Priming of influenza viral RNA transcription by capped heterologous RNAs.
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Amanitins↗
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Mechanisms of action of ribavirin against distinct viruses.
The nucleoside analogue ribavirin has antiviral activity against many distinct viruses both in vitro and in vivo. Five distinct mechanisms have been proposed to explain the antiviral properties of ribavirin. These include both indirect mechanisms (inosine monophosphate dehydrogenase inhibition, immunomodulatory effects) and direct mechanisms (interference with RNA capping, polymerase inhibition, lethal mutagenesis). Recent concerns about bioterrorism have renewed interest in exploring the antiviral activity of ribavirin against unique viruses. In this paper, we review the proposed mechanisms of action with emphasis on recent discoveries, as well as the implications of ribavirin resistance. Evidence exists to support each of the five proposed mechanisms of action, and distinct virus/host combinations may preferentially favour one or more of these mechanisms during antiviral therapy.
Animals↗
The capped U6 small nuclear RNA is transcribed by RNA polymerase III.
U6 RNA is an abundant, capped, small nuclear RNA (snRNA) species associated with heterogeneous nuclear ribonucleoproteins in eukaryotic cells. U4 RNA and U6 RNA are hydrogen bonded in a 1:1 ratio in discrete small nuclear ribonucleoprotein particles that are required in pre-mRNA processing. Previous reports have established that the mRNAs and U1 to U5 U-snRNAs are synthesized by RNA polymerase II. Evidence is presented here for synthesis of U6 RNA by RNA polymerase III. The synthesis of U6 RNA in vitro, using Novikoff hepatoma or HeLa whole cell extracts, was not inhibited at low (1 microgram/ml) concentrations of alpha-amanitin, and only 35% inhibition occurred at 10 micrograms/ml concentration. The in vitro synthesized U6 RNA, like other RNA polymerase III transcripts, was associated with La antigen. The U6 RNA synthesized in vitro by the whole cell extracts was capped, but no other internal post-transcriptional modifications were found. Uridylic acid residues were also added post-transcriptionally to the 3'-end of U6 RNA in vitro. U6 RNA, though capped on its 5'-end, is transcribed by RNA polymerase III; this is the first report of a capped RNA molecule synthesized by RNA polymerase III.
Amanitins↗
The transcription of DNA in chicken mitochondria initiates from one major bidirectional promoter.
Transcription start sites of chicken mitochondrial DNA have been mapped in the control region by direct sequencing of in vitro capped mitochondrial RNA species, by primer extension and by S1 nuclease protection analysis. Transcription of the heavy strand initiates predominantly at a site 156 nucleotides upstream of the tRNA(Phe) gene, i.e. about 135 nucleotides further upstream than the corresponding sites in amphibia and mammals. On the opposite strand, transcription starts predominantly one nucleotide removed from the site in the heavy strand. The L-strand position start site is similar to that found in other vertebrates. The chicken mitochondrial DNA control region thus contains one major transcriptional promoter, whose bidirectional capacity is similar to the situation in amphibia but which contrasts to the mainly unidirectional capacity of mammalian promoters. In chicken mitochondria, the sequence comprising the start sites is A + T rich and contains an almost perfect inverted repeat which can be folded into a cruciform structure. The heavy and light strand initiation sites are flanked on their respective 3' ends by an octanucleotide sequence matching those surrounding the start sites in Xenopus laevis (5'-ACPuTTATA-3'). This motif is found associated with the H-strand start sites in mouse but is not present in human nor bovine mitochondrial DNA promoters.
Animals↗
The cap structure in eukaryotic messenger RNA as a mark of a strand carrying protein information.
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Base Sequence↗
Influenza virus inhibits RNA polymerase II elongation.
The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus, influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding to the CTD of initiating Pol II and subsequent cleavage of the capped 5' end of the nascent transcript, triggers premature Pol II termination.
Chromatin Immunoprecipitation↗
CAP interacts with RNA polymerase in solution in the absence of promoter DNA.
Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation. Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP. Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.
Base Sequence↗
In vivo and in vitro expression of porcine D-amino acid oxidase: in vitro system for the synthesis of a functional enzyme.
In vivo expression of D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, was examined by use of cloned complementary DNA [Fukui, K., Watanabe, F., Shibata, T., & Miyake, Y. (1987) Biochemistry 26, 3612-3618]. RNA blot hybridization analysis revealed that DAO is expressed abundantly in kidney and liver, is expressed significantly in brain, but is not expressed in lung of pig. Three mRNA species were expressed in kidney and liver, but only one was detected in brain. These results show the presence of tissue-specific regulation of DAO gene expression. In vitro expression of a functional enzyme was achieved through the construction of a recombinant plasmid containing an SP6 promoter and a restriction enzyme fragment of cDNA to generate a DAO-specific RNA transcript. The in vitro translation product of the capped RNA transcript showed significant catalytic activity, which was inhibited strongly by benzoate, a potent inhibitor of DAO. The kinetic properties of the in vitro synthesized enzyme were comparable to those of the purified enzyme from porcine kidney. It is now possible to synthesize a functional D-amino acid oxidase in vitro and to investigate its structure-function relationships.
Animals↗
Purification and molecular structure of RNA polymerase from influenza virus A/PR8.
The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.
Animals↗
Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism.
