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RNA interference and human disease.

The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA). The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine.

Animals↗
PubMed2006

RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals.

BACKGROUND: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions. RESULTS: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1alpha as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi. CONCLUSION: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions.

Cell Line↗
PubMed2006

From genome to phenome--RNAi library screening and hit characterization using signaling pathway analysis.

Comprehensive, high-throughput analysis of gene function using RNA interference (RNAi)-based screens is emerging as a significant step forward for preliminary drug-target identification. Until quite recently drug target identification depended heavily on the analysis of changes in gene expression, which in turn needed to be correlated with gene function. The promise of obtaining preliminary 'gene to phenotype' information using a single high-throughput platform is propelling major investment in this area by biotechnology and pharmaceutical companies. This review discusses recent developments in the use of RNAi library screens, and the various assay formats and techniques currently in use for data analysis downstream of such screens.

Animals↗
PubMed2005

[Effect of RNAi-mediated survivin gene silencing on apoptosis of ovarian cancer cell lines SKOV3 and SKOV3/ADM].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) has been widely used in tumor gene therapy, antivirus, and gene drug selection. Survivin gene is highly expressed in ovarian cancer cell line SKOV3 and drug-resistant cell line SKOV3/ADM, and is an ideal target of gene therapy for ovarian cancer. This study was to explore effect of RNAi-mediated Survivin gene silencing on apoptosis of SKOV3 and SKOV3/ADM cells. METHODS: Recombinant plasmid pshRNA-Survivin was transfected into SKOV3 and SKOV3/ADM cells. The expression of Survivin was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of SKOV3 and SKOV3/ADM cells after transfection were evaluated by AO/EB dyeing, TUNEL, and flow cytometry. RESULTS: After transfection of pshRNA-Survivin, mRNA and protein levels of Survivin gene in SKOV3 and SKOV3/ADM cells were obviously reduced; the apoptosis of SKOV3 and SKOV3/ADM cells were significantly higher in transfection group than in control group (P0.01); 48 h after transfection, the apoptosis rate was 14.05% in SKOV3 cells, and 21.02% in SKOV3/ADM cells, no obvious apoptosis was detected in control cells. CONCLUSION: pshRNA-Survivin could reduce the expression of Survivin gene, and induce apoptosis of SKOV3 and SKOV3/ADM cells.

Antineoplastic Agents↗
PubMed2004

RNA interference in crop plants.

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was first discovered in worms, related phenomena such as post-transcriptional gene silencing and coat protein mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. It also offers advantages over mutation-based reverse genetics in its ability to suppress transgene expression in multigene families in a regulated manner.

Animals↗
PubMed2003

In vitro analysis of RNA interference in Drosophila melanogaster.

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.

Animals↗
PubMed2002

Analysis of gene function in somatic mammalian cells using small interfering RNAs.

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.

Animals↗
PubMed2002

Fragile X-related protein and VIG associate with the RNA interference machinery.

RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.

Amino Acid Sequence↗
PubMed2006

Dicer-derived microRNAs are utilized by the fragile X mental retardation protein for assembly on target RNAs.

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

Journal Article↗
PubMed2004

RNA interference, arthropod-borne viruses, and mosquitoes.

RNA interference (RNAi) probably functions as an antiviral mechanism in most eukaryotic organisms. Variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. There are three lines of evidence that show the RNAi pathway exists in Aedes species that transmit arboviruses. The first is that recombinant Sindbis viruses expressing a RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) interfere with DENV-2 replication in Aedes aegypti mosquitoes by a mechanism similar to virus-induced gene silencing described in plants. The second is that transfection of C6/36 (Aedes albopictus) cells with either double-stranded RNA or synthetic small interfering RNAs derived from an arbovirus genome interferes with replication of the homologous virus. The third is that a hairpin DENV-2-specific RNA transcribed from a plasmid can generate virus-resistant C6/36 cells. We hypothesize that genetically modified mosquitoes can be generated that transcribe a flavivirus-specific dsRNA, triggering the RNAi response soon after ingestion of a blood meal. This could induce the RNAi pathway in the midgut prior to establishment of virus infection and profoundly change vector competence. Towards this goal, we are developing transgenic A. aegypti lines that are refractory to DENV by exploiting the RNAi pathway.

Animals↗
PubMed2006

Therapeutic potential of RNA interference against cancer.

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment.

Animals↗
PubMed2004

RNAi in functional genomics.

There has been a lack of powerful tools for systematic analysis of mammalian gene function, but RNA interference (RNAi) may now provide such a strategy. Stable transcription of RNAi triggers from suitable expression cassettes integrated into the host cell genome by viral gene transfer can induce long-term and heritable gene silencing in mammalian cells. However, the use of RNAi as a genetic tool is limited by difficulties in identifying efficient RNAi triggers, the problem of effective delivery and off-target effects, as well as potential genotoxic side effects of viral gene transfer strategies. Recent insights into the molecular mechanisms of silencing processes mediated by either siRNA or miRNA will allow further optimization of RNAi triggers as genetic tools.

Animals↗
PubMed2006

Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.

Animals↗
PubMed2003

Short hairpin RNAs induced RNA interference in human cells.

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is an evolutionarily conserved posttranscriptional gene silencing, in which the introduction of double-stranded RNA into a cell leads to specific suppression of gene expression. RNAi has become an important tool for gene function studies. The aim of this study was to induce RNAi in mammalian cells by short hairpin RNAs (shRNAs) generated from a DNA vector, therefore, to provide a new approach for gene function analysis. METHODS: Dual Luciferase System was used to assess the intracellular effect of the shRNAs generated from a DNA vector. The inhibitory effect of this DNA vector-driven shRNAs at different condition was measured. RESULTS: shRNAs generated from the DNA vector induced RNAi in human cells, and suppressed gene expression in a sequence-specific manner. The inhibitory effect was highly related to the target sites. CONCLUSION: shRNAs induced RNAi in human cells and suppressed gene expression in a sequence-specific manner. This approach may be used for gene function study.

Gene Silencing↗
PubMed2003

Genome-wide RNAi screening in Caenorhabditis elegans.

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.

Animals↗
PubMed2002

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.

Animals↗
PubMed2004

RNA interference: a practical approach.

BACKGROUND: Few new molecular biology techniques have advanced to find practical application as rapidly as RNA interference (RNAi). RNAi denotes the highly specific posttranslational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. The purpose of this review is to present practical guidelines for designing and executing RNAi experiments. MATERIALS AND METHODS: Review of recent literature. RESULT AND CONCLUSION: We summarize the mechanisms underlying RNAi in mammalian cells and focus on practical advice for investigators conducting RNAi experiments. We suggest criteria to help select a suitable target gene sequence, define the structural characteristics of effective siRNAs, discuss transfection strategies, and describe experimental design, including important control methods. RNAi represents a powerful tool for determining the functions of specific genes.

Animals↗
PubMed2006

Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons.

RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

Base Sequence↗