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  1. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  2. [Progress of RNA interference mechanism].

    PubMed

    Yan, Fei; Cheng, Zhuo-Min

    2005-01-01

    RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.

  3. RNA interference and antiviral therapy

    PubMed Central

    Ma, Yan; Chan, Chu-Yan; He, Ming-Liang

    2007-01-01

    RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed. PMID:17876887

  4. Multimodality Imaging of RNA Interference

    PubMed Central

    Nayak, Tapas R.; Krasteva, Lazura K.; Cai, Weibo

    2013-01-01

    The discovery of small interfering RNAs (siRNAs) and their potential to knock down virtually any gene of interest has ushered in a new era of RNA interference (RNAi). Clinical use of RNAi faces severe limitations due to inefficiency delivery of siRNA or short hairpin RNA (shRNA). Many molecular imaging techniques have been adopted in RNAi-related research for evaluation of siRNA/shRNA delivery, biodistribution, pharmacokinetics, and the therapeutic effect. In this review article, we summarize the current status of in vivo imaging of RNAi. The molecular imaging techniques that have been employed include bioluminescence/fluorescence imaging, magnetic resonance imaging/spectroscopy, positron emission tomography, single-photon emission computed tomography, and various combinations of these techniques. Further development of non-invasive imaging strategies for RNAi, not only focusing on the delivery of siRNA/shRNA but also the therapeutic efficacy, is critical for future clinical translation. Rigorous validation will be needed to confirm that biodistribution of the carrier is correlated with that of siRNA/shRNA, since imaging only detects the label (e.g. radioisotopes) but not the gene or carrier themselves. It is also essential to develop multimodality imaging approaches for realizing the full potential of therapeutic RNAi, as no single imaging modality may be sufficient to simultaneously monitor both the gene delivery and silencing effect of RNAi. PMID:23745567

  5. RNA Interference for Antimetastatic Therapy.

    PubMed

    Dahlmann, Mathias; Stein, Ulrike

    2015-01-01

    The suppression of genes involved in tumor progression, metastasis formation, or therapy resistance by RNA interference is a promising tool to treat cancer disease. Efficient delivery of interfering molecules and their sustained presence in tumor cells are required for therapeutic success. This chapter describes a method of systemic application of shRNA expression plasmid via tail vein injection in xenograft mice, causing the sustained reduction of target gene expression in the primary tumor. By choosing S100A4 as a metastasis driving target gene, this therapeutic approach restricted the formation of distant colorectal cancer metastases after intrasplenic transplantation. In vivo imaging of bioluminescent cancer cells allows the monitoring of tumor growth and metastasis formation over time. End point analysis of the trial included scoring of the metastatic burden and the quantification of target gene expression in the tumor. Average S100A4 expression in tumor tissues was reduced by 30 %, causing a 70 % decrease of liver metastases. PMID:26072407

  6. Gene expression: RNA interference in adult mice

    NASA Astrophysics Data System (ADS)

    McCaffrey, Anton P.; Meuse, Leonard; Pham, Thu-Thao T.; Conklin, Douglas S.; Hannon, Gregory J.; Kay, Mark A.

    2002-07-01

    RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

  7. Ethical Perspectives on RNA Interference Therapeutics

    PubMed Central

    Ebbesen, Mette; Jensen, Thomas G.; Andersen, Svend; Pedersen, Finn Skou

    2008-01-01

    RNA interference is a mechanism for controlling normal gene expression which has recently begun to be employed as a potential therapeutic agent for a wide range of disorders, including cancer, infectious diseases and metabolic disorders. Clinical trials with RNA interference have begun. However, challenges such as off-target effects, toxicity and safe delivery methods have to be overcome before RNA interference can be considered as a conventional drug. So, if RNA interference is to be used therapeutically, we should perform a risk-benefit analysis. It is ethically relevant to perform a risk-benefit analysis since ethical obligations about not inflicting harm and promoting good are generally accepted. But the ethical issues in RNA interference therapeutics not only include a risk-benefit analysis, but also considerations about respecting the autonomy of the patient and considerations about justice with regard to the inclusion criteria for participation in clinical trials and health care allocation. RNA interference is considered a new and promising therapeutic approach, but the ethical issues of this method have not been greatly discussed, so this article analyses these issues using the bioethical theory of principles of the American bioethicists, Tom L. Beauchamp and James F. Childress. PMID:18612370

