Concentrations Of dsRNA Research Articles

Diminish the chances of Mediterranean fruit fly Ceratitis capitata eggs surviving by suppressing the expression of the heat shock protein 27 gene (Hsp27).

The hypothesized role of stress-inducible heat shock proteins is to act as a buffer against environmental variations and affect fitness in suboptimal conditions. Our study examining the functions of heat shock protein 27 (Hsp27) in the eggs of the Mediterranean fruit fly (Ceratitis capitata). We utilized double-stranded RNA, specifically targeting the Hsp27 gene, to reduce its expression and assessed the consequent impact on egg viability. The investigation included the examination of early eggs (less than 6 hours post-laying) and late eggs (more than 42 hours post-laying), using varying concentrations (0.02, 0.1, and 0.2 µg/µL) of Hsp27 dsRNA. Control groups of eggs were dsRNA of ATPase, Cctra-2, or phosphate buffer solution (ph-B-S). Down-regulating Hsp27 dsRNA reduced the hatching rate of the eggs compared to the hatching rate in the control groups, especially in the ph-B-S group. The early eggs were more affected than the late eggs after soaking with Hsp27 dsRNA, where the Hsp27 dsRNA decreased the hatching rates to 12.91% early. The tested concentration of 0.2 µg/µL of Hsp27 dsRNA was achieved with the most significant reduction of 2.75 in egg viability. The sex ratio of insects hatched from treated eggs was investigated after hatching. After the down-regulation of Hsp27, there was no significant effect of the Hsp27 down-regulation on the sex ratio.

Novel insights into double-stranded RNA-mediated immunopathology.

Recent progress in human and mouse genetics has transformed our understanding of the molecular mechanisms by which recognition of self double-stranded RNA (self-dsRNA) causes immunopathology. Novel mouse models recapitulate loss-of-function mutations in the RNA editing enzyme ADAR1 that are found in patients with Aicardi-Goutières syndrome (AGS) - a monogenic inflammatory disease associated with increased levels of type I interferon. Extensive analyses of the genotype-phenotype relationships in these mice have now firmly established a causal relationship between increased intracellular concentrations of endogenous immunostimulatory dsRNA and type I interferon-driven immunopathology. Activation of the dsRNA-specific immune sensor MDA5 perpetuates the overproduction of type I interferons, and chronic engagement of the interferon-inducible innate immune receptors PKR and ZBP1 by dsRNA drives immunopathology by activating an integrated stress response or by inducing excessive cell death. Biochemical and genetic data support a role for the p150 isoform of ADAR1 in the cytosol in suppressing the spontaneous, pathological response to self-dsRNA.

Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells.

In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.

Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025–0.041 and 0.211–0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020–0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA’s did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Stimulating effect of double-stranded yeast RNA on the activity of interferon system genes

