dsRNA Digestion Research Articles

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.

Comparative analysis of double-stranded RNA degradation and processing in insects

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within the same order. We identified homologs of key RNAi genes in the genomes of some of these insects and studied their domain architecture. These data suggest that dsRNA digestion by dsRNases and its processing to siRNAs in the cells are among the major factors contributing to differential RNAi efficiency reported among insects.

Double-Stranded RNA of Intestinal Commensal but Not Pathogenic Bacteria Triggers Production of Protective Interferon-β

The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-β (IFN-β) production, which protected mice from experimental colitis. The LAB-induced IFN-β response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-β than LAB, and dsRNA was not involved in pathogen-induced IFN-β induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatoryand protective immune responses.

Construction and advantage of endoribonuclease III-prepared siRNAs (esiRNA) library

Objective To establish KIF4A 3'UTR esiRNA library and analyze the advantage of method in studying the function of KIF4A in SGC-7901 cells.Methods The GST-fusion protein of Escherichia coli endoribonuclease Ⅲ (GST-RNase Ⅲ) was used to digest the double strand RNA (dsRNA),which was transcribed in vitro from a 502bp template of KIF4A genome (T7-KIF4A 3'UTR).The KIF4A esiRNA library generated from the above method and chemically synthesized KIF4A siRNA were then used to transfect SGC-7901 cells at 10 nmol/L and 20 nmol/L.Real-time quantitative PCR and Western Blot were used to detect the mRNA and protein level of KIF4A,respectively.Results The KIF4A esiRNA library was effectively established from dsRNA digestion using GST-RNase Ⅲ.KIF4A expression was significantly reduced in SGC-7901 cells transfected with KIF4A esiRNA or siRNA.In addition,the inhibitory effect of KIF4A esiRNA was more effective than that of chemically synthesized siRNA.Conclusion KIF4A esiRNA library which was obtained using biological method can more effectively inhibited the expression of KIF4A more effectively in SGC-7901 cells than chemically synthesized siRNA.Therefore,esiRNA library can be used as a new and more effective method in studying the function of KIF4A in SGC-7901 cells. Key words: RNA interference; esiRNA library; KIF4A

Production of Cyclin D1 specific siRNAs by double strand processing for gene therapy of esophageal squamous cell carcinoma

BACKGROUND AND AIM: (RNA interference) is a new strategy in gene therapy and biotechnology which provides new viewpoints in treatment of different diseases such as cancer and viral disease s. CCND1 which is a key gene in cell cycle is amplified and over expressed in es ophageal cancer. The aim of this study was to produce siRNAs for CCND1, the key gene in cell cycle. METHODS: dsRNA digestion method was applied by using recombinant human dicer enzyme to cleave in vitro transcribed dsRNA i nto a pool of 22bp siRNA. Total RNA was extracted and cDNA was produced using RT-PCR. T7 promoter was added to both ends of the DNA template by PCR. RNA was produced from both strands of the DNA using T7 RNA polymerase. After annealing both strands, dsRNA was prepared. Finally siRNA pool was produced by dicer treatment . RESULTS: RNA extraction yield from HN5 cell line was 14.69 µ g/10 6 cell. The results from beta actin control gene confirmed the cDNA integrity. After optimization, T7 promoter adding was confirmed using gel electrophoresis and DNA sequencing. After optimization dsRNA yield was improved. The best incubation condition was 18h. Each microgram of dsRNA yielded 0.5 µg siRNA. COCLUSION: dsRNA digestion method includes several steps in which the product of each step is used as the precursor for t he next step. So optimization and increasing the specificity and product yield should be the most important goals of the study, Because the yield of each step has a dir ect relationship with the final product yield namely; siRNA. Optimizing and increasing the yield, dsRNA digestion method could be a rapid, available and pr ofitable method for siRNA generation, and providing large amounts of siRNA.

Double-stranded RNA of intestinal commensal bacteria triggers production of protective IFN-�

Event Abstract Back to Event Double-stranded RNA of intestinal commensal bacteria triggers production of protective IFN-β Tadaomi Kawashima1, 2, Huimin Yan1, 3, Zijin Guo4, Joaquim Carreras1, 5, Ryosuke Uchiyama6, Ryutaro Fukui7, Tsuneyasu Kaisho8, Shizuo Akira8, Kensuke Miyake7, Hiroko Tsutsui6, Takashi Saito4 and Noriko M. Tsuji1* 1 National Institute of Advanced Science and Technology, Biomedical Research Institute, Japan 2 Kikkoman Corporation, Japan 3 The Fifth Hospital of Shijiazhuang, China 4 RIKEN, Japan 5 Tokai University, Japan 6 Hyogo College of Medicine, Japan 7 The University of Tokyo, The Institute of Medical Science, Japan 8 Osaka University, WPI Immunology Frontier Research Center, Japan The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered IFN-β production from dendritic cells (DCs) and protected mice from experimental colitis. Endosomal TLRs are required for this protective mechanism, because Tlr3-/- as well as Unc93b13d mice, in which endosomal TLR3, 7, and 9 are dysfunctional, were not affected by oral administration of LAB in colitis model. While digestion of dsRNA canceled in vivo effects, suggesting TLR3 initiates the anti-inflammatory process, we also found that cooperation of TLR3 and TLR9 maximized IFN-β production in vitro. Compared to LAB, pathogenic bacteria contained less dsRNA and induced much less IFN-β. Moreover, dsRNA was not involved in pathogen-induced IFN-β induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and contribute to the maintenance of immunological homeostasis. Collaborators: Daisuke Kaneko, Ikuko Nishimura Keywords: TLR3, endosome, small intestine, anti-inflammation, Colitis, Lactic acid bacteria, IFN-beta, double-stranded RNA Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Kawashima T, Yan H, Guo Z, Carreras J, Uchiyama R, Fukui R, Kaisho T, Akira S, Miyake K, Tsutsui H, Saito T and Tsuji NM (2013). Double-stranded RNA of intestinal commensal bacteria triggers production of protective IFN-β. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01140 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Aug 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Noriko M Tsuji, National Institute of Advanced Science and Technology, Biomedical Research Institute, Tsukuba, Ibaraki, 305-0046, Japan, nm-tsuji@aist.go.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tadaomi Kawashima Huimin Yan Zijin Guo Joaquim Carreras Ryosuke Uchiyama Ryutaro Fukui Tsuneyasu Kaisho Shizuo Akira Kensuke Miyake Hiroko Tsutsui Takashi Saito Noriko M Tsuji Google Tadaomi Kawashima Huimin Yan Zijin Guo Joaquim Carreras Ryosuke Uchiyama Ryutaro Fukui Tsuneyasu Kaisho Shizuo Akira Kensuke Miyake Hiroko Tsutsui Takashi Saito Noriko M Tsuji Google Scholar Tadaomi Kawashima Huimin Yan Zijin Guo Joaquim Carreras Ryosuke Uchiyama Ryutaro Fukui Tsuneyasu Kaisho Shizuo Akira Kensuke Miyake Hiroko Tsutsui Takashi Saito Noriko M Tsuji PubMed Tadaomi Kawashima Huimin Yan Zijin Guo Joaquim Carreras Ryosuke Uchiyama Ryutaro Fukui Tsuneyasu Kaisho Shizuo Akira Kensuke Miyake Hiroko Tsutsui Takashi Saito Noriko M Tsuji Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by “relocation” of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.

Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue

Background The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. Objectives The aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. Study design dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. Results Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. Conclusions The dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.

The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon β

Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.

Testis Brain Ribonucleic Acid-Binding Protein/Translin Possesses both Single-Stranded and Double-Stranded Ribonuclease Activities

RNA interference (RNAi) is a biological process in which animal and plant cells destroy double-stranded RNA (dsRNA) and consequently the mRNA that shares sequence homology to the dsRNA. Although it is known that the enzyme Dicer is responsible for the digestion of dsRNA into approximately 22 bp fragments, the mechanism through which these fragments are associated with the RNA-induced silencing complex (RISC) is mostly unknown. To find protein components in RISC that interact with the approximately 22 bp fragment, we synthesized a (32)P- and photoaffinity moiety-labeled 22 bp dsRNA fragment and used it as bait to fish out protein(s) directly interacting with the dsRNA fragment. One of the proteins that we discovered by mass spectrometric analysis was TB-RBP/translin. Further analysis of this DNA/RNA binding protein showed that it possesses both ssRNase and dsRNase activities but not DNase activity. The protein processes long dsRNA mainly into approximately 25 bp fragments by binding to the open ends of dsRNA and cutting it with almost no turnover due to its high affinity toward the products. The activity requires physiological ionic strength. However, with single-stranded RNA as substrate, the digestion appeared to be more complete. Both ssRNase and dsRNase activities are inhibited by high levels of common RNase inhibitors. Interestingly, both activities can be enhanced greatly by EDTA.

16] Dictyostetium double-stranded ribonuclease

Publisher Summary As in most other organisms, sense-antisense hybrids cannot be detected by conventional hybridization experiments in Dictyostelium. A dsRNase may therefore cause the degradation of the postulated double-stranded RNA and might be a target for the regulation of antisense mechanisms. By using an assay to identify nucleases that specifically hydrolyze double-stranded RNA, a predominantly cytosolic dsRNase was identified in Dictyostelium extracts. This chapter describes a purification method for Dictyostelium dsRNase (DdsRNase) and presents a partial biochemical characterization of the enzyme. It is nonsequence specific and digests homopolymer duplexes as well as random sequence dsRNA targets generated from various Dictyostelium gene sequences; dsRNA digestion depends on divalent metal ions, with a vast preference for Mg 2+ . DdsRNase has been purified and elutes from a gel-filtration column as a large, approximately 450-kDa multicomponent complex. The enzyme is thus distinct from the RNase III family and other previously described dsRNase activities by structure and biochemical activity.