dsRNA Molecules Research Articles

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of approximately 50-150 microm and extends over approximately 15 microm around the mean penetration depth, which is adjustable between 0 and 50 microm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery. When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

RNA interference

RNA interference

MicroRNAs and cancer

The Nobel Prize in Medicine and Physiology was awarded to the RNA interference (RNAi) field in 2006 because of the huge therapeutic potential this technique harbours. However, the RNAi technology is based on a natural mechanism that utilizes short noncoding RNA molecules (microRNAs) to regulate gene expression. This paper reviews our current knowledge about microRNAs focusing on their involvement in cancer and their potential as diagnostics and therapeutics.

Expression of Long Anti-HIV-1 Hairpin RNAs for the Generation of Multiple siRNAs: Advantages and Limitations

Promoter expressed long-hairpin RNAs (lhRNAs) that can be processed into multiple small interfering RNA (siRNAs) are being considered as effective agents for treating rapidly mutating viruses such as human immunodeficiency virus (HIV). In the present study, we have generated human U6 promoter-driven lhRNAs of 50, 53, and 80 base pairs (bp) targeting contiguous sequences within the tat and rev genes of HIV-1 and evaluated the efficacy of these lhRNAs as well as their processing in cells. By using multiple G:U mismatches in the stems, we have been able to readily incorporate the long-hairpin structures into a lentiviral vector transduction system. Here we show that such long hairpins can be stably and functionally expressed for a long term in HIV-1 susceptible T cells, where they provide potent inhibition of HIV replication against both non-mutant and mutant variants of HIV-1. Our studies provide strong support for the use of the G:U wobble pair containing lhRNAs to generate multiple siRNAs from a single transcript, but we also show that lhRNAs of 80 bp may be the upper size limit for effectively producing multiple, functional siRNAs.

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.

Structural determinants of RNA recognition and cleavage by Dicer

A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.

DETECTION OF MUTATIONS IN AVIAN REOVIRUS dsRNA GENOME BY DENATURING GRADIENT (DGGE) AND CONSTANT DENATURING (CDGE) GEL ELECTROPHORESIS

Polyacrylamide gel electrophoresis techniques performed in constant (CDGE) or gradient (DGGE) denaturing gels that are commonly used to detect DNA mutations were adapted for the detection of mutations directly in dsRNA molecules. The techniques were applied to investigate genomic differences between a highly pathogenic avian reovirus strain (S1133wt) and its cold-adapted non-pathogenic strain (P100) used as a vaccine, in an attempt to identify mutated virulence gene(s) that had led to viral attenuation. Wild and vaccine strains presented identical electrophoretic profiles of their 10-dsRNA genomic segments in standard SDS-PAGE. Under denaturing conditions, migration differences between the two strains were observed in many genes, including M1, M2 and S1, three genes associated with viral pathogenesis. The techniques are fast, simple to perform, and may prove valuable to screen for mutations and polymorphisms in other important dsRNA viruses, such as rotaviruses, birnaviruses and several plant viruses. DOI: http://dx.doi.org/10.17525/vrrjournal.v12i1-2.12

Caracterização de um vírus baciliforme isolado de Solanum violaefolium transmitido pelos ácaros Brevipalpus phoenicis e Brevipalpus obovatus (Acari: Tenuipalpidae)

