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PubMed2004

Dynamics of gene silencing by RNA interference.

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Animals↗
PubMed2006

Moment analysis for kinetics of gene silencing by RNA interference.

RNA interference (RNAi) was quantitatively evaluated from a kinetic viewpoint. A simple kinetic evaluation based on moment analysis was proposed, assuming suppression and recovery phases of gene expression. We defined the area under the curve of the inhibitory effect (AUC(IE)) as an index of the total intensity of RNAi and the mean response time of the inhibitory effect (MRT(IE)) as an index of its duration. The proposed kinetic analysis helps to understand the RNAi effect in a quantitative and time-dependent manner, which will be beneficial for designing RNAi-based gene silencing for both experimental and therapeutic purposes.

Animals↗
PubMed2001

The Caenorhabditis elegans distal-less ortholog ceh-43 is required for development of the anterior hypodermis.

Homeobox genes of the Distal-less (Dll) class are expressed in developing appendages as well as in the central nervous system in invertebrates and vertebrates. Mutant analyses in mice and Drosophila have implicated these genes in outgrowth of structures, cell adhesion, cell migration, and cell fate decisions. We have investigated the expression and function of ceh-43, the Dll ortholog from the nematode Caenorhabditis elegans, by using gfp reporter constructs and double-stranded RNA-mediated interference (RNAi). Our results show that, as in the fly, the C. elegans Dll ortholog seems to play a role in cell adhesion. An antibody against the butterfly Distal-less homeodomain stains the nervous system of C. elegans embryos (Panganiban et al. [1997] Proc Natl Acad Sci USA. 94:5162-5166). GFP expression under the control of the ceh-43 promoter looks similar, although strong expression is primarily confined to the head hypodermis and to neuronal support cells. ceh-43(RNAi) results in 100% lethality at embryonic or early larval stages. At the beginning of morphogenesis, ceh-43(RNAi) embryos start to lose cells through a hole in the head hypodermis. They either rupture anteriorly as elongation proceeds, or they elongate normally to threefold egg length with the pharynx not connected to the mouth. Elongated ceh-43(RNAi) animals die before or soon after hatching with a fluid-filled pseudocoel and large vacuoles. These phenotypes suggest a role for ceh-43 in development of adhesive properties in the head hypodermis that connects the epithelia of the skin and the digestive tract. Furthermore, possible defects in the excretory system may result at least in part from a requirement for ceh-43 in the CAN neurons where ceh-43:gfp is also expressed.

Amino Acid Sequence↗
PubMed2004

Analysis of RING finger genes required for embryogenesis in C. elegans.

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1-12, 2004.

Amino Acid Motifs↗
PubMed2005

Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells.

Activating BRAF mutations and loss of wild-type INK4A expression both occur at high frequencies in melanomas. Here, we present evidence that BRAF and INK4A have different effects on melanogenesis, a marker of melanocytic differentiation. Human melanoma cell line 624Mel harbors mutations in both BRAF and INK4A. The in vitro and in vivo growth of these cells was inhibited by either reduced expression of mutant BRAF using stable retroviral RNA interference (RNAi) or retrovirus-mediated stable expression of wild-type INK4A cDNA. Consistent with the observed growth inhibition, phosphorylation of S780 and S795 in pRB, both CDK4/6 targets, was suppressed in cells expressing either mutant BRAF RNAi or wild-type INK4A. Interestingly, melanoma cells expressing mutant BRAF RNAi had increased pigmentation, produced more mature melanosomes and melanin and expressed higher levels of tyrosinase and tyrosinase-related protein-1, whereas melanogenesis was not induced by wild-type INK4A. We found that the melanocyte lineage-specific master control protein microphthalmia-associated transcription factor was upregulated by inhibition of mutant BRAF, which may be the cause for the melanogenic effect of BRAF RNAi. The results suggest that, although both BRAF and INK4A lesions promote cell growth and tumor formation, mutant BRAF may also induce dedifferentiation in melanoma cells.

Animals↗
PubMed2004

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. METHODS: We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. RESULTS: As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. CONCLUSION: The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.

Base Sequence↗
PubMed2004

RNA interference: a new mechanism by which FMRP acts in the normal brain? What can Drosophila teach us?

