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1.  Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants 
PLoS Pathogens  2015;11(3):e1004711.
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.
Author Summary
RNA interference (RNAi) is a cellular mechanism activated by double-stranded RNA (dsRNA). Cellular dsRNA-specific RNaseIII enzymes (Dicer) recognize dsRNA and process it into double-stranded small interfering RNAs (ds-siRNAs) of 21–25 nucleotides (nt). siRNAs guide RNAi to degrade also single-stranded RNA homologous to the trigger. RNAi regulates gene expression, controls transposons, and represents an important antiviral defense mechanism. Therefore, viruses encode proteins dedicated to countering RNAi. In this study, the RNaseIII enzymes of a fish DNA virus (PPIV) and a plant RNA virus (SPCSV) were compared for suppression of RNAi in non-host organisms. The fish iridovirus RNaseIII suppressed RNAi in a plant and a nematode. It also enhanced accumulation of an RNAi suppressor deficient virus in plants, and suppressed antiviral RNAi and could rescue multiplication of an unrelated, RNAi suppressor-defective virus in nematodes. In contrast, the plant virus RNaseIII could suppress RNAi only in plants. Our results underscore that the active viral RNaseIII enzymes suppress RNAi. Their activity in suppression of RNAi seems to differ for the spectrum of unrelated organisms. Understanding of this novel mechanism of RNAi suppression may inform means of controlling the diseases and economic losses which the RNaseIII-containing viruses cause in animal and plant production.
doi:10.1371/journal.ppat.1004711
PMCID: PMC4352025  PMID: 25747942
2.  Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression 
PLoS ONE  2009;4(12):e8424.
Background
Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules.
Principal Findings
Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite.
Conclusions
Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.
doi:10.1371/journal.pone.0008424
PMCID: PMC2793006  PMID: 20037645
3.  The Ebola Virus VP35 Protein Is a Suppressor of RNA Silencing 
PLoS Pathogens  2007;3(6):e86.
RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.
Author Summary
Cells have evolved mechanisms to protect themselves from virus infection. A well-known antiviral mechanism in mammals is the interferon (IFN) response of the innate immune system. In plants, insects, and worms, RNA silencing or RNA interference (RNAi) is a strong antiviral defence mechanism. It is still debated whether RNAi is also used as an antiviral mechanism in mammals. Many mammalian viruses encode essential factors that suppress the innate antiviral responses of the host. Such innate immunity suppressor proteins, or IFN antagonists, have recently been reported to also suppress RNAi in mammalian cells. We now demonstrate that the Ebola virus VP35 protein, a known IFN antagonist, suppresses RNAi in human cells. In addition, VP35 restores the production of an HIV-1 variant with a defective RNAi suppressor Tat protein. These results indicate that RNAi is part of the innate antiviral defence response in mammals and that viruses need to counteract this response in order to replicate. Whereas RNAi and INF act in concert to prevent the infection of mammalian cells, the invading viruses encode a protein that counteracts both defence mechanisms.
doi:10.1371/journal.ppat.0030086
PMCID: PMC1894824  PMID: 17590081
4.  Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression 
PLoS ONE  2014;9(1):e87517.
In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells—expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.
doi:10.1371/journal.pone.0087517
PMCID: PMC3903663  PMID: 24475301
5.  RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening 
Background
The 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 Findings
We 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.
Conclusions
Transient 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.
Author Summary
RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi.
doi:10.1371/journal.pntd.0000850
PMCID: PMC2957409  PMID: 20976050
6.  An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses 
PLoS Pathogens  2010;6(3):e1000829.
Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.
Author Summary
Nearly all viruses produce dsRNA during their replication cycle. This molecule is not normally found in a healthy host cell and thus functions as a flag, alerting the host to a viral infection. Cells can die by lysis during virus infections, and the intracellular dsRNA is then released into the extracellular space. This dsRNA is stable in the extracellular milieu, and is able to function as a signaling molecule, detected by neighboring cells. This has been observed experimentally, as extracellular dsRNA has been used for years to trigger host antiviral responses. It has also been suggested that extracellular dsRNA plays a role in causing pathological symptoms in virus infected patients. Our data suggests that class A scavenger receptors (SR-As) function as cell surface receptors for dsRNA. SR-As bind extracellular, viral dsRNA and mediate its entry into the cell, where it delivers the dsRNA to other known intracellular dsRNA sensors, activating intracellular antiviral responses. These findings shed new light on how the host detects and responds to virus infection.
doi:10.1371/journal.ppat.1000829
PMCID: PMC2847946  PMID: 20360967
7.  Short hairpin type of dsRNAs that are controlled by tRNAVal promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells 
Nucleic Acids Research  2003;31(2):700-707.
