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Results for “RNA interference (RNAi)”

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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↗
PubMed2004

RNAi, a new therapeutic strategy against viral infection.

RNA interference (RNAi) is an adaptive defense mechanism triggered by double-stranded RNA (dsRNA). It is a powerful reverse genetic tool that has been widely employed to silence gene expression in mammalian and human cells. RNAi-based gene therapies, especially in viral diseases have become more and more interesting and promising. Recently, small interfering RNA (siRNA) can be used to protect host from viral infection, inhibit the expression of viral antigen and accessory genes, control the transcription and replication of viral genome, hinder the assembly of viral particles, and display influences in virus-host interactions. In this review, we attempt to present recent progresses of this breakthrough technology in the above fields and summarize the possibilities of siRNA-based drugs.

Animals↗
PubMed2005

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome.

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.

Acyltransferases↗
PubMed2005

RNA interference-based gene silencing in mice: the development of a novel therapeutical strategy.

RNAi (RNA interference) was originally detected in Caenorhabditis elegans as biological response to exogenous double-stranded RNA (dsRNA), which induces very effective sequence-specific silencing of gene expression. Further investigations revealed that RNAi can occur in many eukaryotic species. Increasing understanding of the biochemical components of RNAi indicates the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer processes the dsRNA to small interfering RNAs (siRNAs) 21-23 nt in length. These siRNAs enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induce destruction of the corresponding mRNAs. Since RNAi has become an excellent strategy for gene silencing, it is tempting to apply this technology to 'knock-down' gene expression in living animals. The generation of transgenic mice from embryonic stem cells expressing small hairpin RNAs (shRNAs) has provided evidence for in vivo application of RNAi. Furthermore, different experimental strategies have been developed to analyze the influence of chemically synthesized siRNAs and of vector-based shRNAs on the expression of different transgenes and endogenous genes in vivo. Recent studies describe the in vivo delivery of siRNAs to inhibit transgene expression in certain organs of adult mice, predominately murine liver. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor-bearing animals with siRNAs are beginning to emerge. They are of utmost interest for systemic diseases such as cancer. In addition, several groups have shown that RNAi can also be used to block the infectivity or suppress the replication of different RNA viruses relevant to human diseases including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). In summary, multiple lines of evidence indicate that RNAi seems to become a powerful tool for the fight against undesirable gene expression in human diseases.

Animals↗
PubMed2006

Conserved ribonuclease, Eri1, negatively regulates heterochromatin assembly in fission yeast.

RNA interference (RNAi) is a conserved silencing mechanism that has widespread roles in RNA degradation, translational repression, and the epigenetic control of chromatin structure [1]. In fission yeast, heterochromatin assembly requires RNAi machinery and is initiated by small interference RNAs (siRNAs) derived from heterochromatic regions and by the RNA-induced transcriptional silencing (RITS) complex [2-7]. Although recent studies have been successful in uncovering the functions of effector complexes in the RNAi pathway [4, 5, 8-10], exactly how heterochromatic siRNAs are processed and function in assembling heterochromatin remains unclear. In this study we focused on a conserved ribonuclease, Eri1, which was originally identified as a negative regulator of RNAi in C. elegans [11], and show the importance of the Eri1 protein in RNAi-mediated heterochromatin assembly in fission yeast. Eri1 specifically degrades double-stranded siRNAs through two functional domains and represses the accumulation of cellular siRNAs in vivo. Deletion of eri1(+) causes an increase in siRNAs associated with the RITS complex and enhances heterochromatic silencing, which is accompanied by increased levels of histone H3-K9 methylation and the Swi6 protein. Our findings suggest that the fission yeast Eri1 controls the accumulation of heterochromatic siRNAs and negatively regulates the RNAi-mediated heterochromatin assembly.

Amino Acid Sequence↗
PubMed2003

Expression profiling reveals off-target gene regulation by RNAi.

RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.

Base Sequence↗
PubMed2005

Two classes of endogenous small RNAs in Tetrahymena thermophila.

Endogenous small RNAs function in RNA interference (RNAi) pathways to guide RNA cleavage, translational repression, or methylation of DNA or chromatin. In Tetrahymena thermophila, developmentally regulated DNA elimination is governed by an RNAi mechanism involving approximately 27-30-nucleotide (nt) RNAs. Here we characterize the sequence features of the approximately 27-30-nt RNAs and a approximately 23-24-nt RNA class representing a second RNAi pathway. The approximately 23-24-nt RNAs accumulate strain-specifically manner and map to the genome in clusters that are antisense to predicted genes. These findings reveal the existence of distinct endogenous RNAi pathways in the unicellular T. thermophila, a complexity previously demonstrated only in multicellular organisms.

