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PubMed2003

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

Base Sequence↗

PubMed2006

Induction of the interferon response by siRNA is cell type- and duplex length-dependent.

Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.

Cell Survival↗

PubMed2003

RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase.

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.

Animals↗

PubMed2003

Inheritance of Oryza sativa endornavirus in F1 and F2 hybrids between japonica and indica rice.

We have found a 14 kbp double-stranded RNA (dsRNA) in many cultivars of japonica rice (Oryza sativa L.) but not in any cultivars of indica rice. This dsRNA is an RNA replicon with plasmid-like properties and is proposed to be a novel dsRNA virus, Oryza sativa endornavirus (OSV). Reciprocal crosses between the OSV-carrier japonica variety (Nipponbare) and the OSV-free indica variety (IR 26 or Kasalath) were performed to investigate whether OSV can be transmitted to F1 hybrids. When IR 26 and Nipponbare were used, efficient transmission of OSV from ova (93%) and pollen (89%) was observed. When Kasalath and Nipponbare were used, the OSV transmission efficiency to F1 progeny was 68% from ova and 20% from pollen. The transmission of OSV to F2 progeny plants was also complicated, showing non-Mendelian inheritance. These results suggest that the dsRNA replicon (OSV) is unstable in indica rice plants.

Chimera↗

PubMed2001

Double-stranded rna elements and virus-like particles in Aspergilli.

Mycoviruses with double-stranded RNA (dsRNA) genomes are frequently encountered in Aspergillus isolates. A detailed study of such dsRNA elements in black Aspergillus isolates collected worldwide was carried out, and the data were analysed. The results indicate that about 10% of black Aspergilli are infected. However, the geographic distribution of infected isolates exhibits large variations; 3-13% of the isolates collected from different continents were found to carry dsRNA elements. Hybridization experiments indicated that electrophoretic banding patterns are not reliable tools for estimating the diversity of these mycovirus genomes. Among strains representing other Aspergillus sections, dsRNA segments indicative of mycovirus infection were observed for the first time in 4 species (A. leporis, A. petrakii, A. fumigatus and A. primulinus). The latter species is able to reproduce sexually. This is the second report on the detection of naturally-occurring dsRNAs in sexually reproducing Aspergillus species. The presence of virus-like particles in these and other Aspergilli was also examined by electron microscopy. Most infected Aspergillus isolates examined were found to carry virus-like particles in the size range 36-40 nm.

Aspergillus↗

PubMed2000

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

Animals↗

PubMed2001

Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN.

Two classes of nucleic acids, bacterial DNA containing unmethylated CpG motifs and dsRNA in viruses, induce the production of type I IFN that contributes to the immunostimulatory effects of these microbial molecules. Thus, it is important to determine which cells produce type I IFN in response to CpG DNA and dsRNA. CD4(+)CD11c(-) type 2 dendritic cell precursors (pre-DC2) were identified as the main producers of type I IFN in human blood in response to viruses. Here we asked whether pre-DC2 also produce type I IFN in response to CpG DNA and dsRNA. Oligodeoxynucleotides containing particular palindromic CpG motifs induced pre-DC2, but not CD11c(+) blood DC or monocytes, to produce IFN-alpha. In contrast, a synthetic dsRNA, polyinosinic polycytidylic-acid, induced CD11c(+) DC, but not pre-DC2 or monocytes, to produce IFN-alphabeta. These data indicate that CpG DNA and polyinosinic-polycytidylic acid stimulate different types of cells to produce type I IFN and that it is important to select oligodeoxynucleotides containing particular CpG motifs to induce pre-DC2 to produce type I IFN, which may play a key role in the strong adjuvant effects of CpG DNA.

Cell Differentiation↗

PubMed2006

TLR3 can directly trigger apoptosis in human cancer cells.

TLRs function as molecular sensors to detect pathogen-derived products and trigger protective responses ranging from secretion of cytokines that increase the resistance of infected cells and chemokines that recruit immune cells to cell death that limits microbe spreading. Viral dsRNA participate in virus-infected cell apoptosis, but the signaling pathway involved remains unclear. In this study we show that synthetic dsRNA induces apoptosis of human breast cancer cells in a TLR3-dependent manner, which involves the molecular adaptor Toll/IL-1R domain-containing adapter inducing IFN-beta and type I IFN autocrine signaling, but occurs independently of the dsRNA-activated kinase. Moreover, detailed molecular analysis of dsRNA-induced cell death established the proapoptotic role of IL-1R-associated kinase-4 and NF-kappaB downstream of TLR3 as well as the activation of the extrinsic caspases. The direct proapoptotic activity of endogenous human TLR3 expressed by cancerous cells reveals a novel aspect of the multiple-faced TLR biology, which may open new clinical prospects for using TLR3 agonists as cytotoxic agents in selected cancers.

