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PubMed1986

Generation of cDNA clones of the bacteriophage phi 6 segmented dsRNA genome: characterization and expression of L segment clones.

Bacteriophage phi 6 has three dsRNA genome segments of about 3.0, 4.0, and 6.4 kbp. More than 90% of the segmented phi 6 dsRNA genome has been cloned as subchromosomal cDNA fragments, generated by reverse transcription of denatured polyadenylated dsRNA, RNA removal, annealing, filling, size fractionation, tailing, and insertion at the PstI site of pBR322. All of the large (L) segment is represented by five overlapping fragments, 98% of the small (S) segment is present in three fragments, and 67% of the medium (M) segment is contained in two fragments. Fragments have been aligned in linear arrays by Southern blot hybridization and restriction enzyme analysis. The orientation of the ordered fragments with respect to genomic RNA and phi 6 transcriptional direction was determined by comparison of terminal DNA sequences with RNA sequences at the genomic ends of phi 6 RNA. Expression of L segment clones using both Escherichia coli minicells and T7 polymerase/promoter vectors indicate that the order of known phi 6 genes on the large chromosome is: 5'--gene 7, gene 2, gene 4, gene 1--3'. cDNA complementation of a ts mutant, ts411, has located this mutation in gene 4.

Bacteriophages↗
PubMed1984

Sequence of the preprotoxin dsRNA gene of type I killer yeast: multiple processing events produce a two-component toxin.

The preprotoxin gene of the 1.9 kb M1 dsRNA genome from type I killer yeast has been sequenced employing a partial-length cDNA derived from an in vivo transcript. A single open reading frame, commencing with AUG at M1 dsRNA bases 14-16, terminates with UAG at 963-965 and codes for a 316 amino acid protein, believed to be identical to the 34 kd preprotoxin species, M1-P1, synthesized by in vitro translation of denatured M1 dsRNA. N-terminal sequencing of M1-P1 confirms this prediction. Secreted toxin is shown to consist of two dissimilar, disulfide-bonded subunits, alpha and beta, of apparent size 9.5 and 9.0 kd, respectively, whose N-terminal sequences are also found in the predicted preprotoxin sequence. Its proposed domains consist of delta, a 44 amino acid N-terminal segment, followed by alpha and beta, which are separated by gamma, a large central glycosylated segment. Processing sites, domain functions, and the potential role of gamma in immunity are discussed.

Base Sequence↗
PubMed1995

Differentiation of cognate dsRNA genome segments of bluetongue virus reassortants by temperature gradient gel electrophoresis.

The analysis of reassortant viruses has been a valuable tool in the investigation of protein interaction and function in double-stranded (ds) RNA virus research. The differentiation of cognate dsRNA genome segments of reassortants is conventionally achieved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, due to a high degree of sequence homology among different bluetongue virus (BTV) serotypes, it is not uncommon to find that certain cognate dsRNA segments cannot be differentiated by SDS-PAGE. Temperature gradient gel electrophoresis (TGGE) has been shown to be a much more sensitive method of differentiating RNA or DNA fragments of high sequence homology. Here we report the preliminary application of TGGE in analysis of genomic reassortants of two BTV serotypes, 1 and 23. While six out of ten genome segments between BTV-1 and BTV-23 could not be resolved by SDS-PAGE, all of them were differentiated by TGGE. The ability of TGGE to distinguish between dsRNA segments of high sequence homology may also make it useful in the search for BTV genes responsible for defined characteristics, such as virulence, by differentiating wild-type and mutated gene segments of viruses displaying altered phenotypes.

Bluetongue virus↗
PubMed2006

Ebp1 is a dsRNA-binding protein associated with ribosomes that modulates eIF2alpha phosphorylation.

dsRNA-binding domains (dsRBDs) characterize an expanding family of proteins involved in different cellular processes, ranging from RNA editing and processing to translational control. Here we present evidence that Ebp1, a cell growth regulating protein that is part of ribonucleoprotein (RNP) complexes, contains a dsRBD and that this domain mediates its interaction with dsRNA. Deletion of Ebp1's dsRBD impairs its localization to the nucleolus and its ability to form RNP complexes. We show that in the cytoplasm, Ebp1 is associated with mature ribosomes and that it is able to inhibit the phosphorylation of serine 51 in the eukaryotic initiation factor 2 alpha (eIF2alpha). In response to various cellular stress, eIF2alpha is phosphorylated by distinct protein kinases (PKR, PERK, GCN2, and HRI), and this event results in protein translation shut-down. Ebp1 overexpression in HeLa cells is able to protect eIF2alpha from phosphorylation at steady state and also in response to various treatments. We demonstrate that Ebp1 interacts with and is phosphorylated by the PKR protein kinase. Our results demonstrate that Ebp1 is a new dsRNA-binding protein that acts as a cellular inhibitor of eIF2alpha phosphorylation suggesting that it could be involved in protein translation control.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Human endometrial epithelial cells cyclically express Toll-like receptor 3 (TLR3) and exhibit TLR3-dependent responses to dsRNA.