Influence of double-stranded RNA (dsRNA) from Saccharomyces cerevisiae yeast upon expression levels of the macrophage genes encoding TLR3 receptor, interferons alpha and beta (IFNα, IFNβ), 2’,5’-oligoadenylate synthetase (OAS) and protein kinase R (PKR) enzymes has been studied in the J774 mouse histiocytic cell culture and in vivo in Balb/c mice. It has been shown that dsRNA exerts a selective activating effect on genes of TLR3 receptor, antiviral proteins IFNα, IFNβ, and OAS, both in vitro and in vivo. With J774 cell culture, the highest induction capacity was observed for the IFNβ gene: 365 to 802-fold. The stimulatory effect was dependent on the dose of dsRNA in the range of 16.9 to 125 μg/ml. The preparation enhanced IFNα gene activity to lesser degree (more than 10-fold), TLR3 and OAS (3 to 4-fold), while the expression levels for these genes were not significantly dependent on the dose of dsRNA. The stimulating effect of dsRNA was dosedependent in murine peritoneal macrophages. The maximum activating effect of the preparation was shown upon administration of the effective antiviral dose (0.5 mg of dsRNA/kg). Five hours after intraperitoneal injection of dsRNA, the highest level of mRNA synthesis was observed for IFNα (54-fold), OAS (43-fold) and TLR3 (28-fold) genes. Expression of the IFNβ gene increased to a lesser degree (9-fold). An increase in the dose of preparation to 1.5 mg/kg led to decrease of the stimulatory effect. Expression levels of the IFNα, TLR3, and OAS genes in that case decreased by 2-4-fold as compared to a lower dose, and the PKR gene expression was 5-fold lower compared to the control. One day after dsRNA administration, a tendency was observed for both experimental groups towards a decreased transcription of macrophage genes, if compared with the 5-hour term. The weakening of gene activity was less pronounced in animals treated with dsRNA at the dose of 1.5 mg/kg. The transcription indices for IFNβ, OAS, and TLR3 genes were much higher during this period (5-10-fold higher than the control values). The dynamics of PKR gene transcription in both experimental systems was significantly different from the expression of other studied genes. The dsRNA preparation at this dose range did not have a pronounced stimulatory effect upon expression of this gene. A moderate increase in PKR gene activity in macrophages of mice was observed only a day following intraperitoneal administration of dsRNA. Concentrations and length of dsRNA molecules are known to be critical factors to the PKR gene activation. An ability to increase the expression of the gene is shown at low dsRNA concentrations (10-7 g/ml and below), while highly polymeric dsRNAs weaken the gene activity. Since the doses and concentrations of dsRNA used in our experiments were significantly different from those mentioned above, it could, in general, affect regulation of PKR gene transcription towards reduction of the stimulatory effect.

Inducing regulated necrosis and shifting macrophage polarization with anti-EMMPRIN antibody (161-pAb) and complement factors.

Treatment of solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME) that prevents effector immune cells from eradicating tumor cells and promotes tumor progression, angiogenesis, and metastasis. Therefore, targeting components of the TME to restore the ability of immune cells to drive anti‐tumoral responses has become an important goal. One option is to induce an immunogenic cell death (ICD) of tumor cells that would trigger an adaptive anti‐tumoral immune response. Here we show that incubating mouse renal cell carcinoma (RENCA) and colon carcinoma cell lines with an anti‐extracellular matrix metalloproteinase inducer polyclonal antibody (161‐pAb) together with complement factors can induce cell death that inhibits caspase‐8 activity and enhances the phosphorylation of receptor‐interacting protein kinase 3 (RIPK3) and mixed‐lineage kinase‐like domain (MLKL). This regulated necrotic death releases high levels of dsRNA molecules to the conditioned medium (CM) relative to the necrotic death of tumor cells induced by H2O2 or the apoptotic death induced by etoposide. RAW 264.7 macrophages incubated with the CM derived from these dying cells markedly enhanced the secretion of IFNβ, and enhanced their cytotoxicity. Furthermore, degradation of the dsRNA in the CM abolished the ability of RAW 264.7 macrophages to secrete IFNβ, IFNγ‐induced protein 10 (IP‐10), and TRAIL. When mice bearing RENCA tumors were immunized with the 161‐pAb, their lysates displayed elevated levels of phosphorylated RIPK3 and MLKL, as well as increased concentrations of dsRNA, IFNβ, IP‐10, and TRAIL. This shows that an antigen‐targeted therapy using an antibody and complement factors that triggers ICD can shift the mode of macrophage activation by triggering regulated necrotic death of tumor cells.

Molecular characterization, expression analysis of 14-3-3 beta/alpha and the effect of RNA interference on ion transporter protein Na+-K+-ATPase, Na+-H+-exchanger and CFTR in turbot (Scophthalmus maximus).

