Expression Of dsRNA Research Articles

Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex inDrosophila melanogaster

The anaphase-promoting complex/cyclosome or APC/C is a key regulator of chromosome segregation and mitotic exit in eukaryotes. It contains at least 11 subunits, most of which are evolutionarily conserved. The most abundant constituents of the vertebrate APC/C are the four structurally related tetratrico-peptide repeat (TPR) subunits, the functions of which are not yet precisely understood. Orthologues of three of the TPR subunits have been identified in Drosophila. We have shown previously that one of the TPR subunits of the Drosophila APC/C, Apc3 (also known as Cdc27 or Mákos), is essential for development, and perturbation of its function results in mitotic cyclin accumulation and metaphase-like arrest. In this study we demonstrate that the Drosophila APC/C associates with a new TPR protein, a genuine orthologue of the vertebrate Apc7 subunit that is not found in yeasts. In addition to this, transgenic flies knocked down for three of the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by RNA interference were established to investigate their function. Whole-body expression of subunit-specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Apc8/Cdc23 silencing induces developmental delay and causes different pupal lethality. Cytological examination showed that these animals had an elevated level of apoptosis, high mitotic index and delayed or blocked mitosis in a prometaphase-metaphase-like state with overcondensed chromosomes. The arrested neuroblasts contained elevated levels of cyclin B but, surprisingly, cyclin A appeared to be degraded normally. Contrary to the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the apparent loss of Apc7 function does not lead to the above abnormalities. Instead, the Apc7 knocked down animals and null mutants are viable and fertile, although they display mild chromosome segregation defects and anaphase delay. Nevertheless, the Apc7 subunit shows synergistic genetic interaction with Apc8/Cdc23 that, together with the phenotypic data, assumes a limited functional role for Apc7. Taken together, these data suggest that the structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex.

Quercetin influences response in Nicotiana megalosiphon infected by satellite-associated cucumber mosaic virus

Quercetin is a prominent flavonoid with a remarkable spectrum of biochemical activities. Its impact on human health, as well as its role in the normal development of plants has been exten-sively studied. In this work the influence of quercetin on satel-lite associated cucumber mosaic virus (CMV) infection and the response of Nicotiana megalosiphon host plants were investigated. Daily continuous flavonoid treatment of infected plants resulted in a significant increase in leaf mass and total protein synthesis. Attenuation of the yellowing symptom was also recorded, accompanied by lower expression of both viral and satellite replicative dsRna. This correlated with the decreased total peroxidase activity established in those plants. Expression of some glycoproteins (38 and 22.5 kDa), absent in healthy plants, were suppressed when infected plants were treated with quercetin. At the same time, some other infection-specific glycoproteins (22, 24 and 29 kDa) were significantly amplified by quercetin. The inability of quercetin to bind Cmv nucleoprotein or isolated ssRna was demonstrated. Quercetin has not shown any effect on Hsp70 synthesis in Cmv-infected plants.

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.

The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

DsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.

Tethering RITS to a Nascent Transcript Initiates RNAi- and Heterochromatin-Dependent Gene Silencing

In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4+ gene silences ura4+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4+ siRNAs to silence a second ura4+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4+, or ura4+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism.

Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA

We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion.

Delivering RNA Interference to the Mammalian Brain

RNA interference (RNAi) is a new modality in gene therapy which can elicit down-regulation of gene expression and has enormous potential in the treatment of neurological diseases. RNAi is a conserved system through which double stranded RNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatus may be artificially triggered by delivery of naked siRNA molecules or by plasmid-based expression of dsRNA. Before these techniques can be used as effective treatments in the brain, efficient methods of in vivo delivery must be devised. This review first describes the mechanism of RNAi, and then critically examines both viral and non-viral methods for delivery of RNAi to the mammalian brain. There have been a number of important recent publications in this field and the progress towards effective in vivo delivery of RNAi to the central nervous system is discussed. Finally, potential problems that must be considered before applying this technology to the human brain are outlined.

Vigilins Bind to Promiscuously A-to-I-Edited RNAs and Are Involved in the Formation of Heterochromatin

The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location . The expression of dsRNA in the nucleus leads to several distinct consequences. First, the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts . Second, dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism . Is RNA editing also connected to heterochromatin? We report that members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work , we find that these proteins localize to heterochromatin and that mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing.

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin ( Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.

Arthritogenic properties of double-stranded (viral) RNA.

