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At least 685 records · Page 38Linked to original sources
PubMed1984

Isolation of a heme-controlled inhibitor of translation that blocks the interaction between messenger rna and eukaryotic initiation factor 2.

A heme-controlled inhibitor of translation was isolated from the S-100 of rabbit reticulocytes by a novel procedure including chromatography on double-stranded ribonucleic acid (dsRNA)-cellulose. The inhibitor thus purified is extremely active and functionally resembles previously studied heme-controlled inhibitor preparations in terms of kinetics and extent of inhibition of translation, relief of inhibition by eukaryotic initiation factor 2 (eIF-2), relief of inhibition by 2-aminopurine, and preferential inhibition of alpha-over beta-globin synthesis. The action of this inhibitor on translation is resistant to treatment with bacterial alkaline phosphatase, micrococcal nuclease, or trypsin and to incubation at 95 degrees C, pH 2 or pH 12. The inhibitor not only is retained on DEAE-cellulose, phosphocellulose, and dsRNA-cellulose but also exhibits a high affinity for the dye Cibacron Blue, properties that suggest that it may be a protein. Unlike previously described heme-controlled inhibitor preparations, or preparations that did not pass over dsRNA-cellulose, the inhibitor recovered upon dsRNA-cellulose chromatography does not exhibit eIF-2 kinase activity. The inhibitor does not block ternary complex formation between eIF-2, methionyl-tRNAfMet, and GTP but inhibits the ability of eIF-2 to form a complex with labeled globin mRNA. In the presence of inhibitor, the formation of mRNA/eIF-2 complexes can be restored effectively by an excess of eIF-2 but not by an excess of mRNA. The inhibitor thus appears to block the interaction between eIF-2 and mRNA not by competing with eIF-2 for a binding site on mRNA but, instead, by acting on eIF-2 itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Animals↗
PubMed1993

A yeast antiviral protein, SKI8, shares a repeated amino acid sequence pattern with beta-subunits of G proteins and several other proteins.

SKI8 is a yeast antiviral gene, essential for controlling the propagation of M double-stranded RNA (dsRNA) and thus for preventing virus-induced cytopathology. Our DNA sequence of SKI8 shows that it encodes a 397 amino acid protein containing two copies of a 31 amino acid repeat pattern first identified in mammalian beta-transducin and Cdc4p of yeast. There are also four copies of this repeat in yeast Mak11p, necessary for M dsRNA propagation, and three copies in the putative product of the Dictyostelium AAC3 gene. Analysis of 36 cases of the repeat unit shows they have a consensus predicted structure: N-helix-sheet-turn-sheet-turn-sheet-helix-C.

Amino Acid Sequence↗
PubMed2000

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae).

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.

Animals↗
PubMed1995

Mechanism of interferon action: double-stranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons.

Treatment of human amnion U cells with interferon increased the steady state level of mRNA encoding the double-stranded (ds) RNA-specific adenosine deaminase (AdD) as measured by Northern gel-blot analysis. A single major dsRNA-specific AdD transcript of approximately 6.7 kb was detected; the transcript was induced by both interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma). Likewise, Western immunoblot analysis revealed that a 150-kDa protein recognized by antiserum prepared against recombinant dsRNA-specific AdD was increased in the human amnion U and neuroblastoma SH-SY5Y cell lines treated with interferon. Both IFN-alpha and IFN-gamma induced the 150-kDa protein. These results, which establish that dsRNA-specific AdD is an IFN-inducible protein in human cells, have implications regarding the possible role of interferon in persistent viral infections.

Adenosine Deaminase↗
PubMed2005

Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Adenosine Triphosphatases↗
PubMed1994

Increased thermal aggregation of proteins in ATP-depleted mammalian cells.

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coli beta-galactosidase, the 70-kDa constitutive heat-shock proteins and the 68-kDa dsRNA-dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock. The heat-induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70-kDa constitutive heat-shock proteins and the 68-kDa protein kinase are ATP-binding proteins but ATP depletion also considerably increases the aggregation of beta-galactosidase to the nuclear pellets, although this enzyme is not known to be an ATP-binding molecule. Insolubilization of all four reporter proteins occurs in ATP-depleted cells even at normal growing temperatures (37 degrees C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular 'free' chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP. Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP-depleted cells and for the ability of mitochondrial uncouplers to trigger a heat-shock response in some cells.

