Search PubMed⌕ Search

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.

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

At least 397 records · Page 22Linked to original sources
PubMed2000

Potential role of PKR in double-stranded RNA-induced macrophage activation.

In this study, the role of the double-stranded (ds) RNA-dependent protein kinase (PKR) in macrophage activation was examined. dsRNA [polyinosinic:polycytidylic acid (poly IC)]-stimulated inducible nitric oxide synthase, interleukin (IL)-1alpha and IL-1beta mRNA expression, nitrite formation and IL-1 release are attenuated in RAW264.7 cells stably expressing dominant negative (dn) mutants of PKR. The transcriptional regulator nuclear factor (NF)-kappaB is activated by dsRNA, and appears to be required for dsRNA-induced macrophage activation. While dnPKR mutants prevent macrophage activation, they fail to attenuate dsRNA-induced IkappaB degradation or NF-kappaB nuclear localization. The inhibitory actions of dnPKR on dsRNA-induced macrophage activation can be overcome by treatment with interferon (IFN)-gamma, an event associated with PKR degradation. Furthermore, dsRNA + IFN-gamma stimulate inducible nitric oxide synthase expression, IkappaB degradation and NF-kappaB nuclear localization to similar levels in macrophages isolated from PKR(-/-) and PKR(+/+) mice. These findings indicate that both NF-kappaB and PKR are required for dsRNA-induced macrophage activation; however, dsRNA-induced NF-kappaB activation occurs by PKR-independent mechanisms in macrophages. In addition, the PKR dependence of dsRNA-induced macrophage activation can be overcome by IFN-gamma.

Animals↗
PubMed1991

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.

Animals↗
PubMed1994

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

Amino Acid Sequence↗
PubMed1980

Double-stranded sequences in RNA of Drosophila melanogaster: relation to mobile dispersed genes.

Double-stranded RNA (dsRNA) sequences were isolated from either total RNA or cytoplasmic poly(A)+RNA of D. melanogaster culture cells using a method described previously /1,2/. Virtually all dsRNA was found to be of high molecular weight (> 200 base pairs) and unable to snap back after RNA melting. Thus, it corresponds to one of dsRNA classes found in mouse cells, namely, to dsRNA-A /3/. Three different cloned DNA fragments of D melanogaster which hybridized to melted dsRNa were selected among 100 randomly taken. All of them efficiently bound poly(A)+RNA and high percentage of total cellular DNA. According to these and other properties, they were assigned to a group of mobile dispersed genes of D. melanogaster. DsRNA hybridizes to all subfragments of the two mobile dispersed genes tested (mdg 1 and mdg 3). Thus, complete transcripts of mobile dispersed genes are present in dsRNA. In total RNA, transcripts from one strand are more abundant than those from another one. DsRNA is heavily enriched in the transcripts from mobile dispersed genes as compared to total or poly(A)+RNA of the cytoplasm. It has been suggested that dsRNA in D. melanogaster is formed as the result of symmetric transcription of mobile dispersed genes. At least in the cytoplasmic fraction, two complementary strands are separated in vivo and may combine during the isolation of RNA.

Chromatography, Gel↗
PubMed2006

The double-stranded RNA-induced apoptosis pathway is involved in the cytopathogenicity of cytopathogenic Bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV), which is classified in the genus Pestivirus, family Flaviviridae, can be divided into two biotypes according to its ability to induce a cytopathic effect in tissue culture cells. The mechanisms through which cytopathogenic (cp) BVDV induces cell death and non-cytopathogenic (ncp) BVDV causes persistent infection without producing cell death remain unclear. Here, it was found that the overexpression of four apoptosis-related cellular mRNAs in cells infected with cpBVDV could also be caused by synthetic dsRNA. In fact, it was found that the amount of dsRNA produced by cpBVDV considerably exceeded the amount yielded by ncpBVDV. To evaluate the possible involvement of dsRNA in the induction of apoptosis, this study examined whether RNAi-mediated depletion of two dsRNA-reactive cellular factors, dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase 1, resulted in the prevention of cpBVDV-induced apoptosis. Although the induction of apoptosis was reduced after the suppression of either factor alone, the simultaneous silencing of both factors resulted in an almost complete inhibition of apoptosis without affecting viral titre. These results showed that dsRNA is the main trigger of apoptosis in cpBVDV-infected cells and that the cytopathogenicity of BVDV depends on the yield potential of dsRNA. In contrast, ncpBVDV yielded minimal levels of dsRNA, thereby establishing a persistent infection without inducing apoptosis. This report supports the significance of viral dsRNA as a trigger of innate immune responses.

