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At least 235 records · Page 13Linked to original sources
PubMed2005

Novel modes of protein-RNA recognition in the RNAi pathway.

Gene silencing mediated by RNA interference (RNAi) depends on short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These RNAs have unique features, namely a defined size of 19-21 base pairs, and characteristic two-nucleotide single-stranded 3' overhangs and 5' monophosphate groups. These molecular features of siRNAs and miRNAs are produced by RNase III enzymes, which are a hallmark of gene silencing induced by double-stranded RNA. Recent structural studies of components of the RNAi pathway, including PAZ, Piwi and RNase III domains, as well as full-length Argonaute and viral p19 proteins, have revealed distinct and novel modes of sequence-independent recognition of the characteristic features of siRNAs and miRNAs in the RNAi pathway.

Binding Sites↗
PubMed2005

The best control for the specificity of RNAi.

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

Animals↗
PubMed2004

Enhancement of RNAi activity by improved siRNA duplexes.

RNA interference (RNAi) is a powerful tool for suppressing the expression of a gene of interest, in which 21-25 nucleotide short interfering RNA (siRNA) duplexes homologous to the silenced gene function as sequence-specific RNAi mediators. The present study shows that newly designed siRNA duplexes, 'fork-siRNA duplexes', whose sense-stranded siRNA elements carry one to four nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, can enhance RNAi activity over conventional siRNA duplexes in cultured mammalian cells.

Base Sequence↗
PubMed2003

Asymmetry in the assembly of the RNAi enzyme complex.

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Adenosine Diphosphate↗
PubMed2004

A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

Adenosine Triphosphate↗
PubMed2003

Mammalian RNAi for the masses.

Just a couple of years ago, only biologists working with plants or Caenorhabditis elegans could use RNA-mediated interference (RNAi) technology to gain insight into gene function. However, the recent groundbreaking discovery that in vitro synthesized, 21- to 23-nucleotide, double-stranded RNAs can act as small interfering RNAs (siRNAs) to elicit gene-specific inhibition in mammalian cells has made RNAi possible in mammalian systems too. Reported only a year ago, mammalian RNAi is already changing our way of studying gene function in higher eukaryotes. And, a recent exciting advance allows delivery of siRNAs into mammalian cells by a DNA vector. In addition to providing a low-cost alternative to the chemically synthesized siRNAs, this DNA-vector-based strategy is capable of mediating stable target gene inhibition, thus allowing gene function analysis over an extended period of time.

Animals↗
PubMed2001

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi.

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.

Animals↗
PubMed2003

Potentials for RNAi in sarcoma research and therapy: Ewing's sarcoma as a model.

Existing data identify EWS-FLI1 as indispensable for sustained Ewing's sarcoma growth and as the ideal therapeutic target in this disease. The siRNA may hold great promises as a fusion gene specific agent. RNAi mediated suppression of EWS-FLI1 is likely to result in an altered tumor cell phenotype including changes in chemosensitivity, and a restored differentiation potential. Thus, RNAi may serve as an adjuvant to chemotherapy. As a therapeutic means however, RNAi is hampered by limitations in the delivery of the agent and emergence of resistant clones. In vitro suppression of EWS-FLI1 expression will allow to define the phenotypic characteristics of dormant tumor cells that may give rise to late relapses, enabling improved diagnosis and treatment even of minimal residual disease.

Animals↗
PubMed2002

RNAi: gene-silencing in therapeutic intervention.

Several rapidly developing RNA interference (RNAi) methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. As such, RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop highly specific dsRNA based gene-silencing therapeutics.

Animals↗
PubMed2006

Gene expression: long-term gene silencing by RNAi.

Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.

Animals↗
PubMed2005

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans.

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

Animals↗
PubMed2004

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25-30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

Gene Expression Regulation↗
PubMed2006

RNAi therapeutics: a potential new class of pharmaceutical drugs.

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.

Animals↗
PubMed2005

Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.

Amyotrophic Lateral Sclerosis↗
PubMed2006

Minimizing the risk of reporting false positives in large-scale RNAi screens.

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Databases, Genetic↗
PubMed2004

RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells.

RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.

Animals↗
PubMed2006

High-throughput RNAi screening by time-lapse imaging of live human cells.

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Automation↗
PubMed2003

RNAi reveals doublecortin is required for radial migration in rat neocortex.

Mutations in the doublecortin gene (DCX) in humans cause malformation of the cerebral neocortex. Paradoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. We used electroporation of plasmids encoding short hairpin RNA to create interference (RNAi) of DCX protein in utero, and we show that DCX is required for radial migration in developing rat neocortex. RNAi of DCX causes both cell-autonomous and non-cell autonomous disruptions in radial migration, and creates two disruptions in neocortical development. First, many neurons prematurely stop migrating to form subcortical band heterotopias within the intermediate zone and then white matter. Second, many neurons migrate into inappropriate neocortical lamina within normotopic cortex. In utero RNAi can therefore be effectively used to study the specific cellular roles of DCX in neocortical development and to produce an animal model of double cortex syndrome.

Animals↗