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1
Weinheimer, Isabel, Kajohn Boonrod, Mirko Moser, Michael Wassenegger, Gabi Krczal, Sarah J. Butcher, and Jari P. T. Valkonen. "Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus." Journal of General Virology 95, no. 2 (February 1, 2014): 486–95. http://dx.doi.org/10.1099/vir.0.058693-0.
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Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III–like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2′-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg2+ was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn2+. Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.
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Grünberg, Sebastian, Baptiste Coxam, Tien-Hao Chen, Nan Dai, Lana Saleh, Ivan R. Corrêa, Nicole M. Nichols, and Erbay Yigit. "E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity." Nucleic Acids Research 49, no. 9 (April 22, 2021): 5265–77. http://dx.doi.org/10.1093/nar/gkab284.
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Abstract Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.
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Iqbal, Munir, Emma Poole, Stephen Goodbourn, and John W. McCauley. "Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA." Journal of Virology 78, no. 1 (January 1, 2004): 136–45. http://dx.doi.org/10.1128/jvi.78.1.136-145.2004.
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ABSTRACT Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-κB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.
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Gupta, Ankush, and Pramod C. Rath. "Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5AIn Vitro." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/817024.
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Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.
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Li, Yize, Shuvojit Banerjee, Yuyan Wang, Stephen A. Goldstein, Beihua Dong, Christina Gaughan, Robert H. Silverman, and Susan R. Weiss. "Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses." Proceedings of the National Academy of Sciences 113, no. 8 (February 8, 2016): 2241–46. http://dx.doi.org/10.1073/pnas.1519657113.
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The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans—OAS1, OAS2, and OAS3—synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.
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Magkouras, Ioannis, Philippe Mätzener, Till Rümenapf, Ernst Peterhans, and Matthias Schweizer. "RNase-dependent inhibition of extracellular, but not intracellular, dsRNA-induced interferon synthesis by Erns of pestiviruses." Journal of General Virology 89, no. 10 (October 1, 2008): 2501–6. http://dx.doi.org/10.1099/vir.0.2008/003749-0.
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Recombinant pestivirus envelope glycoprotein Erns has been shown to interfere with dsRNA-induced interferon (IFN-α/β) synthesis. This study demonstrated that authentic, enzymically active Erns produced in mammalian cells prevented a dsRNA-induced IFN response when present in the supernatant of bovine cells. Strikingly, IFN synthesis of cells expressing Erns was eliminated after extracellular addition, but not transfection, of dsRNA. Importantly, the same applied to cells infected with bovine viral diarrhea virus (BVDV) expressing Erns but lacking the N-terminal protease Npro. Free Erns concentrations circulating in the blood of animals persistently infected with BVDV were determined to be approximately 50 ng ml−1, i.e. at a similar order of magnitude as that displaying an effect on dsRNA-induced IFN expression in vitro. Whilst Npro blocks interferon regulatory factor-3-dependent IFN induction in infected cells, Erns may prevent constant IFN induction in uninfected cells by dsRNA that could originate from pestivirus-infected cells. This probably contributes to the survival of persistently BVDV-infected animals and maintains viral persistence in the host population.
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Weiss, Susan R. "Activation and Antagonism of the OAS–RNase L Pathway." Proceedings 50, no. 1 (June 4, 2020): 14. http://dx.doi.org/10.3390/proceedings2020050014.
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The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, virus replication and spread, leading to apoptotic cell death. Endogenous host dsRNA, which accumulates in the absence of adenosine deaminase acting on RNA (ADAR)1, can also activate RNase L and lead to apoptotic cell death. RNase L activation and antiviral activity during infections with several types of viruses in human and bat cells is dependent on OAS3 but independent of virus-induced interferon (IFN) and, thus, RNase L can be activated even in the presence of IFN antagonists. Differently from other human viruses examined, Zika virus is resistant to the antiviral activity of RNase L and instead utilizes RNase L to enhance its replication factories to produce more infectious virus. Some betacoronaviruses antagonize RNase L activation by expressing 2′,5′-phosphodiesterases (PDEs) that cleave 2-5A and thereby antagonize activation of RNase L. The best characterized of these PDEs is the murine coronavirus (MHV) NS2 accessory protein. Enzymatically active NS2 is required for replication in myeloid cells and in the liver. Interestingly, while wild type mice clear MHV from the liver by 7–10 days post-infection, RNase L knockout mice fail to effectively clear MHV, probably due to diminished apoptotic death of infected cells. We suggest that RNase L antiviral activity stems from direct cleavage of viral genomes and cessation of protein synthesis as well as through promoting death of infected cells, limiting the spread of virus. Importantly, OASs are pattern recognition receptors and the OAS–RNase L pathway is a primary innate response pathway to viruses, capable of early response, coming into play before IFN is induced or when the virus shuts down IFN signaling.
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Boyce, Mark, and Polly Roy. "Recovery of Infectious Bluetongue Virus from RNA." Journal of Virology 81, no. 5 (December 6, 2006): 2179–86. http://dx.doi.org/10.1128/jvi.01819-06.
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ABSTRACT Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.
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Askenase, Philip. "Suppressor T cell exosomes via miR-150* and antigen specific Ig light chains. (50.13)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 50.13. http://dx.doi.org/10.4049/jimmunol.186.supp.50.13.
