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Results for “RNA interference”

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PubMed2004

Effectiveness of RNA interference in transgenic plants.

RNA interference (RNAi) can be used to study gene function by effecting degradation of the targeted transcript. However, the effectiveness of transgene-induced RNAi among multiple target genes has not been compared systematically. To this end, we developed a relative quantitative RT-PCR protocol that allows use of a single internal standard over a wide range of target gene expression levels. Using this method in an analysis of transgenic Arabidopsis thaliana RNAi lines targeting 25 different endogenes revealed that independent, homozygous, single-copy (sc) T4 lines targeting the same gene generally reduce transcript levels to the same extent, whereas multi-copy RNAi lines differed in the degree of target reduction and never exceeded the effect of sc transgenes. The maximal reduction of target transcript levels varied among targets. These observations suggest that each target sequence possesses an inherent degree of susceptibility to dsRNA-mediated degradation.

Arabidopsis↗

PubMed2006

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.

Animals↗

PubMed2004

RNA-based drugs: from RNA interference to short interfering RNAs.

RNA interference consists of a sequence specific post-transcriptional gene silencing phenomenon triggered by a double strand RNA molecule homologous to the silenced gene. The dsRNA is cleaved by DICER enzyme in small dsRNA pieces, named short interfering RNAs (siRNAs). These fragments are thereafter associated to RISC complex where the cleavage of target RNA occurs. The observation that siRNAs can trigger the RNA interference mechanism in mammalian cells represents a fundamental discovery that discloses new horizons in genetic researches in that theoretically each gene can be silenced. The relative simplicity by which active short interfering RNAs can be designed and synthesized explains their widespread use in basic and applied researches, even if appropriate controls that exclude off-target effects are strictly required. The findings that siRNAs are active even when expressed in viral vectors open the possibility that they can be very soon used for gene therapy of several human diseases.

Animals↗

PubMed2003

RNA interference and chemically modified siRNAs.

RNA interference (RNAi) represents one of the most promising new frontiers in drug discovery. Short double-stranded RNA molecules are able to sequence-specifically inhibit expression of genes. This young technology offers both opportunities and challenges to nucleic acid chemists.

RNA Interference↗

PubMed2006

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.

RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.

Amino Acid Sequence↗

PubMed2006

[RNA interference--towards individualized genetic medicine].

RNA interference (RNAi) is an evolutionarily conserved cellular mechanism by which gene expression is suppressed by double-stranded RNA (dsRNA) in a sequence-specific manner. Enzymatically processed dsRNA molecules (siRNAs) target and facilitate cleavage of mRNA. By harnessing the RNAi machinery using synthetic or DNA-encoded RNA effectors, the siRNA technology offers new therapeutic strategies for genetic diseases. We outline the basic mechanisms of RNAi and present state-of-the-art examples of in vivo siRNA delivery towards development of RNAi-based individualized drugs.

Biotechnology↗

PubMed2004

A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans.

In many organisms, introducing double-stranded RNA (dsRNA) causes the degradation of messenger RNA that is homologous to the trigger dsRNA--a process known as RNA interference. The dsRNA is cleaved into short interfering RNAs (siRNAs), which hybridize to homologous mRNAs and induce their degradation. dsRNAs vary in their ability to trigger RNA interference: many mRNA-targeting dsRNAs show weak phenotypes, and nearly all mRNAs of the Caenorhabditis elegans nervous system are refractory to RNA interference. C. elegans eri-1 was identified in a genetic screen for mutants with enhanced sensitivity to dsRNAs. Here we show that eri-1 encodes an evolutionarily conserved protein with domains homologous to nucleic-acid-binding and exonuclease proteins. After exposure to dsRNA or siRNAs, animals with eri-1 mutations accumulate more siRNAs than do wild-type animals. C. elegans ERI-1 and its human orthologue degrade siRNAs in vitro. In the nematode worm, ERI-1 is predominantly cytoplasmic and is expressed most highly in the gonad and a subset of neurons, suggesting that ERI-1 siRNase activity suppresses RNA interference more intensely in these tissues. Thus, ERI-1 is a negative regulator that may normally function to limit the duration, cell-type specificity or endogenous functions of RNA interference.

Amino Acid Sequence↗

PubMed2004

HIV-1-specific RNA interference.

The preclinical development of RNA interference (RNAi) as a novel therapeutic agent for HIV-1 infection is reviewed. RNAi refers to the sequence-specific degradation of RNA that follows the cellular introduction of homologous, short-interfering RNA (siRNA). RNAi has emerged as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Advances in vector design permit the effective expression of siRNA in human cells by transfer of short hairpin RNA expression cassettes. Recent investigations have described the ability of RNAi to decrease the replication of HIV-1 in lymphocytic cells using siRNA targeting viral (eg, Tat, Gag and Rev) and host (eg, CCR5 and CD4) proteins. Can RNAi be used as a form of genetic therapy for HIV-1 and associated infections? There are numerous challenges associated with converting RNAi from a laboratory technique to an antiviral therapeutic. Recent research on the cellular delivery, antiviral durability and gene-silencing specificity of HIV-1-specific RNAi is reviewed.

Genetic Therapy↗

PubMed2005

Delivering RNA interference to the mammalian brain.