To understand the role of 14-3-3 beta/alpha in hypoosmotic regulation of turbot (Scophthalmus maximus), we characterized the 14-3-3 beta/alpha gene and analyzed the tissue distribution and its gene transcriptional patterns in the main expressed tissues under low salt stress. The 14-3-3 beta/alpha cDNA is 892bp in length, incorporating an ORF of 774bp with a putative primary structure of 257 residues. The deduced amino acid sequences shared highly conserved structures with other eukaryotes. Quantitative real-time PCR results showed that the 14-3-3 beta/alpha transcripts were widely expressed in various tissues of turbot, with most abundant in the gill (P<.05), to a lesser extent in the kidney, intestine, brain and spleen, and at low levels in the pituitary and other tissues examined. And the expression of turbot 14-3-3 beta/alpha exhibited a trend of increasing first and then decreasing with the time of stress under low salt stress, and the highest value appeared in 12h (P<.05). After injecting different concentrations of dsRNA, the mRNA expression of 14-3-3 gene decreased significantly during the monitoring period, and the best interference effect was achieved 12h after injecting 4μg/g dsRNA. For the first time, the gene was silenced in fish by intramuscular injection of dsRNA. It also provides a new and effective way to study gene function at the individual level. Moreover, the mRNA interference of 14-3-3 beta/alpha would cause changes in the expression of several ion channel proteins, for example, the decrease of Na+-K+-ATPase and Na+-H+-exchanger and the increase of CFTR. As a result, 14-3-3 beta/alpha appears to be an important molecular regulator for osmosensory signal transduction in gill of turbot.

A genetically enhanced sterile insect technique against the fruit fly, Bactrocera dorsalis (Hendel) by feeding adult double-stranded RNAs

RNAi based sterile insect technique (SIT) is an authentic insect management approach but requires proper target genes. During this study, spermless males were developed by interfering with germ cell differentiation and azoospermia related genes. Data demonstrates significant reductions in the target genes expressions (boul, zpg, dsxM, fzo and gas8) after oral dsRNAs administration. Knock down of target genes significantly affected the reproductive ability of males and reduced egg-hatching as compared to the control group. Furthermore, different combinations of selected gene dsRNAs (boul + zpg, boul + dsxM and zpg + dsxM) were made, which resulted up to 85.40% of male sterility. The most effective combination was selected to prepare different concentrations of dsRNA, 250, 500, 750 and 1000 ng/μl, that caused 18.97%, 38.68%, 58.02% and 85.40% male sterility, respectively. Subsequently, 1000 ng/μl of the same combination of ds-RNAs was used against differently aged adult flies (1, 5, 7, 10 days) which lead to 85.40%, 31.42%, 21.76% and 9.90% male sterility, respectively. SIT developed in this study showed that, boul + zpg combination of dsRNA feeding for 6 hours significantly reduced the number of spermatozoa and viability of sperm in 1-day-old B. dorsalis flies. In short, this study provides an effective SIT technique for long-term B. dorsalis management.

Silencing of JHEH and EcR genes of Plutella xylostella (Lepidoptera: Plutellidae) through double stranded RNA oral delivery

Plutella xylostella (L.), Diamondback moth (DBM), is a notorious pest disrupting cruciferous crops across the world. Overuse of chemically synthesized insecticides as well as application of bio-pesticide like Bacillus thuringiensis has gained progressive resistance against them, resulting in DBM management failures. To overcome this, a more potential, non-chemical and safe technology like Ribonucleic acid interference (RNAi) is to be implemented. RNAi, is a reverse genetic tool with high sequence specificity, elicited by double-stranded RNA (dsRNA) for silencing the target genes. The objective of this study was to silence two candidate genes from DBM viz., Juvenile Hormone Epoxide hydrolase (PxJHEH) and Ecdysteroid receptor (PxEcR) involved in regulating metamorphosis and molting respectively. In this regard, we have custom designed the dsRNAs (500bp) by cloning and sequencing the candidate genes and delivered it orally (non-invasive mode). Further, we have assessed the extent of down regulation of target genes with five concentrations of dsRNA (1, 2, 5, 10 and 20μg/μl) at four time points (24, 48, 72 and 96h). The extent of gene silencing and mortality recorded for both genes were proportional to the dsRNA concentration. Our study projects that PxJHEH and PxEcR genes would be good target for dsRNA mediated gene silencing in management of insect pests such as DBM in near future.