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Abstract Tolerance in contact sensitivity (CS) is due to CD8+ suppressor T cell (Ts) released factor (TsF) that is phenol chloroform extractable (PCE), in RNA fraction of Qiagen DNA/RNA separation column, sensitive to RNase A, not DNase nor trypsin, and also sensitive to RNase III; a property of double stranded RNA (dsRNA) like miRNA. Sizing electrophoresis indicated the active inhibitory RNA was about 75bp; the size of pre-miRNA. TsF RNA also inhibited in vitro IL-2 responses of T cell lines. We concluded that the small suppressive dsRNA was in the miRNA family TsF RNA is transported systematically in vivo in exosomes in the blood plasma of Ts donors. Remarkably, the exosomes act antigen (Ag) specifically due to surface coat of Ag-specific immunoglobulin free light chains, likely derived from activated B-1 B cells, and absorbed to receptors on the exosome surface. Ag specificity of the exosomes that eluted from the Ag column provided enriched exosome RNA for cDNA cloning and deep sequencing to identify candidate miRNA-150*. We postulate that the suppressive Ag-specific exosomes act systemically in an endocrine manner, via the blood, to target Ag specific effector T cells at distant sites, and inhibit their function via transfer of the inhibitory miRNA. This is the first demonstration of systemic immunoregulation by exosomes delivering inhibitory miRNA Ag specifically from donor suppressor T cells to distant target effector T cells in an endocrine manner.
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Mogren, Christina L., and Jonathan Gary Lundgren. "In silico identification of off-target pesticidal dsRNA binding in honey bees (Apis mellifera)." PeerJ 5 (December 13, 2017): e4131. http://dx.doi.org/10.7717/peerj.4131.
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Background Pesticidal RNAs that silence critical gene function have great potential in pest management, but the benefits of this technology must be weighed against non-target organism risks. Methods Published studies that developed pesticidal double stranded RNAs (dsRNAs) were collated into a database. The target gene sequences for these pesticidal RNAs were determined, and the degree of similarity with sequences in the honey bee genome were evaluated statistically. Results We identified 101 insecticidal RNAs sharing high sequence similarity with genomic regions in honey bees. The likelihood that off-target sequences were similar increased with the number of nucleotides in the dsRNA molecule. The similarities of non-target genes to the pesticidal RNA was unaffected by taxonomic relatedness of the target insect to honey bees, contrary to previous assertions. Gene groups active during honey bee development had disproportionately high sequence similarity with pesticidal RNAs relative to other areas of the genome. Discussion Although sequence similarity does not itself guarantee a significant phenotypic effect in honey bees by the primary dsRNA, in silico screening may help to identify appropriate experimental endpoints within a risk assessment framework for pesticidal RNAi.
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Kandasamy, Suresh K., and Ryuya Fukunaga. "Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production." Proceedings of the National Academy of Sciences 113, no. 49 (November 21, 2016): 14031–36. http://dx.doi.org/10.1073/pnas.1612393113.
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The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
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Romano, P. R., S. R. Green, G. N. Barber, M. B. Mathews, and A. G. Hinnebusch. "Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae." Molecular and Cellular Biology 15, no. 1 (January 1995): 365–78. http://dx.doi.org/10.1128/mcb.15.1.365.
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The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.
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Tian, Lan, Min-Sung Kim, Hongzhi Li, Jimin Wang, and Wei Yang. "Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid." Proceedings of the National Academy of Sciences 115, no. 3 (January 2, 2018): 507–12. http://dx.doi.org/10.1073/pnas.1719746115.
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HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.
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von Beck, Troy Alexander, Luis Mena Hernandez, Hongyi Zhou, Jeffrey Skolnick, and Joshy Jacob. "Repurposed antibiotic and antiviral drugs inhibit the immune evasive endoribonuclease of SARS-CoV-2 and restrict coronavirus infection in vitro." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 125.29. http://dx.doi.org/10.4049/jimmunol.208.supp.125.29.
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Abstract Despite the success of SARS-CoV-2 vaccines in curbing viral transmission and severe disease, infections in unvaccinated and immune deficient patients continue to drive significant morbidity and mortality globally. To address the needs of this patient population, multiple antiviral drugs must be developed to expand on the protection currently offered by convalescent plasma, monoclonal antibodies, remdesivir, and other next generation SARS-CoV-2 antivirals which may be contraindicated for some patient groups. To this end, we employed a machine-learning based virtual ligand screening algorithm, FRAGSITE, to screen a library of FDA approved compounds for binding to the SARS-CoV-2 endoribonuclease (nsp15), a structurally conserved and demonstrated coronavirus virulence factor in SARS-CoV-1, HCoV-229E, IBV, MHV, and PEDV. Using recombinant SARS-CoV-2 nsp15, we identified the inhibition of nuclease activity by 11 drugs in vitro; 6 also restrict infection by the related human OC43 coronavirus in human A549 airway epithelial cells as measured by a focus forming assay. Among these 6 candidates, pibrentasvir remained active against HCoV-OC43 at the lowest concentration (IC50 < 0.625μM). Consistent with previous findings, HCoV-OC43 infection of A549 cells did not provoke an innate response to the viral dsRNA as quantified by IFNB1 mRNA production, antiviral stress granule formation, or RNase L activation. Our preliminary results suggest that antiviral stress granule formation is partially restored by nsp15 inhibitor treatment in HCoV-OC43 infected cells, although IFNβ production and RNase L activation remain unchanged. Supported by grants from the Emory School of Medicine
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Gao, Yanning, Shao-an Xue, and Beverly E. Griffin. "Sensitivity of an Epstein-Barr Virus-Positive Tumor Line, Daudi, to Alpha Interferon Correlates with Expression of a GC-Rich Viral Transcript." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7305–13. http://dx.doi.org/10.1128/mcb.19.11.7305.
