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At least 667 records · Page 37Linked to original sources
PubMed2003

C114 is a novel IL-11-inducible nuclear double-stranded RNA-binding protein that inhibits protein kinase R.

We have identified a cDNA (named C114) that encodes novel transcripts induced by IL-11 in mouse 3T3 L1 cells. Northern analysis of RNAs from multiple mouse tissues detects two C114 transcripts of approximately 1.0 and approximately 2.0 kb with the highest expression in liver, testis, brain, and kidney. The C114 cDNA contains an open reading frame of 187 amino acids with a predicted mass of 21 kDa. Three putative nuclear localization signals are predicted at amino acids 83-88, 126-131, and 167-178. Using green fluorescent protein (GFP)-C114 fusion plasmids, amino acids 126-131 are shown to be essential for the nuclear localization of C114. An arginine-rich region (amino acids 98-143) spanning the nuclear localization signals (amino acids 126-131) exhibits a double-stranded RNA (dsRNA) binding activity. Competition experiments with different RNA homopolymers demonstrate that C114 preferentially binds to poly(I.C). Similar to other dsRNA-binding proteins, C114 binds to the dsRNA-activated protein kinase, protein kinase R (PKR), via dsRNA-binding domains of PKR and the N-terminal region of the C114 protein. In vitro kinase assays indicate that C114 inhibits PKR activation via a dsRNA-independent mechanism. Overexpression of C114 protein inhibits the induction of eIF-2alpha phosphorylation following poly(I.C) treatment. This is the first demonstration of a novel PKR modulator induced by a gp130 superfamily cytokine that may play a role in cytokine-mediated biological functions.

3T3 Cells↗
PubMed2004

Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus.

Influenza A is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Toll-like receptor (TLR) 3 was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA may be critical for the outcome of the infection. Here we first compared the activation triggered by either influenza A virus or dsRNA in pulmonary epithelial cells. We established that TLR3 is constitutively expressed in human alveolar and bronchial epithelial cells, and we describe its intracellular localization. Expression of TLR3 was positively regulated by the influenza A virus and by dsRNA but not by other inflammatory mediators, including bacterial lipopolysaccharide, the cytokines tumor necrosis factor-alpha and interleukin (IL)-1beta, and the protein kinase C activator phorbol 12-myristate 13-acetate. We also demonstrated that TLR3 contributes directly to the immune response of respiratory epithelial cells to influenza A virus and dsRNA, and we propose a molecular mechanism by which these stimuli induce epithelial cell activation. This model involves mitogen-activated protein kinases, phosphatidylinositol 3-kinase/Akt signaling, and the TLR3-associated adaptor molecule TRIF but not MyD88-dependent activation of the transcription factors NF-kappaB or interferon regulatory factor/interferon-sensitive response-element pathways. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES (regulated on activation normal T cell expressed and secreted), and interferon-beta and the up-regulation of the major adhesion molecule ICAM-1.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone.

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Cell Extracts↗
PubMed1971

Antibodies to polynucleotides in human sera: antigenic specificity and relation to disease.