Animals↗
PubMed2004

The long processes of short interfering RNAs--RNA interference and its implications in neuronal cells.

Reverse genetics has been greatly advanced by the discovery of RNA interference (RNAi). This intracellular RNA-mediated gene silencing pathway is partially conserved from plants to mammals and offers a new powerful tool for the analysis of gene function. We give a brief overview of the discovery of RNAi, the underlying mechanisms and probable intrinsic roles of the pathway. Recent reports utilizing RNAi for gene silencing approaches in neuronal cells are reviewed and possible delivery techniques for small interfering RNA/double-stranded RNA are discussed.

Animals↗
PubMed2026

Screening, optimization and artificial recombination of dsRNA fragments for RNAi-mediated pest resistance in Apolygus lucorum.

RNA interference (RNAi) is an eco-friendly strategy for pest management, with double-stranded RNA (dsRNA) as the core functional component. In this study, three RNAi target genes (Ubx, wupA and Dpp) with strong lethal effects on Apolygus lucorum were screened via microinjection. The 7-day cumulative mortalities were 56.67 ± 3.33% for dsUbx, 94.44 ± 1.11% for dswupA and 92.22 ± 1.11% for dsDpp. We optimized dsRNA sequences by removing conserved sequences in non-target organisms based on homology alignment and off-target risk analysis. The optimized fragments dswupA-OTE and dsDpp-OTE still exhibited high insecticidal activity, with 7-day cumulative mortalities of 77.78 ± 2.94% and 70.00 ± 1.93%, respectively. We also evaluated the effects of dsRNA length and target sites on RNAi efficiency and screened potent short dsRNA fragments. Novel artificially recombinant dsRNAs were constructed by assembling effective short fragments from different genes, which retained strong insecticidal activity despite shorter sequence length. This study verifies the feasibility of multi-target recombinant dsRNA for pest control and provides a theoretical basis for developing multi-gene RNAi technologies against A. lucorum.

Apolygus lucorum↗
PubMed2005

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro.

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

Carcinoma, Hepatocellular↗
PubMed2006

Cell cycle arrest drastically extends the duration of gene silencing after transient expression of short hairpin RNA.

Targeted gene silencing through RNA interference (RNAi) utilizes short interfering RNA (siRNA) duplexes or vectors expressing short hairpin RNA (shRNA), which is processed in the cells to siRNA. Stable RNAi in mammalian cells is usually achieved through genomic integration of shRNA expressing vectors, but transiently transfected siRNA was also reported to produce long-term silencing in primary mammalian cells. We have developed lentiviral vector LLCEP TU6X for tetracycline/doxycycline-inducible expression of cloned shRNA and a new selectable marker EGFP-Puro, comprising destabilized green fluorescent protein fused with puromycin acetyltransferase. To investigate the stability of gene silencing after transient shRNA expression, LLCEP TU6X vector carrying shRNA against firefly luciferase was transduced into luciferase-expressing human HT1080 fibrosarcoma cells. When doxycycline-induced transcription was followed by the removal of the inducer, EGFP-Puro reverted to basal level within two days, but RNAi activity required six days for full reversion in proliferating cells. When cell division was blocked with mimosine or by inducible expression of cell cycle inhibitors p27 or p21, RNAi effect was undiminished for 4-5 days and maintained at >60% level as late as 21 days after the inducer was removed. In contrast to the phenotypic stability of RNAi, the amount of siRNA in nondividing cells, measured by an RNAse protection assay, decreased approximately 7-fold just one week after transcription shutdown. These results indicate that gene silencing by transiently expressed shRNA is extremely stable in nondividing cells, and that this effect is not merely a consequence of siRNA stability.

Cell Cycle↗
PubMed2004

[Effects of RNA interference on epidermal growth factor receptor expression in SPC-A-1 cells].

