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At least 1,171 records · Page 65Linked to original sources
PubMed2004

GENE impedance: a natural process for control of gene expression and the origin of RNA interference.

Gene expression is controlled by coordinated transcriptional and post-transcriptional mechanisms. Normally, expression of a gene switches on and off in response to specific physiological signals that are triggered by cellular demand for the gene products at a given time. Based on our previous studies and the scientific literature, we hypothesize that when a gene promoter switches to transcriptional repression mode, transcription of the gene ceases, and a small amount of double-stranded RNA (dsRNA) is synthesized by the RNA polymerase switching to the opposite DNA strand at the termination region of the gene. These dsRNA structures, which result from normal transcriptional repression, can then be processed into short interfering RNAs (siRNAs) within the nucleus. These molecules subsequently direct specific cleavage of the cognate mRNAs and interfere with their translation through sequence complementarily. We further hypothesize that cellular defense mechanisms invoked by invading genetic elements could be rooted in this fundamental regulatory pathway that we call "GENE impedance", or simply, GENEi. Here, we present a working model that illustrates how transcription-termination and transcription-arrest can contribute to the regulation of gene expression via GENEi. In our model RNAi is only one component of GENEi, which is a more generalized mechanism of gene regulation.

Animals↗
PubMed1985

The repeated regions of Semliki Forest virus defective-inferfering RNA interferes with the encapsidation process of the standard virus.

Two different defective interfering RNAs of Semliki Forest virus have been cloned and sequenced previously. These molecules have repeated sequence blocks between unique terminal regions. The late gene region of SV40 virus has been replaced with the repeating unit detected in both defective-inferfering (DI) RNAs, and by complementation with a tsA mutant of SV40 a mixed stock of recombinant and helper virus was obtained. Upon infection of monkey kidney cells the recombinant expressed the repeated part of the DI RNA (svDI301 RNA). Superinfection of these cells with standard Semliki Forest virus showed that (i) the synthesis of SFV genomic RNA is marginally if at all affected by the svDI301 RNA, (ii) the svDI301 RNA is not replicated by SFV-RNA-dependent RNA polymerase, and (iii) packaging efficiency of the standard SFV genome RNA into virions is clearly decreased in the presence of svDI301 RNA. These results suggest that the terminal regions of the DI RNA molecule are required for efficient replication while the central repeated elements are involved in encapsidation.

Animals↗
PubMed2003

Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference.

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the beta-arrestins in many seven-membrane-spanning receptor systems.

Arrestins↗
PubMed2002

RNA interference: the new somatic cell genetics?

RNAi is evolving into a powerful tool for manipulating gene expression in mammalian cells with potential utility for investigating gene function, for high-throughput, function-based genetic screens and potentially for development as a therapeutic tool.

Animals↗
PubMed2000

RNA interference demonstrates a role for nautilus in the myogenic conversion of Schneider cells by daughterless.

Schneider SL2 cells activate the myogenic program in response to the ectopic expression of daughterless alone, as indicated by exit from the cell cycle, syncytia formation, and the presence of muscle myosin fibrils. Myogenic conversion can be potentiated by the coexpression of DMEF2 and nautilus with daughterless. In RT-PCR assays Schneider cells express two mesodermal markers, nautilus and DMEF2 mRNAs, as well as very low levels of daughterless mRNA but no twist. Full-length RT-PCR products for nautilus and DMEF2 encode immunoprecipitable proteins. We used RNA-i to demonstrate that both endogenous nautilus expression and DMEF2 expression are required for the myogenic conversion of Schneider cells by daughterless. Coexpression of twist blocks conversion by daughterless but twist dsRNA has no effect. Our results indicate that Schneider cells are of mesodermal origin and that myogenic conversion with ectopic expression of daughterless occurs by raising the levels of daughterless protein sufficiently to allow the formation of nautilus/daughterless heterodimers. The effectiveness of RNA-i is dependent upon protein half-life. Genes encoding proteins with relatively short half-lives (10 h), such as nautilus or HSF, are efficiently silenced, whereas more stable proteins, such as cytoplasmic actin or beta-galactosidase, are less amenable to the application of RNA-i. These results support the conclusion that nautilus is a myogenic factor in Drosophila tissue culture cells with a functional role similar to that of vertebrate MyoD. This is discussed with regard to the in vivo functions of nautilus.

Animals↗
PubMed2004

Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells.

