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At least 109 records · Page 6Linked to original sources
PubMed2006

Betanodavirus B2 is an RNA interference antagonist that facilitates intracellular viral RNA accumulation.

Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

HeLa Cells↗
PubMed2006

Design of expression vectors for RNA interference based on miRNAs and RNA splicing.

RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have designed new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene, phospholipase D2. Moreover, the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted BsmBI sites placed internal to the arms of the new miRNA-based vectors, so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression.

Base Sequence↗
PubMed2003

The effect of RNA interference Down-regulation of RNA editing 3'-terminal uridylyl transferase (TUTase) 1 on mitochondrial de novo protein synthesis and stability of respiratory complexes in Trypanosoma brucei.

Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F(1).F(0)-ATPase complex and on several other mitochondrial proteins.

Animals↗
PubMed2004

RNA interference and double-stranded-RNA-activated pathways.

RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.

Animals↗
PubMed2004

Revealing the world of RNA interference.

The recent discoveries of RNA interference and related RNA silencing pathways have revolutionized our understanding of gene regulation. RNA interference has been used as a research tool to control the expression of specific genes in numerous experimental organisms and has potential as a therapeutic strategy to reduce the expression of problem genes. At the heart of RNA interference lies a remarkable RNA processing mechanism that is now known to underlie many distinct biological phenomena.

Animals↗
PubMed2005

Hepatitis C virus replicons escape RNA interference induced by a short interfering RNA directed against the NS5b coding region.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

Amino Acid Sequence↗
PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗
PubMed2006

RNA-interference effectors and their delivery.

RNA-interference, the mechanism by which the expression of a specific protein can be reduced or eliminated, has emerged as a potential way to treat disease. RNA-interference effectors, such as small interfering RNA and small hairpin RNA, are double-stranded nucleic acid sequences expressly designed to have homology to sections of the target protein's mRNA, and when present in the cytosol trigger hydrolysis of the mRNA through the RNA-interference pathway. Because RNA-interference effectors are macromolecular and typically polyanionic, their efficacy is poor when not accompanied by a delivery vehicle. This review outlines the RNA-interference mechanism and discusses the delivery of RNA-interference effectors, with an emphasis on synthetic polymer-based delivery systems.

Animals↗
PubMed2003

RNA interference. An approach to produce knockout organisms and cell lines.

In various eucaryotic organisms double-stranded RNA causes effective degradation of homologous mRNA molecules by a process called RNA interference. RNA interference is a phenomenon associated with gene suppression via regulatory RNA molecules, which are common in plants, animals, and fungi. The discovery of RNA interference stimulated the development of new approaches for suppression of target gene expression, production of stable knockout cell lines and organisms, and also stimulated studies on possible intracellular functions of this phenomenon.

Animals↗
PubMed2004

Downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells.

RNA interference (RNAi) is a recently observed process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA (mRNA) in animal and plant cells. In several model systems, RNAi had been developed into a useful tool for the investigation of gene function. In order to study the effectiveness of RNAi in mammalian cells, we introduced chemically synthetic 21-nucleotide small interference RNA (siRNA) duplexes into 293T/GFP cells, which were transduced by enhanced green fluorescence protein (EGFP) gene, by means of TransIT-TKO, Oligofectamine reagent, Lipofectamine 2000 respectively. The results demonstrated that EGFP expression was significantly and specifically inhibited by the corresponding dsRNA, but not by unrelated dsRNA. In three different vectors, Lipofectamine 2000 demonstrated the highest transfection efficiency with a 48 h exposure. The decrease in EGFP fluorescence intensity was approximately 80%. Although TransIT-TKO and Oligofectamine displayed similar trends, the transfections were inefficient, and often toxic. The results also exhibited that siRNA inhibited the EGFP gene expression in a dose and time-dependent manner. Therefore, we concluded that the Lipofectamine 2000 was a better transfection reagent for RNAi. RNAi pathway seems operative in mammalian embryo cells. RNAi may be developed into a potential tool for gene therapy.

Cell Line↗
PubMed2005

DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.

In RNA interference, the RNase-III enzyme Dicer processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.

Alleles↗
PubMed2005

Dampening the silencing effect of RNA interference in mammals.

RNAi (RNA interference) refers to the suppression of expression of a target gene (mainly at the post-transcriptional or translational level) induced by small (21-23 nucleotides) RNA molecules, including siRNA (small interfering RNA). Suppression of gene expression by RNAi represents an important part of the regulation of gene expression. Interestingly, recent advancements in RNAi research support the notion that RNAi can be regulated just as an ordinary gene. In this issue of the Biochemical Journal, Hong et al. report their finding that suppression of RNAi is triggered by a high dose of siRNA in mice, and the suppression of RNAi in mice is related to eri-1 (enhanced RNA interference). Eri-1 is an RNaseT enzyme initially found in Caenorhabditis elegans that can degrade double-stranded RNA with 3' overhangs. The results presented by Hong et al. have the potential to be extended and contribute to our knowledge about the regulation of RNAi in mammals.

Animals↗
PubMed2005

Suppression of RNA interference by adenovirus virus-associated RNA.

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.

Adenoviruses, Human↗
PubMed2005

RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription.

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.

Argonaute Proteins↗
PubMedDate not supplied

[The phenomenon of RNA interference and development of organism].

RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNQA interference in stem cell formation and functioning is also considered.

Animals↗
PubMedDate not supplied

Interfering with cancer: a brief outline of advances in RNA interference in oncology.

RNA interference (RNAi) is a potent and ubiquitous gene-silencing mechanism that is generating considerable excitement in the fields of molecular biology and gene therapy. It is now in widespread use for loss-of-function analysis in many diseases including cancer. Nevertheless, RNAi is still in its infancy, with new discoveries appearing on a monthly basis. This article presents a brief outline of the history and recent advances in RNAi with a specific focus on its potential in oncology.

Genetic Therapy↗
PubMed2006

Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA.

RNA interference (RNAi) has become acknowledged as an effective and useful tool to study gene function in diverse groups of cells. We aimed to suppress the expression of the E-cadherin gene during in vitro development of bovine preimplantation embryos using RNAi approach. In this experiment the effect of microinjection of E-cadherin and Oct-4 (as control) double-stranded (ds) RNA on the mRNA and protein expression level of the target E-cadherin gene was investigated. For this, a 496 bp long bovine E-cadherin and 341 bp long Oct-4 dsRNA sample were prepared using in vitro transcription. In vitro produced bovine zygotes were categorized into four treatment groups including those injected with E-cadherin dsRNA, Oct-4 dsRNA, RNase-free water, and uninjected controls. While the injection of E-cadherin dsRNA resulted in the reduction of E-cadherin mRNA and protein levels at the morula and blastocyst stage, the transcript and protein product remained unaffected in the Oct-4 dsRNA, water injected and uninjected control groups. The relative abundance of E-cadherin mRNA in the E-cadherin dsRNA injected morula stage embryos was reduced by 80% compared to the control group (P < 0.05). The Western blot analysis also showed a significant decrease in the E-cadherin protein (119 kDa) in E-cadherin dsRNA injected embryos compared to the other three groups. Microinjection of E-cadherin dsRNA has resulted only 22% blastocyst rate compared to 38%-40% in water injected and uninjected controls. In conclusion, our results indicated the suppression of E-cadherin mRNA and protein has resulted in lower blastocyst rate and the RNAi technology is a promising approach to study the function of genes in early bovine embryogenesis.

Animals↗
PubMed2004

A link between mRNA turnover and RNA interference in Arabidopsis.

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.

Animals↗