Solano-violeta (Solanum violaefolium) é uma planta ornamental rasteira usada para cobrir solos de áreas sombreadas. Um vírus que induz manchas anelares nas folhas desta planta, tentativamente designado Solanum violaefolium ringspot virus - SvRSV, transmitido pelo ácaro Brevipalpus phoenicis (Acari: Tenuipalpidae) foi encontrado em Piracicaba, SP. Trata-se de um vírus baciliforme que se assemelha a outros vírus do tipo citoplasmático transmitidos por Brevipalpus sp. Este trabalho teve como objetivo relatar propriedades biológicas e estabelecer uma caracterização molecular parcial do SvRSV. O vírus pode ser transmitido mecanicamente a várias outras espécies botânicas, causando lesões localizadas. Entre as espécies avaliadas, Datura stramonium mostrou-se a melhor hospedeira experimental. Observou-se também a manifestação de sintomas nestas plantas após infestação das mesmas por B. obovatus previamente alimentado em lesões de SvRSV, confirmando esta outra espécie de ácaro como vetor do vírus. Suas propriedades físicas in vitro foram: temperatura de inativação 40-45 ºC; ponto final de diluição 10-3-10-4; longevidade in vitro 12 dias. Em secções ultrafinas, as partículas do SvRSV mostraram-se levemente mais delgadas e mais longas que as de outros vírus do mesmo grupo. A partir do dsRNA do SvRSV foi construída uma biblioteca de cDNA e foram identificadas duas possíveis regiões codificadoras das proteínas de movimento e replicase viral. Baseado nestas regiões foram desenhados "primers" para amplificação do RNA do SvRSV por RT-PCR. Sondas baseadas nas seqüências obtidas hibridizaram com ss- e dsRNA de D. stramonium infectadas pelo vírus. Ensaios preliminares de RT-PCR e hibridização não resultaram em reação com o vírus da leprose dos citros, tipo citoplasmático (CiLV-C).

Rust of flax and linseed caused byMelampsora lini

Melampsora lini, while of economic importance as the causal agent of rust disease of flax and linseed, has for several decades been the 'model' rust species with respect to genetic studies of avirulence/virulence. Studies by Harold Flor demonstrated that single pairs of allelic genes determine the avirulence/virulence phenotype on host lines with particular resistance genes and led him to propose his famous 'gene-for-gene' hypothesis. Flor's inheritance studies, together with those subsequently carried out by others, also revealed that, in some cases, an inhibitor gene pair and an avirulence/virulence gene pair interact to determine the infection outcome on host lines with particular resistance genes. Recently, avirulence/virulence genes at four loci, AvrL567, AvrM, AvrP4 and AvrP/AvrP123, have been cloned. All encode novel, small, secreted proteins that are recognized inside plant cells. Yeast two-hybrid studies have shown that the AvrL567 proteins interact directly with the resistance gene protein. The molecular basis of Flor's gene-for-gene relationship has now been elucidated for six interacting gene pairs: those involving resistance genes L5, L6, L7, M, P and P2, where both the resistance gene and the corresponding avirulence gene have been cloned. In other inheritance studies it has been shown that M. lini does not possess a (+) and (-) mating system, but may possess a two factor system. Double-stranded (ds) RNA molecules occur in many strains of M. lini: examination of the progeny of one strain that possesses 11 dsRNA molecules revealed that they fall into three transmission units, designated L, A and B. The L unit consists of a single large dsRNA of 5.2 kbp while the A and B units each consist of five dsRNAs in the size range 1.1-2.8 kbp. The three units have different sexual and asexual transmission characteristics. The L unit is encapsidated in a virus-like particle, whereas the other units are not encapsidated. The population and coevolutionary aspects of M. lini on a wild, native Australian host species, Linum marginale, have been extensively investigated. A recent molecular analysis revealed that the M. lini isolates from L. marginale fall into two distinct lineages, one of which is apparently hybrid between two diverse genomes. Isolates in this lineage are largely fixed for heterozygosity, which suggests that sexual recombination does not occur in this lineage.

