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PubMed2006

Combinatorial RNA interference in Caenorhabditis elegans reveals that redundancy between gene duplicates can be maintained for more than 80 million years of evolution.

BACKGROUND: Systematic analyses of loss-of-function phenotypes have been carried out for most genes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. Although such studies vastly expand our knowledge of single gene function, they do not address redundancy in genetic networks. Developing tools for the systematic mapping of genetic interactions is thus a key step in exploring the relationship between genotype and phenotype. RESULTS: We established conditions for RNA interference (RNAi) in C. elegans to target multiple genes simultaneously in a high-throughput setting. Using this approach, we can detect the great majority of previously known synthetic genetic interactions. We used this assay to examine the redundancy of duplicated genes in the genome of C. elegans that correspond to single orthologs in S. cerevisiae or D. melanogaster and identified 16 pairs of duplicated genes that have redundant functions. Remarkably, 14 of these redundant gene pairs were duplicated before the divergence of C. elegans and C. briggsae 80-110 million years ago, suggesting that there has been selective pressure to maintain the overlap in function between some gene duplicates. CONCLUSION: We established a high throughput method for examining genetic interactions using combinatorial RNAi in C. elegans. Using this technique, we demonstrated that many duplicated genes can retain redundant functions for more than 80 million years of evolution. This provides strong support for evolutionary models that predict that genetic redundancy between duplicated genes can be actively maintained by natural selection and is not just a transient side effect of recent gene duplication events.

Animals↗
PubMed2006

Therapeutic biology: checkpoint pathway activation therapy, HIV Tat, and transkingdom RNA interference.

Therapeutic biology is an exciting new frontier in the post-genomic era with the mission to better human health. The explosive increase in the understanding of molecular and regulatory biology has enabled the identification of a plethora of potential targets and pathways for the discovery of new medicines. Conversely, molecularly based therapeutic intervention of biological aberrations in human diseases offers a test of the depth of our understanding of biology. This article discusses three examples of therapeutic biology research. The first concerns the treatment of cancer by activating genome surveillance circuitry, namely checkpoint-pathway activation therapy. The second example is the identification of the HIV Tat protein as both a cause of immune cell activation and apoptosis, and as a vaccine candidate against HIV infection. The third example describes transkingdom RNA interference and its application in the investigational and therapeutic silencing of disease genes.

AIDS Vaccines↗
PubMed2003

Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy.

Calmodulin (CaM), a small calcium-binding protein, is the key mediator of numerous calcium-induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we use the unique possibility offered by the C. elegans model system to inactivate the single CaM gene (cmd-1) through RNA interference (RNAi). We show that the RNAi microinjection approach results in a severe embryonic lethal phenotype. Embryos show disturbed morphogenesis, aberrant cell migration patterns, a striking hyperproliferation of cells and multiple defects in apoptosis. Finally, we show that RNAi delivery by the feeding protocol does not allow the efficient silencing of the CaM gene obtained by microinjection. General differences between the two delivery methods are discussed.

Amino Acid Sequence↗
PubMed2003

Duplexes of 21 nucleotide RNA specific for COX II mediates RNA interference in cultured bovine aortic coronary endothelial cells (BAECs).

The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygenase (COX) II and demonstrate inhibition of the expression of COX II protein and its product PGE. A 21-dsRNA specific for COX II was introduced by lipofectamine into a primary cell culture of bovine aortic coronary endothelial cells (BAECs). BAECs basally express COX I but not COX II, and COX II expression is only apparent after stimulation with phorbol 12-myristate acetate (PMA). We first demonstrated that the lipofected fluorescent dsRNA-COX II is accumulated and localized within the cultured cells. We then demonstrated gene silencing of PMA-induced COX II protein expression by dsRNA-COX II using immuno-histochemistry. Western blot analysis and radioimmunoassay were used to quantitate the percent of inhibition. It was found that lipofected dsRNA-COX II reduced the percent of PMA-induced COX II enzyme by 36% and PGE production by 40%. There was no demonstrable effect of dsRNA-COX II or PMA on COX I expression.

Animals↗
PubMed2006

[Inhibition of HCV IRES controlled reporter gene expression by RNA interference].

OBJECTIVE: To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression. METHODS: Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system. RESULTS: The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid. CONCLUSION: Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.

Gene Expression Regulation↗
PubMed2000

[RNA interference in Escherichia coli cells: the expression of molecules that are complementary to the lon gene mRNA in parallel orientation].

To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.