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ABSTRACT The exquisite sensitivity of the Burkitt’s lymphoma (BL)-derived cell line Daudi to type I interferons has not previously been explained. Here we show that expression of an Epstein-Barr virus (EBV) transcript, designated D-HIT (Y. Gao et al., J. Virol. 71:84–94, 1997), correlates with the sensitivity of different Daudi cell isolates (or that of other EBV-carrying cells, where known) to alpha interferon (IFN-α). D-HIT, transcribed from a GC-rich repetitive region (IR4) of the viral genome, is highly structured, responding to RNase digestion in a manner akin to double-stranded RNA. Comparing EBV-carrying BL cell lines with differing responses to IFN-α, we found the protein levels of the dsRNA-activated kinase, PKR, to be similar, whereas the levels of the autophosphorylated active form of PKR varied in a manner that correlated with endogenous levels of D-HIT expression. In a classical in vitro kinase assay, addition of either poly(I)-poly(C) or an in vitro-transcribed D-HIT homolog stimulated the autophosphorylation activity of PKR from IFN-α-treated cells in both EBV-positive and EBV-negative B lymphocytes. By transfection experiments, these RNAs were shown to reduce cell proliferation and to sensitize otherwise relatively insensitive Raji cells to IFN-α. The data lead to a model wherein the D-HIT viral RNA also serves as a possible transcriptional activator of IFN-α or cellular genes regulated by this cytokine.
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Silvestri, Lynn S., M. Alejandra Tortorici, Rodrigo Vasquez-Del Carpio, and John T. Patton. "Rotavirus Glycoprotein NSP4 Is a Modulator of Viral Transcription in the Infected Cell." Journal of Virology 79, no. 24 (December 15, 2005): 15165–74. http://dx.doi.org/10.1128/jvi.79.24.15165-15174.2005.
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ABSTRACT The outer shell of the rotavirus triple-layered virion is lost during cell entry, yielding a double-layered particle (DLP) that directs synthesis of viral plus-strand RNAs. The plus-strand RNAs act as templates for synthesis of the segmented double-stranded RNA (dsRNA) genome in viral inclusion bodies (viroplasms). The viral endoplasmic reticulum (ER)-resident glycoprotein NSP4 recruits progeny DLPs formed in viroplasms to the ER, where the particles are converted to triple-layered particles (TLPs) via budding. In this study, we have used short interfering RNAs to probe the role of NSP4 in the viral life cycle. Our analysis showed that knockdown of NSP4 expression had no marked effect on the expression of other viral proteins or on the replication of the dsRNA genome segments. However, NSP4 loss of function suppressed viroplasm maturation and caused a maldistribution of nonstructural and structural proteins that normally accumulate in viroplasms. NSP4 loss of function also inhibited formation of packaged virus particles, instead inducing the accumulation of empty particles. Most significant was the observation that NSP4 knockdown led to dramatically increased levels of viral transcription late in the infection cycle. These findings point to a multifaceted role for NSP4 in virus replication, including influencing the development of viroplasms, linking genome packaging with particle assembly, and acting as a modulator of viral transcription. By recruiting transcriptionally active or potentially active DLPs to the ER for conversion to quiescent TLPs, NSP4 acts as a feedback inhibitor down-regulating viral transcription when adequate levels of plus-strand RNAs are available to allow for productive infection.
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Niño-Sánchez, Jonatan, Li-Hung Chen, Jorge Teodoro De Souza, Sandra Mosquera, and Ioannis Stergiopoulos. "Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria." Journal of Fungi 7, no. 2 (February 9, 2021): 125. http://dx.doi.org/10.3390/jof7020125.
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Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.
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Jiang, Xue, Qinfeng Huang, Wenjian Wang, Haohao Dong, Hinh Ly, Yuying Liang, and Changjiang Dong. "Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression." Journal of Biological Chemistry 288, no. 23 (April 24, 2013): 16949–59. http://dx.doi.org/10.1074/jbc.m112.420521.
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A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3′-5′ exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5′ triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.
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Zhuravlyov*, V. S., V. V. Dolgikh, S. A. Timofeev, and F. B. Gannibal. "RNA interference method in plant protection against insect pests." PLANT PROTECTION NEWS 105, no. 1 (April 25, 2022): 28–39. http://dx.doi.org/10.31993/2308-6459-2022-105-1-15219.
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RNA-interference, or suppression of gene expression by small RNAs, was originally described in Caenorhabditis elegans in 1998 and is currently widely considered for use in plant protection. The use of double-stranded RNA molecules as an inducer of the RNA interference pathway in insect pests potentially allows employing them as active ingredients in modern pesticides. Genetically modified crops expressing dsRNA have been developed as commercial products with a great potential in insect pest management. Alternatively, some nontransformative approaches, including foliar spray and chemigation, are also suitable for practical applications. This review explains the mechanism of artificially induced RNA interference and existing strategies for the delivery of small RNAs to target insects within the framework of plant protection.
