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PubMed2004

Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos.

RNA interference (RNAi) refers to the selective degradation of mRNA induced by double-stranded RNA (dsRNA), first discovered in Caenorhabditis elegans. Homology-dependent silencing phenomena related to RNAi have been observed in many species from all eukaryotic kingdoms. RNAi and related mechanisms share several conserved components. The hallmark of these phenomena is the presence of short dsRNA molecules (21-25 bp long), termed short interfering RNA (siRNA), which are generated from dsRNA by the activity of Dicer, a specific type III RNAse. These molecules serve as a template for the recognition and cleavage of the cognate mRNA. As it is beyond the scope of a single review to cover all aspects of RNAi, this review will focus on certain steps of the pathway relevant to mammals and on the use of long dsRNA to specifically silence genes in mammalian cells permissive to this technique, such as oocytes and early embryos.

Animals↗

PubMed1991

Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae. Identification of its single-stranded RNA form as 20 S RNA.

Most strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called W. We obtained W cDNA clones by random priming of denatured W dsRNA followed by reverse transcription. Sequence data of W shows that only one strand ((+)-strand) has coding capacity for a protein with 829 amino acids which spans almost the entire length of the molecule (2.5 kilobases). Within this protein we found a sequence pattern characteristic of RNA dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. W has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC or M1. However, a (+)-strand-specific probe for W hybridized with 20-S RNA. Furthermore, W (+)-strands comigrated with 20 S RNA in strand separation gels. These results suggest that 20 S RNA is a (+)-single-stranded RNA form of W dsRNA itself or a closely related molecule.

Amino Acid Sequence↗

PubMed2026

Molecular characterization of a novel partitivirus harboring an additional third dsRNA segment from Trichoderma harzianum.

We report the complete genome sequence of a novel partitivirus identified from Trichoderma harzianum NFCF092 strain, designated Trichoderma harzianum partitivirus 4 (ThPV4). Unlike canonical members of the family Partitiviridae, which possess a bipartite genome consisting of two double-stranded RNA (dsRNA) segments encoding an RNA-dependent RNA polymerase (RdRP) and a capsid protein (CP), ThPV4 harbors a third dsRNA segment encoding a protein of unknown function. The complete genome consists of dsRNA1 (1,950 bp; encoding the RdRP), dsRNA2 (1,772 bp; encoding the CP), and dsRNA3 (1,629 bp; encoding a protein with unknown function). Sequence analysis shows that each segment possesses a single open reading frame (ORF). The deduced amino acid sequence of the RdRP shows the highest similarity (90.5% identity) to that of Trichoderma gamsii alphapartitivirus 1. Phylogenetic analyses based on the RdRP indicate that ThPV4 clusters within the genus Alphapartitivirus of the family Partitiviridae. To our knowledge. ThPV4 is the first member of the genus Alphapartitivirus identified from T. harzianum to possess an additional, conserved third dsRNA segment.

Phylogeny↗

PubMed2002

[Specific suppression of GFP gene expression by short double stranded RNA in mammalian cells].

BACKGROUND AND OBJECTIVE: Double-stranded RNA (dsRNA) has been shown to interfere the function of specific genes in various species. The current study was designed to investigate whether short interfering RNAs(siRNAs) of green fluorescence protein(GFP) can inhibit the expression of GFP in mammalian cells. METHODS: HeLa cells were grown in standard conditions and transfected with pEGFPC2 and long dsRNA or siRNAs. Fluoroscopy and fluoroscent spectrophotometer were used to evaluate impact of GFPds RNA, Cx26 siRNAs, and longer double-strand RNAs on GFP expression, and flow cytometry was used to evaluate the cell apoptosis. RESULTS: Synthetic siRNAs can inhibite specific expression of GFP gene in human HeLa cell lines and siRNAs have no such specific effect. Longer double-stranded but no siRNAs can induce cell apoptosis. CONCLUSION: These observations may open a path toward the use of siRNAs as a new tool for researching gene function in mammalian cells and a gene-specific therapeutic tool.

Apoptosis↗

PubMed2004

RNA interference by short hairpin RNAs expressed in vertebrate cells.

