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Results for “double stranded RNA (dsRNA)”

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PubMed1995

Double-stranded RNA in rice: a novel RNA replicon in plants.

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5' non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3' non-coding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.

Amino Acid Sequence↗
PubMed2003

RNAse III-mediated degradation of unspliced pre-mRNAs and lariat introns.

Double-stranded RNA (dsRNA) has emerged as a modulator of gene expression, from gene silencing to antiviral responses. Here we show that dsRNA stem-loop structures found in intronic regions of the S. cerevisiae RPS22B and RPL18A transcripts trigger degradation of unspliced pre-mRNAs and lariat introns and can control the level of mRNA produced from these intron-containing genes. The dsRNA regions are cleaved by Rnt1p, the yeast ortholog of RNase III, which creates an entry site for complete degradation by the Xrn1p and Rat1p exonucleases and by the nuclear exosome. These results identify an alternative discard pathway for precursors and products of the splicing machinery and a physiological function for dsRNA in eukaryotic RNA catabolism.

Base Sequence↗
PubMed1990

Adapting the polymerase chain reaction to a double-stranded RNA genome.

We have adapted the polymerase chain reaction (PCR) to a double-stranded RNA (dsRNA) target without possessing unambiguous sequence information. Infectious bursal disease virus of chickens, a member of the binavirus group, has a dsRNA genome which is resistant to denaturation and subsequent enzyme modification. The only published sequence information was for a strain of virus unavailable to us. We have used a quick primer binding assay to select appropriate primers and have combined a simple denaturation method with reverse transcription and subsequent polymerization using the cDNA template to yield amplified product easily detectable by ethidium bromide staining. By varying the times of denaturation, annealing, and polymerization and by reducing the total number of amplification cycles, artifacts have been eliminated when using purified genome as the template. This allowed us to obtain partial sequence information for one viral strain. We have enhanced the utility of our method by optimizing a rapid cell lysis and capsid digestion protocol such that no purification steps are required from initial tissue handling through final PCR product. Total time for all procedures involved no more than 6 h. This technique should be applicable to all other members of the Birnaviradae family and to any other species of dsRNA.

Animals↗
PubMed2001

Presence of double-stranded RNA and virus-like particles in Rhizopus isolates.

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.

RNA, Double-Stranded↗
PubMedDate not supplied

Identification of viruses with bi- and trisegmented double-stranded RNA genome in faeces of children with gastroenteritis.

Polyacrylamide gel electrophoresis of nucleic acid extracted from faecal samples of diarrhoeic children revealed the presence of group A rotavirus in 50 (23.4%) samples and group C rotavirus in 1 (0.5%) sample out of 214 tested. One other sample showed the presence of three bands (with apparent length of 2.92, 2.37 and 1.32 kbp) which by enzymatic digestion analysis, were shown to consist of double-stranded RNA (dsRNA). The sample was shown by electron microscopy to contain virus particles with a diameter of 32-34 nm. On the basis of morphology and genomic characteristics, this virus closely resembles a virus hitherto described only in chickens by Leite et al. in 1990 and tentatively named "picotrirnavirus". From the same group of 214, one sample containing a "picobirnavirus" was also identified. Thus, small icosahedral viruses with either a bior trisegmented dsRNA genome appear to infect humans. However, their pathogenic potential remains to be established.

Child↗
PubMed1995

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae.

Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.

Fusarium↗
PubMed2006

Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA.

We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion.

Animals↗
PubMed2005

RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998; Guo and Kemphues, 1995). The completion of the C. elegans genome in 1998 coupled with the advent of RNAi techniques to knock down gene function ushered in a new age in the field of functional genomics. There are four methods for double-stranded RNA (dsRNA) delivery in C. elegans: (1) injection of dsRNA into any region of the animal (Fire et al., 1998), (2) feeding with bacteria producing dsRNA (Timmons et al., 2001), (3) soaking in dsRNA (Tabara et al., 1998), and (4) in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans.

Animals↗
PubMed2006

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Base Sequence↗
PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed1991

Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA.