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Mishra, Vibhor, Jasleen Singh, Feng Wang, Yixiang Zhang, Akihito Fukudome, Jonathan C. Trinidad, Yuichiro Takagi, and Craig S. Pikaard. "Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV." Proceedings of the National Academy of Sciences 118, no. 13 (March 22, 2021): e2019276118. http://dx.doi.org/10.1073/pnas.2019276118.
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In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV’s 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3′ ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).
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Dumetier, Baptiste, Camille Sauter, Azadeh Hajmirza, Baptiste Pernon, Romain Aucagne, Cyril Fournier, Céline Row, et al. "Repeat Element Activation-Driven Inflammation: Role of NFκB and Implications in Normal Development and Cancer?" Biomedicines 10, no. 12 (December 1, 2022): 3101. http://dx.doi.org/10.3390/biomedicines10123101.
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The human genome is composed of unique DNA sequences that encode proteins and unique sequence noncoding RNAs that are essential for normal development and cellular differentiation. The human genome also contains over 50% of genome sequences that are repeat in nature (tandem and interspersed repeats) that are now known to contribute dynamically to genetic diversity in populations, to be transcriptionally active under certain physiological conditions, and to be aberrantly active in disease states including cancer, where consequences are pleiotropic with impact on cancer cell phenotypes and on the tumor immune microenvironment. Repeat element-derived RNAs play unique roles in exogenous and endogenous cell signaling under normal and disease conditions. A key component of repeat element-derived transcript-dependent signaling occurs via triggering of innate immune receptor signaling that then feeds forward to inflammatory responses through interferon and NFκB signaling. It has recently been shown that cancer cells display abnormal transcriptional activity of repeat elements and that this is linked to either aggressive disease and treatment failure or to improved prognosis/treatment response, depending on cell context and the amplitude of the so-called ‘viral mimicry’ response that is engaged. ‘Viral mimicry’ refers to a cellular state of active antiviral response triggered by endogenous nucleic acids often derived from aberrantly transcribed endogenous retrotransposons and other repeat elements. In this paper, the literature regarding transcriptional activation of repeat elements and engagement of inflammatory signaling in normal (focusing on hematopoiesis) and cancer is reviewed with an emphasis on the role of innate immune receptor signaling, in particular by dsRNA receptors of the RIG-1 like receptor family and interferons/NFκB. How repeat element-derived RNA reprograms cell identity through RNA-guided chromatin state modulation is also discussed.
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Iordanov, Mihail S., Jayashree M. Paranjape, Aimin Zhou, John Wong, Bryan R. G. Williams, Eliane F. Meurs, Robert H. Silverman, and Bruce E. Magun. "Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways." Molecular and Cellular Biology 20, no. 2 (January 15, 2000): 617–27. http://dx.doi.org/10.1128/mcb.20.2.617-627.2000.
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ABSTRACT Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.
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Kniert, Justine, Theodore dos Santos, Heather E. Eaton, Woo Jung Cho, Greg Plummer, and Maya Shmulevitz. "Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly." PLOS Pathogens 18, no. 9 (September 13, 2022): e1010641. http://dx.doi.org/10.1371/journal.ppat.1010641.
Full textAbstract:
Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins μ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a μ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC μ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating μ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.
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Nameth, S. T., and S. L. Cheng. "Identification and Partial Characterization of Endogenous Double-stranded Ribonucleic Acid in Mulberry." Journal of the American Society for Horticultural Science 119, no. 4 (July 1994): 859–61. http://dx.doi.org/10.21273/jashs.119.4.859.
Full textAbstract:
Double-stranded ribonucleic acid (dsRNA) analysis of apparently healthy red mulberry (Morus rubra L.) yielded four distinct dsRNA banding profiles. dsRNA type 1 contained three dsRNA bands with approximate molecular weights (MWs) of 12.0, 1.0, and 0.9 × 106, respectively. dsRNA type 2 contained two dsRNA bands with MWs of 1.0 and 0.9 × 106. dsRNA type 3 contained four dsRNA bands with MWs of 1.0, 0.9, 0.89, and 0.88 × 106. dsRNA type 4 contained three dsRNA bands with MWs of 1.0, 0.88, and 0.87 × 106. No virus particles were associated with any of the samples analyzed. All four types of dsRNA were resistant to DNase I and RNase A in high salt and susceptible to RNase A in low salt. Mulberry dsRNAs were somewhat similar to endogenous dsRNAs (edsRNA) associated with other hosts. This is the first report of edsRNA associated with a deciduous tree.
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Cánovas-Márquez, José Tomás, Sebastian Falk, Francisco E. Nicolás, Subramanian Padmanabhan, Rubén Zapata-Pérez, Álvaro Sánchez-Ferrer, Eusebio Navarro, and Victoriano Garre. "A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA." Nucleic Acids Research 49, no. 9 (April 20, 2021): 5294–307. http://dx.doi.org/10.1093/nar/gkab238.
Full textAbstract:
Abstract Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.
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Kreuze, Jan F., Eugene I. Savenkov, Wilmer Cuellar, Xiangdong Li, and Jari P. T. Valkonen. "Viral Class 1 RNase III Involved in Suppression of RNA Silencing." Journal of Virology 79, no. 11 (June 1, 2005): 7227–38. http://dx.doi.org/10.1128/jvi.79.11.7227-7238.2005.
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ABSTRACT Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.
