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Results for “RNA interference”

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PubMed2004

Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells.

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.

Animals↗

PubMed2006

UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in Arabidopsis.

Aberrant mRNAs containing premature termination codons (PTCs) have the potential to be translated into truncated proteins, which could act to the detriment of the organism by interfering with normal cellular processes. Eukaryotes have mechanisms of mRNA quality control that identify PTC-containing transcripts and target them for destruction, a process known as nonsense-mediated mRNA decay (NMD). Surprising differences have been reported in the mechanisms of NMD between different organisms. UPF1 and UPF3 are structurally unrelated proteins, which function in the NMD pathway in yeast, mammals, Drosophila and Caenorhabditis elegans. Here we show that NMD in plants requires UPF1, as mRNAs containing PTCs become stabilized in upf1-5 mutants. However, in contrast to NMD in mammals, UPF1-dependent NMD is capable of targeting both spliced and unspliced PTC-containing mRNAs. An allelic series of upf1 mutants exhibits a range of unexpected vegetative and floral abnormalities, including jagged leaves, late flowering, fused flowers and seedling lethality. We also show that mutants in UPF3 share these abnormalities. As both UPF1 and UPF3 are required for NMD, the similar phenotypes of the upf1 and upf3 mutants suggest that NMD regulates a common set of genes required for plant development and survival. Finally, gene silencing by an inverted repeat transgene is impaired in upf1-5 mutants, indicating a connection between UPF1 and RNA interference in plants.

Alleles↗

PubMed2003

The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development.

Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

Animals↗

PubMed2005

Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy.

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.

Adenocarcinoma↗

PubMed2004

Intravenous, non-viral RNAi gene therapy of brain cancer.

RNA interference (RNAi) has the potential to knock down oncogenes in cancer, including brain cancer. However, the therapeutic potential of RNAi will not be realised until the rate-limiting step of delivery is solved. The development of RNA-based therapeutics is not practical, due to the instability of RNA in vivo. However, plasmid DNA can be engineered to express short hairpin RNA (shRNA), similar to endogenous microRNAs. Intravenous, non-viral RNAi-based gene therapy is enabled with a new gene-targeting technology, which encapsulates the plasmid DNA inside receptor-specific pegylated immunoliposomes (PILs). The feasibility of this RNAi approach was evaluated by showing it was possible to achieve a 90% knockdown of brain tumour-specific gene expression with a single intravenous injection in adult rats or mice with intracranial brain cancer. The survival of mice with intracranial human brain cancer was extended by nearly 90% with weekly intravenous injections of PILs carrying plasmid DNA expressing a shRNA directed against the human epidermal growth factor receptor. RNAi-based gene therapy can be coupled with gene therapy that replaces mutated tumour suppressor genes to build a polygenic approach to the gene therapy of cancer.

Animals↗

PubMed2005

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

Analysis of Variance↗

PubMed2005

RNA silencing in Drosophila.

Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.

Animals↗

PubMed2004

The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine.

Members of the ezrin-radixin-moesin (ERM) family of proteins have been found to serve as linkers between membrane proteins and the F-actin cytoskeleton in many organisms. We used RNA interference (RNAi) approach to assay ERM proteins of the Caenorhabditis elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. We identify erm-1 as the only ERM protein required for development and show, by multiple RNA interference, that additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. We show that the initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin-cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction.

Actins↗

PubMed2004

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1.

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.

Amino Acid Sequence↗

PubMed2006

RNA knockdown as a potential therapeutic strategy in Parkinson's disease.

Parkinson's disease is a prevalent progressive degenerative disorder of the elderly. There is a current need for novel therapeutic strategies because the standard levodopa pharmacotherapy is only temporarily efficacious. Recently, there have been some high-profile successful preclinical results obtained in animal models of neurological disorders using small interfering RNAs delivered by viral vectors. RNA interference can theoretically be applied to Parkinson's disease since over-expression of various proteins is known to kill the dopamine neurons of the substantia nigra in animal models and in familial forms of Parkinson's disease. Potential RNA interfering strategies and caveats are discussed in this review.

