RNA interactions with different forms of RNA polymerase by gel retardation and crosslinking. Preferred binding of 6S RNA to Eσ70 was confirmed, however weaker binding to Eσ38 was also observed. The crosslinking analysis revealed direct contact between a central 6S RNA sequence element and the β/β′ and σ subunits. Promoter complex formation and in vitro transcription analysis with exponential- and stationary-phase-specific promoters and the corresponding holoenzymes demonstrated that 6S RNA interferes with transcription initiation but does not generally distinguish between exponential- and stationary-phase-specific promoters. Moreover, we show for the first time that 6S RNA acts as a template for the transcription of defined RNA molecules in the absence of DNA. In conclusion, this study reveals new aspects of 6S RNA function. PMID:17332013

  18. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors

    PubMed Central

    Russo, Joseph; Harrington, Andrew W.; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. PMID:26534950

  20. 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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. The Yin and Yang of Hepatitis C: Synthesis and Decay of HCV RNA

    PubMed Central

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

    2016-01-01

    PREFACE Hepatitis C virus (HCV) is an unusual RNA virus that possesses a striking capacity to persist for life 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 host RNA-binding proteins and microRNAs (miRNAs) that influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. We discuss how these processes impact HCV pathogenesis and therapeutic strategies against the virus. PMID:26256788

  2. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

    PubMed

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

    2017-01-26

    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  3. Preparation of long templates for RNA in vitro transcription by recursive PCR.

    PubMed

    Bowman, Jessica C; Azizi, Bahareh; Lenz, Timothy K; Roy, Poorna; Williams, Loren Dean

    2012-01-01

    Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5' T7 promoter region, and confirmation of the amplified DNA sequence. Complications arise in applications where long, nonnative sequences are desired in the final RNA transcript. Here we describe a ligase-independent method for the preparation of long synthetic DNA templates for in vitro RNA transcription. In Recursive PCR, partially complementary DNA oligonucleotides coding for the RNA sequence of interest are annealed, extended into the full-length double-stranded DNA, and amplified in a single PCR. Long insertions, mutations, or deletions are accommodated prior to in vitro transcription by simple substitution of oligonucleotides.

  4. Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

    PubMed Central

    Alic, Nazif; Ayoub, Nayla; Landrieux, Emilie; Favry, Emmanuel; Baudouin-Cornu, Peggy; Riva, Michel; Carles, Christophe

    2007-01-01

    We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 103 over the error rate determined solely by selectivity (1.8 × 10−4). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II. PMID:17553959

  5. Transcription by single molecules of RNA polymerase observed by light microscopy

    NASA Astrophysics Data System (ADS)

    Schafer, Dorothy A.; Gelles, Jeff; Sheetz, Michael P.; Landick, Robert

    1991-08-01

    THE kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general1, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope2,3 suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.

  6. Evolutionary Origins of Two-Barrel RNA Polymerases and Site-Specific Transcription Initiation.

    PubMed

    Fouqueau, Thomas; Blombach, Fabian; Werner, Finn

    2017-09-08

    Evolution-related multisubunit RNA polymerases (RNAPs) carry out RNA synthesis in all domains life. Although their catalytic cores and fundamental mechanisms of transcription elongation are conserved, the initiation stage of the transcription cycle differs substantially in bacteria, archaea, and eukaryotes in terms of the requirements for accessory factors and details of the molecular mechanisms. This review focuses on recent insights into the evolution of the transcription apparatus with regard to (a) the surprisingly pervasive double-Ψ β-barrel active-site configuration among different nucleic acid polymerase families, (b) the origin and phylogenetic distribution of TBP, TFB, and TFE transcription factors, and (c) the functional relationship between transcription and translation initiation mechanisms in terms of transcription start site selection and RNA structure.

  7. Elevated serum microRNA-122/222 levels are potential diagnostic biomarkers in Egyptian patients with chronic hepatitis C but not hepatic cancer.

    PubMed

    Motawi, Tarek M K; Sadik, Nermin A H; Shaker, Olfat G; Ghaleb, Maggy H

    2016-07-01

    MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. Because of their size, specificity, and relative stability in plasma, miRNAs can be used as diagnostic and prognostic biomarkers to monitor liver injury, such as that caused by hepatitis C virus (HCV) and liver cancer. In this study, we investigated miRNA expression patterns from the serum of Egyptian patients with HCV and liver cancer compared with matched healthy controls. Using microarray-based expression profiling followed by real-time quantitative polymerase chain reaction validation, we compared the levels of circulating miRNA-122 and miRNA-222 in serum from patients with hepatitis C virus (n = 40) and liver cancer (n = 60) to matched healthy controls (n = 30). MiRNA SNORD68 was the housekeeping endogenous control. We found that the serum levels of miR-122 and miR-222 were significantly elevated in HCV patients, but not in liver cancer patients, compared with controls. Receiver operating characteristic analysis revealed that miR-122 and miR-222 have a high diagnostic potential in discriminating patients with HCV from controls. Serum miR-222 was significantly higher in HCV patients compared to liver cancer patients. Our results indicate that serum miR-122 and miR-222 are elevated in Egyptian patients with chronic HCV, and these miRNAs have a strong potential to serve as novel biomarkers for liver injury but not specifically for liver cancer.

  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. 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

  10. Counterselection of prokaryotic ribosomal RNA during reverse transcription using non-random hexameric oligonucleotides.

    PubMed

    Gonzalez, J M; Robb, F T

    2007-12-01

    Ribosomal RNA (rRNA) is the major component in total RNA extracts, interfering with the synthesis of cDNA corresponding to messenger RNA (mRNA). In this study, we present a novel strategy for selectively discriminating against rRNA and favoring mRNA from prokaryotes during synthesis of cDNA by reverse transcriptase. Our technique is based on the fact that rRNA sequences, in many species, are G+C rich relative to the genome at large, and highly conserved among prokaryotes. The sequence TTTT is therefore rarely found in rRNA sequences. However, TTTT priming sites are found at a much higher frequency in protein-encoding gene sequences. We designed specific hexamers (HD/DHTTTT) to prime reverse transcription reactions resulting in a selective synthesis of cDNA corresponding to mRNA from prokaryotic total RNA extractions.

  11. MicroRNA-27a regulates basal transcription by targeting the p44 subunit of general transcription factor IIH

    PubMed Central

    Portal, Maximiliano M.

    2011-01-01

    General transcription factor IIH (TFIIH) is a complex RNA polymerase II basal transcription factor comprising 10 different polypeptides that display activities involved in transcription and DNA repair processes. Although biochemical studies have uncovered TFIIH importance, little is known about how the mRNAs that code for TFIIH subunits are regulated. Here it is shown that mRNAs encoding seven of the TFIIH subunits (p34, p44, p52, p62, XPB, CDK7, and p8) are regulated at the posttranscriptional level in a Dicer-dependent manner. Indeed, abolition of the miRNA pathway induces abnormal accumulation, stabilization, and translational activation of these seven mRNAs. Herein, miR-27a was identified as a key regulator of p44 mRNA. Moreover, miR-27a was shown to destabilize the p44 subunit of the TFIIH complex during the G2-M phase, thereby modulating the transcriptional shutdown observed during this transition. This work is unique in providing a demonstration of global transcriptional regulation through the action of a single miRNA. PMID:21558443

  12. An RNA enhancer in a phage transcriptional antitermination complex functions as a structural switch

    PubMed Central

    Su, Leila; Radek, James T.; Labeots, Laura A.; Hallenga, Klaas; Hermanto, Patrick; Chen, Huifen; Nakagawa, Satoe; Zhao, Ming; Kates, Steve; Weiss, Michael A.