Viral dsRNA Inhibitors Prevent Self-association and Autophosphorylation of PKR

Host response to viral RNA genomes and replication products represents an effective strategy to combat viral invasion. PKR is a Ser/Thr protein kinase that binds to double-stranded (ds)RNA, autophosphorylates its kinase domain, and subsequently phosphorylates eukaryotic initiation factor 2α (eIF2α). This results in attenuation of protein translation, preventing synthesis of necessary viral proteins. In certain DNA viruses, PKR function can be evaded by transcription of highly structured virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We probe here the mechanism of PKR inhibition by two viral inhibitor RNAs, EBER I (from Epstein–Barr) and VA I (from human adenovirus). Native gel shift mobility assays and isothermal titration calorimetry experiments confirmed that the RNA-binding domains of PKR are sufficient and necessary for the interaction with dsRNA inhibitors. Both EBER I and VA I are effective inhibitors of PKR activation by preventing trans-autophosphorylation between two PKR molecules. The RNA inhibitors prevent self-association of PKR molecules, providing a mechanistic basis for kinase inhibition. A variety of approaches indicated that dsRNA inhibitors remain associated with PKR under activating conditions, as opposed to activator dsRNA molecules that dissociate due to reduced affinity for the phosphorylated form of PKR. Finally, we show using a HeLa cell extract system that inhibitors of PKR result in translational recovery by the protein synthesis machinery. These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation.

Tissue-specific gene silencing by RNA interference in the whitefly Bemisia tabaci (Gennadius)

The hemipteran whitefly Bemisia tabaci (Gennadius) species complex and the plant viruses they transmit pose major constraints to vegetable and fiber production, worldwide. The whitefly tissue- and developmental-specific gene expression has not been exhaustively studied despite its economic importance. In 2002, a functional genomic project was initiated, which generated several thousands expressed sequence tags (ESTs) and their sequence. This project provides the basic information to design experiments aimed at understanding and manipulating whitefly gene expression. In this communication, for the first time we provide evidence that the RNA interference mechanism discovered in many organisms, including in Hemiptera, is active in B. tabaci. By injecting into the body cavity long double-stranded RNA (dsRNA) molecules, specifically directed against genes uniquely expressed in the midgut and salivary glands, we were able to significantly inhibit the expression of the targeted mRNA in the different organs. Gene expression levels in RNAi-silenced whiteflies were reduced up to 70% compared to whiteflies injected with buffer or with a green fluorescent protein (GFP)-specific dsRNA. Phenotypic effects were observed in B. tabaci ovaries following dsRNA targeting the whitefly Drosophila chickadee homologue. Disruption of whitefly gene expression opens the door to new strategies aimed at curbing down the deleterious effects of this insect pest to agriculture.

Therapeutic Potential of RNA Interference in Neurodegenerative Diseases

RNA interference (RNAi) is a cellular process that eventually leads to the degradation of mRNAs and silencing of gene expression (for detailed reviews see [1–3]). This powerful mechanism of gene silencing at a post-transcriptional level has been identified in a wide spectrum of eukaryotes, including plants, yeast, worms, flies and mammals. The signals that trigger RNAi are small double-stranded (ds)RNA molecules. RNAi-mediated gene silencing is a step-wise biochemical process: the dsRNAs are converted by the cellular enzyme Dicer, to socalled short interfering RNAs (siRNAs). The siRNAs are incorporated into the RNA-induced silencing complex (RISC) and guide the RISC to complementary mRNA molecules, thereby triggering gene-specific mRNA degradation. Small dsRNAs can be generated during infection with RNA viruses. Another important class of RNAi triggers are short hairpin RNAs (shRNAs) and micro RNAs (miRNAs) that form stem-loop structures. miRNAs are generated from longer RNA molecules that are encoded in the genome of worms, flies and mammals. Mature miRNAs bind to the 3 untranslated region of mRNAs and suppress translation of the bound mRNA. In addition, miRNAs have been shown to induce degradation of the target mRNA molecule. To date, more than 500 miRNAs have been identified and approximately 30% of human genes have been postulated to be regulated by miRNAs. RNAi is a potent tool to suppress specific genes in human cells and has great potential for therapeutic use in neurological disorders. Exogenouslyproduced siRNA molecules can be directly used to silence cellular gene expression. However, this requires chemical or enzymatic synthesis, as well as efficient RNA transfer to the target cells (e.g., by RNA transfection or electroporation) [4]. An alternative approach is to produce siRNAs or shRNAs within the target cell from RNA polymerase III transcription units. Presently, the most widespread siRNA/shRNA-expression cassettes contain either the promoter of RNAse P, RNA H1, or small nuclear RNA U6. Transfer of these expression cassettes into neuronal cells can be efficiently achieved by viral vectors in vitro and in vivo. In this editorial, I will focus on viral vectors for the induction of RNAi.

