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PubMed2004

RNA interference: historical overview and significance.

In the early 1990s, attempts to manipulate gene expression by researchers working in three different fields resulted in unanticipated gene silencing. Rather than ignoring such results, these researchers went on to document and further investigate the nature of such silencing, which was named "co-suppression" in plants, "quelling" in fungi, and "RNA interference" (RNAi) in nematodes. By the late 1990s, it was discovered that silencing could be initiated in this diverse set of organisms by exposing cells to double-stranded RNA (dsRNA), which directed the destruction of mRNAs containing similar sequences. Soon afterward, such dsRNA-mediated silencing was employed as a reverse genetic technique to analyze the functions of specific genes in a broad variety of organisms. Biochemical and genetic studies designed to uncover the components of the RNA silencing machinery identified a common core of proteins that serve to amplify the interfering RNA signal and direct endonucleolytic cleavage of target RNAs. A subset of silencing events may also direct DNA methylation of targeted genes. RNA silencing is thought to have evolved as a defense mechanism to suppress viral replication and transposon mobilization. However, additional functions involving the RNAi machinery have been uncovered, including posttranscriptional regulation of endogenous genes, and maintenance of structure and function of heterochromatin. Whereas many researchers have focused on understanding the natural biological functions of RNA silencing, others are testing its utility in antiviral and cancer therapies and in other biotechnological and biomedical applications.

Animals↗
PubMed2005

RNAi and high-content screening in target identification and validation.

The development of effective novel therapeutic agents faces many significant challenges, such as demonstrating that a candidate target plays a critical role in disease progression. RNA interference (RNAi) has proven to be a robust and highly scalable technology, and as such, has become an essential method for studying targets in many disease models. High-content screening (HCS) is a platform for quantitatively measuring cellular features such as transcription factor localization. This is a more powerful method of measuring signal transduction than reporter assays because the image-based data of HCS can eliminate many sources of assay artifacts, and the associated statistical tools are highly effective. While it appears obvious that convergence of technologies is required to establish RNAi screening assays in HCS formats, some challenges arise when combining the approaches. However, combining RNAi and HCS provides significant and unique advantages to a target validation program.

Animals↗
PubMed2005

Genome-wide screening for gene function using RNAi in mammalian cells.

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.

Animals↗
PubMed2004

High-throughput gene silencing using cell arrays.

A recently established transfected cell array (TCA) technology has opened new experimental dimensions in the field of functional genomics. Cell arrays allow for transfection of several thousands different DNA molecules in microarray format. The effects of overexpression of hundreds of proteins on cellular physiology can be observed in a single experiment. The TCA technique has also found its application in RNA interference (RNAi) research. Small interfering RNAs (siRNA) as well as plasmid expressing short hairpin RNAs can be transferred into the cells through the process of reverse transfection. The silencing of numerous genes in spatially separated manner can be thus monitored. This review will provide an overview on current concepts concerning combination of cell array and RNAi for high-throughput loss-of-function studies.

Gene Silencing↗
PubMed2006

Knocking down disease with siRNAs.

Knocking down expression of disease-related genes using small interfering RNAs (siRNAs) has potential for treating a variety of illnesses. This Essay will examine the opportunities for harnessing RNA interference (RNAi) for therapy, as well as the obstacles and possible ways to circumvent them.

Animals↗
PubMed2006

Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery.

Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE.

Animals↗
PubMed2006

Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders.

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Animals↗
PubMed2004

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Cell Division↗
PubMed2005

Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome.

Double-stranded RNA (dsRNA) mediated gene silencing (RNA interference; RNAi) is a powerful tool for investigating gene function. It is usually performed in Caenorhabditis elegans via the injection or oral delivery of dsRNA, but an alternative approach, the expression of RNA hairpins from introduced DNA (hairpin RNAi; hpRNAi) has several advantages: (1) it can be induced systemically or in a tissue-specific manner; (2) because it is heritable, it allows consistent RNAi silencing across a whole population of genetically identical animals; and (3) it can be applied in refractory tissue such as neurons. hpRNAi has not been widely used to investigate gene function because a number of steps are relatively inefficient and labour-intensive. We describe Wormgate, a new cloning system, which facilitates the efficient high-throughput production of hpRNAi constructs using clones from the C. elegans ORFeome library. The combined use of pWormgate2 and the ORFeome library, with a recently developed particle bombardment transformation system, expedites hpRNAi gene silencing. This will be particularly useful for studying those genes that are refractory to the effects of injected or fed dsRNA, such as neural genes. We report the efficient production of hpRNAi constructs using pWormgate2 and also the knockdown of selected genes, including neurally expressed genes that have previously been refractory to RNAi. Further, when combined with the rrf-3 RNAi hypersensitive strain, the Wormgate approach delivered a highly penetrant knockdown phenotype in nearly 100% of worms for a gene that was completely refractory to other RNAi delivery methods.

Administration, Oral↗
PubMed2006

[Construction of RNAi binary vector of soybean receptor-like kinase gene (rlpk2) and its soybean transformation].