    1997-01-01

    Antitermination protein N regulates the transcriptional program of phage λ through recognition of RNA enhancer elements. Binding of an arginine-rich peptide to one face of an RNA hairpin organizes the other, which in turn binds to the host antitermination complex. The induced RNA structure mimics a GNRA hairpin, an organizational element of rRNA and ribozymes. The two faces of the RNA, bridged by a sheared GA base pair, exhibit a specific pattern of base stacking and base flipping. This pattern is extended by stacking of an aromatic amino acid side chain with an unpaired adenine at the N-binding surface. Such extended stacking is coupled to induction of a specific internal RNA architecture and is blocked by RNA mutations associated in vivo with loss of transcriptional antitermination activity. Mimicry of a motif of RNA assembly by an RNA–protein complex permits its engagement within the antitermination machinery. PMID:9303537

  13. Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence.

    PubMed

    Bochkareva, Aleksandra; Yuzenkova, Yulia; Tadigotla, Vasisht R; Zenkin, Nikolay

    2012-02-01

    Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined by the ability of the elongating RNA polymerase to recognize the sequence of the RNA-DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly 'senses' the shape and/or identity of base pairs of the RNA-DNA hybrid. Recognition of the RNA-DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through 'in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA-DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation.

  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. 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.

  16. 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

  17. Binding of transcription termination protein nun to nascent RNA and template DNA.

    PubMed

    Watnick, R S; Gottesman, M E

    1999-12-17

    The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

  18. The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

    PubMed

    Jeronimo, Célia; Robert, François

    2017-10-01

    Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A small RNA targets pokeweed antiviral protein transcript.

    PubMed

    Klenov, Alexander; Neller, Kira C M; Burns, Lydia A; Krivdova, Gabriela; Hudak, Katalin A

    2016-03-01

    Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

  20. Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7.

    PubMed

    Park, Sung Hee; Nguyen, Tu N; Kirkham, Justin K; Lee, Ju Huck; Günzl, Arthur

    2011-11-01

    Trypanosoma brucei has a multifunctional RNA polymerase (pol) I that transcribes ribosomal gene units (RRNA) and units encoding its major cell surface proteins variant surface glycoprotein (VSG) and procyclin. Previous analysis of tandem affinity-purified, transcriptionally active RNA pol I identified ten subunits including an apparently trypanosomatid-specific protein termed RPA31. Another ortholog was identified in silico. No orthologs of the yeast subunit doublet RPA43/RPA14 have been identified yet. Instead, a recent report presented evidence that RPB7, the RNA pol II paralog of RPA43, is an RNA pol I subunit and essential for RRNA and VSG transcription in bloodstream form trypanosomes [18]. Revisiting this attractive hypothesis, we were unable to detect a stable interaction between RPB7 and RNA pol I in either reciprocal co-immunoprecipitation or tandem affinity purification. Furthermore, immunodepletion of RPB7 from extract virtually abolished RNA pol II transcription in vitro but had no effect on RRNA or VSG ES promoter transcription in the same reactions. Accordingly, chromatin immunoprecipitation analysis revealed cross-linking of RPB7 to known RNA pol II transcription units but not to the VSG ES promoter or to the 18S rRNA coding region. Interestingly, RPB7 did crosslink to the RRNA promoter but so did the RNA pol II-specific subunit RPB9 suggesting that RNA pol II is recruited to this promoter. Overall, our data led to the conclusion that RNA pol I transcription in T. brucei does not require the RNA pol II subunit RPB7.

  1. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

    PubMed

    Ferizi, Mehrije; Aneja, Manish K; Balmayor, Elizabeth R; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-12-15

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

  2. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

    PubMed Central

    Ferizi, Mehrije; Aneja, Manish K.; Balmayor, Elizabeth R.; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-01-01

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs. PMID:27974853

  3. Prevalence of hepatitis G virus RNA in a monocentric population of French haemophiliacs.

    PubMed

    Gerolami, V; Halfon, P; Chambost, H; Sicardi, F; Thuret, I; Planells, R; Halimi, G; Fossat, C; Michel, G; Cartouzou, G

    1997-10-01

    Hepatitis G virus (HGV) and hepatitis GB virus (GBV-C) have been reported as possible causes of non-A-E transfusional hepatitis. To assess the prevalence of hepatitis G virus infection in haemophiliacs we retrospectively investigated the presence of viral RNA in 92 patients with and without HCV infection. HGV/GBV-C RNA was reverse transcribed and amplified with primers from the 5' non-coding region of the genome. RNA was detected in 16/92 patients (17.4%). Restriction enzyme analysis revealed that the 16 patients belonged to the HGV-like genotype. Serology with E2-specific antibodies demonstrated that HGV viraemia underestimates previous infection by HGV. 33 patients were positive for HGV; all but two have cleared HGV RNA. 47/92 patients had a marker of prior infection by HGV. No difference between HGV RNA positive and negative patients was observed concerning age, diagnosis, HIV and HCV status. Previous HBV infection correlated with the frequency of HGV infection. There was no difference in alanine aminotransferase levels between HGV positive and negative patients. All 18 patients exposed to only virally inactivated plasma-derived concentrates were negative for both HGV RNA and anti E2 antibodies. Prior exposure to untreated concentrates correlated with HGV viraemia (P=0.03), HGV seropositivity (P=0.0002), and markers of HGV infection (P<0.0001). In haemophiliacs with a past exposure to non-inactivated concentrates, persistence of HCV RNA (53/74 patients) was more frequent than HGV RNA persistence (16/74 patients) although HGV viraemia is more frequent than HCV viraemia in blood donors. This may be related to a greater ability of individuals to clear HGV infection and suggests that hepatitis G virus infection in multi-transfused patients has a better outcome than infection with other blood-borne viruses.

  4. Effect of Soil Clay Content on RNA Isolation and on Detection and Quantification of Bacterial Gene Transcripts in Soil by Quantitative Reverse Transcription-PCR ▿†

    PubMed Central

    Novinscak, A.; Filion, M.

    2011-01-01

    In this study, we evaluated the effect of soil clay content on RNA isolation and on quantitative reverse transcription-PCR (qRT-PCR) quantification of microbial gene transcripts. The amount of clay significantly altered RNA isolation yields and qRT-PCR analyses. Recommendations are made for quantifying microbial gene transcripts in soil samples varying in clay content. PMID:21724880

  5. Ubiquitin mRNA is a major stress-induced transcript in mammalian cells.

    PubMed Central

    Fornace, A J; Alamo, I; Hollander, M C; Lamoreaux, E

    1989-01-01

    Ubiquitin mRNA was found to be an abundant transcript which was induced by heat shock (HS), and certain other stresses in mammalian cells. In Chinese hamster cells, the 2 major ubiquitin transcripts of 2.6 kb and 1.7 kb were induced coordinately, while a minor ubiquitin transcript of 0.8 kb was not induced; the response was similar in human cells with induction of the 2.5 kb Ub C and 1.0 kb Ub B transcripts. A representative ubiquitin cDNA clone, isolated from a cDNA library derived from HS-treated Chinese hamster cells, coded for a typical tandem repeat polyubiquitin transcript. Only a portion of the 5' nontranslated sequence of this clone had homology with the previously published corresponding region in human Ub B mRNA. Oligonucleotide probes complementary to the portion of the 5' nontranslated sequence with homology to the human sequence and also portions with no homology hybridized only to the 1.7 kb transcript. There was coordinate induction of ubiquitin, HSP27, and HSP70 mRNA by HS as determined by both increased RNA and increased transcription. Ubiquitin mRNA was induced by certain DNA damaging agents, in particular the alkylating agent methylmethane sulfonate, or incubation in isoleucine-deficient medium under conditions where the other HSP mRNA were not. Images PMID:2537950

  6. RNA Silencing Induced by an Artificial Sequence That Prevents Proper Transcription Termination in Rice1[W

    PubMed Central

    Kawakatsu, Taiji; Wakasa, Yuhya; Yasuda, Hiroshi; Takaiwa, Fumio

    2012-01-01

    Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5′ or 3′ flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5′ or 3′ end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes. PMID:22843666

  7. RNA silencing induced by an artificial sequence that prevents proper transcription termination in rice.

    PubMed

    Kawakatsu, Taiji; Wakasa, Yuhya; Yasuda, Hiroshi; Takaiwa, Fumio

    2012-10-01

    Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5' or 3' flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5' or 3' end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes.