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Abaturov, A. E., and V. L. Babуch. "MicroRNA biogenesis. Part 2. Formation of mature miRNAs. Maturation of non-canonical miRNAs." CHILD`S HEALTH 16, no. 3 (June 22, 2021): 257–63. http://dx.doi.org/10.22141/2224-0551.16.3.2021.233912.
Full textAbstract:
The scientific review presents the biogenesis of miRNAs. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article shows the stages of formation of mature miRNAs. It is noted that duplex RNAs resulting from DICER-mediated cleavage interact with Argonaute (AGO) proteins to form an effector RNA-induced silencing complex (RISC). It is shown that the deficiency of AGO proteins leads to a significant decrease in the amount of miRs, and overexpression of AGO proteins is accompanied by an increase in the level of miRs. The main stages of assembling a fully functional RISC are presented. The first stage is the loading of duplex miRs on AGO proteins. The second stage is the promotion of duplex miRs. Human diseases associated with processing disorders in the cytoplasm of the cell are presented. Numerous alternative mechanisms involved in the formation of functionally active miRs are is characterized. There are three classes of mirtrons: typical mirtrons, 5’-tailed mirtrons and 3’-tailed mirtrons. Endogenous csRNAs resemble Drosha-independent synthetic csRNAs used to experimentally induce gene knockout. Chimeric hairpins of non-canonical miR genes are transcribed in tandem or as a part of another type of small RNA gene. Thus, the formation of mature miRs occurs due to the formation of the RISC complex. The core of the RISC complex consists of microRNA, AGO and protein with a trinucleotide repeat 6. Loading dsRNA on AGO proteins and subsequent promotion of duplex RNA are the main stages of assembly of a fully functional RISC. Disorders of processing in the cytoplasm of the cell are associated with the development of some human diseases. There are alternative mechanisms involved in the formation of functionally active miRs: mirtrons, endogenous short RNAs containing hairpins, chimeric hairpins.
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Banerjee, Shuvojit, Elona Gusho, Christina Gaughan, Beihua Dong, Xiaorong Gu, Elise Holvey-Bates, Manisha Talukdar, et al. "OAS-RNase L innate immune pathway mediates the cytotoxicity of a DNA-demethylating drug." Proceedings of the National Academy of Sciences 116, no. 11 (February 27, 2019): 5071–76. http://dx.doi.org/10.1073/pnas.1815071116.
Full textAbstract:
Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.
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Daou, Salima, Manisha Talukdar, Jinle Tang, Beihua Dong, Shuvojit Banerjee, Yize Li, Nicole M. Duffy, et al. "A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency." Proceedings of the National Academy of Sciences 117, no. 40 (September 21, 2020): 24802–12. http://dx.doi.org/10.1073/pnas.2006883117.
Full textAbstract:
The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
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Chitrakar, Alisha, Sneha Rath, Jesse Donovan, Kaitlin Demarest, Yize Li, Raghavendra Rao Sridhar, Susan R. Weiss, Sergei V. Kotenko, Ned S. Wingreen, and Alexei Korennykh. "Real-time 2-5A kinetics suggest that interferons β and λ evade global arrest of translation by RNase L." Proceedings of the National Academy of Sciences 116, no. 6 (January 17, 2019): 2103–11. http://dx.doi.org/10.1073/pnas.1818363116.
Full textAbstract:
Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2′,5′-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.
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Zeng, Chun, Xin Yi, Danny Zipris, Hongli Liu, Lin Zhang, Qiaoyun Zheng, Krishnamurthy Malathi, Ge Jin, and Aimin Zhou. "RNase L contributes to experimentally induced type 1 diabetes onset in mice." Journal of Endocrinology 223, no. 3 (October 6, 2014): 277–87. http://dx.doi.org/10.1530/joe-14-0509.
Full textAbstract:
The cause of type 1 diabetes continues to be a focus of investigation. Studies have revealed that interferon α (IFNα) in pancreatic islets after viral infection or treatment with double-stranded RNA (dsRNA), a mimic of viral infection, is associated with the onset of type 1 diabetes. However, how IFNα contributes to the onset of type 1 diabetes is obscure. In this study, we found that 2-5A-dependent RNase L (RNase L), an IFNα-inducible enzyme that functions in the antiviral and antiproliferative activities of IFN, played an important role in dsRNA-induced onset of type 1 diabetes. Using RNase L-deficient, rat insulin promoter-B7.1 transgenic mice, which are more vulnerable to harmful environmental factors such as viral infection, we demonstrated that deficiency of RNase L in mice resulted in a significant delay of diabetes onset induced by polyinosinic:polycytidylic acid (poly I:C), a type of synthetic dsRNA, and streptozotocin, a drug which can artificially induce type 1-like diabetes in experimental animals. Immunohistochemical staining results indicated that the population of infiltrated CD8+T cells was remarkably reduced in the islets of RNase L-deficient mice, indicating that RNase L may contribute to type 1 diabetes onset through regulating immune responses. Furthermore, RNase L was responsible for the expression of certain proinflammatory genes in the pancreas under induced conditions. Our findings provide new insights into the molecular mechanism underlying β-cell destruction and may indicate novel therapeutic strategies for treatment and prevention of the disease based on the selective regulation and inhibition of RNase L.
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Liu, Ruikang, and Bernard Moss. "Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants." Journal of Virology 90, no. 17 (June 22, 2016): 7864–79. http://dx.doi.org/10.1128/jvi.00869-16.
