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At least 37 records · Page 2Linked to original sources
PubMed2005

Development of resistance to RNAi in mammalian cells.

The discovery of RNA interference (RNAi) in C. elegans and in plants has revolutionized current approaches to biology and medicine. RNAi silences genes in a sequence-specific manner through the actions of small pieces of double-stranded RNAs (siRNAs and miRNAs). RNAi has been found as a widespread natural phenomenon in eukaryotic cells and is also being used as a powerful experimental tool to explore gene function. Most importantly, it has many potential therapeutic applications. Viral gene-specific siRNAs are theoretically very promising antiviral inhibitors and have been examined in a broad range of medically important viruses. However, many RNA viruses escape RNAi-mediated suppression by counteracting the RNAi machinery through mutation of the targeted region, by encoding viral suppressors, or both. DNA viruses also counteract the RNAi machinery, preferentially using viral suppressors. Cellular factors may also contribute to RNAi resistance; ADAR1 was the first cellular factor found to be responsible for editing-mediated RNAi resistance. Because siRNAs can be used as potent small-molecule inhibitors of any cellular gene, the best way for a cell to maintain expression of essential genes for its long-term survival is to develop a program to resist the detrimental effects of RNAi.

Animals↗
PubMed2003

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila.

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.

Animals↗
PubMed2003

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.

Animals↗
PubMed2005

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721.

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SVV), were constructed by ligating into the vector, pSuppressorNeo (pSuNeo) digested with restriction enzymes Xba I and Sal I and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SVV, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Apoptosis↗
PubMed2004

Larval RNAi in Tribolium (Coleoptera) for analyzing adult development.

We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.

Animals↗
PubMed2006

RNAi therapy for neurodegenerative diseases.

RNA interference (RNAi) mediates gene silencing in a sequence-specific manner and has proven to be an exceptionally valuable discovery for bench scientists. In the laboratory, RNAi technologies provide efficient means for validating drug targets and for performing reverse genetics to study gene function (Friedman and Perrimon, 2004). Patients may also benefit from RNAi as applications extend to potential human therapies. RNAi-based treatments are being investigated and may provide hope for patients suffering from cancer, viral infections, or genetic diseases for which effective therapies are currently lacking. Notably, several independent studies have demonstrated that RNAi therapy can improve disease phenotypes in various mouse models of human disease. In this chapter, we focus on the potential of RNAi in treating neurologic diseases for which reduction of mutant or toxic gene expression may provide therapeutic benefit. We discuss approaches to achieving RNAi in vivo, progress in the field, and the potential pitfalls associated with RNAi-based therapies.

Animals↗
PubMed2000

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.

Animals↗
PubMed2006

Commercial potential of RNAi.

The commercial potential of RNAi is assessed on the basis of successful translation of technology into applications in three areas: (1) drug discovery and research-currently the biggest segment; (2) potential therapeutic applications; and (3) the role of microRNA in molecular diagnostics. RNAi is an important method for analyzing gene function and identifying new drug targets that use dsRNA to knock down or silence specific genes. Sets of siRNAs focused on a specific gene class (siRNA libraries) have the capacity to greatly increase the pace of pathway analysis and functional genomics. RNAi plays an important role in drug discovery by facilitating target validation. The discovery of the role of microRNA (miRNAs) in various pathological processes opens up possible applications in molecular diagnostics, particularly that of cancer. The advantages of RNAi-based therapeutics over traditional pharmaceuticals include the capability for more specific therapies with small molecule siRNA. Drawbacks include the development of resistance in cancer and viral infections as well as the interferon effect. RNAi is closely related to gene therapy and the vectors developed for gene therapy are also being used for delivery of siRNAs. RNAi, along with other related technologies, will contribute to the development of personalised medicine. Although none of the RNAi-based drugs is in the market yet, some are in clinical trials. By the year 2010 the market for RNAi-based drugs is expected to be worth 3.5 billion dollars and is expected to expand to 10.5 billion dollars by the year 2015.

Chemistry, Pharmaceutical↗
PubMed2001

Antisense RNA regulation of the par post-segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, which are the toxin and antitoxin of the par PSK system respectively. RNAI encodes an open reading frame for a 33 amino acid toxin called Fst. Expression of fst is regulated post-transcriptionally by RNAII. RNAII interacts with RNAI by a unique antisense RNA mechanism involving binding at the 5' and 3' ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem-loop and a set of direct repeat sequences, DRa and DRb, located at the 5' end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI-RNAII complex were analysed by partial digestion with Pb(II) and ribonucleases. Probing data for RNAI and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and terminator sequences are involved in the formation of the RNAI-RNAII complex. Mutant par RNAs were used to show that the binding reaction occurs in at least two steps. The first step is the formation of an initial kissing interaction between the transcriptional terminator stem-loops of both RNAs. The subsequent step(s) involves an initial pairing of the complementary direct repeat sequences followed by complete hybridization of the 5' nucleotides to stabilize the RNAI-RNAII complex.

Amino Acid Sequence↗
PubMed2002

Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs.

The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3'-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3'-end of either strand, using 2'-O,4'-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3'-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3'-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3'-end is recognized by the RNA-induced silencing complex (RISC).

Animals↗
PubMed2006

GenomeRNAi: a database for cell-based RNAi phenotypes.

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

Animals↗
PubMed2004

High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics.

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.

Animals↗
PubMed2003

RNA interference (RNAi) in hematology.