Adenosine Triphosphate↗
PubMed2002

RNA helicase MUT-14-dependent gene silencing triggered in C. elegans by short antisense RNAs.

Posttranscriptional gene silencing in Caenorhabditis elegans results from exposure to double-stranded RNA (dsRNA), a phenomenon designated as RNA interference (RNAi), or from co-suppression, in which transgenic DNA leads to silencing of both the transgene and the endogenous gene. Here we show that single-stranded RNA oligomers of antisense polarity can also be potent inducers of gene silencing. As is the case for co-suppression, antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-14. Our data favor the hypothesis that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded.

Amino Acid Motifs↗
PubMed2004

[Blocking with double- stranded RNA of the expression of Hes5 in rat bone marrow-derived neural stem cells].

OBJECTIVE: To examine the efficiency of exogenous small double-stranded RNA (dsRNA) in knocking down the gene expression at the post-transcription level, and investigate the factors that may influence the transfection. METHOD: The bone marrow stromal cells of SD rat were separated and cultured in vitro, followed by induction of the cells to evolve into neural stem cells using special culture medium prepared by our laboratory. Synthetic dsRNA was then transferred into the cells at varied concentrations, and the results were analyzed by Western blotting. RESULTS: The concentrations ranging from 200 to 300 nmol/L were optimal for specifically blocking the expression of Hes5, whereas the suitable concentrations for the cell survival were between 50 and 200 nmol/L. CONCLUSION: dsRNA is capable of triggering RNA interference in neural stem cells, and at appropriate concentration, it may specifically and effectively knock down endogenous gene expression without sacrificing the viability of the cells.

Animals↗
PubMed2003

Sex determination in Drosophila melanogaster and Musca domestica converges at the level of the terminal regulator doublesex.

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins' 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex ( dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX(M)) and female-specific (MdDSX(F)) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX(F) expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX(M) expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.

Amino Acid Sequence↗
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↗
PubMed1999

Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.

Antigen Presentation↗
PubMed2001

RNAi is antagonized by A-->I hyper-editing.

RNA interference (RNAi) and adenosine to inosine conversion are both mechanisms that respond to double-stranded RNA (dsRNA) and have been suggested to have antiviral roles. RNAi involves processing of dsRNA to short interfering RNAs (siRNAs), which subsequently mediate degradation of the cognate mRNAs. Deamination of adenosines changes the coding capacity of the RNA, as inosine is decoded as guanosine, and alters the structure because A-U base pairs are replaced by I*U wobble pairs. Here we show that RNAi is inhibited if the triggering dsRNA is first deaminated by ADAR2. Moreover, we show that production of siRNAs is progressively inhibited with increasing deamination and that this is sufficient to explain the inhibition of RNAi upon hyper-editing of dsRNAs.

Adenosine↗
PubMed2001

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing.

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A-A or A-C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5' side of the loop, exposing their Watson-Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage.

Base Sequence↗
PubMed2004

A PTEN-like phosphatase with a novel substrate specificity.

We show that a novel PTEN-like phosphatase (PLIP) exhibits a unique preference for phosphatidylinositol 5-phosphate (PI(5)P) as a substrate in vitro. PI(5)P is the least characterized member of the phosphoinositide (PI) family of lipid signaling molecules. Recent studies suggest a role for PI(5)P in a variety of cellular events, such as tumor suppression, and in response to bacterial invasion. Determining the means by which PI(5)P levels are regulated is therefore key to understanding these cellular processes. PLIP is highly enriched in testis tissue and, similar to other PI phosphatases, exhibits poor activity against several proteinaceous substrates. Despite a recent report suggesting a role for PI(5)P in the regulation of Akt, the overexpression of wild-type or catalytically inactive PLIP in Chinese hamster ovary-insulin receptor cells or a dsRNA-mediated knockdown of PLIP mRNA levels in Drosophila S2 cells does not alter Akt activity or phosphorylation. The unique in vitro catalytic activity and detailed biochemical and kinetic analyses reported here will be of great value in our continued efforts to identify in vivo substrate(s) for this highly conserved phosphatase.