Expression of temperature responsive genes in cell cultures derived from Bombyx mori

Insects are heterotherms that exhibit a close relationship between their ecology (especially temperature changes) and physiology. In the present study, selected genes associated with cell death and temperature were examined to determine gene expression in Bombyx mori in high and low temperature environments. We determined the amount of dsRNA, different concentrations of dsRNA, and different type of cells to set the conditions most efficient for RNAi. We then prepared dsRNA transcripts of the genes associated with cell death and temperature response. We analyzed cell damage via Trypan blue staining and found that cell viability was reduced after knockdown of these genes. The special transduced cell lines produced in the present study can be applied in various research fields. We also expect that these cell lines can be used as a research tool for the precise functional analysis of various genes.

Response of various target genes to diet-delivered dsRNA mediated RNA interference in the cotton bollworm, Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera is a highly polyphagous pest infesting a number of economically important crops, annually causing enormous crop losses. Management of this pest is challenging over the years due to various factors including development of resistance to wide spectrum of chemical insecticides. RNA interference (RNAi) has tremendous potential to combat insect pests. However, RNAi mediated silencing efficiency varies from gene to gene, hence successful RNAi mediated pest control requires selection of suitable target gene(s), which are effectively silenced by the exogenous delivery of cognate double-stranded RNA (dsRNA) through midgut. Therefore, we have evaluated the effect of two concentrations of dsRNA delivered through semi-synthetic diet in silencing five important genes, viz. glutathione-S-transferase, cytochrome P450 (both involved in detoxification of host allelochemicals); trypsin, chymotrypsin (both involved in digestion of proteins) and juvenile hormone acid methyl transferase (jhamt) (involved in larval metamorphosis). Extent of silencing was assessed by quantitative real-time PCR (qRT-PCR). Results revealed that above target genes were silenced variably, 20 μg dsRNA treatment having a more pronounced effect than 10 μg in reducing the transcript levels, larval, pupal weight, and pupation. Silencing of chymotrypsin had a more pronounced effect on larval and pupal weight compared to other target genes, while jhamt severely affected pupation. This study demonstrated that target genes have varied sensitivity to RNAi, chymotrypsin, and jhamt were shown to be suitable candidate genes that could be utilized for RNAi mediated management of H. armigera.

Effect of diet delivered various concentrations of double-stranded RNA in silencing a midgut and a non-midgut gene of Helicoverpa armigera

Ribonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (15–18 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.

Long-term effect of systemic RNA interference on circadian clock genes in hemimetabolous insects

RNA interference (RNAi) strategy, which enables gene-specific knock-down of transcripts, has been spread across a wide area of insect studies for investigating gene function without regard to model and non-model insects. This technique is of particular benefit to promote molecular studies on non-model insects. However, the optimal conditions for RNAi are still not well understood because of its variable efficiency depending on the species, target genes, and experimental conditions. To apply RNAi technique to long-running experiments such as chronobiological studies, the effects of RNAi have to persist throughout the experiment. In this study, we attempted to determine the optimal concentration of double-stranded RNA (dsRNA) for systemic RNAi and its effective period in two different insect species, the cricket Gryllus bimaculatus and the firebrat Thermobia domestica. In both species, higher concentrations of dsRNA principally yielded a more efficient knock-down of mRNA levels of tested clock genes, although the effect depended on the gene and the species. Surprisingly, the effect of the RNAi reached its maximum effect 1–2weeks and 1month after the injection of dsRNA in the crickets and the firebrats, respectively, suggesting a slow but long-term effect of RNAi. Our study provides fundamental information for utilizing RNAi technique in any long-running experiment.

RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

BackgroundThe possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.Methodology/Principal FindingsWe investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.ConclusionsTransient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

The optimal concentration of siRNA for gene silencing in primary cultured astrocytes and microglial cells of rats

