SEARCH · Search PubMed

Results for “DsRNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,513 records · Page 84Linked to original sources

PubMed1999

Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR.

The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.

3T3 Cells↗

PubMed1988

Mechanism of interferon action: studies on the activation of protein phosphorylation and the inhibition of translation in cell-free systems.

We describe the ability of reovirus messenger RNA (mRNA) to serve as a template for translation and as an activator of protein phosphorylation in cell-free extracts prepared from untreated and from interferon (IFN)-treated mouse fibroblast L cells. In vitro transcribed reovirus mRNA was purified by column chromatography on CF-11 cellulose. This procedure removed trace amounts of double-stranded RNA (dsRNA) [0.01%-0.1%] present in mRNA preparations purified solely by extensive LiCl precipitation. In the absence of added dsRNA, CF-11 cellulose-purified reovirus mRNA did not detectably activate phosphorylation of either ribosome-associated protein P1 or the alpha subunit of protein synthesis initiation factor eIF-2 in S-10 extracts prepared from L cells; the CF-11 cellulose-purified reovirus mRNA was translated more efficiently than was LiCl-purified reovirus mRNA in these extracts. Highly purified CF-11 reovirus mRNA was, however, translated less efficiently by S-10 extracts prepared from IFN-treated L cells than by extracts prepared from untreated L cells, suggesting that the inefficient translation by IFN-treated extracts was an integral property of reovirus mRNA. Increasing the secondary structure of reovirus mRNA by substituting bromouridine (Br-uridine) for uridine in the mRNA caused an increased inhibition of mRNA binding to ribosomes in extracts prepared from IFN-treated as compared to untreated cells. The mechanism of inhibition of translation of CF-11 cellulose-purified reovirus mRNA in IFN-treated systems remains to be established.

Animals↗

PubMed2006

Induction of interferon synthesis by the PKR-inhibitory VA RNAs of adenoviruses.

In virus-infected cells, double-stranded RNA (dsRNA) activates the transcription factor interferon (IFN) regulatory factor-3 (IRF-3), which stimulates type I IFN (IFN-alpha/beta) gene expression. In addition, dsRNA activates the enzyme RNA-activated protein kinase (PKR), which phosphorylates the eukaryotic initiation factor 2alpha (eIF2beta), thereby inhibiting mRNA translation. Adenoviruses express highly structured RNA molecules termed VA RNAs (VA(I)/VA(II)) known to specifically inhibit PKR. As PKR impairs expression from transfected cDNA constructs, plasmids encoding VA RNAs are widely used as enhancers of transgene expression. Here, we describe induction of IFN synthesis as a novel feature of VA RNAs. Transfection of a VA(I)/VA(II)-expressing plasmid was found to induce type I IFN production, resulting in activation of IFN-dependent genes, such as IFN-stimulated gene 56 (ISG56) or MxA, and the establishment of an antiviral state in transfected cells. Curiously, VA RNAs did not activate IRF-3, suggesting an alternative pathway of IFN induction. These data may be considered when using genetically modified adenoviruses as therapeutic agents and suggest caution in choosing VA RNA constructs as a means to increase expression of a gene of interest.

Adenoviridae↗

PubMed2005

Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-terminal kinase.

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH(2)-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Here, we identified the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response.

Amino Acid Sequence↗

PubMed2006

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

ATP-Binding Cassette Transporters↗

PubMed2005

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly.

The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonas fragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.

Binding Sites↗

PubMed2002

Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene.

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.

Animals↗

PubMed1997

A ribonuclease specific for inosine-containing RNA: a potential role in antiviral defence?

RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I-RNase, that is responsible for the degradation of inosine-containing RNA (I-RNA). I-RNase is Mg2+ dependent and specifically degrades single-stranded I-RNA. Comparison of the Km of the enzyme for I-RNA with the Ki for inhibition by normal RNA suggests a approximately 300-fold preferential binding to I-RNA, which can account for the specificity of degradation. The site of cleavage by I-RNase is non-specific; I-RNase acts as a 3'-->5' exonuclease generating 5'-NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I-RNase is able to degrade RNAs that previously have been modified by the RED-1 double-stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I-RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.

Adenosine Deaminase↗

PubMed2000

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation.

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.

Actins↗

PubMed2001

Protein kinase PKR is required for platelet-derived growth factor signaling of c-fos gene expression via Erks and Stat3.

The double-stranded RNA (dsRNA)-activated protein kinase PKR is an interferon (IFN)-induced enzyme that controls protein synthesis through phosphorylation of eukaryotic initiation factor 2alpha (eIF-2alpha). PKR also regulates signals initiated by diverse stimuli, including dsRNA, IFN-gamma, tumor necrosis factor-alpha, interleukin-1 and lipopolysaccharide, to different transcription factors, resulting in pro-inflammatory gene expression. Stat3 plays an essential role in promoting cell survival and proliferation by different growth factors, including platelet-derived growth factor (PDGF). Here we show that PKR physically interacts with Stat3 and is required for PDGF-induced phosphorylation of Stat3 at Tyr705 and Ser727, resulting in DNA binding and transcriptional activation. PKR-mediated phosphorylation of Stat3 on Ser727 is indirect and channeled through ERKS: Although PKR is pre-associated with the PDGF beta-receptor, treatment with PDGF only modestly activates PKR. However, the induction of c-fos by PDGF is defective in PKR-null cells. Taken together, these results establish PKR as an upstream regulator of activation of Stat3 and as a common mediator of both growth-promoting and growth-inhibitory signals.