The post-transcriptional gene silencing in animals and plants is called RNA interference (RNAi). Guides for the sequence-specific degradation of mRNA are 21-nt small interfering RNAs (siRNAs) that are generated by Dicer-dependent cleavage from longer double-stranded RNAs (dsRNAs). To examine the relationship between the localization of dsRNA and the target cleavage of RNAi in human cells, we constructed five kinds of dsRNA expression vector that were controlled by tRNAVal or U6 promoter. Transcripts of tRNA-dsRNA were consistently localized in the cytoplasm and were efficiently processed by Dicer. In contrast, transcripts of tRNA-dsRNA were not processed in cells that expressed Dicer-directed ribozymes. In addition, transcripts of U6-dsRNA were basically localized in the nucleus and were not significantly processed, unless the transcripts of U6-dsRNAs possessed a microRNA-based loop motif: in the latter case, U6-dsRNAs with a microRNA-based loop were transported to the cytoplasm and were effectively processed. More over, tRNA-dsRNA directed against a mutant k-ras transcript cleaved its target mRNA efficiently in assays of RNAi not only in vitro with a cytoplasmic extract but also in vivo. Therefore, it appears that RNAi in human cells occur in the cytoplasm. Importantly, the same tRNA-dsRNA did not affect the degradation of the normal k-ras mRNA in vitro and in vivo. Our tRNA-dsRNA technology should be a powerful tool for studies of the mechanism of RNAi and the functions of various genes in mammalian cells with potential utility as a therapeutic agent.
PMCID: PMC140522  PMID: 12527779
8.  dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells 
Nucleic Acids Research  2011;40(1):399-413.
Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3′-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.
doi:10.1093/nar/gkr702
PMCID: PMC3245926  PMID: 21908396
9.  Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway 
PLoS Pathogens  2009;5(2):e1000299.
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.
Author Summary
Dengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes aegypti mosquitoes. Understanding the mechanisms mosquitoes use to modulate infections by these agents of serious human diseases should give us critical insights into virus–vector interactions leading to transmission. RNA interference (RNAi) is an innate defense mechanism used by invertebrates to inhibit RNA virus infections; however, little is known about the antiviral role of RNAi in mosquitoes. RNAi is triggered by double-stranded RNA, leading to degradation of RNA with sequence homology to the dsRNA trigger. We show that dengue virus type 2 (DENV2) infection of Ae. aegypti by the natural route generates dsRNA and DENV2-specific small interfering RNAs, hallmarks of the RNAi response; nevertheless, persistent infection of mosquitoes occurs, suggesting that DENV2 circumvents RNAi. We also show that DENV2 infection is modulated by RNAi, since impairment by silencing expression of genes encoding important sensor and effector proteins in the RNAi pathway increases virus replication in the vector and decreases the incubation period before virus transmission. Our findings indicate a significant role for RNAi in determining the mosquito vector's potential for transmitting human diseases.
doi:10.1371/journal.ppat.1000299
PMCID: PMC2633610  PMID: 19214215
10.  Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer 
Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75–80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA) -mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3 %, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06 % and in weight by 73.08 %. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.
doi:10.1186/1479-0556-3-5
PMCID: PMC1187910  PMID: 15987532
RNA interference; epidermal growth factor receptor; double stranded RNA; small interference RNA; non small cell lung cancer
11.  RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase 
Nucleic Acids Research  2002;30(10):e46.
Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.
PMCID: PMC115300  PMID: 12000851
12.  Structural Diversity Repertoire of Gene Silencing Small Interfering RNAs 
Nucleic Acid Therapeutics  2011;21(3):125-131.
Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure–activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3′ overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.
doi:10.1089/nat.2011.0286
PMCID: PMC3198623  PMID: 21749289
13.  Ectopic expression of systemic RNA interference defective protein in embryonic stem cells 
RNA interference (RNAi), a post-transcriptional gene silencing mechanism originally described in C. elegans, involves sequence-specific mRNA degradation mediated by double-stranded RNAs (dsRNAs). Passive dsRNA uptake has been uniquely observed in C. elegans due to the expression of systemic RNA interference defective-1 (SID-1). Here we investigated the ability of ectopic SID-1 expression to enable passive cellular uptake of short interfering RNA (siRNA) or double stranded RNA (dsRNA) in pluripotent mouse embryonic stem cells (mESCs). When SID-1-GFP and the Firefly luciferase reporter gene (luc Fir) were co-expressed in mESCs, lucFir activity could be suppressed by simple incubation with dsRNAs/siRNAs that were designed to specifically target lucFir. By contrast, suppression was not observed in mESCs expressing lucFir and GFP alone or when either GFP- or SID-1-GFP-expressing cells were incubated with control dsRNAs/siRNAs (non-silencing or Renilla luciferase-specific). These results may lead to high-throughput experimental strategies for studying ESC differentiation and novel approaches to genetically inhibit or eliminate the tumorigenicity of ESCs.