Transmissible hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, is associated with cytoplasmic double-stranded-RNA (dsRNA) viruses. The fungal laccase has attracted interest because its activity is reduced in hypovirulent dsRNA-containing strains. A laccase cDNA clone was isolated by screening a cDNA expression library with antibodies against the purified extracellular laccase. The amino acid sequence deduced from part of the cDNA clone revealed high homology to other fungal laccases, especially to the Neurospora crassa laccase. A major laccase transcript 2.3 kb in length was detected in Northern (RNA) blots. In liquid culture, extracellular laccase activity was reduced by about 75% in the hypovirulent (dsRNA-free) strain EP155/2. In contrast, production of biomass was not affected by the dsRNA. Northern blot analysis indicated that dsRNA down regulates laccase biosynthesis by reducing laccase mRNA accumulation. The laccase gene is one of several developmentally regulated genes affected by the presence of dsRNA.

Amino Acid Sequence↗
PubMed2004

Methods for delivery of double-stranded RNA into Caenorhabditis elegans.

The nematode Caenorhabditis elegans is often employed in investigations of diverse aspects of biology, including behavior, development, basic cellular processes, and disease states. The ability to utilize double-stranded RNA (dsRNA) to inhibit specific gene function in this organism has dramatically increased its value for these kinds of studies and has provided more flexibility in experimental design that include procedures. Here, we have collected a set of protocols from the C. elegans community for propagation of C. elegans, techniques for dsRNA preparation, four basic methods for delivery of dsRNA to C. elegans (injection, soaking, feeding, and in vivo delivery), and we suggest schemes that should facilitate detection of specific gene silencing.

Animals↗
PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Animals↗
PubMed2001

The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1.

RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited.

Adenosine Deaminase↗
PubMed2002

Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation.

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.

Arginine↗
PubMed2001

Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.

BACKGROUND: Infection by virus or treatment with double-stranded RNA (dsRNA) results in the activation of transcription factors including IRF-3, IRF-7 and a pleiotropic regulator NF-kappaB by specific phosphorylation. These factors are important in triggering a cascade of antiviral responses. A protein kinase that is yet to be identified is responsible for the activation of these factors and plays a key role in the responses. RESULTS: The signal cascade was analysed using sensitive assays for the activation of IRF-3 and NF-kappaB, and various inhibitors. We found that the activation of IRF-3 and NF-kappaB by dsRNA or virus involves a process that is sensitive to Geldanamycin. Although the induction of NF-kappaB by dsRNA/virus and TNF-alpha involves common downstream pathways including IKK activation, the upstream, Geldanamycin-sensitive process was unique to the dsRNA/virus-induced signal. By an in vitro assay using cell extract, we found an inducible protein kinase activity with physiological specificity of IRF-3 phosphorylation. Furthermore, the same extract specifically phosphorylated IRF-7 in a similar manner. CONCLUSIONS: Double-stranded RNA or virus triggers a specific signal cascade that results in the activation of the IRF-3/-7 kinase we detected, which corresponds to the long-sought signalling machinery that is responsible for triggering the early phase of innate response. The signal branches to a common NF-kappaB activation cascade, thus resulting in the activation of a set of critical transcription factors for the response.

Benzoquinones↗
PubMed1983

Regulation of the antiviral and anticellular activities of interferon by exogenous double-stranded RNA.

The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation of dsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5' oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Yeast virology.

The three families of double-stranded RNA (dsRNA) viruses and two families of retroviruses (retrotransposons) of the yeast Saccharomyces cerevisiae are all transmitted between cells only by cell fusion, probably reflecting the high frequency of mating of yeast cells in nature. One dsRNA virus and two retroviruses apparently use ribosomal "frameshifting" to produce major coat protein-polymerase fusion proteins. This mechanism allows regulation of the relative amounts of major coat protein and fusion protein that are made and avoids the possibility of mutant virus genomes being generated by splicing. Moreover, the fusion protein structure suggests a possible mechanism of genome packaging. The recent development of in vitro replication, transcription, and integration systems for these viruses, and the ease with which classical genetic and molecular studies are executed in yeast, are yielding detailed information about the roles of cellular and viral components in the viral replication cycles and the host defensive response. A host defense system against yeast dsRNA viruses is known and there is evidence to suggest a system active against the retroviruses.

DNA Transposable Elements↗