ATP-Dependent Proteases↗
PubMed2006

RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4-2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4-2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4-2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Animals↗
PubMed2004

Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference.

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

RNA interference screening in ticks for identification of protective antigens.

Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important arthropod vector of pathogens in North America. Development of vaccines directed against tick proteins may effect reduction of tick infestations and transmission of tick-borne pathogens. The limiting step for the development of tick vaccines has been the identification of tick protective antigens. Reverse vaccinology approaches aimed at reducing animal experimentation while allowing for the rapid screening of pools of potential tick vaccine candidates would greatly facilitate progress towards the development of tick vaccines. Herein, we describe the screening of Ixodes scapularis cDNAs for identification of tick protective antigens using RNA interference (RNAi). The results of the RNAi screening were similar to those obtained previously using expression library immunization and demonstrated that RNAi could serve as a more rapid and cost-effective tool for vaccine antigen discovery in ticks and in other nonmodel organisms.

Animals↗
PubMed2004

Knockdown of Chk1, Wee1 and Myt1 by RNA interference abrogates G2 checkpoint and induces apoptosis.

Mammalian cells undergo cell cycle arrest in response to DNA damage due to the existence of multiple checkpoint response mechanisms. One such checkpoint pathway operating at the G(1) phase is frequently lost in cancer cells due to mutation of the p53 tumor suppressor gene. However, cancer cells often arrest at the G(2) phase upon DNA damage, due to activation of another checkpoint pathway that prevents the activation Cdc2 kinase. The kinases, Chk1, Wee1, and Myt1 are key regulators of this G(2) checkpoint, which act directly or indirectly to inhibit Cdc2 activity. Here we show that RNA interference (RNAi)-mediated downregulation of Wee1 kinase abrogated an Adriamycin trade mark -induced G(2) checkpoint in human cervical carcinoma Hela cells that are defective in G(1) checkpoint response. Wee1 downregulation sensitized HeLa cells to Adriamycin trade mark -induced apoptosis. Downregulation of Chk1 kinase in Hela cells also caused a significant amount of cell death in dependent of DNA damage. In contrast, Myt1 downregulation also abrogated Adriamycin trade mark -induced G(2) arrest but did not cause substantial apoptosis. Reduction in Wee1, Chk1, or Myt1 levels did not sensitize normal human mammary epithelial cells (HMEC) cells to Adriamycin trade mark -induced apoptosis unlike the situation in Hela cells. Our study reveals distinct roles for Chk1, Wee1, and Myt1 in G(2) checkpoint regulation. The data reported here support the attractiveness of Wee1 and Chk1 is as molecular targets for abrogating the G(2) DNA damage checkpoint arrest, a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.

Apoptosis↗
PubMed2004

RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine.

Ribonucleotide reductase is emerging as an important determinant of gemcitabine chemoresistance in human cancers. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with gemcitabine chemoresistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances gemcitabine-induced cytotoxicity in vitro. We demonstrate the ability of systemically administered RRM2 siRNA to suppress tumoral RRM2 expression in an orthotopic xenograft model of pancreatic adenocarcinoma. Synergism between RRM2 siRNA and gemcitabine results in markedly suppressed tumor growth, increased tumor apoptosis and inhibition of metastasis. Our findings confirm the importance of RRM2 in pancreatic adenocarcinoma gemcitabine chemoresistance. This is the first demonstration that systemic delivery of siRNA-based therapy can enhance the efficacy of an anticancer nucleoside analog.

Adenocarcinoma↗
PubMed2005

Interfering with the brain: use of RNA interference for understanding the pathophysiology of psychiatric and neurological disorders.

Psychiatric and neurological disorders are among the most complex, poorly understood, and debilitating diseases in medicine. The burgeoning advances in functional genomic technologies have led to the identification of a vast number of novel genes that are potentially implicated in the pathophysiology of such disorders. However, many of these candidate genes have not yet been functionalized and require validation in vivo. Traditionally, abrogating gene function is one of the primary means of examining the physiological significance of a given gene product. Several methods have been developed for gene ablation or knockdown, however, with limited levels of success. The recent discovery of RNA interference (RNAi), as a highly efficient method for gene knockdown, has been one of the major breakthroughs in molecular medicine. In vivo application of RNAi is further demonstrating the promise of this technology. Recent efforts have focused on applying RNAi-based knockdown to understand the genes implicated in neuropsychiatric disorders. However, the greatest challenge with this approach is translating the success of RNAi from mammalian cell cultures to the brain in animal models of disease and, subsequently, in patients. In this review, we describe the various methods that are being developed to deliver RNAi into the brain for down-regulating gene expression and subsequent phenotyping of genes in vivo. We illustrate the utility of various approaches with a few successful examples and also discuss the potential benefits and pitfalls associated with the use of each delivery approach. Appropriate tailoring of tools that deliver RNAi in the brain may not only aid our understanding of the complex pathophysiology of neuropsychiatric disorders, but may also serve as a valuable therapy for disorders, where there is an immense unmet medical need.