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Development of strategies for conditional RNA interference

RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.

Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

Double-stranded RNA viruses infecting Epichloe festucae

Some isolates of Epichloë festucae are asymptomatically infected by Epichloë festucae virus 1 (EfV1), a member of the family Totiviridae. This virus has a genome composed by a 5109 bp molecule of double stranded RNA (dsRNA). In addition, a 3 kbp dsRNA molecule which could be the genome of another virus (EfV2) was frequently found in isolates of the fungal endophyte. In a survey of two populations of E. festucae it was found that 73% of the isolates were infected by one or both viruses. Although both viruses are very efficiently transmitted to conidia, a barrier to virus transmission occurs during ascospore formation. Keywords: Epichloë festucae, virus, dsRNA, Totivirus

RNA Interference: Big Applause for Silencing in Stockholm

Eight years ago, Craig Mello, Andrew Fire, and their coworkers provided the first demonstration that double-stranded RNA (dsRNA) triggers the gene-silencing technique that we now call RNA interference (RNAi). For this landmark discovery, Mello and Fire are honored with this year's Nobel Prize in Physiology or Medicine.

Production of dsRNA for RNAi in Avian Embryos

INTRODUCTIONThis protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos.

Injection of dsRNA and Electroporation in Avian Embryos

INTRODUCTIONThis protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi-cell competence changes with differentiation. The half-life of the target protein product may also affect the time point of injection and electroporation-proteins with slow turnover may require RNAi at earlier stages, preferably before the onset of expression. Check the quality of the dsRNA used for injections and make sure that the buffer used for injection does alter development or survival of the embryo. Tris buffers and buffers containing glycerol are not compatible with in vivo injections. Salt concentration and pH should be in the physiological range. Always use the same buffers for injection and electroporation of test and control embryos.

Molecular Characterization of a Partitivirus from Ophiostoma Himal-ulmi

The complete nucleotide sequences of two double-stranded (ds) RNA molecules, S1 (1,744 bp) and S2 (1,567 bp), isolated from an isolate HP62 of the Himalayan Dutch elm disease fungus, Ophiostoma himal-ulmi, were determined. RNA S1 had the potential to encode a protein, P1, of 539 amino acids (62.7 kDa), which contained sequence motifs characteristic of RNA-dependent RNA polymerases (RdRps). A database search showed that P1 was closely related to RdRps of members of the genus Partitivirus in the family Partitiviridae. RNA S2 had the potential to encode a protein, P2, of 430 amino acids (46.3 kDa), which was related to capsid proteins of members of the genus Partitivirus. Virus particles isolated from isolate HP62 were shown to be isometric with a diameter of 30 nm, and to contain dsRNAs S1 and S2 and a single capsid protein of 46 kDa. N-terminal sequencing of tryptic peptides derived from the capsid protein proved unequivocally that it is encoded by RNA S2 and corresponds to protein P2. It is concluded that O. himal-ulmi isolate HP62 contains a new member of the genus Partitivirus, which is designated Ophiostoma partitivirus 1. A phylogenetic tree of RdRps of members of the family Partitiviridae showed that there are least two RdRp lineages of viruses currently classified in the genus Partitivirus. One of these lineages contained viruses with fungal hosts and viruses with plant hosts, raising the possibility of horizontal transmission of partitiviruses between plants and fungi. The partitivirus RdRp and capsid proteins appear to have evolved in parallel with the capsid proteins evolving much faster than the RdRps.