RNA interference (RNAi) is now established as a general method to silence gene expression in a variety of organisms. Double-stranded RNA (dsRNA), when introduced to cells, interferes with the expression of homologous genes, disrupting their normal function. In mammals, transient delivery of synthetic short interfering RNAs (siRNAs), which resemble the processed form of standard double stranded RNAi triggers, is effective in silencing mammalian genes. Issues related to transfer efficiency and duration of the silencing effect, however, restrict the spectrum of the applications of siRNAs in mammals. These shortcomings of siRNAs have been solved by the cellular expression of short hairpin RNAs (shRNAs) from DNA vectors. shRNAs are indistinguishable from siRNAs in terms of efficacy and mechanism but can be produced within cells from standard mammalian expression vectors. In this way, shRNA expression makes possible the creation of continuous cell lines and transgenic animals in which suppression of a target gene is stably maintained by RNAi. As a result, the types of RNAi-based gene function analysis that can be carried out in mammals have been greatly expanded. We describe methods for the construction and transfer of stable shRNA expressing vectors suitable for generating loss of function alleles in mammalian cells in vitro or in vivo.

Alleles↗

PubMed2004

Putative replication intermediates in endornavirus, a novel genus of plant dsRNA viruses.

Oryza sativa endornavirus (OSV) belongs to a new genus (Endornavirus) and family (Endoviridae) with members containing large double-stranded RNA (dsRNA) replicons with plasmid-like properties. Analysis of products obtained from in vitro reaction of the OSV RNA-dependent RNA polymerase revealed a rapid increase of a population of the non-coding strand RNA molecules with a head-to-tail composition. Northern hybridization of total RNA from OSV-carrier cells with riboprobes specific for the coding strand RNA, revealed two types of RNA molecules (i) with a site specific nick and (ii) full-length unnicked molecules. Quantitative analyses of these RNAs showed about 50-fold higher amounts of full-length unnicked molecules in cultured cells in which the OSV copy number increases compared with those found in the seedling cells. Both the head-to-tail linked non-coding strand and the full-length coding strand molecules were also found in wild rice and broad beans infected with other endornaviruses indicating that the presence of these unique types of RNA molecules should be considered as a characteristic feature of Endoviridae .

Base Sequence↗

PubMed1990

Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry.

We studied changes in the distribution pattern of relative RNA content during the in vitro aging of TIG-3 cells by flow cytometry (FACS III). Propidium iodide (PI) does not stain total cellular RNA, but it intercalates specifically into double-helical regions of both DNA and RNA. In applying this principle to RNA, we stained double-stranded RNA (dsRNA) in whole cells with PI after DNA digestion with DNase. The results showed that dsRNA distribution patterns were relatively constant at 7-75 population doublings (PD) but were significantly altered after 77 PD. The distribution patterns were similar as those for cell volume measured with a Coulter Counter. The total cellular dsRNA contents increased linearly at the senescent phase of their in vitro life span. In contrast, the mean dsRNA contents (50% dsRNA contents) rapidly increased to 77-79 PD, but decreased somewhat at 81-83 PD. Two-dimensional histograms of the dsRNA contents versus cell size were little altered from 25 PD to 75 PD. However, a population with relatively larger cell volume and weaker fluorescence intensity appeared and increased after 79 PD. This cell population group may be categorized as "terminal senescent cells" that no more divide in respect that the dsRNA content decreases in spite of the increase of total RNA content.

Cell Survival↗

PubMed2000

3' poly(A) is dispensable for translation.

In wild-type cells, the 3' poly(A) structure is necessary for translation of mRNA and for mRNA stability. The superkiller 2 (ski2), ski3, ski6, ski7, and ski8 mutations enhance the expression of the poly(A)(-) mRNAs of yeast RNA viruses. Ski2p is a DEVH-box RNA helicase and Slh1p resembles Ski2p. Both repress L-A double-stranded RNA (dsRNA) virus copy number, further suggesting that their functions may overlap. We find that slh1Delta ski2Delta double mutants are healthy (in the absence of viruses) and show normal rates of turnover of several cellular mRNAs. The slh1Delta ski2Delta strains translate electroporated nonpoly(A) mRNA with the same kinetics as polyA(+) mRNA. Thus, the translation apparatus is inherently capable of efficiently using nonpoly(A) mRNA even in the presence of normal amounts of competing poly(A)(+) mRNA, but is normally prevented from doing so by the combined action of the nonessential proteins Ski2p and Slh1p.