  8. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

    PubMed

    Min, Irene M; Waterfall, Joshua J; Core, Leighton J; Munroe, Robert J; Schimenti, John; Lis, John T

    2011-04-01

    Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

  9. Importance of steric effects on the efficiency and fidelity of transcription by T7 RNA polymerase.

    PubMed

    Ulrich, Sébastien; Kool, Eric T

    2011-11-29

    DNA-dependent RNA polymerases such as T7 RNA polymerase (T7 RNAP) perform the transcription of DNA into mRNA with high efficiency and high fidelity. Although structural studies have provided a detailed account of the molecular basis of transcription, the relative importance of factors like hydrogen bonds and steric effects remains poorly understood. We report herein the first study aimed at systematically probing the importance of steric and electrostatic effects on the efficiency and fidelity of DNA transcription by T7 RNAP. We used synthetic nonpolar analogues of thymine with sizes varying in subangstrom increments to probe the steric requirements of T7 RNAP during the elongation mode of transcription. Enzymatic assays with internal radiolabeling were performed to compare the efficiency of transcription of modified DNA templates with a natural template containing thymine as a reference. Furthermore, we analyzed effects on the fidelity by measuring the composition of RNA transcripts by enzymatic digestion followed by two-dimensional thin layer chromatography separation. Our results demonstrate that hydrogen bonds play an important role in the efficiency of transcription but, interestingly, do not appear to be required for faithful transcription. Steric effects (size and shape variations) are found to be significant both in insertion of a new RNA base and in extension beyond it.

  10. 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

  11. Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA damage.

    PubMed

    Cullinane, C; Mazur, S J; Essigmann, J M; Phillips, D R; Bohr, V A

    1999-05-11

    The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

  12. PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi.

    PubMed

    Wen, L M; Xu, P; Benegal, G; Carvalho, M R; Buck, G A

    2000-10-01

    In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli, showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.

  13. FDM: a graph-based statistical method to detect differential transcription using RNA-seq data.

    PubMed

    Singh, Darshan; Orellana, Christian F; Hu, Yin; Jones, Corbin D; Liu, Yufeng; Chiang, Derek Y; Liu, Jinze; Prins, Jan F

    2011-10-01

    In eukaryotic cells, alternative splicing expands the diversity of RNA transcripts and plays an important role in tissue-specific differentiation, and can be misregulated in disease. To understand these processes, there is a great need for methods to detect differential transcription between samples. Our focus is on samples observed using short-read RNA sequencing (RNA-seq). We characterize differential transcription between two samples as the difference in the relative abundance of the transcript isoforms present in the samples. The magnitude of differential transcription of a gene between two samples can be measured by the square root of the Jensen Shannon Divergence (JSD*) between the gene's transcript abundance vectors in each sample. We define a weighted splice-graph representation of RNA-seq data, summarizing in compact form the alignment of RNA-seq reads to a reference genome. The flow difference metric (FDM) identifies regions of differential RNA transcript expression between pairs of splice graphs, without need for an underlying gene model or catalog of transcripts. We present a novel non-parametric statistical test between splice graphs to assess the significance of differential transcription, and extend it to group-wise comparison incorporating sample replicates. Using simulated RNA-seq data consisting of four technical replicates of two samples with varying transcription between genes, we show that (i) the FDM is highly correlated with JSD* (r=0.82) when average RNA-seq coverage of the transcripts is sufficiently deep; and (ii) the FDM is able to identify 90% of genes with differential transcription when JSD* >0.28 and coverage >7. This represents higher sensitivity than Cufflinks (without annotations) and rDiff (MMD), which respectively identified 69 and 49% of the genes in this region as differential transcribed. Using annotations identifying the transcripts, Cufflinks was able to identify 86% of the genes in this region as differentially transcribed

  14. FDM: a graph-based statistical method to detect differential transcription using RNA-seq data

    PubMed Central

    Singh, Darshan; Orellana, Christian F.; Hu, Yin; Jones, Corbin D.; Liu, Yufeng; Chiang, Derek Y.; Liu, Jinze; Prins, Jan F.

    2011-01-01

    Motivation: In eukaryotic cells, alternative splicing expands the diversity of RNA transcripts and plays an important role in tissue-specific differentiation, and can be misregulated in disease. To understand these processes, there is a great need for methods to detect differential transcription between samples. Our focus is on samples observed using short-read RNA sequencing (RNA-seq). Methods: We characterize differential transcription between two samples as the difference in the relative abundance of the transcript isoforms present in the samples. The magnitude of differential transcription of a gene between two samples can be measured by the square root of the Jensen Shannon Divergence (JSD*) between the gene's transcript abundance vectors in each sample. We define a weighted splice-graph representation of RNA-seq data, summarizing in compact form the alignment of RNA-seq reads to a reference genome. The flow difference metric (FDM) identifies regions of differential RNA transcript expression between pairs of splice graphs, without need for an underlying gene model or catalog of transcripts. We present a novel non-parametric statistical test between splice graphs to assess the significance of differential transcription, and extend it to group-wise comparison incorporating sample replicates. Results: Using simulated RNA-seq data consisting of four technical replicates of two samples with varying transcription between genes, we show that (i) the FDM is highly correlated with JSD* (r=0.82) when average RNA-seq coverage of the transcripts is sufficiently deep; and (ii) the FDM is able to identify 90% of genes with differential transcription when JSD* >0.28 and coverage >7. This represents higher sensitivity than Cufflinks (without annotations) and rDiff (MMD), which respectively identified 69 and 49% of the genes in this region as differential transcribed. Using annotations identifying the transcripts, Cufflinks was able to identify 86% of the genes in this region

  15. Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase.

    PubMed

    Deshpande, Aishwarya P; Patel, Smita S

    2012-01-01

    Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP "sensor" to regulate gene expression. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

  16. A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis.

    PubMed

    Weisenberger, D; Scheer, U

    1995-05-01

    When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.

  17. Upstream binding factor-dependent and pre-rRNA transcription-independent association of pre-rRNA processing factors with rRNA gene.

    PubMed

    Ueshima, Shuhei; Nagata, Kyosuke; Okuwaki, Mitsuru

    2014-01-03

    The nucleolus is the ribosome biogenesis center. The nucleolar structure is disrupted upon entry into mitosis and is formed in early G1 phase. To understand the molecular mechanisms of nucleolar assembly and disassembly, we have studied the mechanism of association between factors involved in pre-ribosome RNA (rRNA) processing and rRNA gene chromatin (r-chromatin). We found that the pre-rRNA transcription-processing linking factor Nopp140 and pre-rRNA processing factors such as DKC1 and fibrillarin (FBL) associate with r-chromatin during interphase, while Nopp140, DKC1, and FBL were released from r-chromatin in mitosis. The association of these factors with r-chromatin was found to be restored independent of pre-rRNA transcription in early G1 phase, but a mature nucleolar structure was not formed, suggesting that nucleolar assembly can be divided into at least two steps with respect to pre-rRNA transcription. Moreover, we found that the r-chromatin association of Nopp140, DKC1, and FBL was dependent on the transcription factor upstream binding factor (UBF). However, we demonstrated that UBF alone was not sufficient to recruit these pre-rRNA processing factors to r-chromatin. Thus, UBF is necessary but not sufficient for the associations between pre-rRNA processing factors and r-chromatin. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. 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.