Animals↗
PubMed2003

The sex peptide of Drosophila melanogaster: female post-mating responses analyzed by using RNA interference.

Mating induces profound changes in female insect behavior and physiology. In Drosophila melanogaster, mating causes a reduction in sexual receptivity and an elevation in egg production for at least 5 days. Injection of the seminal fluid sex peptide (SP) induces both responses in virgin females, but only for 1-2 days. The role of SP in eliciting the responses to mating remains to be elucidated. Functional redundancy between seminal fluid components may occur. In addition, mating with spermless males results in brief (1- to 2-day) post-mating responses, indicating either that there is a "sperm effect" or that sperm act as carriers for SP or other seminal fluid components. Here we used RNA interference to suppress SP expression, to determine whether SP is required to elicit full post-mating responses, the magnitude of responses due to other seminal fluid components, and whether SP accounts for the "sperm effect." Receptivity was higher and egg production lower in females mated to SP knock-down males than in controls. Comparison with virgins showed that the responses were brief. SP is therefore required for normal magnitude and persistence of postmating responses. Sperm transfer and use were normal in mates of SP knock-down males, yet their post-mating responses were briefer than after normal matings, and similar to those reported in mates of spermless son-of-tudor males. The prolonged "sperm effect" on female receptivity and egg production is therefore entirely attributable to SP, but sperm are necessary for its occurrence.

Animals↗
PubMed2004

Selective degradation of cyclin B1 mRNA in rat oocytes by RNA interference (RNAi).

Cyclic adenosine monophosphate (cAMP) keeps oocytes in meiotic arrest, thereby preventing activation of the key regulators of meiosis, p34cdc2/cyclin B1, (known as maturation-promoting factor (MPF)) and Erk 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. The activity of MAPK in oocytes is upregulated by Mos. We previously demonstrated that Mos translation in rat oocytes is negatively regulated by a PKA-mediated cAMP action, which inhibits c-mos mRNA polyadenylation and is associated with the suppression of p34 cdc2 kinase. The goal of the present study was to provide definitive evidence that Mos translation is subjected to MPF regulation. In order to inhibit MPF activity, we employed the double-stranded (ds) RNA interference (RNAi) of gene expression. We demonstrated that the introduction of cyclin B1 dsRNA into rat oocytes selectively depleted the corresponding mRNA, further ablating its protein product. These oocytes, which exhibit low MPF activity, failed to elongate the c-mos mRNA poly(A) tail, did not accumulate Mos and were unable to activate MAPK. We conclude that an active MPF in rat oocytes is necessary for c-mos mRNA polyadenylation and Mos translation.

Animals↗
PubMed2003

Electroporation and RNA interference in the rodent retina in vivo and in vitro.

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

Animals↗
PubMed2006

RNA interference of the Arabidopsis putative transcription factor TCP16 gene results in abortion of early pollen development.

Pollen development is a fundamental and essential biological process in seed plants. Pollen mother cells generated in anthers undergo meiosis, which gives rise to haploid microspores. The haploid cells then develop into mature pollen grains through two mitotic cell divisions. Although several sporophytic and gametophytic mutations affecting male gametogenesis have been identified and analyzed, little is known about the underlying molecular mechanism. In this study, we investigated the function of the TCP16 gene, which encodes a putative transcription factor. Expression analysis of the promoter::GUS fusion gene revealed that TCP16 transcription occurred predominantly in developing microspores. GUS expression began at the tetrad stage and markedly increased in an early unicellular stage. Transgenic plants harboring a TCP16 RNA interference (RNAi) construct generated equal amounts of normal and abnormal pollen grains. The abnormal pollen grains exhibited morphological abnormality and degeneration of genomic DNA. The defective phenotype of the RNAi plants was first detectable at the middle of the unicellular stage. Our results therefore suggest that TCP16, a putative transcription factor, plays a crucial role in early processes in pollen development.

Arabidopsis↗
PubMed2006

Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach.

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

Adaptor Proteins, Signal Transducing↗