Plant receptor-like kinases (RLKs) play important roles in plant growth, development and responses to environmental stress. RNA interference (RNAi) is a powerful tool to study functions of RLKs. In this study, a 312bp 3'end cDNA fragment of the soybean receptor-like kinase gene rlpk2 was chosen to construct the rlpk2-RNAi expression cassette. The binary vector pART27-R2 harboring rlpk2-RNAi expression cassette was constructed through three times subcloning via mid-clone vector and then transformed into Agrobacterium LBA4404. Three independent transgenic plants were obtained by Agrobactium-mediated soybean cotyledon transformation method. RT-PCR analysis showed that rlpk2 gene was successfully knocked down in all transgenic plants. It was found that photosynthesis activities of rlpk2-RNAi transgenic leaves were much improved, suggesting rlpk2 may function as a negative regulator of soybean leaf functions and/or chloroplast structure.

Gene Expression Regulation, Plant↗
PubMed2006

Retraction of synapses and dendritic spines induced by off-target effects of RNA interference.

RNA interference (RNAi), which allows selective gene silencing, has been proposed for functional genomic analysis and for the treatment of human disease. However, induction of RNAi in mammalian cells by expression of double-stranded RNA can activate innate antiviral response pathways that perturb off-target gene expression. The activation and functional consequences of these effects in neurons are unknown. We find that expression of subsets of short hairpin RNAs (shRNAs) in rat hippocampal pyramidal neurons can have off-target effects that reduce the complexity of dendritic arbors and trigger the loss of dendritic spines. Morphological changes are accompanied by electrophysiological perturbations in passive membrane properties and a decrease in the number and strength of excitatory and inhibitory synapses. These perturbations depend on the shRNA sequence and are independent of the identity of the targeted protein. Our results indicate that off-target effects of RNAi severely perturb neuronal structure and function and may lead to the functional withdrawal of affected cells from the brain circuitry.

Adaptation, Physiological↗
PubMed2006

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells.

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.

Cell Line, Tumor↗
PubMed2004

Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells.

BACKGROUND: RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. RESULTS: Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. CONCLUSION: These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

Animals↗
PubMed2004

Adenovirus vector-mediated doxycycline-inducible RNA interference.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Adenoviridae↗
PubMed2004

Endogenous inhibitors of RNA interference in Caenorhabditis elegans.

In eukaryotes, double-stranded RNAs (dsRNAs) or short, interfering dsRNAs (siRNAs) can reduce the accumulation of a sequence-related mRNA, often resulting in a loss-of-function phenotype-a process termed RNA interference (RNAi). Unfortunately, some mRNAs are resistant to the effects of dsRNA. Experiments designed to unravel RNAi mechanisms in Caenorhabditis elegans have led to the identification of two worm proteins, RRF-31,2 and, now, ERI-1,3 that can inhibit RNAi responses. Animals defective in either protein can display enhanced RNAi phenotypes for mRNAs that were previously resistant to dsRNA. Since ERI-1 is a conserved protein, development of procedures to enhance RNAi effectiveness in other systems may be possible.

Animals↗
PubMed2006

RNA interference-mediated inhibition of brain-derived neurotrophic factor expression increases cocaine's cytotoxicity in cultured cells.

Previous studies showed that cocaine exposure decreased brain-derived neurotrophic factor (BDNF) function and resulted in neuronal cell death. To investigate a role of BDNF in cocaine's cytotoxicity, an RNA interference (RNAi) approach was used. Transfection of neuroblastoma SK-N-AS cells or primary rat hippocampal neurons with the small double-stranded interfering RNA (siRNA) targeting BDNF mRNA, but not the scrambled siRNA, resulted in reductions in levels of BDNF mRNA and proteins by more than 70% in the transfected cells as compared with the control group, suggesting an RNAi-mediated, sequence-specific gene silencing. The results also showed that cocaine-induced cytotoxicity, assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide) assay, was more pronounced in the cells transfected with the siRNA than in the cells transfected with the scrambled siRNA or in the cells treated with Lipofectamine 2000 alone (the control group), suggesting that inhibition of BDNF expression enhances cocaine's cytotoxicity. Together with previous studies showing that cocaine suppresses BDNF expression, the present data suggest that the drug-induced reduction of BDNF productions may make neurons more vulnerable to cocaine's toxic effects and precipitate cocaine-induced central nervous system damages.

Animals↗
PubMed2006

RNAi: a potential therapy for the dominantly inherited nucleotide repeat diseases.

Genetic diseases that are accompanied by central nervous system involvement are often fatal. Among these are the autosomal dominant neurogenetic diseases caused by nucleotide repeat expansion. For example, Huntington's disease (HD) and spinal cerebellar ataxia are caused by expansion of a tract of CAGs encoding glutamine. In HD and the other CAG-repeat expansion diseases, the expansion is in the coding region. Myotonic dystrophy is caused by repeat expansions of CUG or CCTG in noncoding regions, and the mutant RNA is disease causing. Treatments for these disorders are limited to symptomatic intervention. RNA interference (RNAi), which is a method for inhibiting target gene expression, provides a unique tool for therapy by attacking the fundamental problem directly. In this review, we describe briefly several representative disorders and their respective molecular targets, and methods to accomplish therapeutic RNAi. Finally, we summarize studies performed to date.

Animals↗
PubMed2001

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Animals↗