Adaptor Proteins, Signal Transducing↗

PubMed1996

The regulation of the protein kinase PKR by RNA.

A model is presented for the regulation of the double-stranded RNA (dsRNA)-activated mammalian protein kinase PKR, which is involved in protein synthesis inhibition and the antiviral response in cells. A series of previous findings abut PKROs behavior are reviewed, including its effects on translation; the activation of its protein kinase activity; binding sites for PKR on RNA; PKROs protein domains, which include two double-stranded RNA binding motifs (dsRBMs); and the likelihood of PKR dimer formation. The model which emerges to account for many of these observations includes the suggestion that PKR dimers form which are stabilized and rearranged upon binding to dsRNA regions 60 bp or longer. The hypothesis includes protein conformational changes within each member of a PKR dimer bound to dsRNA which re-position an inhibitory polypeptide domain and thus allow kinase activation. Also considered are ways in which PKR interacts with imperfectly duplexed, highly structured RNA molecules.

Animals↗

PubMed2004

Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode.

The influenza virus nonstructural protein 1 encoded by influenza A virus (NS1A protein) is a multifunctional protein involved in both protein-protein and protein-RNA interactions. NS1A binds nonspecifically to double-stranded RNA (dsRNA) and to specific protein targets, and regulates several post-transcriptional processes. The N-terminal structural domain corresponding to the first 73 amino acids of the NS1 protein from influenza A/Udorn/72 virus [NS1A(1-73)] possesses all of the dsRNA binding activities of the full-length protein. Both NMR and X-ray crystallography of this domain have demonstrated that it is a symmetric homodimer which forms a six-helix chain fold, a unique structure that differs from that of the predominant class of dsRNA-binding domains, termed dsRBDs, that are found in a large number of eukaryotic and prokaryotic proteins. Here we describe biophysical experiments on complexes containing NS1A(1-73) and a short 16 bp synthetic dsRNA duplex. From sedimentation equilibrium measurements, we determined that the dimeric NS1A(1-73) binds to the dsRNA duplex with a 1:1 stoichiometry, yielding a complex with an apparent dissociation constant (K(d)) of approximately 1 microM. Circular dichroism and nuclear magnetic resonance (NMR) data demonstrate that the conformations of both NS1A(1-73) and dsRNA in the complex are similar to their free forms, indicating little or no structural change in the protein or RNA upon complex formation. NMR chemical shift perturbation experiments show that the dsRNA-binding epitope of NS1A(1-73) is associated with helices 2 and 2'. Analytical gel filtration and gel shift studies of the interaction between NS1A(1-73) and different double-stranded nucleic acids indicate that NS1A(1-73) recognizes canonical A-form dsRNA, but does not bind to dsDNA or dsRNA-DNA hybrids, which feature B-type or A/B-type intermediate conformations, respectively. On the basis of these results, we propose a three-dimensional model of the complex in which NS1A(1-73) sits astride the minor groove of A-form RNA with a few amino acids in the helix 2-helix 2' face forming an electrostatically stabilized interaction with the phosphodiester backbone. This mode of dsRNA binding differs from that observed for any other dsRNA-binding protein.

Carbon Isotopes↗

PubMed2005

RNA interference: from gene silencing to gene-specific therapeutics.

In the past 4 years, RNA interference (RNAi) has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. By harnessing an evolutionary conserved endogenous biological pathway, first identified in plants and lower organisms, double-stranded RNA (dsRNA) reagents are used to bind to and promote the degradation of target RNAs, resulting in knockdown of the expression of specific genes. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21-23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability and delivery in vivo, while minimising off-target and nonspecific effects, are developed.

Animals↗

PubMed2006

Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs.

Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain and a C-terminal kinase domain. Upon binding double-stranded RNA (dsRNA), PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2alpha (eIF-2alpha). Phosphorylation of eIF-2alpha results in attenuation of protein translation by the ribosome in either a general or an mRNA-specific manner. Therefore, the interaction between PKR and dsRNAs represents a crucial host cell defense mechanism against viral infection. Viruses can circumvent PKR function by transcription of virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We present here a biophysical characterization of the interactions between human PKR and two viral inhibitor RNAs, EBER(I) (from Epstein-Barr virus) and VA(I) (from human adenovirus). Autophosphorylation assays confirmed that both EBER(I) and VA(I) are inhibitors of PKR activation, and profiled the kinetics of the inhibition. Binding affinities of dsRNAs to PKR double-stranded RNA-binding domains (dsRBDs) were determined by isothermal titration calorimetry and gel electrophoresis. A single stem-loop domain from each inhibitory RNA mediates the interaction with both dsRBDs of PKR. The binding sites on inhibitor RNAs and the dsRBDs of PKR have been mapped by NMR chemical shift perturbation experiments, which indicate that inhibitors of PKR employ similar surfaces of interaction as activators. Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition.

Adenoviruses, Human↗

PubMed2005

[Inhibitory effect of hTERT dsRNA on telomerase activity in lung carcinoma cell line A549].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is a new technology in gene study. The mechanism of RNAi is that double-stranded RNA (dsRNA) can band target mRNA and decompose it. This study was to assess possibility and specificity of dsRNA on suppressing human telomerase reverse transcriptase (hTERT) in lung carcinoma cells, investigate its effect on cell proliferation to confirm whether it has unspecific killing activity on mammalian cells, and explore its application in lung cancer research and treatment. METHODS: Sequences of 2 exons and 1 intron of hTERT gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) or PCR. The sense and antisense cDNA sequences were connected in a tandem manner, and the whole fragment was inserted into pCI-neo mammalian expression vector to construct the dsRNA expression vector, and then transfected into lung carcinoma cell line A549. The expression of hTERT was detected by RT-PCR and Western blot. Telomerase activity was measured by telomerase repeat amplification protocol (TRAP). Cell morphology was observed, and cell proliferation was assessed under invert microscope. RESULTS: After transfection of 2 exon fragments of hTERT dsRNA, mRNA and protein expression of hTERT and telomerase activity in A549 cells were suppressed, cell proliferation was markedly inhibited. Meanwhile, dsRNA didn't show unspecific toxic activity on A549 cells. CONCLUSIONS: hTERT dsRNA can specifically silent hTERT gene, inhibit telomerase activity and proliferation of A549 cells. hTERT dsRNA might be a potential method of gene therapy for lung cancer.

Adenocarcinoma↗

PubMed1997

Analysis of Xenopus dsRNA adenosine deaminase cDNAs reveals similarities to DNA methyltransferases.

We isolated two similar, but distinct, cDNA classes that encode Xenopus double-stranded RNA (dsRNA) adenosine deaminase. The longest, full-length open reading frame (ORF) predicts a 1,270-amino acid protein of 138,754 Da that is similar in size and about 50% identical to proteins encoded by mammalian cDNAs, yet larger than the 120-kDa protein purified from Xenopus eggs. Alignments of the Xenopus and mammalian ORFs show N-terminal heterogeneity, three conserved dsRNA binding motifs (dsRBMs), and strongly conserved carboxyl termini. Consistent with the observation of two cDNA classes, northern analyses of Xenopus oocyte poly A+ RNA show at least three mRNA species. Multiple nuclear polyadenylation hexamers and putative cytoplasmic polyadenylation elements were found in the 3' UTRs of cDNAs corresponding to the largest mRNA. In vitro translation experiments show that the cDNAs encode active deaminases and that the entire N-terminus and first dsRBM are dispensable for deaminase activity. Importantly, an analysis of the C-termini of five known dsRNA adenosine deaminases, and two putative deaminases, reveals motifs that are strikingly similar to the conserved motifs of the DNA-(adenine-N6alpha)-aminomethyltransferases and the DNA-(cytosine-5)-methyltransferases.

Adenosine Deaminase↗

PubMed2026

Chitosan-dsRNA improves tissue stability and delivery for RNAi-mediated Varroa destructor control.