Amino Acid Sequence↗
PubMed2005

A conserved major groove antideterminant for Saccharomyces cerevisiae RNase III recognition.

Rnt1p, the only known Saccharomyces cerevisiae RNase III double-stranded RNA endonuclease, plays important roles in the processing of precursors of ribosomal RNAs and small nuclear and nucleolar RNAs and in the surveillance of unspliced pre-mRNAs. Specificity of cleavage by Rnt1p relies on the presence of RNA tetraloop structures with the consensus sequence AGNN at the top of the target dsRNA. The sequences of 79 fungal RNase III substrates were inspected to identify additional conserved sequence elements or antideterminants that may contribute to Rnt1p recognition of the double-stranded RNA. Surprisingly, U-A sequences at the base pair adjacent to the conserved terminal tetraloop (closing base pair) were found to be absent from all but one inspected sequence. Analysis of chemically modified variants of the closing base pair showed that the presence of exocyclic groups in the major groove of purines 3' to the last nucleotide of the tetraloop inhibits Rnt1p cleavage without strongly inhibiting Rnt1p binding. We propose that these groups interfere with the recognition of the RNA substrate by the catalytic domain of Rnt1p. These results identify exocyclic groups of purines in the major groove downstream of the tetraloop as a major antideterminant in S. cerevisiae RNase III activity, and suggest a rationale for their apparent counter selection in RNA processing sites.

Adenine↗
PubMed2003

Viral dsRNA activates mucin transcription in airway epithelial cells.

Double-stranded (ds) RNA is a biologically active component of many viruses including rhinoviruses infecting the upper respiratory tract. Mucus production is a common symptom of such infections. Here, we show that mucin, the glycoprotein subunit of mucus gels, is transcriptionally upregulated in an NF-kappaB- and p38-dependent manner when homogeneous cultures of epithelial cells are exposed to dsRNA. Furthermore, upstream of p38 in this system, dsRNA stimulates the extracellular release of ATP and activation of cell surface ATP receptors, which are G protein-coupled. This results in the stimulation of phospholipase C and protein kinase C. These findings suggest that ATP receptor antagonists could be used to modulate mucus production induced by virus.

Cell Line↗
PubMed1996

Host genotype controls the ability of the ISGF3 complex to activate transcription of IFN-inducible genes.

C57BL/6 mice are unable to express the Ifi 202 type genes upon injection in vivo of multiple dsRNA, poly rl:rC, or IFN-treatment in vitro. For this purpose the 5' terminal flanking region (called the b segment of 804 bp) was linked to a heterologous reporter gene chloramphenicol acetyl transferase (CAT) and transfected into NIH3T3 cells or BLK cells derived from the C57BL/6 strain. IFN-alpha induced strong CAT activity in NIH3T3 but not in BLK cells. This lack of transcription activation was not due to a defect in STAT factor activity, since IFN-alpha treatment in the presence of IFN-gamma priming induced translocation of the ISGF3 into the nucleus, and binding to the ISRE (IFN-Stimulated Response Element) of the 202 gene even in C57BL/6 derived cells. Surprisingly when three tandem copies of the 202 ISRE (42 bp) were linked to a heterologous promoter (c-fos promoter) driving the reporter CAT gene, activation was also observed in C57BL/6 cells upon IFN-treatment. Finally, another IFN-inducible gene, namely the Mx, was activated in C57BL/6 mice. Thus, the primary defect of the C57BL/6 strain leading to an impaired Ifi 202 type gene response to IFN appears to be an inability of the ISGF3 complex to activate the endogenous promoter. Altogether these results suggest that unidentified nuclear factors related to the host genotype control the ability of the STAT factors to activate transcription upon IFN-treatment.

3T3 Cells↗