  19. An Ago2-associated capped transcriptional start site small RNA suppressing adenovirus DNA replication.

    PubMed

    Kamel, Wael; Akusjärvi, Göran

    2017-08-24

    Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3'end. The MLP-TSS-sRNA is highly stable in cells and functionally active, downregulating complementary targets in a sequence and dose dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and pre-terminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and pre-terminal protein mRNA expression. As a consequence of this the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. 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

  1. The role of the lid element in transcription by E. coli RNA polymerase.

    PubMed

    Toulokhonov, Innokenti; Landick, Robert

    2006-08-25

    The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAP's RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing sigma), the lid interacts with sigma region 3 and encloses an extended linker between sigma region 3 and sigma region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Deltalid RNAP exhibited dramatically reduced activity during initiation from -35-dependent and -35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Deltalid RNAP. Importantly, Deltalid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.

  2. A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

    PubMed

    Karataylı, Ersin; Altunoğlu, Yasemin Çelik; Karataylı, Senem Ceren; Alagöz, S Gökçe K; Cınar, Kubilay; Yalçın, Kendal; Idilman, Ramazan; Yurdaydın, Cihan; Bozdayı, A Mithat

    2014-05-01

    Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Production of infectious RNA transcripts from full-length cDNA clones representing two subgroups of peanut stunt virus strains: mapping satellite RNA support to RNA1.

    PubMed

    Hu, C C; Sanger, M; Ghabrial, S A

    1998-08-01

    Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV-ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups.

  4. Gene regulation by the act of long non-coding RNA transcription

    PubMed Central

    2013-01-01

    Long non-protein-coding RNAs (lncRNAs) are proposed to be the largest transcript class in the mouse and human transcriptomes. Two important questions are whether all lncRNAs are functional and how they could exert a function. Several lncRNAs have been shown to function through their product, but this is not the only possible mode of action. In this review we focus on a role for the process of lncRNA transcription, independent of the lncRNA product, in regulating protein-coding-gene activity in cis. We discuss examples where lncRNA transcription leads to gene silencing or activation, and describe strategies to determine if the lncRNA product or its transcription causes the regulatory effect. PMID:23721193

  5. 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.

  6. Transcriptional properties and splicing of the flamenco piRNA cluster

    PubMed Central

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

    2014-01-01

    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

  7. 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

  8. Tentative Mapping of Transcription-Induced Interchromosomal Interaction using Chimeric EST and mRNA Data

    PubMed Central

    Unneberg, Per; Claverie, Jean-Michel

    2007-01-01

    Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or “noise” in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology. PMID:17330142

  9. The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators

    PubMed Central

    Rabhi, Makhlouf; Espéli, Olivier; Schwartz, Annie; Cayrol, Bastien; Rahmouni, A Rachid; Arluison, Véronique; Boudvillain, Marc

    2011-01-01

    In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity. PMID:21673658

  10. The RNA Export Factor, Nxt1, Is Required for Tissue Specific Transcriptional Regulation

    PubMed Central

    Jiang, Jianqiao; White-Cooper, Helen

    2013-01-01

    The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. Nxt1 is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many Drosophila melanogaster testis-specific mRNAs require Nxt1 for their accumulation. The transcripts that require Nxt1 also depend on a testis-specific transcription complex, tMAC. We show that loss of Nxt1 leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in Nxt1 mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in Drosophila primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires Nxt1. We additionally find that transcripts from intron-less genes are more sensitive to loss of Nxt1 function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex. PMID:23754955

  11. The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

    PubMed Central

    Balajee, Adayabalam S.; Machwe, Amrita; May, Alfred; Gray, Matthew D.; Oshima, Junko; Martin, George M.; Nehlin, Jan O.; Brosh, Robert; Orren, David K.; Bohr, Vilhelm A.

    1999-01-01

    Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype

  12. The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research

    PubMed Central

    Rocheleau, Lynda; Pelchat, Martin

    2006-01-01

    Background Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs. Description The Subviral RNA database is a web-based environment that facilitates the research and analysis of viroids, satellite RNAs, satellites viruses, the human hepatitis delta virus, and related RNA sequences. It integrates a large number of Subviral RNA sequences, their respective RNA motifs, analysis tools, related publication links and additional pertinent information (ex. links, conferences, announcements), allowing users to efficiently retrieve and analyze relevant information about these small RNA agents. Conclusion With its design, the Subviral RNA Database could be considered as a fundamental building block for the study of these related RNAs. It is freely available via a web browser at the URL: . PMID:16519798

  13. The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research.

    PubMed

    Rocheleau, Lynda; Pelchat, Martin

    2006-03-06

    Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs. The Subviral RNA database is a web-based environment that facilitates the research and analysis of viroids, satellite RNAs, satellites viruses, the human hepatitis delta virus, and related RNA sequences. It integrates a large number of Subviral RNA sequences, their respective RNA motifs, analysis tools, related publication links and additional pertinent information (ex. links, conferences, announcements), allowing users to efficiently retrieve and analyze relevant information about these small RNA agents. With its design, the Subviral RNA Database could be considered as a fundamental building block for the study of these related RNAs. It is freely available via a web browser at the URL: http://subviral.med.uottawa.ca.

  14. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader.

    PubMed

    Kriner, Michelle A; Groisman, Eduardo A

    2017-01-25

    Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5' leader mRNA of the Salmonella corA gene can adopt two mutually exclusive conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

    PubMed

    Fairley, Jennifer A; Mitchell, Louise E; Berg, Tracy; Kenneth, Niall S; von Schubert, Conrad; Silljé, Herman H W; Medema, René H; Nigg, Erich A; White, Robert J

    2012-02-24

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader

    PubMed Central

    Kriner, Michelle A.; Groisman, Eduardo A.

    2017-01-01

    Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5′ leader mRNA of the Salmonella corA gene can adopt two mutually exclusive conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals. PMID:28123036

  17. Intergenic and repeat transcription in human, chimpanzee and macaque brains measured by RNA-Seq.

    PubMed

    Xu, Augix Guohua; He, Liu; Li, Zhongshan; Xu, Ying; Li, Mingfeng; Fu, Xing; Yan, Zheng; Yuan, Yuan; Menzel, Corinna; Li, Na; Somel, Mehmet; Hu, Hao; Chen, Wei; Pääbo, Svante; Khaitovich, Philipp

    2010-07-01

    Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq) to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20-28% of non-ribosomal transcripts correspond to annotated exons and 20-23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40-48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20%) represents 3'UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

  18. Intergenic and Repeat Transcription in Human, Chimpanzee and Macaque Brains Measured by RNA-Seq

    PubMed Central

    Xu, Ying; Li, Mingfeng; Fu, Xing; Yan, Zheng; Yuan, Yuan; Menzel, Corinna; Li, Na; Somel, Mehmet; Hu, Hao; Chen, Wei; Pääbo, Svante; Khaitovich, Philipp

    2010-01-01

    Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq) to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20–28% of non-ribosomal transcripts correspond to annotated exons and 20–23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40–48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20%) represents 3′UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits. PMID:20617162

  19. E-Cadherin Is Transcriptionally Activated via Suppression of ZEB1 Transcriptional Repressor by Small RNA-Mediated Gene Silencing

    PubMed Central

    Mazda, Minami; Nishi, Kenji; Naito, Yuki; Ui-Tei, Kumiko

    2011-01-01

    RNA activation has been reported to be induced by small interfering RNAs (siRNAs) that act on the promoters of several genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3 cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were found in the coding region in addition to the 3′ untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes, which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs. Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of the siRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription. Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo. PMID:22205962

  20. Interactive cooperation and hierarchical operation of microRNA and transcription factor crosstalk in human transcriptional regulatory network.