BACKGROUND: Varroa destructor is an ectoparasitic mite and a major threat to honey-bee colony health worldwide. RNA interference (RNAi) offers a potentially species-specific approach for mite control, but practical application is limited by double-stranded RNA (dsRNA) degradation and inefficient delivery to mites. This study evaluated coatomer protein I (COPI) complex subunits as RNAi targets and tested whether chitosan-based dsRNA formulation could improve dsRNA stability, tissue uptake, and delivery from honey-bees to mites. RESULTS: Direct microinjection of dsRNAs targeting COPB, COPD, and COPE significantly reduced target-gene expression and mite survival compared with the double-stranded green fluorescent protein (dsGFP) control, with 72-h survival rates of 8.0%, 12.7%, and 5.3%, respectively, compared with 40.7% in the control group (all log-rank P < 0.0001). Chitosan-conjugated dsRNA remained detectable for longer periods than naked dsRNA in honey-bee tissue fluids, and CNP-Cy3-dsGFP was detected in the honey-bee midgut and fat body. A qualitative fluorescence observation in V. destructor was consistent with host-to-mite dsRNA transfer. Ingestion of COP-targeted chitosan-dsRNAs reduced mite survival, whereas honey-bee survival and expression of honey-bee COP orthologs were not affected. In silico analysis detected no contiguous ≥19-nt matches between Varroa COP dsRNAs and the honey-bee transcriptome or genome. CONCLUSION: COPI subunits are promising RNAi targets in V. destructor, and chitosan formulation may improve dsRNA persistence and uptake while supporting honey-bee-mediated delivery to mites. These laboratory findings support further evaluation of chitosan-formulated dsRNA as a potentially species-selective strategy for Varroa management, while broader safety assessment and field validation remain necessary. © 2026 Society of Chemical Industry.

COPI complex↗

PubMed2002

Short interfering RNA confers intracellular antiviral immunity in human cells.

Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. In plants, it serves as an antiviral defence mechanism. Animal cells also possess this machinery but its specific function is unclear. Here we demonstrate that dsRNA can effectively protect human cells against infection by a rapidly replicating and highly cytolytic RNA virus. Pre-treatment of human and mouse cells with double-stranded, short interfering RNAs (siRNAs) to the poliovirus genome markedly reduces the titre of virus progeny and promotes clearance of the virus from most of the infected cells. The antiviral effect is sequence-specific and is not attributable to either classical antisense mechanisms or to interferon and the interferon response effectors protein kinase R (PKR) and RNaseL. Protection is the result of direct targeting of the viral genome by siRNA, as sequence analysis of escape virus (resistant to siRNAs) reveals one nucleotide substitution in the middle of the targeted sequence. Thus, siRNAs elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses.

Animals↗

PubMed2004

Efficient gene silencing by expression of double stranded RNA in Neurospora crassa.

In Neurospora crassa, sequence-specific inhibition of endogenous genes can be induced by the introduction of transgenic DNA homologous to the target gene, through the mechanism of post-transcriptional gene silencing (PTGS) known as quelling. The application of this strategy to inactivate genes in N. crassa has, to date, been restricted by a limited silencing efficiency and instability of the silenced phenotype. In this study we show that the use of constructs that express hairpin double stranded RNA (dsRNA) permits efficient gene silencing by-passing limiting events in the quelling triggering process occurring upstream of dsRNA production. We found that silenced strains expressing a hairpin RNA displayed higher phenotypic stability compared with quelled strains. Moreover, we show that gene silencing can be modulated by expressing the double stranded RNA from an inducible promoter. Together these results make this method suitable for producing hypomorphic mutants in N. crassa.

Base Sequence↗

PubMed2006

Dynamics of dsRNA mycoviruses in black Aspergillus populations.

Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes ranging from 0.8 to 4.4 kb in size. In one strain the virus shows clear visible effects on its host with non-sporulating sectors. We quantified the fitness costs of these and more 'cryptic' virus infections on mycelial growth rate and spore production, and on competitive ability with respect to other strains under different growth conditions. Mycovirus infection proved detrimental in all these measures. The reduced success in interference competition due to mycovirus infection belies co-evolution of mycovirus and host to a mutually beneficial symbiosis, like in killer virus systems in yeast and smut and agrees more to recent infections. For a stable virus infection frequency in the black Aspergillus population, fitness costs and spontaneous loss should be balanced with new infections. Implications of even small viral fitness effects combined with the observed transmission limits for host and mycovirus are discussed.

Aspergillus↗