Apoptosis↗

PubMed2002

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene.

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies.

Animals↗

PubMed1999

RNA-dependent RNA polymerase activity associated with Eimeria necatrix virus particles containing either double-stranded or single-stranded RNA.

Single-stranded (ss) RNA containing and double-stranded (ds) RNA containing virus particles of Eimeria necatrix were isolated by centrifugation through a CsCl gradient. RNA from the gradient fractions was identified as single-stranded or double-stranded by probing northern blots with digoxigenin-labeled riboprobes. These probes were generated with SP6 and T7 RNA polymerases from a partial cDNA clone derived from 5.6-kb viral dsRNA of E. necatrix. RNA-dependent RNA polymerase (RDRP) activity was identified in these CsCl-purified virus particles. The polymerase products of the ssRNA particles consisted of dsRNA indicating replicase activity, whereas the polymerase products of the dsRNA particles consisted of ssRNA indicating transcriptase. activity. RNase treatment in high salt solution (0.3 M NaCl) of the pooled RDRP products revealed that the products consisted of both RNase-resistant dsRNA and RNase-sensitive ssRNA. These results show that both replicase and transcriptase activities were present in the purified virus. The digoxigenin-labeled products hybridized to both SP6 and T7 transcripts confirming the presence of both activities.

Animals↗

PubMed2001

RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs.

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21-25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma bruceiby expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 x g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20-30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24-26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24-26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24-26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Actins↗

PubMed2002

[The use of RNAi as a technique to study the functions of heart-related genes in Drosophila].

RNAi is a recently developed method to block the activity of cellular genes by artificially providing sense and anti-sense RNA corresponding to a target gene. By inducing rapid degradation of the corresponding endogenous mRNA and blocking new mRNA synthesis, RNAi leads to post-transcriptional gene silencing. Now this phenomenon has been claimed to exist in C. elegans, Drosophila, buds, fungi and plants and is being used to study the functions of some special genes or the known genes at specific time point. It is extremely useful for those genes or organisms that their mutants are not easily obtained. The Drosophila heart related genes, tinman and wingless, have been shown to play an important role in coordinating the early formation of heart progenitor cells and precursors, yet the late function is still unexplored. In this experiment, we took the advantage of RNAi technique, microinjected tinman and wingless dsRNA into the early embryos in Drosophila respectively and got these two genes' RNAi phenotypes, which were very similar to that of their mutant, showing heart tube defects or no heart precursors formation. tinman dsRNA even caused visceral mesoderm defects and the somatic muscles disruption, yet wingless dsRNA only affected heart precursors and had no effect on visceral mesoderm and somatic muscles, indication that the heart-related genes dsRNA interference worked effectively and exclusively in Drosophila.

Animals↗

PubMed2002

Sodium pentosan polysulfate reduces urothelial responses to inflammatory stimuli via an indirect mechanism.

PURPOSE: Sodium pentosan polysulfate has been promoted as a urothelial cytoprotective agent for treating interstitial cystitis. The nuclear transcription factor nuclear factor kappaB is thought to have a role in mediating the urothelial inflammatory response of interstitial cystitis. We further defined a possible cytoprotective effect of sodium pentosan polysulfate by characterizing the effect of the drug on the expression of nuclear factor kappaB. MATERIALS AND METHODS: For cell culture human urothelial cells were incubated in various concentrations of sodium pentosan polysulfate for 16 hours in keratinocyte serum-free medium. They were subsequently treated with the known nuclear factor kappaB stimulants tumor necrosis factor-alpha, lipopolysaccaride (LPS) and double-stranded RNA (dsRNA). Each stimulant was then incubated with sodium pentosan polysulfate separately and the mixture was used to treat cultured urothelial cells. For electrophoretic mobility shift assay total cell extracts were prepared and run in electrophoretic mobility shift assays using a radiolabeled nuclear factor kappaB consensus sequence as a probe. Western blot analysis was done to assess nuclear factor kappaB activation by measuring degradation of the inhibitory subunit of the nuclear factor kappaB complex. RESULTS: Nuclear factor kappaB activation by tumor necrosis factor-alpha, LPS and dsRNA was unaltered when cultured cells were incubated in sodium pentosan polysulfate before treatment. In contrast, nuclear factor kappaB activation by LPS and dsRNA was suppressed when the stimulants were incubated with sodium pentosan polysulfate before cell treatment. This suppressive effect was confirmed by Western blot analysis. CONCLUSION: Sodium pentosan polysulfate may have a nonspecific effect against the viral (dsRNA) and bacterial (LPS) activation of nuclear factor kappaB. The observed clinical effect of sodium pentosan polysulfate may be mediated by nonspecific binding of sodium pentosan polysulfate molecules and the inflammatory stimulants of urothelial activation. These findings suggest a mechanism of action for sodium pentosan polysulfate that occurs in the urine rather than at the mucosal membrane by direct interaction of the drug with potential interstitial cystitis inducing inflammatory agents.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1992

