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Results for “RNA interference”

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PubMed2003

Requirement for a nuclear function of beta-catenin in Wnt signaling.

Wnt signaling stabilizes beta-catenin, which in turn influences the transcription of Wnt-responsive genes in conjunction with T-cell factor (TCF) transcription factors. At present, there are two models for the actions of beta-catenin. The conventional nuclear model suggests that beta-catenin acts in the nucleus to form a heterodimeric transcriptional factor complex with TCF, with TCF providing DNA-specific binding and the C and N termini of beta-catenin stimulating transcription. The alternative cytoplasmic model postulates that beta-catenin exports TCF from the nucleus to relieve its repressive activity or activates it in the cytoplasm. We have generated modified forms of beta-catenin and used RNA interference against endogenous beta-catenin to distinguish between these models in cultured mammalian and Drosophila cells. We show that the VP16 transcriptional activation domain can replace the C terminus of beta-catenin without loss of function and that the function of beta-catenin is compromised by fusion to a transcriptional repressor domain from histone deacetylase, favoring the direct effects of beta-catenin in the nucleus. Furthermore, membrane-tethered beta-catenin requires interaction with the adenomatous polyposis coli protein but not with TCF for its function, whereas untethered beta-catenin requires binding to TCF for its signaling activity. Importantly, by using RNA interference, we show that the signaling activity of membrane-tethered beta-catenin, but not free beta-catenin, requires the presence of endogenous beta-catenin, which is able to accumulate in the nucleus when stabilized by the binding of the beta-catenin degradation machinery to the membrane-tethered form. All of these data support a nuclear model for the normal function of beta-catenin.

Animals↗

PubMed2005

[Construction of FAS-targeted RNAi-retroviral vector and its identification for biological activity].

This study was aimed to construct mouse Fas-targeting si RNA-expressing recombinant retroviral vector in order to explore the therapeutic potential of Fas inhibition by siRNA in the treatment of aplastic anemia and to provide a basia for extensive development of RNA interference techninque. The U6(+) 27 promoter cassette and siFas sequence were obtained by PCR method. The U6-siFas fragment was cloned into the multiple restriction site of pLXSN-EGFP and directly downstream of EGFP gene. The resultant retroviral vector pLXSN/EGFP-siFas was packaged using PA317 cell line and tittered using NIH3T3 cell line. P815 cells were infected by the retroviral vector. EGFP expression in P815 was observed under fluorescent microscope and Fas inhibition effect was detected by immunohistochemistry. The results indicated that successfully constructed retrovirus vector pLXSN/EGFP-siFas was could not only deliver siRNA into mammalian cells efficiently and inhibit Fas expression in P815 cells, but also could express EGFP as marker and neomycine resistance gene to allow antibiotic selection. It is concluded that the successful construction of this retroviral vector would greatly facilitate the application of RNA interference and lay the foundation for therapeutic study of Fas inhibition in the treatment of aplastic anemia.

Anemia, Aplastic↗

PubMed2005

[SiRNA expression cassettes targeting VEGF effectively inhibit the growth of laryngeal squamous cell carcinoma cell lines].

OBJECTIVE: To study the effects of siRNA expression cassettes (SECs) targeting VEGF in vitro on cultured Hep-2 cells, the observation of the expression of VEGF, the screening of the best interference sequences, and the exploration of the application of RNA interference on tumor gene therapy in the future. METHODS: On the basis of the principle of target sequence of siRNA, four interference sequences of VEGF were designed, and the downstream gene-specific primers of the SECs were synthesized. The RNAi transcription kit used U6 RNA-based polymerase III promoter and modified terminator for high level, precise siRNA expression inside target cells. The Hep-2 cells was transfected in growth period of index with the PCR products of the four sites separately, and began to observe and measure the results of RNA interference in 48 hours. RESULTS: The transfected 1366-site cells created, turned into the round shape and began to shed off, whereas morphology of the cells of other groups had not obviously changed. Performing the agarose gels electrophoresis with RT-PCR products, compared with the contrast groups, some cells VEGF mRNA of 1366-site were suppressed obviously, the ratio of OD was 0. 05 while the expression of VEGF of the cell of other groups had not obviously changed. Western blot revealed that VEGF expression was decreased obviously post transfection using 1366-site SECs. Flow cytometry showed that apoptosis rate of 1366-site transfected cells is 43%, and apoptosis rate of the rest three site transfected cells scarcely changed. Similar results were obtained in three independent experiments. CONCLUSIONS: The study suggested that siRNA expression cassettes (SECs) targeting VEGF 1366-site can effectively inhibit the growth laryngeal squamous cell carcinoma cell lines (Hep-2).