The multidrug resistance (MDR) mediated by P-glycoprotein (P-gp), the MDR1 gene product, is one of the major obstacles in leukemia treatment. The present study was designed to explore a MDR1-targeted small interfering RNA (si-MDR1) approach for reversal of P-gp-mediated MDR in the MDR human leukemia cell line k562/A02. It was found that si-MDR1 significantly inhibited MDR1 expression at both mRNA and protein levels. Depletion of MDR1 by si-MDR1 correlated with the increased sensitivity of the cells to cytotoxic agents and with the enhanced intracellular retention of daunorubicin (DNR). One base-pair mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression. These findings indicate that siRNA specifically and efficiently interferes with the expression of mdr1 and could be used as a molecularly defined therapeutic approach for MDR in the treatment of leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Inhibition of 2-5A synthetase expression by antisense RNA interferes with interferon-mediated antiviral and antiproliferative effects and induces anchorage-independent cell growth.

It has been shown previously that the IFN-induced enzyme 2-5A synthetase is sufficient to induce antiviral and antiproliferative effects in transfected cells expressing the protein. In this study, the possibility that this enzyme is also essential in generating these biological activities was investigated. For this purpose, a plasmid, pMSas-NEO, was constructed. This plasmid carries an active neomycin-resistant gene. In addition, it contains a metallothionein promoter fused to an inverted 180-bp fragment derived from the 5' end of cDNA encoding the 43-kDa isoform of murine 2-5A synthetase. NIH/3T3 mouse fibroblasts were transfected with the plasmid, about 50 neomycin-resistant clones were isolated, and two, clone 11 and clone 22, were chosen for further studies. One clone transfected only with the neomycin-resistant gene, clone Neo, was used as a control. The results show that in the case of clone 11, the combined treatment of IFN and ZnCl2 reduced significantly the level of the IFN-induced 2-5A synthetase activity, the amount of the 40-, 43-, and 71-kDa 2-5A synthetase isoforms and the level of the 1.7-kb specific RNA transcript. An even stronger effect on these parameters was observed with clone 22 cells. No difference in PKR activity was evident under the same conditions with all three clones tested. Most important, the combined treatment of IFN and ZnCl2 reversed the IFN-mediated antiproliferative and antiviral activities, as determined by the kinetics of cell growth, thymidine incorporation, cloning efficiency, and infection with mengovirus. Strikingly, the growth of colonies in soft agar were observed in both clone 11 (small colonies) and clone 22 (large colonies) cells, particularly following treatment with ZnCl2. We conclude that 2-5A synthetase is an essential component in the IFN-induced biological activities and that interference with its function results in anchorage-independent growth of the transfected cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Mutations at positions 13 and/or 914 in Escherichia coli 16S ribosomal RNA interfere with the initiation of protein synthesis.

Mutations at positions 13 (U-->A) and/or 914 (A-->U) of Escherichia coli 16S rRNA severely affect cell growth and protein synthesis, when expressed in vivo in a vector encoding an rrn operon under control of an inducible promoter. In vitro assays using extension inhibition indicate that the mutations interfere with the formation of the 30S translational initiation complex, which can account for their effect on cell growth. The two mutations destabilize an adjacent pseudoknot helix in which bases 17-19 pair to bases 916-918. This was shown by the increased binding of an oligodeoxyribonucleotide probe complementary to one strand of the pseudoknot helix, and by the increased reactivity to kethoxal of base G917 within this helix. These observations suggest that this pseudoknot helix participates in the formation of the 30S translational initiation complex.

Adenine↗
PubMed2005

Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells.

Xenotransplantation from pig to human being is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal were capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.

Animals↗
PubMed2006

Induction of silencing effect of Swedish mutant amyloid precursor protein by RNA interference.

Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP (APPwt) and Swedish mutant APP (APPswe). One siRNA targeting APPwt and the other siRNA targeting APPswe were designed. All these siRNAs were endogenously expressed by siRNAs expressing plasmids. COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector. The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein (GFP). It was found that the siRNAs silenced APPwt and APPswe to different degrees. siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.

Amyloid beta-Protein Precursor↗
PubMed1995

Decreasing the distance between the two conserved sequence elements of histone pre-messenger RNA interferes with 3' processing in vitro.