    PubMed

    Gov, Esra; Arga, Kazim Yalcin

    2016-12-01

    Transcriptional regulation of gene expression is an essential cellular process that is arranged by transcription factors (TFs), microRNAs (miRNA) and their target genes through a variety of mechanisms. Here, we set out to reconstruct a comprehensive transcriptional regulatory network of Homo sapiens consisting of experimentally verified regulatory information on miRNAs, TFs and their target genes. We have performed topological analyses to elucidate the transcriptional regulatory roles of miRNAs and TFs. When we thoroughly investigated the network motifs, different gene regulatory scenarios were observed; whereas, mutual TF-miRNA regulation (interactive cooperation) and hierarchical operation where miRNAs were the upstream regulators of TFs came into prominence. Otherwise, biological process specific subnetworks were also constructed and integration of gene and miRNA expression data on ovarian cancer was achieved as a case study to observe dynamic patterns of the gene expression. Meanwhile, both co-operation and hierarchical operation types were determined in active ovarian cancer and process-specific subnetworks. In addition, the analysis showed that multiple signals from miRNAs were integrated by TFs. Our results demonstrate new insights on the architecture of the human transcriptional regulatory network, and here we present some lessons we gained from deciphering the reciprocal interplay between miRNAs, TFs and their target genes.

  1. Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription.

    PubMed

    Kutky, Meherzad; Hudak, Katalin A

    2017-10-05

    HIV-1 (human immunodeficiency virus) transcription is primarily controlled by the virally encoded Tat (transactivator of transcription) protein and its interaction with the viral TAR (transcription response element) RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In the present study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant (Phytolacca americana), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and specificity protein 1, which modulate HIV-1 transcription by binding to the viral LTR (5'-long terminal repeat), were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  2. Primary microRNA processing is functionally coupled to RNAP II transcription in vitro

    PubMed Central

    Yin, Shanye; Yu, Yong; Reed, Robin

    2015-01-01

    Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing. PMID:26149087

  3. Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis.

    PubMed

    Andrews, Laura M; Jones, Mark R; Digman, Michelle A; Gratton, Enrico

    2013-03-01

    Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

  4. Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis

    NASA Astrophysics Data System (ADS)

    Andrews, Laura M.; Jones, Mark R.; Digman, Michelle A.; Gratton, Enrico

    2013-03-01

    Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

  5. Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity

    PubMed Central

    Samai, Poulami; Goldberg, Gregory W.; Hatoum-Aslan, Asma; Marraffini, Luciano A.

    2015-01-01

    SUMMARY Immune systems must recognize and destroy different pathogens that threat the host. CRISPR-Cas immune systems protect prokaryotes from viral and plasmid infection utilizing small CRISPR RNAs that are complementary to the invader's genome and specify the targets of RNA-guided Cas nucleases. Type III CRISPR-Cas immunity requires target transcription and whereas genetic studies demonstrated DNA targeting, in vitro data have shown crRNA-guided RNA cleavage. The molecular mechanism behind this disparate activities is not known. Here we show that transcription across the targets of the Staphylococcus epidermidis type III-A CRISPR-Cas system results in the cleavage of the target DNA and its transcripts, mediated by independent active sites within the Cas10-Csm ribonucleoprotein effector complex. Immunity against plasmids and DNA viruses requires DNA but not RNA cleavage activity. Our studies reveal a highly versatile mechanism of CRISPR immunity that can defend microorganisms against diverse DNA and RNA invaders. PMID:25959775

  6. Sequence organization of the Acanthamoeba rRNA intergenic spacer: identification of transcriptional enhancers.

    PubMed Central

    Yang, Q; Zwick, M G; Paule, M R

    1994-01-01

    The primary sequence of the entire 2330 bp intergenic spacer of the A.castellanii ribosomal RNA gene was determined. Repeated sequence elements averaging 140 bp were identified and found to bind a protein required for optimum initiation at the core promoter. These repeated elements were shown to stimulate rRNA transcription by RNA polymerase I in vitro. The repeats inhibited transcription when placed in trans, and stimulated transcription when in cis, in either orientation, but only when upstream of the core promoter. Thus, these repeated elements have characteristics similar to polymerase I enhancers found in higher eukaryotes. The number of rRNA repeats in Acanthamoeba cells was determined to be 24 per haploid genome, the lowest number so far identified in any eukaryote. However, because Acanthamoeba is polyploid, each cell contains approximately 600 rRNA genes. Images PMID:7984432

  7. 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

  8. 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.

  9. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

    PubMed

    Goh, Wee Siong Sho; Falciatori, Ilaria; Tam, Oliver H; Burgess, Ralph; Meikar, Oliver; Kotaja, Noora; Hammell, Molly; Hannon, Gregory J

    2015-05-15

    MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

  10. Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR

    PubMed Central

    2016-01-01

    Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver. PMID:28164071

  11. Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR.

    PubMed

    Lee, Jae Man

    2016-12-01

    Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver.

  12. Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template.

    PubMed

    Donahue, B A; Yin, S; Taylor, J S; Reines, D; Hanawalt, P C

    1994-08-30

    A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.

  13. A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

    PubMed

    Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

    2016-11-17

    Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories

    PubMed Central

    Caudron-Herger, Maïwen; Cook, Peter R.; Rippe, Karsten; Papantonis, Argyris

    2015-01-01

    While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)+ and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts. PMID:25897132

  15. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

    PubMed Central

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3′ end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells. PMID:25764216

  16. Control of Transcriptional Fidelity by Active Center Tuning as Derived from RNA Polymerase Endonuclease Reaction*

    PubMed Central

    Sosunova, Ekaterina; Sosunov, Vasily; Epshtein, Vitaly; Nikiforov, Vadim; Mustaev, Arkady

    2013-01-01

    Precise transcription by cellular RNA polymerase requires the efficient removal of noncognate nucleotide residues that are occasionally incorporated. Mis-incorporation causes the transcription elongation complex to backtrack, releasing a single strand 3′-RNA segment bearing a noncognate residue, which is hydrolyzed by the active center that carries two Mg2+ ions. However, in most x-ray structures only one Mg2+ is present. This Mg2+ is tightly bound to the active center aspartates, creating an inactive stable state. The first residue of the single strand RNA segment in the backtracked transcription elongation complex strongly promotes transcript hydrolytic cleavage by establishing a network of interactions that force a shift of stably bound Mg2+ to release some of its aspartate coordination valences for binding to the second Mg2+ thus enabling catalysis. Such a rearrangement that we call active center tuning (ACT) occurs when all recognition contacts of the active center-bound RNA segment are established and verified by tolerance to stress. Transcription factor Gre builds on the ACT mechanism in the same reaction by increasing the retention of the second Mg2+ and by activating the attacking water, causing 3000–4000-fold reaction acceleration and strongly reinforcing proofreading. The unified mechanism for RNA synthesis and degradation by RNA polymerase predicts that ACT also executes NTP selection thereby contributing to high transcription fidelity. PMID:23283976

  17. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    PubMed

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

  18. Activation of DNA replication in yeast by recruitment of the RNA polymerase II transcription complex.

    PubMed

    Stagljar, I; Hübscher, U; Barberis, A

    1999-05-01

    Activators of transcription are known to also play an important and direct role in activating DNA replication. However, the mechanism whereby they stimulate replication has remained elusive. One model suggests that, in the context of replication origins, transcriptional activators work by interacting with replication factors. We show that a defined, single interaction between a DNA-bound derivative of the activator Gal4 and Gal11P, a mutant form of the RNA polymerase II holoenzyme component Gal11, suffices for stimulating DNA replication as it does for transcription. Moreover, recruitment of TBP, which can activate transcription from a gene promoter, also stimulates DNA replication from an origin site. These results strongly argue that transcriptional activators may not necessarily need to contact DNA replication factors directly, but can stimulate replication by recruiting the RNA polymerase II transcription complex to DNA.