Apoptosis↗

PubMed2006

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Binding Sites↗

PubMed2006

Inhibition of multiple strains of Venezuelan equine encephalitis virus by a pool of four short interfering RNAs.

RNA interference, mediated by short interfering RNAs (siRNAs), has been shown to have activity against a wide range of viruses and is a promising new antiviral therapy. Using multiple siRNAs that target conserved areas of the genome allows for increased chances of antiviral activity against different viral strains and also helps to prevent the emergence of escape mutants. In this study, four siRNAs were designed to target areas of conserved sequence between divergent strains of Venezuelan equine encephalitis virus (VEEV). A pool of these siRNAs inhibited the replication of all six strains of VEEV tested. A single nucleotide mismatch at the extreme 3' end of one of the siRNA sense strands did not affect antiviral activity but other mutations were not tolerated. Two strains of VEEV were tested for their abilities to overcome the inhibitory effects of RNA interference following 10 consecutive incubations in the presence of siRNAs. One strain remained susceptible throughout the course of the experiment but the other strain became resistant to the activity of siRNAs. Sequence analysis of the siRNA target sites in this strain showed that no mutations had been generated, indicating that the virus may had become resistant in some other manner. In the absence of effective antiviral drugs and vaccines to combat VEEV infection, these siRNAs offer a potential new therapeutic approach but, as with all antimicrobial agents, caution needs to be exercised with respect to the generation of resistance.

Animals↗

PubMed2006

[Effects of siRNA targeted to survivin in suppressing proliferation and inducing apoptosis in breast cancer MCF-7 cells].

OBJECTIVE: Blocking the expression of survivin with RNA interference techniques, the effects of suppressing proliferation and inducing apoptosis of breast cancer MCF-7 cells were investigated. METHODS: A siRNA eukaryotic expression vector against survivin was constructed and transfected into breast cancer MCF-7 cells with lipofectamine 2000. The changes of survivin expression were detected by semi-quantitive RT-PCR and immunohistochemistry. The effect of suppressing proliferation of MCF-7 cell was detected by MTT assay. The effect of inducing MCF-7 cell apoptosis was detected by TUNEL assay. RESULTS: The sequence-specific siRNA can efficiently block the expression of survivin both at mRNA and protein levels. The expression inhibition rate was 64.9% at mRNA level detected by semi-quantitive RT-PCR and 79.7% at protein level detected by immunohistochemistry. Blocking the expression of survivin can suppress proliferation of MCF-7 cells significantly. At 24 and 48 h after the cells were reseeded, the proliferation inhibition rates were 31.6% and 33.0%, respectively. At 24 h after transfection, apoptosis was induced in 12.9% of the cells as detected by TUNEL assay. CONCLUSION: Blocking the expression of survivin with RNA interference technology can significantly suppress proliferation of MCF-7 cells and induce apoptosis to a certain degree. RNAi targeted to survivin has a potential value in gene therapy of breast cancer.

Apoptosis↗

PubMed2004

A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei.

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN(2) in T. brucei.

Amino Acid Sequence↗

PubMed2004

Dicer is essential for formation of the heterochromatin structure in vertebrate cells.

RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. The biological function of the RNAi-related pathway in vertebrate cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken-human hybrid DT40 cell line that contains human chromosome 21. We show that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from alpha-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.

Animals↗

PubMed2005

Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

Algorithms↗

PubMed2004

Identification of powdery mildew-induced barley genes by cDNA-AFLP: functional assessment of an early expressed MAP kinase.

Gene expression analysis by cDNA-AFLP in barley ( Hordeum vulgare L.) after powdery mildew ( Blumeria graminis f.sp. hordei , Bgh ) inoculation revealed 615 (3.7%) of 16 500 screened cDNA fragments being differentially regulated 4 and/or 12 h after inoculation. Of these transcript derived fragments (TDFs), 120 were sequenced, and for 28 out of 29 tested, induction was confirmed via RT-PCR. Most TDFs did not show any homology to sequences with known functions, others showed homology to genes involved in primary and secondary metabolism, pathogen response, redox regulation, and signal transduction. TDFs with homology to a MAP kinase ( PWMK1 ), a WRKY transcription factor, a heparanase, an immunophilin, a cytochrome P450, and a receptor-like protein kinase were isolated as full length cDNAs. Knockdown by RNA interference via biolistic delivery of sequence specific double stranded RNA to leaf segments tagged two of these genes as possible candidates being causally involved in the outcome of the barley- Bgh interaction. Knockdown of the receptor-like protein kinase and the WRKY transcription factor increased resistance to the fungus, while knockdown of PWMK1 only led to a slightly enhanced susceptibility of epidermal cells to Bgh . This suggests that the receptor-like protein kinase and the WRKY protein are candidates for negative regulators of powdery mildew resistance. Based on expression analyses, PWMK1 appears to be more generally involved in stress response.