RNA interference (RNAi) is a new modality in gene therapy which can elicit down-regulation of gene expression and has enormous potential in the treatment of neurological diseases. RNAi is a conserved system through which double stranded RNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatus may be artificially triggered by delivery of naked siRNA molecules or by plasmid-based expression of dsRNA. Before these techniques can be used as effective treatments in the brain, efficient methods of in vivo delivery must be devised. This review first describes the mechanism of RNAi, and then critically examines both viral and non-viral methods for delivery of RNAi to the mammalian brain. There have been a number of important recent publications in this field and the progress towards effective in vivo delivery of RNAi to the central nervous system is discussed. Finally, potential problems that must be considered before applying this technology to the human brain are outlined.

Animals↗

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↗

PubMed2003

RNA interference: a potential tool against Kaposi's sarcoma-associated herpesvirus.

PURPOSE OF REVIEW: RNA interference is a conserved cellular function that controls viral infection, the expression of transposable elements, repetitive sequences and genes in embryonic development. Originally described as an antiviral mechanism in plants, known as posttranscriptional gene silencing, it is now appreciated that this phenomenon occurs in all living cells. Double-stranded RNA, when acting as part of RNA interference, reduces expression of genes with sequence similarity, but has no effect on the expression of genes of unrelated sequence. Studies of RNA interference in mammalian cells have demonstrated that exogenous genes delivered by DNA transfection as well as endogenous gene expression can be suppressed by the delivery of RNA interference. We discuss here the potential for exploiting this phenomenon to prevent or treat viral infections, in particular Kaposi's sarcoma-associated herpesvirus. RECENT FINDINGS: There have been several studies showing that RNA interference can be exploited to target a wide range of human viruses, including HIV-1, human T cell leukaemia virus-1, human papillomavirus, hepatitis B, hepatitis C and the polio virus. RNA interference is effective in mammalian cells and can be delivered by various methods. Double-stranded RNA has been injected into the tail veins of mice to block both virally and chemically induced hepatitis. SUMMARY: A greater understanding of RNA interference allows us to exploit this phenomenon in order to study the functions of genes in mammalian cells, and also to target the expression of mutated cellular or viral genes. New delivery techniques should be developed to allow the process to be used as a therapeutic tool against viruses and malignancies in humans.

Antiretroviral Therapy, Highly Active↗

PubMed2003

RNA interference with small hairpin RNAs transcribed from a human U6 promoter-driven DNA vector.

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of organisms. Recently, a small-interference RNA (siRNA) technology has been reported to produce post-transcriptional gene silencing in mammalian cells. In the present study, we constructed a human U6 promoter-driven mammalian expression vector to produce hairpin double-stranded RNA and transfected this into a human cell line. Using this siRNA system, we were able to knock down the gene expression of an enhanced green fluorescence protein. This result indicates that the plasmid vector-based siRNA system is a promising method to downregulate gene expression in human cells.

Cell Line↗

PubMed2005

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse.

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.

Adenosine Deaminase↗

PubMedDate not supplied

[RNA-interference, induced by transient and continuous expression of hairpin RNA in cells from Drosophila and mammals].

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system, RNAi activity was found to vary depending on IR length ass well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

Animals↗

PubMed2005

Dissecting RNA-interference pathway with small molecules.

RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that occurred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a decrease in target RNA cleavage by activated RNA-induced silencing complex (RISC*). Interestingly, when activated RISC*, which harbors unwound antisense siRNA, was treated with ATPA-18 in vitro, target RNA cleavage was not affected, indicating that this compound inhibited siRNA unwinding or steps upstream of unwinding in the RNAi pathway. Our results also establish the timing of siRNA unwinding and show that siRNA helicase activity is required for RNAi. ATPA-18 analogs will therefore provide a new class of small molecules for studying RNAi mechanisms in a variety of model organisms and deciphering in vivo genetic functions through reverse genetics.

Cell Line↗

PubMed2005

RNA interference for treating haematological malignancies.

RNA interference (RNAi) is a method for silencing gene expression. It is relatively gene-specific, potent, and minimally toxic. For these reasons, RNAi holds great promise for the treatment of haematological malignancies. Much has already been learned about RNAi in the laboratory, although many fundamental questions about its mechanisms remain to be elucidated. For human trials, major hurdles to be overcome include the induction of a nonspecific immune response to RNAi, the selection of the most appropriate targets, the design of more specific molecules, and the assurance of efficient delivery and safety in patients. Translational research efforts are currently well on their way to solving these problems, and will be reviewed here.

Animals↗

PubMed2004

Molecular medicine for the brain: silencing of disease genes with RNA interference.

The recent discovery of RNA interference (RNAi) has revolutionised biological research and now holds promise as a potential therapy for human diseases. Currently untreatable neurological diseases are especially attractive targets. Scientists have already succeeded in using RNAi to suppress dominant disease genes in vitro; in some cases, this suppression has been allele-specific, silencing the disease-causing allele while maintaining expression of the normal allele. The challenge now is to bring this powerful technology in vivo to animal models to suppress disease genes and correct disease phenotypes. In the confrontation of this challenge, research should benefit from recent advances in viral and non-viral delivery of therapy to the brain.

Animals↗

PubMed2005

RNA interference: ready to silence cancer?

RNA interference (RNAi) is considered the most promising functional genomics tool recently developed. As in other medical fields, this biotechnology might revolutionize the approach to dissecting the biology of cancer, ultimately speeding up the discovery pace of novel targets suitable for molecularly tailored antitumor therapies. In addition, preclinical results suggest that RNAi itself might be used as a therapeutic weapon. With the aim of illustrating not only the potentials but also the current limitations of RNAi as a tool in the fight against cancer, here we summarize the physiology of RNAi, discuss the main technical issues of RNAi-based gene silencing, and review some of the most interesting preclinical results obtained so far with its implementation in the field of oncology.

Animals↗