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ABSTRACTVaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent double-stranded RNA (dsRNA) accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type VACV and that they coprecipitated with antibody. An E3 deletion mutant induced protein kinase R (PKR) and eukaryotic translation initiation factor alpha (eIF2α) phosphorylation earlier and more strongly than a decapping enzyme mutant even though less dsRNA was made, leading to more profound effects on viral gene expression. Human HAP1 and A549 cells were genetically modified by clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knockout (KO) HAP1 cells in which RNase L is intrinsically inactive but only with a double knockout (DKO) of PKR and RNase L in A549 cells, indicating that both pathways decreased replication equivalently and that no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than that of wild-type virus) in DKO A549 cells but not in DKO HAP1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but nonpermissive for VACV; however, wild-type and mutant viruses replicated in triple-KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L was sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant, particularly in HAP1 DKO, cells indicated additional translational defects.IMPORTANCEViruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded RNA (dsRNA) triggers in host cells. We showed that the dsRNA produced in excess in cells infected with a vaccinia virus (VACV) decapping enzyme mutant and by wild-type virus colocalized with the viral E3 protein in cytoplasmic viral factories. Novel human cell lines defective in either or both protein kinase R and RNase L dsRNA effector pathways and/or the cellular 5′ exonuclease Xrn1 were prepared by CRISPR-Cas9 gene editing. Inactivation of both pathways was necessary and sufficient to allow full replication of the E3 mutant and reverse the defect cause by inactivation of Xrn1, whereas the decapping enzyme mutant still exhibited defects in gene expression. The study provided new insights into functions of the VACV proteins, and the well-characterized panel of CRISPR-Cas9-modified human cell lines should have broad applicability for studying innate dsRNA pathways.
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Ji, Xinhua. "RNA Biogenesis: Mechanism and Evolution of RNase III." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C210. http://dx.doi.org/10.1107/s2053273314097897.
Full textAbstract:
RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer are representative members of the family. The bacterial enzyme possesses a single RNase III domain (RIIID) followed by a dsRNA-binding domain (dsRBD); Rnt1p is defined by the presence of an N-terminal domain (NTD), a RIIID, and a dsRBD; Drosha contains N-terminal P-rich and RS-rich domains followed by two RIIIDs and a dsRBD; and Dicer possesses N-terminal helicase, DUF283, and PAZ domains followed by two RIIIDs and a dsRBD. It is the N-terminal extension beyond the RIIID that distinguishes eukaryotic RNase IIIs from the bacterial enzyme. My lab has been studying the structure and mechanism of RNase III enzymes since 1996. We have reported a total of eleven crystal structures of bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex (Gan et al., Cell, 124:355-366, 2006), and thereby well characterized the mechanism of action for the bacterial enzyme (Court et al., Annu Rev Genet, 47:405-431, 2013). We have also determined the crystal structure of yeast Rnt1p post-cleavage complex, the first structure of a eukaryotic RNase III complexed with RNA in a catalytically meaningful manner (Liang et al., Molecular Cell, 54:431-444, 2014, featured article on the issue cover). Strikingly, the NTD and dsRBD of Rnt1p function as two rulers for substrate selection. This unusual mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs, including Drosha and Dicer.
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Yan, Wei, Lang Ma, Paula Stein, Stephanie A. Pangas, Kathleen H. Burns, Yuchen Bai, Richard M. Schultz, and Martin M. Matzuk. "Mice Deficient in Oocyte-Specific Oligoadenylate Synthetase-Like Protein OAS1D Display Reduced Fertility." Molecular and Cellular Biology 25, no. 11 (June 1, 2005): 4615–24. http://dx.doi.org/10.1128/mcb.25.11.4615-4624.2005.
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ABSTRACT The double-stranded RNA (dsRNA)-induced interferon response is a defense mechanism against viral infection. Upon interferon activation by dsRNA, 2′,5′-oligoadenylate synthetase 1 (OAS1A) is induced; it binds dsRNA and converts ATP into 2′,5′-linked oligomers of adenosine (called 2-5A), which activate RNase L that in turn degrades viral and cellular RNAs. In a screen to identify oocyte-specific genes, we identified a novel murine cDNA encoding an ovary-specific 2′,5′-oligoadenylate synthetase-like protein, OAS1D, which displays 59% identity with OAS1A. OAS1D is predominantly cytoplasmic and is exclusively expressed in growing oocytes and early embryos. Like OAS1A, OAS1D binds the dsRNA mimetic poly(I-C), but unlike OAS1A, it lacks 2′-5′ adenosine linking activity. OAS1D interacts with OAS1A and inhibits the enzymatic activity of OAS1A. Mutant mice lacking OAS1D (Oas1d −/−) display reduced fertility due to defects in ovarian follicle development, decreased efficiency of ovulation, and eggs that are fertilized arrest at the one-cell stage. These effects are exacerbated after activation of the interferon/OAS1A/RNase L pathway by poly(I-C). We propose that OAS1D suppresses the interferon/OAS/RNase L-mediated cellular destruction by interacting with OAS1A during oogenesis and early embryonic development.