Fragile X syndrome is the most common heritable form of mental retardation caused by loss-of-function mutations in the FMR1 gene. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. Recent work in Drosophila melanogaster has shown that the fly homolog of FMR1 (dFMR1) plays an important role in regulating neuronal morphology, which may underlie the observed deficits in behaviors of dFMR1 mutant flies. Biochemical analysis has revealed that dFMR1 forms a complex that includes ribosomal proteins and, surprisingly, Argonaute2 (AGO2), an essential component of the RNA-induced silencing complex (RISC) that mediates RNA interference (RNAi) in Drosophila. dFMR1 also associates with Dicer, another essential processing enzyme of the RNAi pathway. Moreover, both a micro-RNA (miRNA) and short interfering RNAs (siRNAs) can coimmunoprecipitate with dFMR1. Together these findings suggest that dFMR1 functions in an RNAi-related apparatus to regulate the expression of its target genes at the level of translation. These findings raise the possibility that Fragile X syndrome may be the result of a protein synthesis abnormality caused by a defect in an RNAi-related apparatus. Because the core mechanisms of complex behaviors such as learning and memory and circadian rhythms appear to be conserved, studies of Fragile X syndrome using Drosophila as a model provide an economy-of-scale for identifying biological processes that likely underlie the abnormal morphology of dendritic spines and behavioral disturbances observed in Fragile X patients.

Animals↗
PubMed2000

mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans.

The Caenorhabditis elegans gene mag-1 can substitute functionally for its homolog mago nashi in Drosophila and is predicted to encode a protein that exhibits 80% identity and 88% similarity to Mago nashi (P. A. Newmark et al., 1997, Development 120, 3197-3207). We have used RNA-mediated interference (RNAi) to analyze the phenotypic consequences of impairing mag-1 function in C. elegans. We show here that mag-1(RNAi) causes masculinization of the germ line (Mog phenotype) in RNA-injected hermaphrodites, suggesting that mag-1 is involved in hermaphrodite germ-line sex determination. Epistasis analysis shows that ectopic sperm production caused by mag-1(RNAi) is prevented by loss-of-function (lf) mutations in fog-2, gld-1, fem-1, fem-2, fem-3, and fog-1, all of which cause germ-line feminization in XX hermaphrodites, but not by a her-1(lf) mutation which causes germ-line feminization only in XO males. These results suggest that mag-1 interacts with the fog, fem, and gld genes and acts independently of her-1. We propose that mag-1 normally allows oogenesis by inhibiting function of one or more of these masculinizing genes, which act during the fourth larval stage to promote transient sperm production in the hermaphrodite germ line. When the Mog phenotype is suppressed by a fog-2(lf) mutation, mag-1(RNAi) also causes lethality in the progeny embryos of RNA-injected, mated hermaphrodites, suggesting an essential role for mag-1 during embryogenesis. The defective embryos arrest during morphogenesis with an apparent elongation defect. The distribution pattern of a JAM-1::GFP reporter, which is localized to boundaries of hypodermal cells, shows that hypodermis is disorganized in these embryos. The temporal expression pattern of the mag-1 gene prior to and during morphogenesis appears to be consistent with an essential role of mag-1 in embryonic hypodermal organization and elongation.

Animals↗
PubMed2000

The NED-8 conjugating system in Caenorhabditis elegans is required for embryogenesis and terminal differentiation of the hypodermis.

This work has identified the enzymes involved in the activation and conjugation of the ubiquitin-like protein NED-8 in Caenorhabditis elegans. A C. elegans conjugating enzyme, UBC-12, is highly specific in its ability to utilize NED-8 as a substrate. Immunostaining shows that NED-8 is conjugated in vivo to a major target protein with a conjugate size of 90 kDa. While the amount of this conjugate is developmentally regulated with reduced levels in the larval stages, the mRNA encoding C. elegans UBC-12 is constitutively produced throughout development, as is NED-8 itself. The importance of the NED-8 conjugating system in C. elegans was determined by RNA interference (RNAi) assays using double-stranded RNA encoding NED-8, UBC-12, or the NED-8 activating enzyme component ULA-1. The progeny of both ned-8 and ubc-12 RNAi-treated hermaphrodites either arrested during embryonic development or underwent abnormal postembryonic development. The effect on postembryonic development was pleiotropic, the most frequent gross abnormality being vulval eversion during the L4 stage. Individuals with an everted vulva either burst at the L4 to adult molt or gave rise to adults incapable of egg laying. Additionally, both ned-8 and ubc-12 RNAi induced a striking abnormality in the alae, structures produced by the lateral hypodermal seam cells in the adult nematode. Affected alae were patchy and frequently diverged around a central space. Vulval defects were also produced by RNAi directed at C. elegans ula-1. This is the first demonstration of a requirement for NED-8 conjugation in metazoan development.

Amino Acid Sequence↗
PubMed1999

Instability of pUC19 in Escherichia coli transcription termination factor mutant, rho026.