Interleukin-1 alpha mediates an alternative pathway for the antiproliferative action of poly(I.C) on human endothelial cells.

The antiproliferative effect of double-stranded RNA (dsRNA) on human tumor and normal cells has been well established. However, the genes involved in the dsRNA-induced antiproliferative response and the molecular mechanisms by which this occurs remain less well defined. We have studied the ability of synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I.C)) to modify human umbilical vein endothelial cell (HUVEC) growth and report that poly(I.C) induces a dose-dependent inhibition of HUVEC proliferation in vitro. In addition, the mRNA levels for the cytokines interleukin-1 alpha (IL-1 alpha) and interferon-beta 1 are induced in poly(I.C)-treated cells. Moreover, the growth inhibitory effects of poly(I.C) are relieved when cells are grown in the presence of an IL-1 alpha antisense oligonucleotide to the human IL-1 alpha transcript. Thus, the effects of poly(I.C) appear to be mediated, in part, through the function of IL-1 alpha, suggesting an alternative pathway for dsRNA-mediated inhibition of human endothelial cell growth.

Base Sequence↗

PubMed2003

[RNA interference and its promising future].

RNA interference(RNAi) is the process of sequence-specific,post-transcriptional gene silencing initiated by double-stranded RNA(dsRNA). Recent studies showed small interfering RNAs (siRNA) generated by Dicer from longer dsRNA specifically suppressed expression of genes in somatic and embryo cells. An RdRP activity might provide amplification by replication of long trigger dsRNAs or copying of short siRNAs in a primer-independent manner. The siRNA primed RdRP reaction converts target mRNA into dsRNA, as well as possibly replicating trigger dsRNA. Both products then serve as DICER substrates, initiating the RdRP chain reaction. The article reviewed possible mechanisms of RNAi and pointed out the promising future of RNAi as a tool for studying gene function and used in gene-specific therapeutics.

English Abstract↗

PubMedDate not supplied

[Great potential of small RNAs: RNA interference and microRNA].

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

Humans↗

PubMed1990

Electrophoresis of the complementary strands of the double-stranded Kemerovo virus RNAs in agarose-urea gel.

Single-stranded (ss)RNAs derived from 10 double-stranded (ds)RNA segments of Kemerovo virus (KV) were separated into 13 RNA bands by agarose-urea gel electrophoresis. The complementary strands of the dsRNA segments 1, 9 and 10 displayed different electrophoretic mobility. An attempt was made to determine the origin of the ssRNA bands. The ssRNA bands originating from the dsRNA segments 1, 2, 3, 9 and 10 were identified unequivocally, while those originating from the dsRNA segments 4, 5, 6, 7 and 8 were characterized partially. The minus RNA strands of the dsRNA segments 9 and 10 exhibited higher electrophoretic mobilities as their complementary plus RNA strands.

Electrophoresis, Agar Gel↗

PubMed1987

[Comparative study of interferon inducers obtained from natural sources].

Interferon-inducing and antiviral effects of natural dsRNA preparations of phage phi 6 and yeast cells were studied in the culture of murine cells L-929 and on random bred albino mice. Both the preparations showed interferon inducing activity in the cell culture. However, for realization of their effect modification of the surface cell membrane by polycation exchange resin (DEAE-dextran) was required. The interferon-inducing activity of both of the natural dsRNA in the mice was high. The maximum interferon titers (1280-5120 units/ml) in blood serum were observed 4-6 hours after the inductor intraperitoneal administration. The interferon-inducing activity of the phage dsRNA was high in the cell culture and yeast dsRNA--in mice, respectively. Both the inductors had antiviral activity and protected 15 to 38.9 per cent of the experimental animals from the effect of 100 LD50 of the murine encephalomyocarditis virus and 10 LD50 of the influenza virus A/Aichi 2/68 (H3N2).

Animals↗