Animals↗
PubMed2006

Rotavirus double-stranded RNA induces apoptosis and diminishes wound repair in rat intestinal epithelial cells.

BACKGROUND: Recent studies have shown that toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA). Rotaviruses, having a dsRNA genome, infect intestinal epithelial cells (IEC) and cause acute gastroenteritis in young children. The aim of the present study was to clarify the pathophysiological function of rotavirus dsRNA in IEC. METHODS: Expression of TLR3 mRNA or protein in IEC cell lines (IEC-6, HT-29, Caco-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis or immunohistochemistry. Induction of cytokines (TNF-alpha, interferon-beta, interleukin-6) mRNA and activation of signal proteins (ERK1/2 MAPK and IkappaB-alpha) in IEC after stimulation with rotavirus dsRNA were assessed by RT-PCR or Western blot analysis. IEC-6 cells were wounded and cell migration into wound areas after stimulation with rotavirus dsRNA (1-25 microg/mL) was assessed. Induction of apoptosis after stimulation with rotavirus dsRNA was also assessed. RESULTS: Expression of TLR3 mRNA and TLR3 protein was detected in IEC. Expression of TLR3 mRNA in IEC-6 tended to be up-regulated by exposure to IFN-gamma. Induction of cytokine mRNA and activation of the signal proteins were detected after stimulation with rotavirus dsRNA. Apoptosis was induced and epithelial migration into the wound area was dose-dependently diminished (44.1-94.4%, P < 0.01) by exposure to rotavirus dsRNA. Diminishment of wound repair was suppressed by anti-TLR3 antibody or caspase inhibitor. CONCLUSION: Rotavirus dsRNA induces severe apoptosis and diminishes wound repair in IEC through TLR3, which might be involved in the pathogenesis of rotavirus-induced enteritis.

Animals↗
PubMed1974

Viral infection and host defense.

Double-stranded RNA, made as an intermediary substance in the replication of most, if not all, viruses, may play a much more important role in the pathogenesis and the recovery from virus infections than has hitherto been suspected. Apparently, dsRNA is used by both the challenge virus and the host cell in an attempt to gain "molecular control." Double-stranded RNA exerts a set of effects, which may be well balanced, not only at the level of the individual cell but also at the complex assemblage of these cells termed the organism (Fig. 1). In the cell, interferon synthesis is triggered, although interferon mRNA translation may not occur if dsRNA shuts off protein synthesis too quickly. In the whole organism, the disease severity will depend on how certain toxic reactions evoked by infection (such as cell necrosis and fever) are counterbalanced by an increase in the host defense mechanisms (for example, immune responsiveness and interferon production). Many aspects of the response, relating to either progress of, or recovery from, the disease, can be explained on the basis of a dsRNA. In addition to drawing attention to the biodynamic role of dsRNA, our hypothesis suggests specific experimental vectors designed to enhance our information on the molecular basis of the morbid process which occurs with viral infection. Finally, we suggest that, although the dsRNA molecule may be viewed as a rather simple unit structure, the opportunity for further diversity in the biological activity of a given dsRNA molecule always exists. Namely, each deviation from a perfectly double-helical arrangement introduces the possibility for emphasizing one biological reactivity at the expense of another. This latter structure-activity property may partially account for the extreme apparent diversity, commonly encountered, in the presentations of virologic illness. Appendix note added in proof. Subsequent to submission of this text, we have found that the potent mitogen effect of dsRNA for lymphocytes (murine and human) is also exquisitively sensitive to the fidelity in base pairing of the input polymer pair (59). For example, infrequent "loops" (one nucleotide per 20 base pairs) in an otherwise perfectly helical rI(n) (.) rC(n) molecule [for example, rI(n) (.) r(C(19,)U)(n)] strongly changes its mitogenic properties. This observation, which supports our thesis that a "fine structure" term can be developed for other reactions triggered by dsRNA's in biological systems, emphasizes that diverse biological effects may be encountered with an ostensibly uniform family of dsRNA's.

