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PubMed2005

ADAR1 interacts with NF90 through double-stranded RNA and regulates NF90-mediated gene expression independently of RNA editing.

The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 was recently demonstrated to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. Here we report a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins.

Adenosine Deaminase↗

PubMed1980

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus.

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.

Cells, Cultured↗

PubMed1981

Similar sets of terminal oligonucleotides from reovirus double-stranded RNA and viral messenger RNA synthesized in vitro.

Reovirus genomic double-stranded RNA (dsRNA) and viral messenger RNA synthesized in vitro were labeled by periodate oxidation and [3H]borohydride reduction. The 3H label was incorporated into 3'-terminal C residues and 5'-terminal N7-methylguanosine residues. Analysis of ribonuclease digests of the 3H-labeled RNA indicated that the minus strands of dsRNA contained the common 3'-terminal sequence ... PypPupGpC and that the plus strands contained heterogeneous sequence at both the 5' termini and 3'termini that corresponded to sequences at the 5' termini and 3' termini of mRNA. These results suggest that the plus strands of dsRNA and mRNA synthesized in vitro are essentially identical sets of molecules.

Base Sequence↗

PubMed2001

Presence of double-stranded RNA and virus-like particles in Rhizopus isolates.

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.

RNA, Double-Stranded↗

PubMed1992

Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae.

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.

Base Sequence↗

PubMed2004

Exogenous siRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3.

RNA interference (RNAi) is a powerful method that specifically suppresses gene expression in a sequence-dependent manner whose machinery is found in organisms from fungi to mammals. Mammalian cells have developed a sequence-independent system of gene suppression often induced by viral replication that includes the recognition of double-stranded RNA (dsRNA) through Toll-like receptor 3 (TLR3) and induction of type I interferon synthesis. Interferon activates the transcription of a set of genes including dsRNA-activated protein kinase that suppresses protein synthesis and 2'-5'-oligoadenylate synthetase, which generates a product that activates RNase L to cleave RNA in a sequence-independent manner. We observed that 21-bp dsRNA, a key mediator of RNAi, not only induces sequence-specific gene suppression, but also signals TLR3 to induce type I interferon and activates sequence-independent suppression of protein synthesis and enhancement of mRNA degradation. This sequence-independent suppression was demonstrated for both an exogenously administered reporter gene as well as during the targeting of viral genes in the course of acute herpes simplex virus type I infection of keratinocytes. As TLR3 is expressed by many primary cell types and cell lines, this sequence-independent suppression should be considered in the design of experiments using small interfering RNA-mediated gene suppression.

Cell Line↗

PubMed1983

Mechanisms underlying systemic invasion of pea plants by pea enation mosaic virus.

Leaves of Pisum sativum L.cv. 'Perfected Wales' were inoculated with pea enation mosaic virus, at the 3rd and 4th nodes as early as size permitted. Systemic virus spread was then measured over a period of approximately 9 days by assaying the infectivity of 1-cm stem pieces above the 4th and below the 3rd node. The presence of virus antigen and double-stranded (ds) RNA in these stem pieces was measured by radioimmunoassay over the same period. Relative infectivity and amounts of dsRNA in infected tissue were lowest approximately 3 days after inoculation and sharply increased thereafter. A transient plasmodesmatal abnormality was observed 4-6 days after inoculation. The transient nature of the abnormality, which was preceded (24 h) by low infectivity and dsRNA concentrations, may indicate a plant defense reaction that is subsequently overcome. The rise in dsRNA concentration, starting the 4th day after inoculation, coincided with the increase of vesicular material (cytopathological structures) in the phloem of infected plants. Vesicular material was found in sieve elements that were mature at the time of inoculation. Their presence in this tissue can only be explained by transport of the vesicles from infected sites.

Cytopathogenic Effect, Viral↗

PubMed1984

Characterization of the Palyam serogroup viruses (Reoviridae: Orbivirus).

31 Palyam serogroup viruses were examined by complement-fixation and plaque-reduction neutralization tests and by PAGE of the segmented, double-stranded (ds) RNA genome. Although the viruses were indistinguishable by complement-fixation tests, 10 distinct virus serotypes were identified by plaque-reduction neutralization methods. Palyam group viruses which were distinct by the neutralization test had unique dsRNA profiles, whereas those agents which were indistinct by the neutralization test had identical dsRNA profiles. 20 isolations of 3 Palyam serotypes were made from bovines and Culicoides midges in Australia over a 9-year period. When the genome of these isolates was examined electrophoretically, the dsRNA profiles of virus isolates within a given serotype were identical.

Animals↗

PubMed1992

Differentiation of biologically distinct cucumber mosaic virus isolates by PAGE of double-stranded RNA.

Electrophoresis on 5% polyacrylamide was used to analyze dsRNAs of 26 cucumber mosaic virus isolates propagated in Nicotiana tabacum. There was variation between isolates in the migration of each of the dsRNAs 1, 2, and 3. Comparison of the dsRNA profiles enabled each isolate to be allocated to 1 of 7 distinct dsRNA profile types. Two distinct and readily distinguishable isolates were mixed in planta and dsRNA from these infected plants compared with in vitro mixtures of them. All bands from both types were present, indicating that the differences were real and reproducible. This method is of value as a means of classifying cucumber mosaic virus isolates, as it more closely reflects a range of biological characteristics than do other methods currently used.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Selective silencing of angiotensin receptor subtype 1a (AT1aR) by RNA interference.

Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT1a-, AT1b-, and AT2-receptors. Because of a high degree of similarity among these G-protein-coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT1a receptor (AT1aR) subtype. A dsRNA, AT1 47, was found to be highly selective and efficient in reducing the levels of AT1aR subtype. Transfection of AT1aR-expressing CHO cells with dsRNA AT1 47 resulted in an 80% decrease in the AT1aR expression. In contrast, dsRNA AT1 47 showed no significant effects on both AT1bR and AT2R subtypes. Thus, AT1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed2006

JAZ mediates G1 cell-cycle arrest and apoptosis by positively regulating p53 transcriptional activity.

We previously identified JAZ as a novel zinc finger (ZF) protein by screening a murine interleukin-3 (IL-3)-dependent NFS/N1.H7 myeloid cell cDNA library. JAZ is a member of a new class of ZFPs that is evolutionarily conserved and preferentially binds to dsRNA, but its function was unknown. Now, we report that the stress of IL-3 growth factor withdrawal up-regulates JAZ expression in hematopoietic cells in association with p53 activation and induction of cell death. Biochemical analysis reveals that JAZ associates with p53 to stimulate its transcriptional activity in p53-expressing cells, but not in p53-null cells unless complemented with p53. JAZ functions to mediate G1 cell-cycle arrest followed by apoptosis in a p53-dependent mechanism that is associated with up-regulation of p21 and BAX, dephosphorylation of Rb, and repression of cyclin A. Of importance, siRNA "knockdown" of endogenous JAZ inhibits p53 transcriptional activity, decreases the G1/G0 population, and attenuates stress-induced cell death. While JAZ directly binds p53 in vitro in a mechanism requiring p53's C-terminal regulatory domain but independent of dsRNA, the dsRNA-binding ZF domains are required for JAZ's stimulatory role of p53 in vivo by dictating its nuclear localization. Thus, JAZ is a novel negative regulator of cell growth by positively regulating p53.

Active Transport, Cell Nucleus↗

PubMed2005

Functional complementation of RNA interference mutants in trypanosomes.

BACKGROUND: In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments. RESULTS: Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility. CONCLUSIONS: This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.

Animals↗

PubMed2003

Calcium-independent phospholipase A2 mediates CREB phosphorylation in double-stranded RNA-stimulated endothelial cells.

One of the products of a calcium-independent phospholipase A2 (iPLA2) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA2, the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA2. dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated by iPLA2.

Animals↗

PubMed2005

A base-specific recognition signal in the 5' consensus sequence of rotavirus plus-strand RNAs promotes replication of the double-stranded RNA genome segments.

Replication of the segmented double-stranded (ds)RNA genome of rotavirus requires the viral RNA-dependent RNA polymerase (RdRP) to use 11 different (+)RNAs as templates for (-) strand synthesis. Complementary sequences proximal to the 5' and 3' termini are predicted to direct cyclization of the (+)RNAs by forming panhandle structures from which short highly conserved terminal sequences protrude as single-stranded tails. Cell-free replication assays indicate that such structural organization of the 5'- and 3'-ends is required for efficient dsRNA synthesis. Multiple specifically recognized elements exist at the 3'-end that promote dsRNA synthesis including RdRP-recruitment signals and a (-) strand initiation sequence. In contrast to the 3'-end, the role of the 5'-end has been less well defined. In this study, we determined that the 5'-end contains a base-specific recognition signal that plays an important role in the assembly of the RdRP and cofactors into a stable initiation complex for (-) strand synthesis. The 5' recognition signal is associated with the G2 residue of the 5'-consensus sequence, a residue that shows absolute conservation among all rotavirus groups (A, B, and C) examined to date. From our results, we suggest that rotavirus (+)RNA cyclization, although likely initiated by 5'- 3' nucleotide complementarity, may be stabilized by RdRP-dependent bridging. Given that synthesis of the (-) strand on the (+)RNA template will disrupt 5'-3' nucleotide interactions, RdRP-dependent bridging may be the sole mechanism by which the dsRNA product can be held in the necessary cyclized conformation required for efficient multiple rounds of transcription.

Base Sequence↗

PubMed2005

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III.

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.

Base Sequence↗

PubMed2006

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system.

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.

ATP-Binding Cassette Transporters↗

PubMed2003

A transient RNA interference assay system using Arabidopsis protoplasts.

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.

Arabidopsis↗

PubMed2003

Custom knock-outs with hairpin RNA-mediated gene silencing.

Hairpin (hpRNA)-mediated gene silencing exploits a cellular mechanism that recognizes double-stranded RNA (dsRNA) and subjects it and its corresponding mRNA to a sequence-specific degradation. This phenomenon is known as posttranscriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals. dsRNA, when introduced into plant cells through hpRNA constructs, results in severe reduction of the target mRNA-the silencing effect being stably inherited over many generations. While hpRNA constructs can be made using conventional plasmids, use of generic vectors such as pHANNIBAL makes it more convenient to silence a number of genes simultaneously. Vectors, such as pHELLSGATE, that are based on the Gateway(R) technology are suitable for high-throughput gene silencing. The specificity of dsRNA silencing, it's ability to simultaneously silence multiple genes combined with the availability of high-throughput silencing vectors enables the researcher to generate custom knockout plants.

Base Sequence↗