Histone mRNA 3' end formation requires the presence of two cis-acting conserved sequence elements: a stem-loop structure upstream from the site of cleavage and a purine-rich region downstream from the site of cleavage called the histone downstream element (HDE). Possible interactions between these two elements and their respective binding factors were investigated by a series of deletions (1-7 nt) in the region between the two. The efficiency of processing decreased as the stem-loop and the HDE were moved closer together. In contrast with the documented ability of the U7 snRNP to direct cleavage at a fixed distance from the HDE in insertion mutants (Scharl & Steitz, 1994), all deletion substrates for which processing was observed were cleaved at or 1-nt upstream from the wild-type site. The reason for the inability of the system to cleave closer to the stem-loop remains unclear, but the removal of stem-loop binding protein(s) (SLBP) did not activate upstream cleavage events. Thus, although the processing machinery measures the distance between the cleavage site and the HDE of mammalian histone pre-mRNAs, there is a barrier limiting how far upstream cleavage can occur. These data allow a reevaluation of the sites of 3' end processing in known histone pre-mRNAs.

Animals↗
PubMed2004

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells.

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Knock-down of GFRalpha4 expression by RNA interference affects the development of retinal cell types in three-dimensional histiotypic retinal spheres.

PURPOSE: To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina. METHODS: Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFRalpha4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours. RESULTS: In contrast to control cultures, retinal spheres transfected with GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased. CONCLUSIONS: These data showed for the first time that GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.

Amacrine Cells↗
PubMed2005

RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.

We used siRNA against the alpha-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after beta-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific alpha-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by approximately 30%. alpha-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0-200 microg/kg body wt pSi-4, and alpha-ENaC mRNA and protein expressions were analyzed. To reach IC(50) for alpha-ENaC mRNA expression, 32 microg/kg body wt pSi-4 was needed, and to reach IC(50) for alpha-ENaC protein expression, 59 microg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in beta-ENaC expression, at either mRNA or protein level, as well as no changes in alpha(1)-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, alpha-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal alpha-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney alpha- and beta-ENaC mRNA and protein expression. Thus we provide conclusive evidence that beta-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.

Absorption↗
PubMed2005

An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor.

OBJECTIVES: To study the inhibitory effects of plasmid-based siRNA targeting human epidermal growth factor receptor (EGFR) on tumor proliferation and invasion of TJ905 glioblastoma cells. METHODS: Two siRNA expression constructs targeting human EGFR extracellular domain (516-536) and catalytic domain (2400-2420) were transfected into TJ905 cell as mediated by Lipofectamine. Immunofluorescence assay and Western blotting were used to detect EGFR expression. Cell cycle was analyzed by flow cytometry, cell proliferative activity was measured by MTT assay. The expression and enzymatic activity of MMP9 were measured by Western blotting and gelatin zymography. Cell invasive capability was evaluated by Transwell method. RESULTS: The expression of EGFR was knocked-down by 90 and 92, respectively in siRNA constructs transfected groups as indicated by immunofluorescence assay and Western blotting. The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNAs transfected cells than that in parental cells and the cells transfected with empty vector. Compared to parental cells and the cells transfected with empty vector, the survival rates of glioma cells transfected with the siRNAs dramatically dropped down from the first day after implantation (P<0.05) as indicated by MTT assay. Meanwhile, the expression and enzymatic activity of MMP9 decreased significantly in siRNAs transfected in TJ905 cells, and cell invasive potential was also greatly inhibited in the Transwell study. CONCLUSION: The siRNA expression constructs targeting EGFR could specifically suppress EGFR expression, inhibit cell growth, induce cell cycle arrest and suppress invasion. The plasmid-based siRNA targeting human EGFR approach should be a new strategy for gene therapy of malignant gliomas.

Blotting, Western↗
PubMed2006

Effects of small interference RNA against PTP1B and TCPTP on insulin signaling pathway in mouse liver: evidence for non-synergetic cooperation.

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Two closely-related protein tyrosine phosphatases, PTP1B and TCPTP both showed abilities to negatively regulate insulin receptor signaling. In order to test whether these two phosphatases can act synergistically, hydrodynamic injection was applied to deliver small interfering RNA (siRNA) of PTP1B and/or TCPTP to mouse liver. By measuring insulin-sensitive reporter gene expression and plasma glucose of diabetic mice, we found siRNA of PTP1B or TCPTP alone can sensitize insulin signal transduction, but combined treatment of both siRNAs had no better effects than siRNA of PTP1B. These results suggested siRNA of PTP1B and TCPTP can strengthen insulin signaling, but their effects do not appear to be synergistic in mouse liver.

Alloxan↗