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At least 883 records · Page 49Linked to original sources
PubMed2005

Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA (shRNA) transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.

Cell Line↗
PubMed2005

Functional genomic analysis of RNA interference in C. elegans.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Amino Acid Motifs↗
PubMed2006

Innate immune defense through RNA interference.

RNA interference (RNAi, also known as RNA silencing) has recently emerged as a fundamental and widespread regulator of gene expression. New developments in this field implicate RNAi in the innate immune response to infection in plants and animals. Evidence from plants, tissue culture cells, and Caenorhabditis elegans-based systems previously suggested that RNAi plays a role in the defense against viral infection, but definitive evidence using viruses and whole animals has been lacking. Two recent reports now show that both Drosophila embryos and adult flies mount a substantial innate immune response to insect viruses that requires the RNAi machinery. This innate response is distinct from known bacterial and fungal defense systems provided by the Toll and immune deficiency (Imd) pathways, thus defining a previously unrecognized strategy to fight viral infection. Whether RNAi, aside from its function in counteracting viruses, is also used to fight bacterial infection remained enigmatic. New evidence, however, now shows that in Arabidopsis, the bacterial component, flagellin, induces the expression of a specific microRNA, which in turn leads to the down-regulation of the signaling pathways that are implicated in disease susceptibility. This down-regulation then increases the plant's resistance to infection. Whether RNAi mechanisms also exist for combating bacterial diseases in animals remains an intriguing question for future studies.

Animals↗
PubMed2004

Selection and characterization of RNA interference-deficient trypanosomes impaired in target mRNA degradation.

Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with alpha-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

Alleles↗
PubMed2006

Optimization of feline immunodeficiency virus vectors for RNA interference.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

Cell Line, Tumor↗
PubMed2004

Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes.

RNA interference (RNAi) is an antiviral mechanism that is activated when double-stranded RNA is cleaved into fragments, called short interfering RNA (siRNA), that prime an inducible gene silencing enzyme complex. We applied RNAi against a herpes simplex virus type 1 (HSV-1) gene, glycoprotein E, which mediates cell-to-cell spread and immune evasion. In an in vitro model of infection, human keratinocytes were transfected with siRNA specific for glycoprotein E and then infected with wild-type HSV-1. RNAi-mediated gene silencing reproduced the small plaque phenotype of a gE-deletion mutant virus. The specificity of gene targeting was demonstrated by flow cytometry and Northern blot analyses. Exogenous siRNA can suppress HSV-1 glycoprotein E expression and function during active infection in vitro through RNAi. This work establishes RNAi as a genetic tool for the study of HSV and provides a foundation for development of RNAi as a novel antiviral therapy.

Blotting, Northern↗
PubMed2004

Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition.

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

Base Sequence↗
PubMed2005

Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Cell Cycle Proteins↗
PubMed2003

RNA interference: biology, mechanism, and applications.

Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

Animals↗
PubMed2005

RNA interference in biology and disease.

RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens. In fact, recent studies reported in this journal and others have demonstrated success using RNAi to address the role of oncogene expression in leukemia cell lines and to validate the therapeutic potential of RNAi for treating these blood disorders. In order to advance these applications and gain an appreciation for the future of RNAi both in basic research and in the treatment of diseases caused by aberrant gene expression, it is important to have an understanding of the process of RNAi and its limitations.

Gene Expression Regulation, Neoplastic↗
PubMed2005

Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer.

BACKGROUND: The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. METHODS: AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. RESULTS: Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a beta-lactamase reporter gene, a 10-12 fold shift or 2.5-3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. CONCLUSION: EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways.

Antibodies, Monoclonal↗
PubMed2003

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Animals↗
PubMed2006

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference.

BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.

Cloning, Molecular↗
PubMed2006

Minireview: ribonucleic acid interference for the identification of new targets for the treatment of metabolic diseases.

Over the past years, RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a tool to identify new targets for therapeutic intervention. This review focuses on the current understanding of RNAi biology, how RNAi has been used to study the role of different genes in the pathogenesis of diabetes and obesity, and the use of RNAi screens for the identification of new targets for metabolic diseases. Also reviewed are the in vivo proof of principle experiments that provide the validation of these new targets for the development of RNAi-based therapeutics for diabetes.

Adenoviridae↗
PubMed2000

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans.

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.

Amino Acid Sequence↗
PubMed2003

Gene silencing in Caenorhabditis elegans by transitive RNA interference.

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.

Animals↗
PubMed2004

Induction and biochemical purification of RNA-induced silencing complex from Drosophila S2 cells.

The discovery of RNA interference (RNAi) has greatly simplified the process of suppressing genes in many experimental systems, including Caenorhabditis elegans, Drosophila, and mammalian cells. A sequence-specific nuclease complex, called the RNA-induced silencing complex (RISC), can be purified from cells undergoing RNAi. RISC shows RNase activity when exposed to RNAs homologous to the input double-stranded RNA (dsRNAs) but lacks activity in the presence of nontargeted RNAs. We describe the induction of RNAi by dsRNA in cultured Drosophila Schneider-2 (S2) cells and detail procedures for RISC purification from these cells. This purification approach has allowed us to identify several RISC components, including siRNAs, Argo naute 2 (Ago-2), Drosophila Fragile X related protein (dFXR), Vasa intronic gene (VIG), and the micrococcal nuclease family member Tudor-SN (Drosophila CG7008). RNAi is carried out by an endogenous pathway important for normal development in many organisms. In fact, organisms express hundreds of different microRNAs (miRNAs), small hairpin RNAs that function through the RNAi pathway to suppress expression of endogenous genes. The function of miRNAs is poorly understood, and most of their targets are unknown. Purified RISC complexes contain short interfering RNAs and endogenously expressed miRNAs and will be useful for studying many aspects of the RNAi machinery.

Animals↗
PubMed2004

RNA interference silencing the transcriptional message: aspects and applications.

RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomenon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.

Agriculture↗