Viral infections often lead to arthralgias and overt arthritic states. The inflammatogenic compound of the viruses giving rise to such an outcome has to date not been identified. Because expression of dsRNA is a common feature of all viruses, we decided to analyze whether this property leads to the induction of arthritis. Histological signs of arthritis were evident already on day 3 following intra-articular administration of dsRNA. Arthritis was characterized by infiltration of macrophages into synovial tissue. It was not dependent on acquired immune responses because SCID mice also raised joint inflammation. NF-kappa B was activated upon in vitro exposure to dsRNA, indicating its role in the induction/progression of arthritis. Importantly, we found that dsRNA arthritis was triggered through IL-1R signaling because mice being deficient for this molecule were unable to develop joint inflammation. Although dsRNA is typically recognized by Toll-like receptor 3, Toll-like receptor 3 knockout mice developed arthritis, indicating that some other receptors are instrumental in the inducing of inflammation. Our results from in vitro experiments indicate that proinflammatory cytokines and chemokines stimulating monocyte influx were readily triggered in response to stimulation with dsRNA. These findings demonstrate that viral dsRNA is clearly arthritogenic. Importantly, macrophages and their products play an important role in the development of arthritis triggered by dsRNA.

Double-stranded RNA-mediated gene silencing in fission yeast.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression in a variety of organisms by invoking post-transcriptional degradation of homologous mRNA. Here we show that dsRNA-mediated gene regulation also occurs in the fission yeast Schizosaccharomyces pombe. We present evidence that: (i) reporter gene silencing is significantly enhanced when additional non-coding sense RNA is co-expressed with antisense RNA; (ii) expression of a panhandle RNA also silences target gene expression; (iii) expression of dsRNA is associated with siRNAs; (iv) a novel host-encoded factor which enhances antisense RNA gene silencing also enhances panhandle RNA-mediated gene inhibition. Both the exogenously introduced lacZ and c-myc genes are shown to be susceptible to dsRNA- mediated gene silencing in this model. Taken together, these data indicate that RNA-mediated gene silencing can occur through a RNAi-like mechanism in fission yeast.

Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei.

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.

The occurrence of CMV-specific short Rnas in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus.

Expression or introduction of double-stranded (ds)RNA in eukaryotic cells can trigger sequence-specific gene silencing of transgenes, endogenes, and viruses. Transgenic plants producing dsRNAs with homology to viral sequences are likely to exhibit pathogen-derived resistance to the virus. Cucumber mosaic virus (CMV), a very widespread virus with over 1,000 host species, has the natural ability to suppress silencing in order to establish infection. Here, we report the generation of transgenic tobacco lines, where a DNA transgene containing an inverted repeat of CMV cDNA had been introduced. Expression of this DNA construct delivered an RNA transcript that is able to form an intramolecular double strand. Transgenic plants were challenged with CMV. Three categories of plants could be discriminated: susceptible plants, which typically reacted with milder symptoms than the wild-type control; a "recovery" phenotype, in which newly emerging leaves were free of symptoms; and plants that showed complete resistance. Northern analysis showed that the expression of CMV dsRNA caused, in some transgenic lines, the generation of short RNAs characteristic of posttranscriptional gene silencing. Those lines were CMV resistant. The correlation between the detection of short RNAs and virus resistance provides a molecular marker that makes it possible to predict success in attempts to engineer virus resistance by dsRNA.

RNA silencing

RNA silencing

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila.

The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. When expressed under the control of a heat-inducible promoter, dsRNA interfered efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generated defects in larval molting and metamorphosis, resulting in animals that failed to pupariate or prepupae that died with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor had no effect on puparium formation, but led to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that expressed either EcR or betaFTZ-F1 dsRNA showed defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. These results confirm and extend our understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila.

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

Biotype‐specific expression of dsRNA in the sweetpotato whitefly

AbstractThis study was conducted to evaluate the effect of two different biotypes of the sweetpotato whitefly, Bemisia tabaci (Gennadius), on the induction of squash silverleaf (SSL), and to determine if double‐stranded RNA (dsRNA) occurs in geographically remote populations of the two biotypes. Recently collected B‐biotype whiteflies from Florida, Arizona, Mississippi, and Texas (SPW‐B) all contained a 7.0 kb dsRNA molecule. Kb dsRNA molecule. Laboratory colonies of A‐biotype whiteflies that were originally collected in 1981 from cotton in Arizona and California did not contain the 7.0 Kb dsRNA. When the two biotypes were compared only the SPW‐B induced rapid onset, grade 5, SSL. DsRNA similar to that found in adult SPW‐B was concentrated in whitefly nymphs, but host plant leaf tissue did not contain any consistent dsRNA molecules. SPW‐A only induced low‐grade SSL and progeny of SPW‐A that were fed on pumpkin plants displaying SSL did not acquire the ability to express dsRNA or induce SSL. Our data suggest that dsRNA is not directly involved in the induction of SSL and that SSL is a host‐specific response to a feeding injury induced by B‐biotype whiteflies. The origin and source of the 7.0 Kb dsRNA molecule remains enigmatic but its expression is constant in the whitefly biotype that is responsible for the induction of SSL and several other plant disorders in the U.S.

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his −); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu −, his −); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho −). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of tranformed cells from obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 μs duration at 4°C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.

Transmission of killer activity into laboratory and industrial strains ofSaccharomyces cerevisiaeby electroinjection

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.