Mammalian capping enzymes are bifunctional proteins with both RNA 5'-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5'-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO43- and independent of Mg2+, providing additional evidence for phosphate removal from RNA 5' ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211-597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5' cap formation.
3T3 Cells↗
Cloning and characterization of 4EHP, a novel mammalian eIF4E-related cap-binding protein.
All eukaryotic mRNAs (except organellar) are capped at their 5' end. The cap structure (m7GpppN, where N is any nucleotide) is extremely important for the processing and translation of mRNA. Several cap-binding proteins that facilitate these processes have been characterized. Here we describe a novel human cytoplasmic protein that is 30% identical and 60% similar to the human translation initiation factor 4E (eIF4E). We demonstrate that this protein, named 4E Homologous Protein (4EHP), binds specifically to capped RNA in an ATP- and divalent ion-independent manner. The three-dimensional structure of 4EHP, as predicted by homology modeling, closely resembles that of eIF4E and site-directed mutagenesis analysis of 4EHP strongly suggests that it shares with eIF4E a common mechanism for cap binding. A putative function for 4EHP is discussed.
Amino Acid Sequence↗
Isolation and characterization of a new 110 kDa human nuclear RNA-binding protein (p110nrb).
RNA-protein interactions play key roles in many fundamental cellular processes such as RNA processing, RNA transport, and RNA translation. During our attempts to isolate the human U6 small nuclear RNA capping enzyme, we identified a new 110 kDa nuclear RNA-binding protein, designated p110nrb. The full-length cDNA clone for p110nrb was characterized, and it encodes a 963 amino acid polypeptide. It is a highly acidic protein (pI 5.28) and the carboxyl terminal portion contains two conserved RNP motifs. A databank search found a putative C. elegans protein that might be the p110nrb homologue. The p110nrb was overexpressed as a glutathione S-transferase fusion protein in insect Sf9 cells, purified by affinity chromatography and injected into rabbits to produce specific polyclonal antibodies. Immunofluorescent staining showed that p110nrb is distributed evenly throughout the nucleoplasm. Northern blots showed that the mRNA is expressed in all tissues examined. An in vitro RNA-binding assay showed that p110nrb bound to RNA. These data suggest that p110nrb may play a role in the metabolism of nuclear RNA.
Amino Acid Sequence↗
RNA polymerase of influenza virus: role of NP in RNA chain elongation.
Ribonucleoprotein (RNP) cores of influenza virus A/PR/8/34 were dissociated into RNA polymerase (PB1-PB2-PA complex)-associated genome RNA and nuclear protein (NP) fractions by CsCl centrifugation. The RNA polymerase-RNA complexes were capable of catalyzing the endonucleolytic cleavage of capped RNA, the initiation of primer-dependent RNA synthesis, and the synthesis of small-sized RNA, but were unable to synthesize template-sized RNA. By adding the NP protein to the RNA polymerase-RNA complexes, RNP (RNA polymerase-RNA-NP) complexes were reconstituted; they synthesized template-sized transcripts as did native RNP cores. These observations are consistent with the model where viral RNA polymerase is composed of the three P proteins while NP is essential for the elongation of RNA chains. RNP was completely dissociated into RNA-free proteins (PB1, PB2, PA, and NP) and a protein-free genome RNA fraction by centrifugation in cesium trifluoroacetate (CsTFA) and glycerol. By mixing the protein and RNA fractions, primer-dependent RNA-synthesizing activity was regained. These complexes, however, produced only small-sized RNA, presumably due to incorrect assembly of NP on viral RNA.
DNA-Directed RNA Polymerases↗
RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8.
Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P1, P2, and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation.
DNA-Directed RNA Polymerases↗
Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein.
Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
Base Sequence↗
Purification and characterization of a 5' to 3' exoribonuclease from rabbit reticulocytes that degrades capped and uncapped RNAs.
The cytoplasm of mammalian cells of undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5' to 3' manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat-labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10-200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5' end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate-terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate-limiting step involves the positioning of the enzyme at the 5' end of the substrate and/or cleavage of the first internucleotide bond.
Animals↗
Nuclear snRNA and nuclear function (discovery of 5' cap structures in RNA).
In view of the fact that eukaryotic gene expression starts in the nucleus, it is important to have a thorough understanding of nuclear macromolecular structure and function. Newly discovered snRNAs are eukaryotic cell specific and have unique subnuclear compartmental localizations. There are over 200 nucleolar-specific RNAs that include some abundant U3, U8, and U13 RNAs. Extranucleolar-nuclear-specific RNAs (snRNA) are 4.5S RNA I, II, III, 5S RNA III, U1, U2, U4, U5, and U6, in addition to over 500 different RNA species reported up to now. In particular, some snoRNAs and snRNAs have trimethylguanosine cap structures that are not present in bacteria. They have crucial roles in gene expression, such as transcription (U3 snoRNA), processing (U3, U8, U13, U14, U22, and 7-2/MRP), methylation (U14-16, U18, U20-21, and U24-63), pseudouridylation (E2, E3, U19, U23, and U64-72), and hnRNA splicing (U1, U2, U4, U5, and U6 snRNA).
Base Sequence↗
A simple assay for determining the capping efficiencies of RNA polymerases used for in vitro transcription.
We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.
Boronic Acids↗