The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion of rop, also known as rom, and to an ori mutation that impedes RNAI:RNAII interaction. pUC19, unlike pBR322, fails to transform E. coli rho mutant rho026 cells. Here we identify two features of pUC19 that contribute to this transformation defect. (1) The pUCori mutation is involved because replacing the pUCori with that of pBR322 restored transformation. (2) Transcription from the lac promoter in pUC19 is important, since deletion or inversion of the promoter or insertion of a transcription terminator (lambdat0) downstream of it restored transformation. Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in the rho026 strain. We suggest that in rho026 cells pUC19 RNAI:RNAII interaction is more impeded than in rho+ cells and Rop/Rom may confer stability by protecting RNAI against RNase E activity because expression of a rom gene inserted into pUC19 restored transformation.

Bacterial Proteins↗
PubMed2006

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

Animals↗
PubMed2007

RNA interference in mice.

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for functional genomics in mammalian cells. Furthermore, RNAi holds promise as a simple, fast and cost-effective approach to studying mammalian gene function in vivo and as a novel therapeutic approach. This review provides an overview of the progress of RNAi in vivo, with emphasis on systemic/local siRNA delivery, viral shRNA vectors, shRNA vector transgenic mice and conditional systems to control shRNA vectors. Taken together, the data from 80 in vivo studies show that RNAi is a useful tool that offers new opportunities for functional genomics in mice.

Animals↗
PubMed1986

Effect of relA function on the replication of plasmid pBR322 in Escherichia coli.

Replication of the plasmid pBR322, and the accumulation and life time of its primer transcript, RNAII, and replication inhibitor, RNAI, were measured in an isogenic relA+/relA pair of E. coli strains during exponential growth, or following amino acid starvation, or during treatment with chloramphenicol. (1) The synthesis rates of RNAI and RNAII decreased during inhibition of protein synthesis in either strain, i.e. their promoters are not under stringent control; (2) during amino acid starvation, RNAI and RNAII lifetimes increased in complex, rel-dependent patterns; (3) the changes in RNAI and RNAII synthesis and accumulation had no immediate effect on the rate of plasmid replication; (4) continued plasmid replication requires a protein which is synthesized during amino acid deprivation or treatment with low concentrations of chloramphenicol in relA+, but not in relA bacteria.

Bacterial Proteins↗
PubMed2006

Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1alpha gene.

OBJECTIVES: Hypoxia is associated with human non-small cell lung cancers (NSCLC), which are highly resistant to chemotherapy. The hypoxia inducible factor (HIF) as a transcription factor in response to hypoxia indicates that it could be a novel, tumor-specific target for anticancer therapy. We hypothesized that disruption of HIF pathway through lentiviral vector-mediated HIF-1alpha RNA interference (RNAi) could reverse the hypoxia-induced resistance to chemotherapy. METHODS: We transfected Human NSCLC cell lines, SPCA1 and A549 with HIF-1alpha specific RNAi lentiviral vectors as well as controls. HIF-1alpha silenced cells [SPCA1/HIF-1alpha(-) and A549/HIF-1alpha(-)] were screened by blasticidin. They were incubated in 19 or 0.5% O2 for 16 h followed by the assessment of chemosensitivity to cisplatin and doxorubicin with MTT and clonogenic assays. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of HIF-1alpha mRNA and protein, respectively. Moreover, flow cytometry was used to monitor the expression of P-glycoprotein. RESULTS: Exposure of SPCA1 and A549 cells to 0.5% O2 significantly increased resistance to cisplatin and doxorubicin, in contrast to cells incubated in normoxia. Transduction of SPCA1 with HIF-1alpha RNAi vector resulted in sequence specific silencing with 87.2 and 84.6% decreases of HIF-1alpha mRNA transcription and 97.3 and 94.8% of protein expressions in normoxia and hypoxia, respectively. Correspondingly, they are 89.2, 89.9% and 97.2, 88.4% decreases in A549 cells. Hypoxia-induced resistance to cisplatin and doxorubicin were reversed in SPCA1/HIF-1alpha(-) and A549/HIF-1alpha(-) cells. There was no significant P-glycoprotein increase induced by hypoxia in NSCLC cells. CONCLUSIONS: Our studies demonstrated that hypoxia-induced chemoresistance to cisplatin and doxorubicin in NSCLC cells is through the HIF pathway. MDR1 regulation may not be involved in hypoxia-induced chemoresistance. Combining delivery of HIF-1alpha RNAi lentiviral vector with cisplatin-related chemotherapy regimens may enable us to develop more effective strategy for NSCLC therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

On the origin and functions of RNA-mediated silencing: from protists to man.

Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.

Animals↗
PubMed2002

Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference.

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named "Dicer", functions in the RNA interference (RNAi) pathway by producing 21--23 bp dsRNAs which target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences; and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse Chromosome (Chr) 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway.

Amino Acid Sequence↗
PubMed2003

A simple "soaking method" for RNA interference in the planarian Dugesia japonica.

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.

Animals↗
PubMed2006

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference.

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.

Cell Line, Tumor↗