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Azevedo, Andréia Cristiane Souza, Daniel Ricardo Sosa-Gómez, Marcos Rodrigues Faria, and Maria Helena Pelegrinelli Fungaro. "Effects of double-stranded RNA on virulence of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against the silverleaf whitefly, Bemisia tabaci strain B (Homoptera: Aleyrodidae)." Genetics and Molecular Biology 23, no. 1 (March 2000): 61–63. http://dx.doi.org/10.1590/s1415-47572000000100010.
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Bands of double-stranded RNA (dsRNA) were detected in three out of twelve isolates of Paecilomyces fumosoroseus. Identity of these bands was confirmed by RNAse, DNAse and S1 nuclease treatments. The cure of dsRNA for one isolate (P92) was successfully carried out for a single conidium subculture. Isogenic strains, with or without dsRNA, were submitted to virulence tests against the whitefly Bemisia tabaci strain B. In contrast to findings for some phytopathogenic fungi, these dsRNA fragments did not cause hypovirulence in P. fumosoroseus.
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Whelan, Jillian N., Nicholas A. Parenti, Joshua Hatterschide, David M. Renner, Yize Li, Hanako M. Reyes, Beihua Dong, Erick R. Perez, Robert H. Silverman, and Susan R. Weiss. "Zika virus employs the host antiviral RNase L protein to support replication factory assembly." Proceedings of the National Academy of Sciences 118, no. 22 (May 24, 2021): e2101713118. http://dx.doi.org/10.1073/pnas.2101713118.
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Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.
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Brettmann, Erin A., Jahangheer S. Shaik, Haroun Zangger, Lon-Fye Lye, F. Matthew Kuhlmann, Natalia S. Akopyants, Dayna M. Oschwald, et al. "Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response." Proceedings of the National Academy of Sciences 113, no. 43 (October 18, 2016): 11998–2005. http://dx.doi.org/10.1073/pnas.1615085113.
Full textAbstract:
ManyLeishmania(Viannia) parasites harbor the double-stranded RNA virusLeishmania RNA virus 1(LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in bothLeishmania braziliensisandLeishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lackscis- ortrans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For bothL. braziliensisandL. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3–dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence ofL. braziliensis, theLeishmaniaspecies responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of otherLeishmaniaviruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi–viral relationships in evolution: one of balance rather than elimination.
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Streitenfeld, Hein, Amanda Boyd, John K. Fazakerley, Anne Bridgen, Richard M. Elliott, and Friedemann Weber. "Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs." Journal of Virology 77, no. 9 (May 1, 2003): 5507–11. http://dx.doi.org/10.1128/jvi.77.9.5507-5511.2003.
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ABSTRACT Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
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Lamontagne, Bruno, Annie Tremblay, and Sherif Abou Elela. "The N-Terminal Domain That Distinguishes Yeast from Bacterial RNase III Contains a Dimerization Signal Required for Efficient Double-Stranded RNA Cleavage." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1104–15. http://dx.doi.org/10.1128/mcb.20.4.1104-1115.2000.
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ABSTRACT Yeast Rnt1 is a member of the double-stranded RNA (dsRNA)-specific RNase III family identified by conserved dsRNA binding (dsRBD) and nuclease domains. Comparative sequence analyses have revealed an additional N-terminal domain unique to the eukaryotic homologues of RNase III. The deletion of this domain from Rnt1 slowed growth and led to mild accumulation of unprocessed 25S pre-rRNA. In vitro, deletion of the N-terminal domain reduced the rate of RNA cleavage under physiological salt concentration. Size exclusion chromatography and cross-linking assays indicated that the N-terminal domain and the dsRBD self-interact to stabilize the Rnt1 homodimer. In addition, an interaction between the N-terminal domain and the dsRBD was identified by a two-hybrid assay. The results suggest that the eukaryotic N-terminal domain of Rnt1 ensures efficient dsRNA cleavage by mediating the assembly of optimum Rnt1-RNA ribonucleoprotein complex.
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Nganvongpanit, Korakot, Heike Müller, Franca Rings, Michael Hoelker, Danyel Jennen, Ernst Tholen, Vitezslav Havlicek, Urban Besenfelder, Karl Schellander, and Dawit Tesfaye. "Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA." Reproduction 131, no. 5 (May 2006): 861–74. http://dx.doi.org/10.1530/rep.1.01040.
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RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2,in vitroproduced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.
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Zhang, Lin, Luxi Chen, Jing Chen, Weimin Shen, and Anming Meng. "Mini-III RNase-based dual-color system for in vivo mRNA tracking." Development 147, no. 22 (October 22, 2020): dev190728. http://dx.doi.org/10.1242/dev.190728.
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ABSTRACTMini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5β-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.
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Iordanov, Mihail S., John Wong, John C. Bell, and Bruce E. Magun. "Activation of NF-κB by Double-Stranded RNA (dsRNA) in the Absence of Protein Kinase R and RNase L Demonstrates the Existence of Two Separate dsRNA-Triggered Antiviral Programs." Molecular and Cellular Biology 21, no. 1 (January 1, 2001): 61–72. http://dx.doi.org/10.1128/mcb.21.1.61-72.2001.
