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At least 343 records · Page 19Linked to original sources
PubMed2002

Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans.

Recently, a set of 766 genes that are enriched in the ovary as compared to the soma was identified by microarray analysis [1]. Here, we report a functional analysis of 98% of these genes by RNA interference (RNAi). Over half the genes tested showed at least one detectable phenotype, most commonly embryonic lethality, consistent with the expectation that ovary transcripts would be enriched for genes that are essential in basic cellular and developmental processes. We find that essential genes are more likely to be conserved and to be highly expressed in the ovary. We extend previous observations and find that fewer than the expected number of ovary-expressed essential genes are present on the X chromosome. We characterized early embryonic defects for 161 genes and used time-lapse microscopy to systematically describe the defects for each gene in terms of 47 RNAi-associated phenotypes. In this paper, we discuss the use of these data to group genes into "phenoclusters"; in the accompanying paper, we use these data as one component in the integration of different types of large-scale functional analyses. We find that phenoclusters correlate well with sequence-based functional predictions and thus may be useful in predicting functions of uncharacterized genes.

Animals↗
PubMed2003

RNAi: an ever-growing puzzle.

In recent years, sequence-specific gene silencing has been an area of increasing focus, both because of its interesting biology and because of its power as an experimental tool. A growing understanding of one such phenomenon, RNA interference (RNAi), has provided clues that many homology-dependent gene-silencing mechanisms share a common trigger, double-stranded RNA. Recent findings that RNAi and related pathways are involved not only in the response to exogenous pathogenic and endogenous parasitic nucleic acids but also in basic cellular processes, such as gene regulation and heterochromatin formation, have further fueled interest in this rapidly expanding field.

Animals↗
PubMed2003

Exposing oncogenic dependencies for cancer drug target discovery and validation using RNAi.

Oncogenesis occurs through the acquisition and selection of multiple somatic mutations--each contributing to the growth, survival and spread of the cancer. Key attributes of the malignant phenotype, such as unchecked proliferation and cell survival, can often be "reversed" by the selective diminution of dominant oncogenes by chemical or genetic means (e.g. beta-catenin in colorectal carcinomas; bcr-abl in chronic myelogenous leukemias (CMLs)). These observations suggest that the products of oncogenes, or of secondary genes that mediate and maintain tumor phenotypes, might be revealed through the systematic disruption of each and every gene in tumor-derived cells. Some of these genes may encode proteins amenable to therapeutic intervention, thus fueling the cancer drug discovery process. However, a functional assessment of each known or predicted gene in mammalian cells is a daunting task and represents the rate-limiting step in drug target identification and validation. In this regard, RNA interference (RNAi) by small interfering RNAs (siRNA) holds great promise as the "tool of choice" to mediate the selective attenuation of mammalian gene expression and protein function. Here, we review strategies by which RNAi might be used to determine the genetic alterations that contribute to malignant transformation via large-scale cell-based screens, and propose how this information can be used in conjunction with small molecule screens to identify pathways critical to cancer cell survival.

Animals↗
PubMed2003

Making a better RNAi vector for Drosophila: use of intron spacers.

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure.

Animals↗
PubMed2002

RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila.

Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21--25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing.

ATP-Binding Cassette Transporters↗
PubMed2003

Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening.

New opportunities in mammalian functional genomics are emerging through the combination of high throughput technology and methods that allow manipulation of gene expression in living cells. Here we describe the application of an RNAi-based forward genomics approach toward understanding the biology and mechanism of TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumor cells when bound to its cognate receptors. In addition to detecting well-characterized genes in the apoptosis pathway, we uncover several modulators including DOBI, a gene required for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, and MIRSA, a gene that acts to limit TRAIL-induced apoptosis. Moreover, our data suggest a role for MYC and the WNT pathway in maintaining susceptibility to TRAIL. Collectively, these observations offer several insights on how TRAIL mediates the selective killing of tumor cells and demonstrate the utility of large-scale RNAi screens in mammalian cells.

Apoptosis↗
PubMed2002

Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing.

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.

Alternative Splicing↗
PubMed2003

A micrococcal nuclease homologue in RNAi effector complexes.

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

Animals↗
PubMed2004

A large-scale RNAi screen in human cells identifies new components of the p53 pathway.

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.

Cell Division↗
PubMed2005

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance.

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

Apoptosis↗
PubMed2004

RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia.

The dominant polyglutamine expansion diseases, which include spinocerebellar ataxia type 1 (SCA1) and Huntington disease, are progressive, untreatable, neurodegenerative disorders. In inducible mouse models of SCA1 and Huntington disease, repression of mutant allele expression improves disease phenotypes. Thus, therapies designed to inhibit expression of the mutant gene would be beneficial. Here we evaluate the ability of RNA interference (RNAi) to inhibit polyglutamine-induced neurodegeneration caused by mutant ataxin-1 in a mouse model of SCA1. Upon intracerebellar injection, recombinant adeno-associated virus (AAV) vectors expressing short hairpin RNAs profoundly improved motor coordination, restored cerebellar morphology and resolved characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice. Our data demonstrate in vivo the potential use of RNAi as therapy for dominant neurodegenerative disease.

Adenoviridae↗
PubMed2006

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

3' Untranslated Regions↗
PubMed2006

Enzymatically prepared RNAi libraries.

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.

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↗
PubMed2004

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells.

Antiapoptotic genes such as bcl-2 or xIAP may be responsible for resistance to apoptosis induced by cytotoxic drugs. The aim of this study was to investigate if downregulation of bcl-2 or xIAP by RNA interference (RNAi) would sensitize MCF-7 cells to etoposide and doxorubicin. FITC-siRNAs uptake was verified by fluorescence microscopy and downregulation of Bcl-2 or XIAP was confirmed by Western Blotting. Both siRNAs reduced the number of viable cells and increased cellular apoptosis. Treatment with siRNAs followed by treatment with etoposide or doxorubicin further reduced the number of viable cells, when compared to either of the treatments alone. Therefore, downregulation of bcl-2 or xIAP by RNAi enhances the effects of etoposide and doxorubicin.

Antibiotics, Antineoplastic↗
PubMed2004

RNAi, a new therapeutic strategy against viral infection.

RNA interference (RNAi) is an adaptive defense mechanism triggered by double-stranded RNA (dsRNA). It is a powerful reverse genetic tool that has been widely employed to silence gene expression in mammalian and human cells. RNAi-based gene therapies, especially in viral diseases have become more and more interesting and promising. Recently, small interfering RNA (siRNA) can be used to protect host from viral infection, inhibit the expression of viral antigen and accessory genes, control the transcription and replication of viral genome, hinder the assembly of viral particles, and display influences in virus-host interactions. In this review, we attempt to present recent progresses of this breakthrough technology in the above fields and summarize the possibilities of siRNA-based drugs.

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↗
PubMed2004

RNAi in a postmodern, postgenomic era.

As dsRNA-mediated interference (RNAi) libraries become more widely available, genome-wide loss of function experiments in cell culture are likely to become a routine laboratory tool. RNAi screens can then be conducted to test gene functions in different assays in a variety of cell types and across species. In comparing these large phenotypic data sets, researchers will gain a new perspective, enabling them to see each gene as part of a fluid network that is remodelled in response to changes in environmental and developmental context, and with the passage of evolutionary time.

Gene Library↗