BackgroundSmall interfering RNAs (siRNAs) have been used to knockdown specific gene expression in various cells. Astrocytes and microglial cells play a key role in fundamental central nervous system functions and in chronic neuroinflammation. The aims of this study were to determine the optimal concentration of siRNA demonstrating efficient transfection and inhibition of gene expression via RNA interference (RNAi) and lower cytotoxicity, in primary cultured astrocytes and microglial cells of rats.MethodsAstrocytes and microglial cells were isolated from the cerebral cortices of 2-day-old rats. Both the cells were transfected using transfection reagent (Lipofectamine™ 2000), and fluorescein-labeled double-stranded RNA (dsRNA) or siRNA targeting green fluorescent protein. Transfection efficiency and cytotoxicity of dsRNA, and the degrees of RNAi induced by siRNA in these cells, were evaluated at various concentrations of RNA.ResultsTransfection efficiencies of dsRNA in both astrocytes and microglial cells were significantly higher (P < 0.05) at the concentrations of 20, 40, and 80 nM than at the concentrations of 0, 5, and 10 nM. There were no significant cytotoxicities within the applied concentrations of dsRNA (0-80 nM). The degrees of RNAi induced by siRNA were significantly higher (P < 0.05) at the concentrations of 5, 10, 20, 40, 80 nM, and 20, 40, 80 nM in astrocytes and microglial cells, respectively, compared with the control (0 nM).ConclusionsThe siRNA concentration of 20 nM may be appropriate to induce RNAi in both astrocytes and microglial cells, while demonstrating low cytotoxicity, high transfection efficiency, and effective RNAi.

In C. elegans, High Levels of dsRNA Allow RNAi in the Absence of RDE-4

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.

Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions

Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription–polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.

TheCaenorhabditis elegans rsd-2andrsd-6Genes Are Required for Chromosome Functions During Exposure to Unfavorable Environments

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.

Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi.

We describe the successful use of the reverse genetic technique RNA interference (RNAi) to investigate gene function in the human filarial nematode parasite Brugia malayi. We used fluorescently labelled double stranded RNA (dsRNA) to demonstrate that 300 bp molecules are able to enter adult females in culture while they remain excluded from microfilariae (mf). We have developed an optimised microvolume culture system to allow the exposure of parasites to high concentrations of dsRNA for extended periods. Culturing of adult female parasites in this system for 24h does not significantly reduce parasite lifespan or mf release in culture. Three B. malayi genes, beta-tubulin (Bm-tub-1), RNA polymerase II large subunit (Bm-ama-1) and B. malayi mf sheath protein 1/mf22 (Bm-shp-1) were targeted by soaking adult female B. malayi in dsRNA complementary to these transcripts in the optimised culture system. Targeting of the two housekeeping genes Bm-tub-1 and Bm-ama-1 led to a reduction in the levels of their transcripts, as assessed by reverse transcriptase coupled PCR (RT-PCR), and resulted in parasite death in culture. In contrast, targeting of the Bm-shp-1 gene was not lethal to adult females in culture. A marked reduction in mf release was observed for shp-1 RNAi parasites compared to controls and in addition 50% of mf released did not have fully elongated sheaths. This "short" phenotype correlated with the loss of the stockpiled shp-1 transcript from developing mf in treated adult female gonads. From these data we conclude that RNAi may be a useful method for assessment of drug target potential of genes identified in filarial gene discovery projects.

Double-stranded RNA regulates IL-4 expression.

dsRNA, as genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses, can act to signal the immune system of the presence of viral infections. Although most viral infections are associated with strong Th1 immune responses, Th2-type responses have also been observed. In this study, we characterize the effects of dsRNA on the induction of Th2 responses in human lymphocytes. We report that in addition to the well-known Th1-inducing capabilities of dsRNA, treatment of human lymphocytes with low concentrations of dsRNA (0.1-1 microg/ml) leads to the expression of the prototypic Th2 cytokine IL-4. This induction was accompanied with the concentration-dependent activation of NF-kappaB and NF-AT2 but not NF-AT1. In addition, dsRNA can directly activate an IL-4 promoter-driven chloramphenicol acetyltransferase reporter gene in transiently transfected Jurkat cells. These results are the first demonstration of a non-TCR-associated activator of NF-AT in human cells and suggest that dsRNA directly influences IL-4 gene expression through its effect on NF-AT activation. Our data provide support for the idea that dsRNA at low concentrations in vivo may induce a Th2-dominant response that is not optimal for protective immunity to the virus.