The specificities of anti-polynucleotide antibodies found in human sera were studied using several immunological procedures. Anti-native DNA (NDNA) antibodies and certain anti-double-stranded RNA (DSRNA) antibodies were found to react with single-stranded DNA (SDNA), and anti-NDNA antibodies were observed to react more avidly with SDNA than with NDNA in most sera tested. Antibodies to NDNA showed no preferential reactivity with NDNA or SDNA derived from mammalian tissue, bacterial, or viral sources. Precipitating antibodies reactive with individual bases, with common determinants of bases, and with common determinants of SDNA and NDNA were detected utilizing synthetic polydeoxyribonucleotides. Antibodies to DSRNA were also heterogeneous and reactive with both Poly A . Poly U and Poly I . Poly C in addition to reactivity with Poly A and SDNA. In contrast, antibodies to a ribonucleo-protein determined by hemagglutination and by precipitation showed no reaction with NDNA, SDNA, or DSRNA. Serial studies of serum specimens from patients with systemic lupus erythematosus (SLE) indicated that anti-NDNA antibodies were closely associated with disease activity. Titers of antibodies to SDNA or DSRNA were also frequently increased during these periods but in addition showed peaks during quiescent periods. Anti-NDNA antibodies were detected in most patients' sera at sometime during the course of the disease. Three patients were observed with active SLE, who did not develop anti-NDNA antibodies, even in the presence of severe renal disease. Evidence that other antigen-antibody systems may also play a role in the pathogenesis of the renal disease was particularly apparent in these patients. Anti-ribonucleoprotein antibodies were not well correlated with the peaks of antibody activity of other polynucleotide antibodies, suggesting that an independent immunogen was responsible for induction of these antibodies. The close association of certain populations of anti-polynucleotide antibodies during the course of active SLE, the presence of cross-reacting antigenic determinants of SDNA, NDNA, and DSRNA, the preferential avidity of anti-NDNA antibodies for SDNA, and the frequent increase of anti-SDNA antibodies in SLE and other diseases associated with active tissue destruction suggest that SDNA is a ubiquitous antigen that may stimulate the formation of antibodies reactive with a variety of polynucleotides.

Animals↗
PubMed1987

Trichomonas vaginalis phenotypic variation occurs only among trichomonads infected with the double-stranded RNA virus.

Trichomonas vaginalis isolates were examined for the presence of viral double-stranded RNA (dsRNA) and the property of phenotypic variation. Only the heterogeneous isolates composed of mAb-reactive and -nonreactive organisms, as determined by indirect immunofluorescence and flow cytofluorometry, and capable of phenotypic variation possessed the dsRNA. Both the positive and negative phenotype subpopulations separated from the heterogeneous parent contained equal amounts of the dsRNA. Loss of the dsRNA upon prolonged in vitro cultivation always correlated with the lack of expression of the major immunogen. The data indicate a relationship between the presence of the dsRNA and the ability of the pathogenic human trichomonads to express immunogens on their surfaces and to undergo phenotypic variation.

Animals↗
PubMed1990

The binding of the 69- and 100-kD forms of 2',5'-oligoadenylate synthetase to different polynucleotides.

Three major forms of 2',5' oligoadenylate (2-5A) synthetase are induced in interferon (IFN)-treated human cells: 40-46, 69, and 100 kD. Here we studied the binding and activation of the 69- and 100-kD forms to single-stranded (ss) or double-stranded (ds) RNAs. The 69- and 100-kD form enzymes purified by immunoaffinity chromatography, were shown to be activated by synthetic dsRNAs poly(I).poly(C) or poly(A).poly(U) whereas ssRNAs poly(I), poly(C), poly(A), poly(U), and poly(G) had no effect. Both enzymes were also partially purified by binding to dsRNA or ssRNA-Sepharose. The synthetases bound to dsRNA Sepharose were partially activated but required the addition of soluble poly(I).poly(C) for maximal activity. The synthetases bound to ssRNA-Sepharose manifested no activity but became activated in the presence of soluble dsRNA, poly(I).poly(C). However, activation of such ssRNA-bound enzymes by dsRNA did not result in their dissociation from the ssRNA-Sepharose. These results indicate the presence of different polynucleotide binding sites on the 69- and 100-kD forms of 2-5A synthetase: a specific dsRNA binding site essential for activation and another polynucleotide binding site or sites which, although not specific, might be important for the optimal conformation of these proteins in cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Double-stranded RNA and interferon-alpha induce transcription through different molecular mechanisms.

Double-stranded (ds) RNA stimulates the synthesis of several mRNAs known to be induced by type I interferons (IFNs). In this report, it is shown that the IFN-alpha stimulated genes (ISGs) 15, 54, 56, and GBP are transcriptionally induced by dsRNA. Transcriptional stimulation occurred in the presence of the protein synthesis inhibitor cycloheximide (CHX), indicating that inducibility was directly mediated by dsRNA through the action of preformed proteins. ISGF-3, the protein complex mediating primary transcriptional induction of ISGs by IFN-alpha, was not activated by dsRNA in the presence of CHX. Additionally, DNA-binding activity of ISGF-2/IRF-1, a protein involved in the regulation of the IFN-beta gene and ISGs, did not correlate with dsRNA-induced transcriptional induction of ISGs. This suggests that dsRNA and IFN-alpha induce ISGs through different molecular mechanisms.