Toll-like receptor 3 (TLR3) responds to dsRNA, a product of most viral life cycles, and initiates production of proinflammatory and antiviral cytokines. The role of TLR3 in human mucosal immunity of the endometrium has not been examined. The effects of TLR3 ligation in endometrial epithelium could be significant as the endometrium is a significant site for viral entry and infection. Additionally, the cytokine milieu plays an essential role in normal functions of the endometrium such as uterine cycle progression, epithelial proliferation and shedding, and embryo implantation. In this study, we demonstrated cycle dependent expression of functional TLR3 in primary endometrial epithelial tissue and expression of intracellular TLR3 in human endometrial epithelial cell lines. We established that stimulation of TLR3-positive cell lines and primary human endometrial epithelial cells with dsRNA leads to TLR3-dependent expression of interleukin (IL)-6, IL-8, interferon (IFN)-inducible protein 10, RANTES, and IFN-beta. These results indicate that the cytokine profile of human endometrial epithelial cells can be modified through TLR3 stimulation. Our findings suggest that TLR3 is involved in the immune responses of endometrial epithelial cells after exposure to dsRNA and has the potential to alter the cytokine milieu and influence the outcome and consequences of infection.

Adolescent↗
PubMed2026

dsRNAscan maps human dsRNAome, revealing conservation, intermolecular dsRNA, and correlates of ADAR dependency.

The human transcriptome contains millions of A-to-I editing sites arising from an unclear number of poorly characterized dsRNAs. Editing sites reveal the presence of dsRNA, but this method is limited by transcription levels, read depth, and ADAR expression and cannot identify unedited dsRNA. To address these limitations, we developed dsRNAscan. Applying dsRNAscan to the human genome predicted 5 million dsRNAs, mostly in repetitive and intergenic regions. Machine learning models trained on A-to-I editing and RNA structure-probing data identified ∼2.4 million high-confidence predictions, which were enriched at dsRNA-binding protein binding sites. Additionally, we predicted hundreds of dsRNAs conserved across vertebrates and observed thousands of editing-enriched regions suspected to arise from intermolecular dsRNAs formed with sense-antisense transcripts. Quantifying expression of intramolecular and intermolecular dsRNAs accessible to cytoplasmic immune sensors revealed that their ratio correlated with ADAR dependency across cancer cell lines. The human dsRNAome is available as a resource at https://dsrna.chpc.utah.edu/.

A-to-I RNA editing↗
PubMed2001

Mechanisms of beta-cell death in response to double-stranded (ds) RNA and interferon-gamma: dsRNA-dependent protein kinase apoptosis and nitric oxide-dependent necrosis.

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate beta-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs beta-cell function and induces beta-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates beta-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-gamma stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-gamma on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and N(G)-monomethyl-L-arginine, attenuate poly IC + IFN-gamma-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. N(G)-monomethyl-L-arginine fails to prevent poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-gamma is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-gamma-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway.

Animals↗
PubMed2002

Specific interference with gene expression and gene function mediated by long dsRNA in neural cells.

Double-stranded (ds) RNA-induced sequence-specific interference with gene expression, RNA interference (RNAi), has been extensively used in invertebrates, allowing for efficient and high-throughput gene silencing and gene function analysis. In vertebrates, however, use of RNAi to study gene function has been limited due to non-specific effects induced by double-stranded RNA (dsRNA)-dependent protein kinase and interferon activation. dsRNA-induced specific inhibition of vertebrate gene expression has only been shown in embryonic and non-differentiated mammalian cells. In this report, we demonstrate dsRNA-induced specific interference of gene expression and gene function in partially as well as fully differentiated mouse neuroblastoma cells. Specific silencing was observed in the expression of an integrated transgene coding for green fluorescent protein and a variety of endogenous genes. Moreover, we show that RNAi-mediated inhibition of poly (ADP-ribose) polymerase (PARP) expression induced cellular resistance to oxygen-glucose deprivation, consistent with the role of PARP in ischemia-induced brain damage. Our results indicate that RNAi can be used as a powerful tool to study gene function in neural cells.

Animals↗
PubMed2002

2',5'-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity.

Recently, the presence of 2',5'-linked oligoadenylates and a high 2',5'-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2-5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2-5A synthetases, the 2-5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2-5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells. These results indicate that the 2-5A synthetase from G. cydonium may be active per se or is activated by some other mechanism. The sponge enzyme is capable of synthesizing a series of 2-5A oligomers ranging from dimers to octamers. The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates. By its product profile and kinetics of formation, the sponge 2-5A synthetase behaves like a specific isoform of enzymes of the 2-5A synthetase family.