  19. Viral miRNA targeting of bicistronic and polycistronic transcripts.

    PubMed

    Zhu, Ying; Huang, Yufei; Jung, Jae U; Lu, Chun; Gao, Shou-Jiang

    2014-08-01

    Successful viral infection entails a choreographic regulation of viral gene expression program. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes numerous miRNAs that regulate viral life cycle. However, few viral targets have been identified due to the lack of information on KSHV 3' untranslated regions (3'UTRs). Recent genome-wide mapping of KSHV transcripts and 3'UTRs has revealed abundant bicistronic and polycistronic transcripts. The extended 3'UTRs of the 5' proximal genes of bicistronic and polycistronic transcripts offer additional regulatory targets. Indeed, a genome-wide screening of KSHV 3'UTRs has identified several bicistronic and polycistronic transcripts as the novel targets of viral miRNAs. Together, these works have expanded our knowledge of the unique features of KSHV gene regulation program and provided valuable resources for the research community.

  20. Viral miRNA Targeting of Bicistronic and Polycistronic Transcripts

    PubMed Central

    Zhu, Ying; Huang, Yufei; Jung, Jae U.; Lu, Chun; Gao, Shou-Jiang

    2014-01-01

    Successful viral infection entails a choreographic regulation of viral gene expression program. Kaposi’s sarcoma–associated herpesvirus (KSHV) encodes numerous miRNAs that regulate viral life cycle. However, few viral targets have been identified due to the lack of information on KSHV 3′ untranslated regions (3′UTRs). Recent genome-wide mapping of KSHV transcripts and 3′UTRs has revealed abundant bicistronic and polycistronic transcripts. The extended 3′UTRs of the 5′ proximal genes of bicistronic and polycistronic transcripts offer additional regulatory targets. Indeed, a genome-wide screening of KSHV 3′UTRs has identified several bicistronic and polycistronic transcripts as the novel targets of viral miRNAs. Together, these works have expanded our knowledge of the unique features of KSHV gene regulation program and provided valuable resources for the research community. PMID:24821460

  1. Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation.

    PubMed

    Arimbasseri, Aneeshkumar G; Rijal, Keshab; Maraia, Richard J

    2014-01-01

    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.

  2. Dynamics and rRNA transcriptional activity of lactococci and lactobacilli during Cheddar cheese ripening.

    PubMed

    Desfossés-Foucault, Émilie; LaPointe, Gisèle; Roy, Denis

    2013-08-16

    Cheddar cheese is a complex ecosystem where both the bacterial population and the cheese making process contribute to flavor and texture development. The aim of this study was to use molecular methods to evaluate the impact of milk heat treatment and ripening temperature on starter lactococci and non-starter lactic acid bacteria (NSLAB) throughout ripening of Cheddar cheese. Eight Cheddar cheese batches were manufactured (four with thermized and four with pasteurized milk) and ripened at 4, 7 and 12°C to analyze the bacterial composition and rRNA transcriptional activity reflecting the ability of lactococci and lactobacilli to synthesize proteins. Abundance and rRNA transcription of lactococci and lactobacilli were quantified after DNA and RNA extraction by using quantitative PCR (qPCR) and reverse transcription-quantitative PCR (RT-qPCR) targeting the 16S rRNA gene, respectively. Results showed that lactococci remained dominant throughout ripening, although 16S rRNA genome and cDNA copies/g of cheese decreased by four and two log copy numbers, respectively. Abundance and rRNA transcription of Lactobacillus paracasei, Lactobacillus buchneri/parabuchneri, Lactobacillus rhamnosus, Lactobacillus brevis, and Lactobacillus coryniformis as well as total lactobacilli were also estimated using specific 16S rRNA primers. L. paracasei and L. buchneri/parabuchneri concomitantly grew in cheese made from thermized milk at 7 and 12°C, although L. paracasei displayed the most rRNA transcription among Lactobacillus species. This work showed that rRNA transcriptional activity of lactococci decreased throughout ripening and supports the usefulness of RNA analysis to assess which bacterial species have the ability to synthesize proteins during ripening, and could thereby contribute to cheese quality. © 2013.

  3. Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

    PubMed

    Jackson, D P; Percy, D H; Morris, V L

    1984-09-01

    When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

  4. Coupled pre-mRNA and mRNA dynamics unveil operational strategies underlying transcriptional responses to stimuli

    PubMed Central

    Zeisel, Amit; Köstler, Wolfgang J; Molotski, Natali; Tsai, Jonathan M; Krauthgamer, Rita; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Soen, Yoav; Jung, Steffen; Yarden, Yosef; Domany, Eytan

    2011-01-01

    Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude. PMID:21915116

  5. Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos.

    PubMed Central

    Kamakaka, R T; Tyree, C M; Kadonaga, J T

    1991-01-01

    We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Krüppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of approximately 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired. Images PMID:1992453

  6. Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

    PubMed

    Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri; Artsimovitch, Irina; Lucks, Julius B

    2017-04-08

    RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.

  7. COMBINING PROTEIN AND mRNA QUANTIFICATION TO DECIPHER TRANSCRIPTIONAL REGULATION

    PubMed Central

    Xu, Heng; Sepúlveda, Leonardo A.; Figard, Lauren; Sokac, Anna Marie; Golding, Ido

    2015-01-01

    We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription-factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo. PMID:26098021

  8. Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription

    PubMed Central

    Tavenet, Arounie; Suleau, Audrey; Dubreuil, Géraldine; Ferrari, Roberto; Ducrot, Cécile; Michaut, Magali; Aude, Jean-Christophe; Dieci, Giorgio; Lefebvre, Olivier; Conesa, Christine; Acker, Joël

    2009-01-01

    Human PC4 and the yeast ortholog Sub1 have multiple functions in RNA polymerase II transcription. Genome-wide mapping revealed that Sub1 is present on Pol III-transcribed genes. Sub1 was found to interact with components of the Pol III transcription system and to stimulate the initiation and reinitiation steps in a system reconstituted with all recombinant factors. Sub1 was required for optimal Pol III gene transcription in exponentially growing cells. PMID:19706510

  9. Structural basis for transcription elongation by bacterial RNA polymerase.

    PubMed

    Vassylyev, Dmitry G; Vassylyeva, Marina N; Perederina, Anna; Tahirov, Tahir H; Artsimovitch, Irina

    2007-07-12

    The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

  10. 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

    2001-06-08

    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.

  11. 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.

  12. Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

    PubMed Central

    Burke, Emily; Mahoney, Nicole M.; Almo, Steven C.; Barik, Sailen

    2000-01-01

    Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome. PMID:10623728

  13. LIN-42, the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription

    PubMed Central

    Aguirre-Chen, Cristina; Hammell, Christopher M.

    2014-01-01

    During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression. PMID:25032706

  14. LIN-42, the Caenorhabditis elegans PERIOD homolog, negatively regulates microRNA transcription.

    PubMed

    Perales, Roberto; King, Dana M; Aguirre-Chen, Cristina; Hammell, Christopher M

    2014-07-01

    During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression.

