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PubMedDate not supplied

Vectors for RNAi technology in poplar.

The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar. A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the beta-glucuronidase (GUS) reporter gene system. These gene cassettes are driven by the CaMV-35S promoter. To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants. Agrobacterium-mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene. From these results we conclude that RNAi is also functional in poplar.

Base Sequence↗
PubMed2002

Stable suppression of gene expression by RNAi in mammalian cells.

In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, approximately 22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (approximately 500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of approximately 22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable "knock-down" cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.

Animals↗
PubMed2002

RNAi functions in cultured mammalian neurons.

In a wide range of organisms, double-stranded RNA triggers posttranscriptional gene silencing or RNA interference (RNAi). Small interfering RNAs, the 21-nt double-stranded RNA intermediates of this natural pathway, have became a powerful tool to knock down specific gene expression in mammalian cell lines and potentially will be useful for the analysis of loss-of-function phenotypes. In mammalian primary neuronal cultures, where genetic manipulations are especially difficult, RNAi might be developed into a highly efficacious tool to study the roles of specific genes in neuron development and functioning. Neurons, however, have been considered the most resistant to RNAi. We report here an application of RNAi to postmitotic primary neuronal cultures. Synthetic siRNA can be readily introduced into neurons and effectively inhibit the expression of endogenous and transfected genes.

Animals↗
PubMed1986

Defective RNA splicing in the absence of adenovirus-associated RNAI.

We have analyzed late gene expression in 293 cells infected with an adenovirus type 5 mutant dl331, which is defective in production of the low molecular weight virus-associated (VA) RNAI. The results show that several steps in late gene expression are affected. In addition to the previously characterized defect in late mRNA translation, mutant infected cells also show an aberrant selection of RNA splice sites and a substantially reduced L2, L3, and L5 mRNA accumulation. Normal or even slightly elevated amounts of mRNA from region L1 are produced. However, the L1 pre-mRNA is spliced only to generate the mRNA encoding the Mr 52,000-55,000 polypeptide and no detectable mRNA for polypeptide IIIa. Cotransfection of a plasmid encoding VA RNAI complemented the splicing defect in trans, suggesting that the abnormalities are due to the absence of VA RNAI, rather than to a cis-acting change in the nuclear precursor RNA. In a HeLa cell variant, which allows late protein synthesis also in the absence of VA RNAI, because of a lack of eukaryotic initiation factor 2 alpha kinase expression, a normal repertoire of late mRNA was produced. We conclude that a soluble factor, most likely a late viral protein, controls differential RNA splicing and late mRNA accumulation during an adenovirus infection.

Adenoviruses, Human↗
PubMed2006

Cellular phenotyping by RNAi.

A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.

Animals↗
PubMed2001

RNAi is antagonized by A-->I hyper-editing.

RNA interference (RNAi) and adenosine to inosine conversion are both mechanisms that respond to double-stranded RNA (dsRNA) and have been suggested to have antiviral roles. RNAi involves processing of dsRNA to short interfering RNAs (siRNAs), which subsequently mediate degradation of the cognate mRNAs. Deamination of adenosines changes the coding capacity of the RNA, as inosine is decoded as guanosine, and alters the structure because A-U base pairs are replaced by I*U wobble pairs. Here we show that RNAi is inhibited if the triggering dsRNA is first deaminated by ADAR2. Moreover, we show that production of siRNAs is progressively inhibited with increasing deamination and that this is sufficient to explain the inhibition of RNAi upon hyper-editing of dsRNAs.

Adenosine↗
PubMed2002

Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library.

RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.

Animals↗
PubMed2005

RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella.

Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database.

Amino Acid Sequence↗
PubMed2003

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.

Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.

Animals↗
PubMed2005

Inhibition of hepatitis B virus replication by various RNAi constructs and their pharmacodynamic properties.

The strategy of RNA interference (RNAi)-based gene silencing has been suggested to have great potential in treating viral diseases. It provides new hope of being able to complement the limited therapeutic options currently available for chronic hepatitis B virus (HBV) infection. To advance such a strategy towards clinical use, the effects of various parameters on the anti-HBV efficiency of RNAi need to be well-defined. In this study, the efficacy and pharmacodynamic properties of different RNAi target sequences and constructs were examined. Several sequences were found to be effective in cell and animal models, achieving inhibition rates of approximately 80-90 %. Methyl-modified small interfering RNA (siRNA) molecules were found to be more stable inside cells than natural siRNA molecules and offered longer-lasting inhibitory effects. Both were effective at rather low doses (an equimolar ratio with HBV preS2-S protein expression vector). Plasmid DNA vectors were less dose-responsive, but their effectiveness in vivo lasted longer, for approximately 1 month. By analysing these different parameters and their possible mechanisms, some important issues in RNAi therapeutics that should assist the future development of clinical applications have been addressed.

Animals↗
PubMed2002

RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E.

Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. Here we show that RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindle-E, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte.

Adenosine Triphosphatases↗
PubMed2003

High-throughput selection of effective RNAi probes for gene silencing.

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.

Animals↗
PubMed2005

VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection.

Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.

Animals↗
PubMed2005

Genome-wide screening for gene function using RNAi in mammalian cells.

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.

Animals↗
PubMed2002

A microRNA in a multiple-turnover RNAi enzyme complex.

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.

Adenosine Triphosphate↗
PubMed2004

RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins.

At the silent mating-type interval of fission yeast, the RNA interference (RNAi) machinery cooperates with cenH, a DNA element homologous to centromeric repeats, to initiate heterochromatin formation. However, in RNAi mutants, heterochromatin assembly can still occur at low efficiency. Here, we report that Atf1 and Pcr1, two ATF/CREB family proteins, act in a parallel mechanism to the RNAi pathway for heterochromatin nucleation. Deletion of atf1 or pcr1 alone has little effect on silencing at the mating-type region, but when combined with RNAi mutants, double mutants fail to nucleate heterochromatin assembly. Moreover, deletion of atf1 or pcr1 in combination with cenH deletion causes loss of silencing and heterochromatin formation. Furthermore, Atf1 and Pcr1 bind to the mating-type region and target histone H3 lysine-9 methylation and the Swi6 protein essential for heterochromatin assembly. These analyses link ATF/CREB family proteins, involved in cellular response to environmental stresses, to nucleation of constitutive heterochromatin.

Activating Transcription Factor 1↗
PubMed2006

Distinct populations of primary and secondary effectors during RNAi in C. elegans.

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

Animals↗
PubMed2001

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Animals↗