Animals↗

PubMed2005

Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti.

Reverse genetics is a powerful tool for understanding gene functions and their interactions in the mosquito innate immunity. We took the transgenic approach, in combination with the RNA interference (RNAi) technique, to elucidate the role of mosquito REL1, a homolog of Drosophila Dorsal, in regulation of Toll immune pathway in the mosquito Aedes aegypti. By transforming the mosquitoes with DeltaREL1-A or a double-stranded RNA construct of REL1 driven by the female fat body-specific vitellogenin (Vg) promoter with the pBac[3xP3-EGFP, afm] vector, we generated two different transgenic mosquito strains, one with overexpressed AaREL1 and the second with AaREL1 knockdown. Both strains had a single copy of the respective transgene, and the expression in both transgenic mosquitoes was highly activated by blood feeding. Vg-DeltaREL1-A transgenic mosquitoes activate Toll immune pathway in the fat body by blood feeding. The overexpression of both isoforms, AaREL1-A and AaREL1-B, in Vg-DeltaREL1-A transgenic mosquitoes resulted in the concomitant activation of Aedes Spätzle1A and Serpin-27A, independent of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of an Aedes homolog to Drosophila cactus, an IkappaB inhibitor of Drosophila Toll pathway. The effect of the transgenic RNAi knockdown of AaREL1 on mosquito innate immunity was revealed by increased susceptibility to the entomopathogenic fungus Beauveria bassiana and the reduced induction of Spz1A and Serpin-27A gene expression after fungal challenge. These results have proven that AaREL1 is a key downstream regulator of Toll immune pathway in the mosquito A. aegypti.

Aedes↗

PubMed2007

RNAi pathway is functional in peripheral nerve axons.

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

Animals↗

PubMed2006

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

Plant Viruses↗

PubMed2004

[Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene].

We identified the inv gene that encodes left and right asymmetry and regulates kidney development based on the information of the inv mutant mouse. However, functional properties and the modulator of gene expression of inv have been unclear. We used the tissue injury model for assessing the functional roles of inv in ischemia reperfusion injury (IRI). The kidney tissue taken from rats with IRI showed reciprocal changes in mRNA expression of inv: a 0.25-fold decrease at 6 hours and then a gradual increase to a maximum 1.8-fold rise at 10 days of reperfusion. Next, oxidative stress was induced by exposing mouse inner medullary collecting duct (mIMCD-3) cells to hydrogen peroxide(H2O2) in the medium. Real-time PCR showed that mRNA expression of inv decreased 0.52-fold at 3 hours with 0.2 mM H2O2 in the medium, and then increased 3.1-fold at 24 hours with 0.1 mM H2O2 in the medium. RNA interference (RNAi) is a powerful tool to inhibit gene expression in experimental model systems. We knocked down inv gene expression in mIMCD-3 cells using RNAi to investigate the function of the inv gene. We designed a small interfering RNA (siRNA) to target the coding region of inv (inv-siRNA) and random-sequence scrambled siRNA(control siRNA). mIMCD-3 cells transfected with either the inv-siRNA or control siRNA were observed by microscopy. The cells transfected with inv-siRNA progressively lost cell-to-cell contact and the cell population significantly diminished approximately 48 hours post-transfection. The changes in gene expression profile were observed at time points (36 hours) using real-time PCR-based gene screening with categorized primer sets. Several genes related to structural protein of the matrix were downregulated. In contrast, repairing related genes were upregulated. In conclusion, gene expression of inv was modulated under oxidative stress and the inv gene may play a role in repairing and regenerating renal epithelial cells.

Animals↗

PubMed2004

In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.