doi:10.1016/j.bbrc.2007.03.187
PMCID: PMC2464293  PMID: 17434453
SID-1; RNA interference; gene transfer; ectopic expression; embryonic stem cells
14.  Phenotypic Screen of Early-Developing Larvae of the Blood Fluke, Schistosoma mansoni, using RNA Interference 
RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1α), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced (∼1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.
Author Summary
RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in human blood flukes, Schistosoma spp., although its application as a functional genomics tool in early intramolluscan larval stages has been limited to single gene analyses. Accelerating gene discovery efforts over the past 10 years have resulted in extensive, ever-increasing databases of genomic, transcriptomic and EST sequences. Unfortunately, our understanding of the function of the vast majority of these genes has not kept pace with their discovery, and this represents a significant barrier and the next real challenge for investigators of schistosomes, and other parasitic helminths. In the present study, we selected an array of 32 genes expressed in S. mansoni sporocysts to evaluate their susceptibility to double-stranded (ds)RNA treatment and to begin characterizing morphological phenotypes associated with a potential RNAi effect. Results demonstrate that gene knockdown and/or resulting phenotypes are highly transcript-dependent (specific dsRNA sequence used) and vary with time post-dsRNA exposure. Because of this potential variability in both transcript and phenotype expression in response to dsRNA treatment, our findings illustrate that, although a RNAi-type approach holds great promise as a functional reverse-genetics tool for larval schistosomes, its application requires caution in the design and execution of experiments and interpretation of results.
doi:10.1371/journal.pntd.0000502
PMCID: PMC2719580  PMID: 19668375
15.  Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA 
Nucleic Acids Research  2002;30(7):1664-1669.
Double-stranded RNA (dsRNA) induces gene-specific silencing in organisms from fungi to animals, a phenomenon known as RNA interference (RNAi). RNAi represents an evolutionarily conserved system to protect against aberrant expression of genes and a powerful tool for gene manipulation. Despite reports that RNAi can be induced in vertebrates, severe sequence-non-specific effects of long dsRNA have been documented in various systems. It has recently been shown in cultured mammalian cells that small interfering RNAs (siRNAs) of 21–23 nt can mediate RNAi but bypass the non-specific response induced by longer dsRNAs. However, the effectiveness of siRNAs has not been demonstrated in living vertebrates. In addition, the mechanism of siRNA suppression of gene expression in vertebrate cells remains to be elucidated. Here we show that synthetic 21 nt siRNAs can specifically inhibit the expression of exogenously introduced as well as endogenous genes in the embryos of Xenopus laevis. siRNAs significantly reduced the steady-state amount of both the mRNA and protein of the cognate gene target. Moreover, co-injection of siRNA with the target RNA transcript specifically suppressed the activity of the latter. Taken together, our findings establish siRNA-mediated post-transcriptional suppression of gene expression in Xenopus embryos.
PMCID: PMC101847  PMID: 11917028
16.  Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors 
Background
RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.
Results
As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.
Conclusions
Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.
doi:10.1186/1471-2199-5-9
PMCID: PMC514603  PMID: 15291968
17.  Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells 
Cell Cycle  2010;9(17):3552-3564.
The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNβ). This response can be mediated by a number of dsRNA sensors, including TLR3, MDA5, RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA, and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors, TLR3 and MDA5, are not expressed in hES cells and iPS cells. PKR is expressed in hES cells, but is not activated by transfected dsRNA. In addition, RIG-I is expressed, but fails to respond to dsRNA because its signaling adapter, MITA/STING, is not expressed. Finally, the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts, cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together, our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling.
doi:10.4161/cc.9.17.12792
PMCID: PMC3047619  PMID: 20814227
dsRNA; interferon; innate immunity; pluripotency; stem cells
18.  Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin 
PLoS ONE  2011;6(5):e20250.
RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.
doi:10.1371/journal.pone.0020250
PMCID: PMC3102679  PMID: 21637842
19.  RNAi Dynamics in Juvenile Fasciola spp. Liver Flukes Reveals the Persistence of Gene Silencing In Vitro 
Background
Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).