OBJECTIVE: To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress epidermal growth factor receptor (EGFR) expression in non-small-cell lung carcinoma (NSCLC) cells. METHODS: SPC-A-1 cells were transfected using chemically synthesized double stranded RNA (dsRNA) formulated with Lipofectamine 2000. The EGFR numbers were determined by both Western blot and flow cytometry. The antiproliferative effects of dsRNA-EGFR were assessed using cell counts and colony assay. Cell cycle analysis was carried out via flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT. RESULTS: Sequence specific siRNAs targeting EGFR down-regulated EGFR expression significantly. Compared with the control group, dsRNA-EGFR reduced the cell numbers by 78.3% and decreased the colonies by 66.8%. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to the control. Based on the value of IC50 obtained by Origin 6.0 software, we concluded that dsRNA-EGFR increased the sensitivity of SPC-A-1 to cisplatin by seven-fold. CONCLUSIONS: Sequence specific siRNAs targeting EGFR was capable of suppressing EGFR expression, and therefore, significantly inhibiting cellular proliferation and inducing cell cycle arrest. The finding from chemosensitivity assay further revealed that dsRNA-EGFR was associated with an addictive or synergistic effect on tumor growth inhibition when combined with cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGFR overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer.

Antineoplastic Agents↗
PubMed2004

[Specific inhibition of hTERT gene expression by short interfering RNAs in gastric cancer SGC7901 cell].

OBJECTIVE: Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells. In present research, we investigated whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress human telomerase catalytic unit (hTERT) gene expression in gastric SGC7901 cells. METHODS: As a pilot study, we utilized green fluorescent protein (GFP) plasmid pCX-GFP (5 510 bp) as a reporter system and generated constructs SHi-pU6-GFP expressing small hairpin RNA (shRNA) specific for green fluorescence protein (GFP) in K562 and SGC7901 cell respectively. Furthermore, we constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene and transfected in SGC7901 by using Lipofectamine trade mark 2000. The expression of hTERT gene was detected by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) assay. RESULTS: Our pilot study showed the short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into SGC7901 cell. The constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene was proved to be the same as designed by restriction endonuclease analysis. pU6-hTERT-siRNAs were successfully transferred into SGC7901 cell and their stable expression were obtained. The expression of hTERT gene were specific inhibited by pU6-hTERT-siRNAs in SGC7901 cell. CONCLUSIONS: Short hairpin RNAs (shRNAs) could induce sequence-specific hTERT gene silencing in SGC7901 cell. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to cancer gene therapy.

Catalytic Domain↗
PubMed2005

[Application of RNAi in gene-deficient models].

RNA interference (RNAi), a process of sequence-specific gene silence,can effectively and specifically suppress the activity of corresponding mRNAs in a gene-dependent manner induced by double-stranded RNA (dsRNA). This powerful technology has been widely employed to manipulate gene expression in mammalian and human cells, elucidate signal transduction pathways and identify gene functions in a whole-genome scale. Simultaneously, it also displays a bright and fascinating future in the research and development of RNAi-based drugs for various diseases such as viral infections, cancers, metabolic disorders and genetic diseases. In present review, we attempt to recapitulate the application of this breakthrough technology in establishing gene-deficient models and show the alluring foreground of RNAi-based gene therapy in these diseases.

Animals↗
PubMed2004

Intravenous, non-viral RNAi gene therapy of brain cancer.

RNA interference (RNAi) has the potential to knock down oncogenes in cancer, including brain cancer. However, the therapeutic potential of RNAi will not be realised until the rate-limiting step of delivery is solved. The development of RNA-based therapeutics is not practical, due to the instability of RNA in vivo. However, plasmid DNA can be engineered to express short hairpin RNA (shRNA), similar to endogenous microRNAs. Intravenous, non-viral RNAi-based gene therapy is enabled with a new gene-targeting technology, which encapsulates the plasmid DNA inside receptor-specific pegylated immunoliposomes (PILs). The feasibility of this RNAi approach was evaluated by showing it was possible to achieve a 90% knockdown of brain tumour-specific gene expression with a single intravenous injection in adult rats or mice with intracranial brain cancer. The survival of mice with intracranial human brain cancer was extended by nearly 90% with weekly intravenous injections of PILs carrying plasmid DNA expressing a shRNA directed against the human epidermal growth factor receptor. RNAi-based gene therapy can be coupled with gene therapy that replaces mutated tumour suppressor genes to build a polygenic approach to the gene therapy of cancer.

Animals↗
PubMed2004

RNAi: ancient mechanism with a promising future.

RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man. Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means. RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation. We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes. Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients.

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↗
PubMed2006

Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila.

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.

Animals↗