The combination of emergent RNA interference (RNAi) technology with in ovo electroporation in the chick embryo has the potential to provide a powerful and rapid means for functional analyses of novel genes in vivo. In this study, we show that electroporation of short 21-bp RNA duplexes (siRNAs) is a quick and simple method for silencing exogenous and endogenous gene expression in vivo. Quantitative comparisons with two other RNAi protocols that use long double-stranded RNA duplexes and endonuclease-digested duplexes (esiRNAs) demonstrate that siRNAs are significantly more effective at reducing gene expression. Furthermore, we also find that much higher amounts of siRNA are required for silencing of endogenous gene expression relative to plasmid-borne reporter constructs. In short, these results demonstrate that siRNAs are the most effective type of double-stranded RNA duplex for silencing gene expression and suggest that there might be important differences between silencing endogenous and exogenous genes. Finally, we review the parameters for each of these RNA-based methods of RNAi and the controls required to analyze RNAi data in the context of the developing vertebrate embryo.

Animals↗

PubMed2006

Role of cytosolic malate dehydrogenase in oocyte maturation and embryo development.

OBJECTIVE: To elucidate the function of cytosolic malate dehydrogenase (Mor2) in oocyte maturation and embryo development using RNA interference (RNAi). DESIGN: Experimental animal study. SETTING: Research unit of university. ANIMAL(S): Female 4-week-old (C57/BL6) mice. INTERVENTION(S): Isolation of immature germinal vesicle (GV) oocytes or fertilized pronucleus (PN) embryos, microinjection of Mor2 double-stranded RNA (dsRNA), and reverse transcription and polymerase chain reaction (RT-PCR) analysis to investigate Mor2-specific messenger RNA (mRNA) knockdown. MAIN OUTCOME MEASURE(S): Relative changes in mRNA levels after microinjection of Mor2 dsRNA and in rates of oocyte maturation and preimplantation embryo development. RESULT(S): Mor2 mRNA mostly was knocked down in germinal vesicle- and metaphase I (MI)-arrested oocytes, compared with metaphase II (MII)-developed oocytes, after microinjection of Mor2 dsRNA and in vitro culture for 16 hours. In vitro oocyte maturation was significantly decreased (34%), compared with noninjected (73.4%) and buffer-injected (67.5%) control groups. The rate of blastocyst development (48.1%) was lower in the Mor2 dsRNA-injected group than in buffer-injected control (88.2%). CONCLUSION(S): In the present study, the function of Mor2 was analyzed with the aid of RNAi. On the basis of the data obtained, we propose that Mor2 is an essential factor for oocyte maturation and embryo development in mouse.

Animals↗

PubMed2006

Nuclear localization of poly(A)+ mRNA following siRNA reduction of expression of the mammalian RNA helicases UAP56 and URH49.

UAP56 is a eukaryotic RNA helicase that is important for mRNA splicing and nuclear export. Although most eukaryotes have a single protein corresponding to UAP56, we have shown previously that in human and mouse cells there is a second protein, URH49, which is 90% identical to UAP56. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), suggesting that both proteins have similar functions. However, the two helicases have different expression profiles in different tissues and in growth-stimulated cells, which raises the possibility that they might be involved in the splicing and export of non-identical populations of mRNA. In the present study, we have used RNA interference to further explore the functions of these two helicases. Reducing the expression of either URH49 or UAP56 in HeLa cells had little effect on cell proliferation or expression of a co-transfected gene. However, analysis of poly(A)+ RNA localization by fluorescent in situ hybridization revealed a speckled pattern of RNA accumulation throughout the nucleus. Reducing the expression of both helicases resulted in a major reduction in reporter gene expression as well as cell death within 72 h. We also observed a more prominent speckled pattern of nuclear poly(A)+ RNA accumulation as well as reduced accumulation in the cytoplasmic compartment. These observations suggest that both helicases have essential but largely overlapping functions in the processing and export of mammalian mRNAs.

Active Transport, Cell Nucleus↗

PubMed2006

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression.

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.

Apoptosis↗