RNA interference (RNAi), an evolutionary highly conserved process of post-transcriptional gene silencing, can be triggered by small interfering RNAs (siRNAs) that mediate sequence-specific mRNA degradation. Since the first reports in 1998, RNAi has rapidly been developed into an effective tool to specifically knock down gene expression in a wide variety of target cells. Accordingly, RNAi is currently used for both systematic functional genomics in several organisms and for specific therapeutic intervention in preclinical models of different diseases characterized by aberrant gene expression. However, since siRNAs are not replicated in mammalian cells during the process of RNAi, kinetic aspects of RNAi-induced gene silencing that eventually depend on the intracellular level of siRNA must be considered for each analytical or therapeutic application in these cells. We summarize here some aspects of siRNA design and delivery, of RNAi kinetics, potential side effects, and limitations of siRNA-mediated gene silencing, as well as putative RNAi targets for functional and/or therapeutic intervention in hematopoietic cells.

Animals↗
PubMed2005

Design of shRNAs for RNAi-A lesson from pre-miRNA processing: possible clinical applications.

RNA interference has become the tool of choice to analyse the loss-of-function of individual genes and has been exploited to identify complex regulatory pathways following genomic screening. RNAi has both admirers and detractors, but is undeniably a technique with great potential, which has come a long way in the short time since its discovery. RNAi utilises cellular machinery associated with the processing of naturally occurring micro RNA (miRNAs). Effective use of RNAi requires detailed knowledge of the individual steps and the proteins involved, as well as the similarities and distinctions between miRNA and siRNA pathways. RNAi was originally induced by the introduction of long double stranded RNAs (dsRNAs) into cells in which the RNA was cleaved into short RNAs which effectively interfered with a transcription of cognate mRNA. More recently an introduction of short approximately 22 nucleotide RNA duplexes has become the standard in short-term experiments, but is insufficient for long-term knock-down assays. Long-term expression of siRNAs has been achieved by in vivo transcription from plasmids coding for short hairpin RNAs (shRNAs). The cellular processing of shRNAs shares common features with the biogenesis of naturally occurring miRNA such as cleavage by nuclear RNase Drosha, export from the nucleus, processing by a cytoplasmic RNase Dicer, and incorporation into the RNA-induced silencing complex (RISC). Each step has a crucial influence on the efficiency of RNAi and their consideration should be a part of a standard experimental design. RNAi has moved from a purely experimental technique to the stage of potential clinical applications. The possible use of RNAi in the treatment of spinocerebellar ataxia or amyotrophic lateral sclerosis, with its advantages and pitfalls and possible extensions to other diseases are discussed.

Animals↗
PubMed2005

RNAi mechanisms in Caenorhabditis elegans.

RNA interference (RNAi) is a form of gene silencing induced by double stranded RNA (dsRNA) that is processed into short interfering RNAs (siRNAs). RNAi can induce both post-transcriptional and transcriptional gene silencing. In Caenorhabditis elegans, there are several distinct pathways where post-transcriptional or/and transcriptional RNAi mechanisms are involved. RNAi in C. elegans is also systemic and heritable. This review will discuss RNAi related pathways, features of RNAi in C. elegans and possibilities of endogenous gene regulation by RNAi.

Animals↗
PubMed2006

Application of RNAi inducible TNFRI knockdown cells to the analysis of TNFalpha-induced cytotoxicity.

RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies. Tumor necrosis factor receptor I (TNFRI) was chosen as a target gene because its signaling has been implicated in xenobiotic-induced toxicity. We optimized the design and performance of a vector-based RNAi experiment and then investigated viability of both HeLa and HepG2 cells exposed to TNFalpha. In addition, we examined gene expression profile of TNFRI knockdown HeLa cells after TNFalpha treatment, and then protein expression levels for several apoptosis-related genes of the cells. In both HeLa and HepG2 cells, TNFalpha exposure resulted in significantly reduced susceptibility of the knockdown cells to the cytotoxicity as compared with those of mock-transfected cells. Furthermore, the gene expression profiling and western blotting revealed that several genes including apoptosis and/or NF-kappaB pathway were downregulated in the knockdown HeLa cells. These results suggest that downregulation of the TNFRI gene in both HeLa and HepG2 cells by RNAi participates in resistance to TNFalpha-induced cytotoxicity. Therefore, this study raises the possibility that RNAi-based gene silencing in mammalian cells may be a valuable tool for elucidating the relationships between phenotypic changes and target gene functions in response to xenobiotic-induced cytotoxicity. Further exposure study using xenobiotics needs to be done to validate the potential utility of RNAi technology.

Bleomycin↗
PubMed2002

Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.

RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.

Animals↗
PubMed2004

Oncology studies using siRNA libraries: the dawn of RNAi-based genomics.

High-throughput, human cell-based applications of RNA-mediated interference (RNAi) have emerged in recent years as perhaps the most powerful of a 'second wave' of functional genomics technologies. The available reagents and methodologies for RNAi screening studies now enable a wide range of different scopes and scales of investigation, from single-parameter assays applied to focused subsets of genes, to comprehensive genome-wide surveys based on rich, multiparameter readouts. As such, RNAi-based screens are offering important new avenues for the discovery and validation of novel therapeutic targets for several disease areas, including oncology. By enabling a 'clean' determination of gene function, that is the creation of direct causal links between gene and phenotype in human cells, RNAi investigations promise levels of pathophysiological relevance, efficiency, and range of applicability never before possible on this scale. The field of oncology, with its many assays using readily transfectable cell lines, has offered particularly fertile ground for showcasing the potential of RNAi-based genomics. However, like any other technology before it, RNAi is not without its own challenges, limitations, and caveats. Many of these issues stem directly from the choice of silencing reagent to be used in such studies, and the design of the overall screening strategy. Here, we discuss the basic design issues, potential advantages, and technical challenges of large-scale RNAi screens based on the use of chemically synthesized siRNA libraries.

Gene Library↗