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ABSTRACT Double-stranded RNA (dsRNA) of viral origin triggers two programs of the innate immunity in virus-infected cells. One is intended to decrease the rate of host cell protein synthesis and thus to prevent viral replication. This program is mediated by protein kinase R (PKR) and by RNase L and contributes, eventually, to the self-elimination of the infected cell via apoptosis. The second program is responsible for the production of antiviral (type I) interferons and other alarmone cytokines and serves the purpose of preparing naive cells for the viral invasion. This second program requires the survival of the infected cell and depends on the expression of antiapoptotic genes through the activation of the NF-κB transcription factor. The second program therefore relies on ongoing transcription and translation. It has been proposed that PKR plays an essential role in the activation of NF-κB by dsRNA. Here we present evidence that the dsRNA-induced NF-κB activity and the expression of beta interferon and inflammatory cytokines do not require either PKR or RNase L. Our results indicate, therefore, that the two dsRNA-activated programs are separate and can function independently of each other.
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Catalanotto, Caterina, Massimiliano Pallotta, Paul ReFalo, Matthew S. Sachs, Laurence Vayssie, Giuseppe Macino, and Carlo Cogoni. "Redundancy of the Two Dicer Genes in Transgene-Induced Posttranscriptional Gene Silencing in Neurospora crassa." Molecular and Cellular Biology 24, no. 6 (March 15, 2004): 2536–45. http://dx.doi.org/10.1128/mcb.24.6.2536-2545.2004.
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ABSTRACT RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent gene silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transgenes induces sequence-specific mRNA degradation. These phenomena share a common genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like genes by mutation in the fungus Neurospora crassa eliminates transgene-induced gene silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in gene silencing induced by transgenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of silencing.
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Głów, Dawid, Dariusz Pianka, Agata A. Sulej, Łukasz P. Kozłowski, Justyna Czarnecka, Grzegorz Chojnowski, Krzysztof J. Skowronek, and Janusz M. Bujnicki. "Sequence-specific cleavage of dsRNA by Mini-III RNase." Nucleic Acids Research 43, no. 5 (January 29, 2015): 2864–73. http://dx.doi.org/10.1093/nar/gkv009.
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Patterson, N. A., and M. Kapoor. "Detection of plasmid-like DNA and double-stranded RNA elements in some Canadian isolates of the oilseed rape pathogen Leptosphaeria maculans." Canadian Journal of Microbiology 42, no. 9 (September 1, 1996): 977–82. http://dx.doi.org/10.1139/m96-126.
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Nine Canadian isolates of Leptosphaeria maculans were examined for the presence of plasmid-like elements. A single extrachromosomal DNA element, with an estimated size of 9 kb, was detected in undigested genomic DNA of a virulent isolate, Fairview 1. This element was susceptible to hydrolysis by DNAse I and exonuclease III, and it was shown to hybridize to DNA of all virulent isolates. In addition, another virulent strain, Saskatoon 8, was observed to contain four double-stranded RNA (dsRNA) segments, ranging from approximately 500 bp to 2.4 kb. The latter segments (dsRNA) were resistant to DNAse I and exonuclease III treatment, and to RNAse A in high-ionic-strength buffer, but were susceptible to RNAse A in the presence of low-ionic-strength buffer.Key words: Leptosphaeria maculans, Canola, double-stranded RNA, plasmids.
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Budt, Matthias, Lars Niederstadt, Ralitsa S. Valchanova, Stipan Jonjić, and Wolfram Brune. "Specific Inhibition of the PKR-Mediated Antiviral Response by the Murine Cytomegalovirus Proteins m142 and m143." Journal of Virology 83, no. 3 (November 19, 2008): 1260–70. http://dx.doi.org/10.1128/jvi.01558-08.
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ABSTRACT Double-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2α, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR- and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2α phosphorylation during infection with an m142- and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2α kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (γ34.5) rescued the replication defect of an MCMV strain with deletions of both m142 and m143. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142- and m143-deficient MCMV strain replicated to high titers in fibroblasts lacking PKR but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.
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Ludwig, Holger, Yasemin Suezer, Zoe Waibler, Ulrich Kalinke, Barbara S. Schnierle, and Gerd Sutter. "Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara." Journal of General Virology 87, no. 5 (May 1, 2006): 1145–55. http://dx.doi.org/10.1099/vir.0.81623-0.
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Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-ΔE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2′-5′-oligoadenylate synthetase (2′-5′OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2′-5′OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2α (eIF2α) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-ΔE3L-infected mouse embryonic fibroblasts from Pkr 0/0 mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.
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Magg, Thomas, Tsubasa Okano, Lars M. Koenig, Daniel F. R. Boehmer, Samantha L. Schwartz, Kento Inoue, Jennifer Heimall, et al. "Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency." Science Immunology 6, no. 60 (June 18, 2021): eabf9564. http://dx.doi.org/10.1126/sciimmunol.abf9564.
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Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon–induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2′-5′-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell–derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L–mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.
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Rath, Sneha, Jesse Donovan, Gena Whitney, Alisha Chitrakar, Wei Wang, and Alexei Korennykh. "Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15916–21. http://dx.doi.org/10.1073/pnas.1513034112.
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Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.
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Zografidis, Aris, Filip Van Nieuwerburgh, Anna Kolliopoulou, Konstantinos Apostolou-Karampelis, Steven R. Head, Dieter Deforce, Guy Smagghe, and Luc Swevers. "Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection." Journal of Virology 89, no. 22 (September 2, 2015): 11473–86. http://dx.doi.org/10.1128/jvi.01695-15.
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ABSTRACTThe lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkwormBombyx moriagainst both persistent and pathogenic infection ofB. moricytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.IMPORTANCEThis work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV;Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.