Cycloheximide↗
PubMed2004

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.

DNA, Complementary↗
PubMed2003

Inducible systemic RNA silencing in Caenorhabditis elegans.

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

Animals↗
PubMed2003

An ADAR that edits transcripts encoding ion channel subunits functions as a dimer.

In this report, we establish that Drosophila ADAR (adenosine deaminase acting on RNA) forms a dimer on double-stranded (ds) RNA, a process essential for editing activity. The minimum region required for dimerization is the N-terminus and dsRNA-binding domain 1 (dsRBD1). Single point mutations within dsRBD1 abolish RNA-binding activity and dimer formation. These mutations and glycerol gradient analysis indicate that binding to dsRNA is important for dimerization. However, dimerization can be uncoupled from dsRNA-binding activity, as a deletion of the N-terminus (amino acids 1-46) yields a monomeric ADAR that retains the ability to bind dsRNA but is inactive in an editing assay, demonstrating that ADAR is only active as a dimer. Different isoforms of ADAR with different editing activities can form heterodimers and this can have a significant effect on editing in vitro as well as in vivo. We propose a model for ADAR dimerization whereby ADAR monomers first contact dsRNA; however, it is only when the second monomer binds and a dimer is formed that deamination occurs.

Adenosine Deaminase↗
PubMed2000

A new double-stranded RNA-binding protein that interacts with PKR.

We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death.

Amino Acid Motifs↗
PubMed2002

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.

Animals↗
PubMed2003

Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies.

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.

Animals↗
PubMed2005

dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference.

Off-target effects are one of the most serious problems in RNA interference (RNAi). Here, we present dsCheck (http://dsCheck.RNAi.jp/), web-based online software for estimating off-target effects caused by the long double-stranded RNA (dsRNA) used in RNAi studies. In the biochemical process of RNAi, the long dsRNA is cleaved by Dicer into short-interfering RNA (siRNA) cocktails. The software simulates this process and investigates individual 19 nt substrings of the long dsRNA. Subsequently, the software promptly enumerates a list of potential off-target gene candidates based on the order of off-target effects using its novel algorithm, which significantly improves both the efficiency and the sensitivity of the homology search. The website not only provides a rigorous off-target search to verify previously designed dsRNA sequences but also presents 'off-target minimized' dsRNA design, which is essential for reliable experiments in RNAi-based functional genomics.

Algorithms↗
PubMed1990

Rotavirus RNA replication: single-stranded RNA extends from the replicase particle.

The rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used non-denaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SA11-infected MA104 cells and resolved on 0.6% agarose gels containing 50 mM-Tris-glycine pH 8.8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.

Animals↗
PubMed2000

Molecular characterization of the genome of a partitivirus from the basidiomycete Rhizoctonia solani.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungus Rhizoctonia solani isolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5' ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem-loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86-2275; molecular mass 86 kDa) and 683 aa (bases 79-2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses from Fusarium poae and Atkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.

Amino Acid Sequence↗
PubMed1999

Targeted mRNA degradation by double-stranded RNA in vitro.

Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

3' Untranslated Regions↗
PubMed2006

Structural basis for non-catalytic and catalytic activities of ribonuclease III.

Ribonuclease III (RNase III) represents a highly conserved family of double-stranded (ds) RNA-specific endoribonucleases, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. Bacterial RNase III, containing an endonuclease domain followed by a dsRNA-binding domain, is the most extensively studied member of the family. It can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves dsRNA or as a binding protein that binds but does not cleave dsRNA. The available biochemical and structural data support the existence of two distinct forms of the RNase III-dsRNA complex which reflect the dual activities of the protein. The information revealed by the structures of bacterial RNase III provides insight into the mechanism of dsRNA processing by all members of the family.

Animals↗