  15. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    PubMed

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  16. Transcription and cap trimethylation of a nematode spliced leader RNA in a cell-free system.

    PubMed Central

    Maroney, P A; Hannon, G J; Nilsen, T W

    1990-01-01

    Maturation of a fraction of mRNAs in nematodes involves the acquisition of a common 5' terminal spliced leader sequence derived from a nonpolyadenylylated spliced leader RNA by trans splicing. We have developed a cell-free system prepared from Ascaris lumbricoides embryos that accurately and efficiently synthesized the spliced leader RNA of A. lumbricoides. Transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethylguanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA. Images PMID:2300557

  17. 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.

  18. Differential expression of viral PAMP receptors mRNA in peripheral blood of patients with chronic hepatitis C infection

    PubMed Central

    Atencia, Rafael; Bustamante, Francisco J; Valdivieso, Andrés; Arrieta, Arantza; Riñón, Marta; Prada, Alvaro; Maruri, Natalia

    2007-01-01

    Background Pathogen-associated molecular patterns (PAMP) receptors play a key role in the early host response to viruses. In this work, we determined mRNA levels of two members of the Toll-like Receptors family, (TLR3 and TLR7) and the helicase RIG-I, all of three recognizing viral RNA products, in peripheral blood of healthy donors and hepatitis C virus (HCV) patients, to observe if their transcripts are altered in this disease. Methods IFN-α, TLR3, TLR7 and RIG-I levels in peripheral blood from healthy controls (n = 18) and chronic HCV patients (n = 18) were quantified by real-time polymerase chain reaction. Results Our results show that IFN-α, TLR3, TLR7 and RIG-I mRNA levels are significantly down-regulated in patients with chronic HCV infection when compared with healthy controls. We also found that the measured levels of TLR3 and TLR7, but not RIG-I, correlated significantly with those of IFN-α Conclusion Monitoring the expression of RNA-sensing receptors like TLR3, TLR7 and RIG-I during the different clinical stages of infection could bring a new source of data about the prognosis of disease. PMID:18021446

  19. Influence of secondary structure on recovery from pauses during early stages of RNA transcription.

    PubMed

    Klopper, A V; Bois, J S; Grill, S W

    2010-03-01

    The initial stages of transcription by RNA polymerase are frequently marked by pausing and stalling events. These events have been linked to an inactive backtracked state in which the polymerase diffuses along the template DNA. We investigate theoretically the influence of RNA secondary structure in confining this diffusion. The effective confinement length peaks at transcript lengths commensurate with early stalling. This finite-size effect accounts for slow progress at the beginning of transcription, which we illustrate via stochastic hopping models for backtracking polymerases.

  20. Influence of secondary structure on recovery from pauses during early stages of RNA transcription

    NASA Astrophysics Data System (ADS)

    Klopper, A. V.; Bois, J. S.; Grill, S. W.

    2010-03-01

    The initial stages of transcription by RNA polymerase are frequently marked by pausing and stalling events. These events have been linked to an inactive backtracked state in which the polymerase diffuses along the template DNA. We investigate theoretically the influence of RNA secondary structure in confining this diffusion. The effective confinement length peaks at transcript lengths commensurate with early stalling. This finite-size effect accounts for slow progress at the beginning of transcription, which we illustrate via stochastic hopping models for backtracking polymerases.

  1. SOAPfuse: an algorithm for identifying fusion transcripts from paired-end RNA-Seq data

    PubMed Central

    2013-01-01

    We have developed a new method, SOAPfuse, to identify fusion transcripts from paired-end RNA-Seq data. SOAPfuse applies an improved partial exhaustion algorithm to construct a library of fusion junction sequences, which can be used to efficiently identify fusion events, and employs a series of filters to nominate high-confidence fusion transcripts. Compared with other released tools, SOAPfuse achieves higher detection efficiency and consumed less computing resources. We applied SOAPfuse to RNA-Seq data from two bladder cancer cell lines, and confirmed 15 fusion transcripts, including several novel events common to both cell lines. SOAPfuse is available at http://soap.genomics.org.cn/soapfuse.html. PMID:23409703

  2. Polyester: simulating RNA-seq datasets with differential transcript expression

    PubMed Central

    Frazee, Alyssa C.; Jaffe, Andrew E.; Langmead, Ben; Leek, Jeffrey T.

    2015-01-01

    Motivation: Statistical methods development for differential expression analysis of RNA sequencing (RNA-seq) requires software tools to assess accuracy and error rate control. Since true differential expression status is often unknown in experimental datasets, artificially constructed datasets must be utilized, either by generating costly spike-in experiments or by simulating RNA-seq data. Results: Polyester is an R package designed to simulate RNA-seq data, beginning with an experimental design and ending with collections of RNA-seq reads. Its main advantage is the ability to simulate reads indicating isoform-level differential expression across biological replicates for a variety of experimental designs. Data generated by Polyester is a reasonable approximation to real RNA-seq data and standard differential expression workflows can recover differential expression set in the simulation by the user. Availability and implementation: Polyester is freely available from Bioconductor (http://bioconductor.org/). Contact: jtleek@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25926345

  3. Dysregulation of global microRNA expression in splenic marginal zone lymphoma and influence of chronic hepatitis C virus infection.

    PubMed

    Peveling-Oberhag, J; Crisman, G; Schmidt, A; Döring, C; Lucioni, M; Arcaini, L; Rattotti, S; Hartmann, S; Piiper, A; Hofmann, W-P; Paulli, M; Küppers, R; Zeuzem, S; Hansmann, M-L

    2012-07-01

    The precise molecular pathogenesis of splenic marginal zone lymphoma (SMZL) is still unknown. Clinical and epidemiological data suggest that chronic hepatitis C virus (HCV) infection may have an etiological role in a subset of cases.We performed a large-scale microRNA (miRNA) expression profiling analysis of 381 miRNAs by quantitative reverse transcription PCR (Q-RT-PCR) of 26 microdissected splenic tissue samples (7 HCV(+) SMZL; 8 HCV(-) SMZL and 11 non-neoplastic splenic controls). Single assay Q-RT-PCR and miRNA in situ hybridization (miRNA-ISH) were used to confirm the results in an independent cohort. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a distinct signature of SMZL compared with the normal splenic marginal zone. Supervised analysis revealed differentially expressed miRNAs, including miRNAs with previously recognized tumor suppressive or oncogenic potential. Five miRNAs were found significantly overexpressed in SMZL, including miR-21, miR-155 and miR-146a, whereas seven miRNAs showed significantly reduced expression, including miR-139, miR-345, miR-125a and miR-126. Furthermore, we identified miR-26b, a miRNA known to have tumor suppressive properties, as significantly downregulated in SMZL arising in HCV-positive patients (P=0.0016). In conclusion, there is a characteristic dysregulation of miRNA expression in SMZL with a possible implication in its molecular tumorigenesis.

  4. MicroRNA panels as disease biomarkers distinguishing hepatitis B virus infection caused hepatitis and liver cirrhosis.

    PubMed

    Jin, Bo-Xun; Zhang, Yong-Hong; Jin, Wen-Jing; Sun, Xiang-Ying; Qiao, Gui-Fang; Wei, Ying-Ying; Sun, Li-Bo; Zhang, Wei-Hong; Li, Ning

    2015-10-12

    An important unresolved clinical issue is to distinguish hepatitis B virus (HBV) infection caused chronic hepatitis and their corresponding liver cirrhosis (LC). Recent research suggests that circulating microRNAs are useful biomarkers for a wide array of diseases. We analyzed microRNA profiles in the plasmas of a total of 495 chronic hepatitis B (CHB) patients, LC patients and healthy donors and identified 10 miRNAs that were differentially expressed between CHB and LC patients. Our logistic models show that three panels of miRNAs have promising diagnostic performances in discriminating CHB from LC. Blinded tests were subsequently conducted to evaluate the diagnostic performances in clinical practice and a sensitivity of 85% and specificity of 70% have been achieved in separating CHB from LC pateints. The expression levels of some circulating miRNAs were significantly correlated with HBV DNA load and liver function, such as prothrombin activity (PTA) and levels of alanin aminotransferase (ALT), albumin (ALB) and cholinesterase (CHE). Our results provide important information for developing novel diagnostic tools for distinguishing chronic HBV hepatitis and their corresponding cirrhosis.