Methodology/Principal Findings
Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.
Conclusions/Significance
In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.
Author Summary
RNA interference (RNAi) is a method for selectively silencing (or reducing expression of) mRNA transcripts, an approach which can be used to interrogate the function of genes and proteins, and enables the validation of potential targets for anthelmintic drugs or vaccines, by investigating the impact of silencing a particular gene on parasite survival or behaviour. This study focuses on liver fluke parasites, which cause serious disease in both humans and animals. We have only a handful of drugs with which to treat these infections, to which flukes are developing resistance, and no anti-fluke vaccines have yet been developed. New options for treatment and control of liver fluke parasites are sorely needed, and RNAi is a powerful tool in the development of such treatments. This study developed a set of simple methods for triggering RNAi in juvenile liver fluke, which show that although robust transcriptional suppression can be readily achieved across all targets tested, protein suppression occurs only after a target-specific lag period (likely related to protein half-life), which may require >25 days under current in vitro maintenance conditions. These findings are important for researchers aiming to employ RNAi in investigations of liver fluke biology and target validation.
doi:10.1371/journal.pntd.0003185
PMCID: PMC4177864  PMID: 25254508
20.  Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference 
Scientific Reports  2015;5:8700.
RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells.
doi:10.1038/srep08700
PMCID: PMC4346973  PMID: 25731667
21.  Sequestration and Protection of Double-Stranded RNA by the Betanodavirus B2 Protein 
Journal of Virology  2006;80(14):6822-6833.
Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.
doi:10.1128/JVI.00079-06
PMCID: PMC1489041  PMID: 16809288
22.  The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase 
PLoS Biology  2010;8(10):e1000496.
The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.
The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.
Author Summary
Small RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.
doi:10.1371/journal.pbio.1000496
PMCID: PMC2950127  PMID: 20957187
23.  RNAi induced in mammalian and Drosophila cells via transfection of dimers and trimers of small interfering RNA 
Twenty one base pair long small interfering RNAs (siRNAs) are widely in use in mammalian RNAi experiments. The present study assesses the capability of 43 and 63bp dsRNAs with two 2nt long 3′-overhangs to induce RNAi in mammalian and Drosophila cells. Human Dicer was found to cleave these dsRNAs from their ends to generate two or three monomeric siRNA units, each 21-22bp in length. When, in 43bp dsRNA, there was present a highly-effective siRNA sequence in frame with respect to the Dicer digestion, considerably high RNAi activity was noted to be induced in mouse embryonic stem E14TG2a, human HeLa, Chinese hamster CHO-K1 or Drosophila S2 cells. In contrast, RNAi depending on 63bp dsRNA, containing a highly effective siRNA sequence in frame with respect to Dicer digestion, varied considerably depending on cell lines used. While there was no appreciable RNAi in HeLa cells associated with relatively strong interferon response, a significant level of RNAi was noted in E14TG2a, CHO-K1 and S2 cells, in all of which interferon response induction was but slight, if at all. It would thus follow that siRNA oligomers with sequence of a highly functional siRNA monomer unit in frame with respect to dicer digestion should serve as a good RNAi agent in Drosophila and certain mammalian cells.
PMCID: PMC2737202  PMID: 19771208
RNAi; siRNA; dsRNA; interferon response; mammalian cells; Drosophila
24.  MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans 
PLoS Genetics  2010;6(4):e1000903.
RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.
Author Summary
Due to its intrinsic characteristics, RNA interference (RNAi) has become one of the most widely used tools in cell biology and has revolutionized approaches to elucidate gene function. The process, also known as RNA silencing, is triggered by dsRNA molecules that are cleaved by Dicer proteins into small interfering RNAs (siRNAs). The rde-1 gene from Caenorhabditis elegans was one of the first genes found in association with this mechanism and encodes the only Argonaute protein in worms, which is by itself essential for the classical RNAi pathway triggered by exogenously introduced dsRNA. However, little is known about endogenous functions of RDE-1. Here we show that RDE-1 binds to many classes of small RNAs, including microRNAs. We show that miR-243 is efficiently bound by RDE-1 and triggers regular RNAi on an endogenous target, implying that many RNA species, including miRNAs, are constantly being screened against the transcriptome using the canonical exogenous RNAi pathway.
doi:10.1371/journal.pgen.1000903
PMCID: PMC2851571  PMID: 20386745
25.  Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi 
Nucleic Acids Research  2014;42(13):8732-8744.
RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.
doi:10.1093/nar/gku528
PMCID: PMC4117760  PMID: 24939903

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