2',5'-Oligoadenylate Synthetase↗
PubMed2003

Functional cloning of genes encoding dsRNA binding proteins.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

Cloning, Molecular↗
PubMed1999

Abnormal levels and minimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells.

The interferon induced, dsRNA-activated, protein kinase, PKR, is a key regulator of translational initiation, playing an important role in the regulation of cell proliferation, apoptosis and transformation. PKR levels correlate inversely with proliferative activity in several human tumor systems. This inverse relationship breaks down in human invasive ductal breast carcinomas which exhibit high levels of PKR (Haines et al., Tumor Biol. 17 (1996) 5-12). Consistent with the data from human tumors, the levels of PKR in several breast carcinoma cell lines, MCF7, T47D, BT20, MDAMB231 and MDAMB468, are paradoxically high compared to those found in the normal breast cell lines MCF10A and Hs578Bst. The activity of affinity- or immuno-purified PKR from MCF7, T47D, and BT20 cells appears to be severely attenuated, as judged by its ability to autophosphorylate, or phosphorylate eIF2 alpha. Furthermore, the activity of the kinase from breast carcinoma cells is refractory to stimulation by dsRNA or heparin. However, PKR from breast carcinoma cells remains functional with respect to its ability to bind dsRNA. The activity of PKR from MCF10A cells is reduced by prior incubation with extracts from MCF7 cells, suggesting that MCF7 extracts contain a transdominant inhibitor of PKR. Deregulation of PKR may therefore provide a mechanism for the development or maintenance of a transformed phenotype of human breast carcinomas, mimicking the effects of manipulation of PKR or eIF2 activity observed in experimental systems. Thus, breast carcinomas may provide the first indication of a role for PKR in the pathogenesis of a naturally occurring human cancer.

Breast↗
PubMed2002

The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR.

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

Animals↗
PubMed2001

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.

Amino Acid Sequence↗
PubMed2006

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA.

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Antigen Presentation↗
PubMed2003

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.

Animals↗
PubMed2005

A PKR-like eukaryotic initiation factor 2alpha kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is induced as part of the IFN response in mammals and acts to shut down protein synthesis by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). In fish, a PKR-like kinase activity has been detected, but the enzyme responsible has eluded characterization. Here, we describe a PKR-like kinase from zebrafish. Phylogenetic analysis shows that the C-terminal kinase domain is more closely related to the kinase domain of PKR than to any of the other three known eIF2alpha kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Zalpha domains. Zalpha domains specifically bind dsDNA and RNA in the left-handed Z conformation, often with high affinity. They have been found previously in two other IFN-inducible proteins, the dsRNA editing enzyme, ADAR1, and Z-DNA binding protein 1 (ZBP1), as well as in the poxvirus virulence factor, E3L. This previously undescribed kinase, designated PKZ (protein kinase containing Z-DNA binding domains), is transcribed constitutively at low levels and is highly induced after injection of poly(inosinic)-poly(cytidylic) acid, which simulates viral infection. Binding of Z-DNA by the Zalpha domain of PKZ was demonstrated by circular dichroism. PKZ inhibits translation in transfected cells; site-directed mutagenesis indicates that this inhibition depends on its catalytic activity. Identification of a gene combining Zalpha domains with a PKR-like kinase domain strengthens the hypothesis that the ability to bind left-handed nucleic acid plays a role in the host response to viruses.

Alternative Splicing↗
PubMed2006

The dsRNA binding site of human Toll-like receptor 3.

Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.

Binding Sites↗
PubMed2026

Detection of dsRNA in Soil-Derived Ascomycetes and Characterization of Cladosporium cladosporioides Partitivirus 1 Isolate IPBL11.

Mycoviruses can induce phenotypic and physiological changes in their fungal hosts, making them valuable biological resources. To harness this potential, it is crucial to gather comprehensive information on their distribution patterns, genomic and structural characteristics, and interactions with host fungi. In this study, we screened 64 ascomycete isolates collected from various soil environments in Korea to detect the presence of double-stranded RNA (dsRNA) elements. We identified dsRNA bands in three of these isolates. Among them, we determined the complete genome sequence of a bipartite dsRNA virus found in Cladosporium anthropophilum. Phylogenetic analyses based on the RNA-dependent RNA polymerase (RdRP) and capsid protein (CP) sequences indicated that this virus belongs to the genus Gammapartitivirus within the family Partitiviridae. Comparative sequence analyses suggested that this virus is best classified as a new isolate of Cladosporium cladosporioides partitivirus 1, which we designated as Cladosporium cladosporioides partitivirus 1 isolate IPBL11 (CcPV1-IPBL11).

Cladosporium anthropophilum↗