  5. Studying the association of microRNA-210 level with chronic hepatitis B progression.

    PubMed

    Song, G; Jia, H; Xu, H; Liu, W; Zhu, H; Li, S; Shi, J; Li, Z; He, J; Chen, Z

    2014-04-01

    We studied the relationship between hypoxia and microRNA-210 (miR-210) levels, the miR-210 levels in patients with hepatitis B and the roles of miR-210 in liver inflammation. We used the concanavalin A (Con A) murine hepatitis model and inflammation, hypoxia and miR-210 levels were examined. In these patients, we studied serum miR-210 levels and clinical indexes related to hepatitis in 90 patients with different stages of chronic hepatitis B and 30 controls. Two functional assays of miR-210 in vitro under hypoxic condition were conducted. The animal experiments indicated that the liver and serum miR-210 levels significantly increased with liver hypoxia and inflammation. In humans, serum miR-210 levels enhanced with hepatitis severity and were related to serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB) and prothrombin activity (PTA) levels. The miR-210 functional assays showed that miR-210 elevation might be related to the decreases in HepG2.2.15 cell dehydrogenase activity and HBV replication under hypoxic conditions. Because the liver inflammation causes liver hypoxia which also results in liver and serum miR-210 level elevation, the serum miR-210 level may serve as a molecular biomarker for the severity of hepatitis and increases in liver miR-210 that we see may be a response of hepatocytes to hypoxia during hepatitis progression.

  6. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    PubMed Central

    Khaja, Fatima; Jayawardena, Dulari; Kuzmis, Antonina; Önyüksel, Hayat

    2016-01-01

    Since its discovery, small interfering RNA (siRNA) has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA) not easily accessed by conventional drugs. Hence, RNA interference (RNAi) therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs). This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF), an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP), showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  7. Hepatitis C Test

    MedlinePlus

    ... Hepatitis C Antibody; Anti-HCV; HCV-PCR; HCV-RNA; Hepatitis C Viral Load Formal name: Viral Hepatitis C Antibody Screen; Viral Hepatitis C RNA by PCR; Hepatitis C Virus Genotype Related tests: ...

  8. Heat shock transcription factor 1 is a key determinant of HCC development by regulating hepatic steatosis and metabolic syndrome

    PubMed Central

    Jin, Xiongjie; Moskophidis, Demetrius; Mivechi, Nahid F.

    2011-01-01

    Hepatocellular carcinoma (HCC) occurrence and progression is linked tightly to progressive hepatic metabolic syndrome associated with insulin resistance, hepatic steatosis, and chronic inflammation. Heat shock transcription factor 1 (HSF1), a major transactivator of stress proteins, increases survival by protecting cells against environmental stressors. It has been implicated in the pathogenesis of cancer, but specific mechanisms by which HSF1 supports cancer development remain elusive. We propose a pathogenic mechanism whereby HSF1 activation promotes growth of pre-malignant cells and HCC development by stimulating lipid biosynthesis and perpetuating chronic hepatic metabolic disease induced by carcinogens. Our work shows that inactivation of HSF1 impairs cancer progression, mitigating adverse effects of carcinogens on hepatic metabolism by enhancing insulin sensitivity and sensitizing activation of AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis and inhibitor of lipid synthesis. HSF1 is a potential target for the control of hepatic steatosis, hepatic insulin resistance, and HCC development. PMID:21723507

  9. 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

  10. Genetically regulated hepatic transcripts and pathways orchestrate haematological, biochemical and body composition traits

    PubMed Central

    Ponsuksili, Siriluck; Trakooljul, Nares; Hadlich, Frieder; Haack, Fiete; Murani, Eduard; Wimmers, Klaus

    2016-01-01

    The liver is the central metabolic organ and exhibits fundamental functions in haematological traits. Hepatic expression, haematological, plasma biochemical, and body composition traits were assessed in a porcine model (n = 297) to establish tissue-specific genetic variations that influence the function of immune-metabolism-correlated expression networks. At FDR (false discovery rate) <1%, more than 3,600 transcripts were jointly correlated (r = |0.22–0.48|) with the traits. Functional enrichment analysis demonstrated common links of metabolic and immune traits. To understand how immune and metabolic traits are affected via genetic regulation of gene expression, eQTLs were assessed. 20517 significant (FDR < 5%) eQTLs for 1401 transcripts were identified, among which 443 transcripts were associated with at least one of the examined traits and had cis-eQTL (such as ACO1 (6.52 × 10−7) and SOD1 (6.41 × 10−30). The present study establishes a comprehensive view of hepatic gene activity which links together metabolic and immune traits in a porcine model for medical research. PMID:28000754

  11. Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription.

    PubMed

    Chan, B; Weidemaier, K; Yip, W T; Barbara, P F; Musier-Forsyth, K

    1999-01-19

    We report here the direct measurement of intra-tRNA distances during annealing of the tRNA primer to the HIV RNA genome. This key step in the initiation of retroviral reverse transcription involves hybridization of one strand of the acceptor arm of a specific lysine tRNA to the primer binding site on the RNA genome. Although the mechanism of tRNA unwinding and annealing is not known, previous studies have shown that HIV nucleocapsid protein (NC) greatly accelerates primer/template binary complex formation in vitro. An open question is whether NC alone unwinds the primer or whether unwinding by NC requires the RNA genome. We monitored the annealing process in solution by using fluorescence resonance energy transfer (FRET). Distance measurements demonstrate unequivocally that the tRNA acceptor stem is not substantially unwound by NC in the absence of the RNA genome, that is, unwinding is not separable from hybridization. Moreover, FRET measurements show that both heat- and NC-mediated annealing result in an approximately 40-A increase in the separation of the two ends of the tRNA acceptor arm on binding to the template. This large increase in separation of the two ends suggests a complete displacement of the nonhybridized strand of the acceptor stem in the initiation complex.

  12. 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

  13. RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination

    PubMed Central

    Figueroa-Bossi, Nara; Schwartz, Annie; Guillemardet, Benoit; D’Heygère, François; Bossi, Lionello; Boudvillain, Marc

    2014-01-01

    RNA-binding protein CsrA is a key regulator of a variety of cellular processes in bacteria, including carbon and stationary phase metabolism, biofilm formation, quorum sensing, and virulence gene expression in pathogens. CsrA binds to bipartite sequence elements at or near the ribosome loading site in messenger RNA (mRNA), most often inhibiting translation initiation. Here we describe an alternative novel mechanism through which CsrA achieves negative regulation. We show that CsrA binding to the upstream portion of the 5′ untranslated region of Escherichia coli pgaA mRNA—encoding a polysaccharide adhesin export protein—unfolds a secondary structure that sequesters an entry site for transcription termination factor Rho, resulting in the premature stop of transcription. These findings establish a new paradigm for bacterial gene regulation in which remodeling of the nascent transcript by a regulatory protein promotes Rho-dependent transcription attenuation. PMID:24888591

  14. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

    PubMed

    Tillett, D; Burns, B P; Neilan, B A

    2000-03-01

    A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of tr