Animals↗

PubMed2001

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity.

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.

Adenosine Triphosphatases↗

PubMed2001

A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III.

RNases III are a family of double-stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN tetraloops, AGAA and AGUU, using NMR spectroscopy. Both tetraloops have the same overall structure, in which the backbone turn occurs on the 3' side of the syn G residue in the loop, with the first A and G in a 5' stack and the last two residues in a 3' stack. A non-bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various tetraloop sequences defines a novel family of RNA tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.

Base Sequence↗

PubMed2002

Two classes of short interfering RNA in RNA silencing.

RNA silencing is a eukaryotic genome defence system that involves processing of double-stranded RNA (dsRNA) into 21-26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA-directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a green fluorescent protein (GFP) transgene are in short (21-22 nt) and long (24-26 nt) size classes, whereas those from endogenous retroelements are only in the long class. Viral suppressors of RNA silencing and mutations in Arabidopsis indicate that these classes of siRNA have different roles. The long siRNA is dispensable for sequence-specific mRNA degradation, but correlates with systemic silencing and methylation of homologous DNA. Conversely, the short siRNA class correlates with mRNA degradation but not with systemic signalling or methylation. These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals.

Adaptation, Physiological↗

PubMed2002

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP.

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.

Adenosine Triphosphate↗

PubMed1994

Translational maintenance of frame: mutants of Saccharomyces cerevisiae with altered -1 ribosomal frameshifting efficiencies.

A special site on the (+) strand of the L-A dsRNA virus induces about 2% of ribosomes translating the gag open reading frame to execute a -1 frameshift and thus produce the viral gag-pol fusion protein. Using constructs in which a -1 ribosomal frameshift at this site was necessary for expression of lacZ we isolated chromosomal mutants in which the efficiency of frameshifting was increased. These mutants comprise eight genes, named mof (maintenance of frame). The mof1-1, mof2-1, mof4-1, mof5-1 and mof6-1 strains cannot maintain M1 dsRNA at 30 degrees, but, paradoxically, do not lose L-A. The mof2-1, mof5-1 and mof6-1 strains are temperature sensitive for growth at 37 degrees, and all three show striking cell cycle phenotypes. The mof2-1 strains arrest with mother and daughter cells almost equal in size, mof5-1 arrests with multiple buds and mof6-1 arrests as single large unbudded cells. mof2-1 and mof5-1 strains are also Pet-. The mof mutations show differential effects on various frameshifting signals.

Base Sequence↗

PubMed1976

Suppression of the killer phenotype in Ustilago maydis.

Nineteen sensitive cell lines of U. maydis were crossed with three killer strains and sample progenies were screened for killer segregation patterns. Crosses involving 11 lines gave killer frequencies ranging from 71%-100% of the progeny and 4:0 segregations in tetrads. Segregations in some crosses involving each of the remaining 8 lines gave killer frequencies from 0%-58% and mixed tetrads containing both non-killer and killer meiotic products. Many of the killers were unstable on further culture. Killer suppression showed varying degrees of specificity, appeared to be cytoplasmically determined for at least one strain, and was associated with possession of dsRNA in this strain and one other. No dsRNA was detected in two other suppressive strains. There was no evidence for segregation of nuclear maintainer genes for any of the killer determinants.

Basidiomycota↗

PubMed2004

RNA interference: from model organisms towards therapy for neural and neuromuscular disorders.

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology and we address problems to be solved in developing RNAi-based therapies.

Animals↗

PubMed1985

Detection of rotaviruses by nucleic acid hybridization with cloned DNA of simian rotavirus SA11 genes.

We developed a dot-blot hybridization assay to detect rotaviral RNA sequences in tissue culture or in clinical samples. 32P-labeled cloned cDNA probes of the simian rotavirus SA11 specifically detected rotaviral RNA sequences and were more sensitive for detecting SA11 than was the commercial enzyme-linked immunosorbent assay Rotazyme test. A full-length probe of SA11 gene 6 detected 2.5 X 10(5) SA11 particles or approximately 0.27 ng of purified SA11 dsRNA. Combined probes from genes 6 and 9 detected 0.135 ng of purified SA11 dsRNA. The assay detected group A rotaviruses from different subgroups and serotypes, but the sensitivity of RNA detection varied from 0.5 to 31 ng when RNA from heterologous strains of virus was analyzed. An analysis of coded stool samples correctly identified 31 (91%) of 34 samples positive for rotavirus by electron microscopy and 100% of 26 samples negative for rotavirus by electron microscopy. Preliminary experiments also showed the assay has potential to directly characterize (subgroup and serotype) rotaviral isolates.

Cloning, Molecular↗