Ascomycota↗

PubMed2005

Human RISC couples microRNA biogenesis and posttranscriptional gene silencing.

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.

Adenosine Triphosphate↗

PubMed2004

In search of antisense.

In recent years, natural antisense transcripts (NATs) have been implicated in many aspects of eukaryotic gene expression including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation and RNA editing. Moreover, there is growing evidence to suggest that antisense transcription might have a key role in a range of human diseases. Consequently, there have been several recent attempts to identify novel NATs. To date, approximately 2500 mammalian NATs have been found, indicating that antisense transcription might be a common mechanism of regulating gene expression in human cells. There are increasingly diverse ways in which antisense transcription can regulate gene expression and evidence for the involvement of NATs in human disease is emerging. A range of bioinformatic resources could be used to assist future antisense research.

Alternative Splicing↗

PubMed2005

CHR11, a chromatin-remodeling factor essential for nuclear proliferation during female gametogenesis in Arabidopsis thaliana.

Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2/SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A. thaliana chromatin-remodeling protein 11 (CHR11) encodes an imitation of switch (ISWI)-like chromatin-remodeling protein abundantly expressed during female gametogenesis and embryogenesis in Arabidopsis. To determine the function of CHR11 in wild-type plants, we introduced a hairpin construct leading to the production of double-stranded RNA, which specifically degraded the endogenous CHR11 mRNA by RNA interference (RNAi). Transcription of the RNAi-inducing hairpin RNA was driven by either a constitutive cauliflower mosaic virus 35S promoter (CaMV35S) acting at most stages of the sporophytic phase or a newly identified specific promoter acting at the onset of the female gametophytic phase (pFM1). All adult transformants that constitutively lacked sporophytic CHR11 activity showed reduced plant height and small cotyledonary embryos with limited cell expansion. In contrast, RNAi lines in which CHR11 was specifically silenced at the onset of female gametogenesis (megagametogenesis) had normal height and embryo size but had defective female gametophytes arrested before the completion of the mitotic haploid nuclear divisions. These results show that CHR11 is essential for haploid nuclear proliferation during megagametogenesis and cell expansion during the sporophytic phase, demonstrating the functional versatility of SWI2/SNF2 chromatin-remodeling factors during both generations of the plant life cycle.

Adenosine Triphosphatases↗

PubMed2005

Sustained activation of phosphatidylinositol 3-kinase/Akt/nuclear factor kappaB signaling mediates G protein-coupled delta-opioid receptor gene expression.

Expression of the delta-opioid receptor gene (dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt (protein kinase B)/NF-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.

Active Transport, Cell Nucleus↗

PubMed2002

Rotavirus gene silencing by small interfering RNAs.

RNA interference is an evolutionarily conserved double-stranded RNA-triggered mechanism for suppressing gene expression. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, from which the spike protein VP4 projects. Here, we show that a small interfering RNA corresponding to the VP4 gene efficiently inhibits the synthesis of this protein in virus-infected cells. A large proportion of infected cells had no detectable VP4 and the yield of viral progeny was reduced. Most of the virus particles purified from these cells were triple-layered, but lacked VP4, and were poorly infectious. We also show that VP4 might not be required for the last step of virus morphogenesis. The VP4 gene silencing was specific, since the synthesis of VP4 from rotavirus strains that differ in the target sequence was not affected. These findings offer the possibility of carrying out reverse genetics in rotaviruses.

Capsid Proteins↗

PubMed2003

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway.

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.

Cell Line↗

PubMed2006

The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster.

Most organisms have evolved defense mechanisms to protect themselves from viruses and other pathogens. Arthropods lack the protein-based adaptive immune response found in vertebrates. Here we show that the central catalytic component of the RNA-induced silencing complex (RISC), the nuclease Argonaute 2 (Ago-2), is essential for antiviral defense in adult Drosophila melanogaster. Ago-2-defective flies are hypersensitive to infection with a major fruit fly pathogen, Drosophila C virus (DCV), and with Cricket Paralysis virus (CrPV). Increased mortality in ago-2 mutant flies was associated with a dramatic increase in viral RNA accumulation and virus titers. The physiological significance of this antiviral mechanism is underscored by our finding that DCV encodes a potent suppressor of RNA interference (RNAi). This suppressor binds long double-stranded RNA (dsRNA) and inhibits Dicer-2-mediated processing of dsRNA into short interfering RNA (siRNA), but does not bind short siRNAs or disrupt the microRNA (miRNA) pathway. Based on these results we propose that RNAi is a major antiviral immune defense mechanism in Drosophila.

Animals↗

PubMed2006

The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Animals↗