Animals↗
PubMed2006

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

Plant Viruses↗
PubMed1980

Virus-specific RNA synthesis in cells infected by infectious pancreatic necrosis virus.

Pulse-labeling experiments with [(3)H]uridine revealed that the rate of infections pancreatic necrosis virus-specific RNA synthesis was maximal at 8 to 10 h after infection and was completely diminished by 12 to 14 h. Three forms of RNA intermediates were detected: (i) a putative transcription intermediate (TRI) which comigrated in acrylamide gels with virion double-stranded RNA (dsRNA) after RNase treatment; (ii) a 24S genome length mRNA which could be resolved into two bands by polyacrylamide gel electrophoresis; and (iii) a 14S dsRNA component indistinguishable from virion RNA by gradient centrifugation and gel electrophoresis. The TRI (i) was LiCl precipitable; (ii) sedimented slightly faster and broader (14 to 16S) than the 14S virion dsRNA; (iii) had a lower electrophoretic mobility in acrylamide gels than dsRNA, barely entering acrylamide gels as a heterogenous component; (iv) yielded genome-sized pieces of dsRNA after RNase digestion; and (v) was the most abundant RNA form early in the infectious cycle. The 24S single-stranded RNA was thought to be the viral mRNA since it: (i) became labeled during short pulses; (ii) was found in the polysomal fraction of infected cells; and (iii) hybridized to denatured viral RNA, forming two segments of RNase-resistant RNA that comigrated with virion dsRNA in gels. The 24S mRNA component was formed before the synthesis of dsRNA, and radioactivity could be chased from 24S single-stranded RNA to dsRNA, indicating that 24S RNA may serve as template for the synthesis of complementary strands to form dsRNA. Similar to reovirus, infectious pancreatic necrosis viral 24S mRNA contained no polyadenylic acid tracts.

Journal Article↗
PubMed2005

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Animals↗
PubMed2006

Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

Animals↗
PubMed1998

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain.

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

Cell Division↗
PubMed1999

Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.

Animals↗
PubMed1987

L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus- strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism. In this paper, we show that mutations in mak10 or the pet18 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.

Genotype↗
PubMed2002

Double-stranded RNA induces the synthesis of specific chemokines by bronchial epithelial cells.

Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1alpha. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-kappaB and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.

Bronchi↗
PubMed2005

Single-molecule measurements of the persistence length of double-stranded RNA.

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.

Biophysics↗
PubMed1979

[Melting and reassociation of double-stranded RNA of the encephalomyocarditis virus].

The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.

Encephalomyocarditis virus↗
PubMed1980

[Reiterated genes scattered among the chromosomes of Drosophila melangoaster with varying locations. V. Nature of self-complementary RNA sequences transcribed from chromosomal DNA].

Double-stranded RNA (dsRNA) sequences were isolated from either total RNA or cytoplasmic poly(A)+RNA of Drosophila melanogaster culture cells. Virtually all dsRNA was found to be of high molecular weight (> 200 base pairs) and unable to snap back after RNA melting. Thus, it corresponds to one of dsRNA classes found in mouse cells, namely, to dsRNA-A. Three different cloned DNA fragments of D. melanogaster which hybridized to melted dsRNA were selected among 100 randomly taken. All of them efficiently bound poly(A)+RNA and high percentage of total cellular DNA. According to these and other properties, they were assigned to a group of mobile dispersed genes (mdg) of D. melanogaster. dsRNA hybridizes to all subfragments of the two mobile dispersed genes tested (mdg1 and mdg3). Thus, complete transcripts of mobile dispersed genes are present in dsRNA. In total RNA transcripts from one strand are more abundant than those from another one, dsRNA is heavily enriched in the transcripts from mobile dispersed genes as compared to total RNA or poly(A)+RNA of the cytoplasm. It has been suggested that dsRNA in D. melanogaster is formed as a result of symmetric transcription of mobile dispersed genes. At least in the cytoplasmic fraction, two complementary strands are separated in vivo and may combine during the isolation of RNA.

Chromatography, Gel↗