Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.     

SID-1 is a dsRNA-selective dsRNA-gated channel

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.     

Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1

RNA interference (RNAi) is a biological phenomenon that silences the expression of genes of interest. Passive double-stranded RNA (dsRNA) uptake has been uniquely observed in Caenorhabditis elegans due to the expression of systemic RNAi defective-1 (SID-1). We report that ectopic expression of CeSID-1 endows the Sf9 cells with a capacity for soaking RNAi. Soaking the Sf9-SID1 with dsRNA corresponding to either exogenous or endogenous target genes induced a significant decrease in the amount of mRNA or protein. These results enabled us to modify the target proteins of baculovirus expression vector system in both quantities and posttranslational modifications. The current low-cost and high-efficiency RNAi system is useful for high-throughput gene function analysis and mass production of recombinant protein.     

Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

Background

RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects.

Results

We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans.

Conclusion

Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches.     

Uptake of Extracellular Double-Stranded RNA by SID-2

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C.?elegans apical intestinal membrane protein SID-2 is required in C.?elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.     

Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review

RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans’ SID-1 protein and an ‘alternative’ endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control.     

Establishment of Caenorhabditis elegans SID-1-Dependent DNA Delivery System in Cultured Silkworm Cells

The systemic RNA interference defective-1 (SID-1) can transport double-stranded RNA (dsRNA) into cytosol across the cytoplasmic membrane. We report here that ectopic expression of Caenorhabditis elegans SID-1 allows BmN4 cells to import extracellular plasmid dsDNA into cells via the direct soaking method. Interestingly, BmN4-SID1 cells incorporate dsRNA and plasmid DNA simultaneously. Furthermore, the ectopic SID-1 allows us to establish a stably transformed cell line by the simple soaking method. Our results provide an alternative method for silkworm gene function analysis with low cost and low cell toxicity.     

Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.     

Evolution of susceptibility to ingested double-stranded RNAs in Caenorhabditis nematodes

Background

The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus.

Principal Findings

We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species.

Conclusions

The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies.     

Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell

The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAi-mediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV–Bme21 BEVS.     

RNA interference in the Colorado potato beetle,Leptinotarsa decemlineata: Identification of key contributors

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using ³²P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.     

A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.     

Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle,Leptinotarsa decemlineata

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.     

Spreading silence with Sid

RNA interference (RNAi) has been shown to spread from cell to cell in plants and in Caenorhabditis elegans, but it does not spread in other organisms, such as Drosophila. A recent report demonstrates that a membrane channel, encoded by the gene sid-1, is responsible for the spreading of RNAi between cells.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.