  8. Generation of siRNA Nanosheets for Efficient RNA Interference

    NASA Astrophysics Data System (ADS)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  9. Generation of siRNA Nanosheets for Efficient RNA Interference

    PubMed Central

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-01-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances. PMID:27120975

  10. Generation of siRNA Nanosheets for Efficient RNA Interference.

    PubMed

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-01-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances. PMID:27120975

  11. Transgenic inhibitors of RNA interference in Drosophila.

    PubMed

    Chou, Yu-ting; Tam, Bergin; Linay, Fabien; Lai, Eric C

    2007-01-01

    RNA silencing functions as an adaptive antiviral defense in both plants and animals. In turn, viruses commonly encode suppressors of RNA silencing, which enable them to mount productive infection. These inhibitor proteins may be exploited as reagents with which to probe mechanisms and functions of RNA silencing pathways. In this report, we describe transgenic Drosophila strains that allow inducible expression of the viral RNA silencing inhibitors Flock House virus-B2, Nodamura virus-B2, vaccinia virus-E3L, influenza A virus-NS1 and tombusvirus P19. Some of these, especially the B2 proteins, are effective transgenic inhibitors of double strand RNA-induced gene silencing in flies. On the other hand, none of them is effective against the Drosophila microRNA pathway. Their functional selectivity makes these viral silencing proteins useful reagents with which to study biological functions of the Drosophila RNA interference pathway.

  12. RNA interference: genetic wand and genetic watchdog.

    PubMed

    Bosher, J M; Labouesse, M

    2000-02-01

    In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi. PMID:10655601

  13. Symbiont-mediated RNA interference in insects.

    PubMed

    Whitten, Miranda M A; Facey, Paul D; Del Sol, Ricardo; Fernández-Martínez, Lorena T; Evans, Meirwyn C; Mitchell, Jacob J; Bodger, Owen G; Dyson, Paul J

    2016-02-24

    RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. PMID:26911963

  14. Symbiont-mediated RNA interference in insects

    PubMed Central

    Whitten, Miranda M. A.; Facey, Paul D.; Del Sol, Ricardo; Fernández-Martínez, Lorena T.; Evans, Meirwyn C.; Mitchell, Jacob J.; Bodger, Owen G.

    2016-01-01

    RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. PMID:26911963

  15. RNA Interference: Biology, Mechanism, and Applications

    PubMed Central

    Agrawal, Neema; Dasaradhi, P. V. N.; Mohmmed, Asif; Malhotra, Pawan; Bhatnagar, Raj K.; Mukherjee, Sunil K.

    2003-01-01

    Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. PMID:14665679

  16. Inhibition of Henipavirus infection by RNA interference.

    PubMed

    Mungall, Bruce A; Schopman, Nick C T; Lambeth, Luke S; Doran, Tim J

    2008-12-01

    Nipah virus (NiV) and Hendra virus (HeV) are recently emerged zoonotic paramyxoviruses exclusively grouped within a new genus, Henipavirus. These viruses cause fatal disease in a wide range of species, including humans. Both NiV and HeV have continued to re-emerge sporadically in Bangladesh and Australia, respectively. There are currently no therapeutics or vaccines available to treat Henipavirus infection and both are classified as BSL4 pathogens. RNA interference (RNAi) is a process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. Small interfering RNAs (siRNAs) mediate RNAi by inhibiting gene expression of homologous mRNA and our preliminary studies suggest RNAi may be a useful approach to developing novel therapies for these highly lethal pathogens. Eight NiV siRNA molecules (four L and four N gene specific), two HeV N gene specific, and two non-specific control siRNA molecules were designed and tested for their ability to inhibit a henipavirus minigenome replication system (which does not require the use of live virus) in addition to live virus infections in vitro. In the minigenome assay three out of the four siRNAs that targeted the L gene of NiV effectively inhibited replication. In contrast, only NiV N gene siRNAs were effective in reducing live NiV replication, suggesting inhibition of early, abundantly expressed gene transcripts may be more effective than later, less abundant transcripts. Additionally, some of the siRNAs effective against NiV infection were only partially effective inhibitors of HeV infection. An inverse correlation between the number of nucleotide mismatches and the efficacy of siRNA inhibition was observed. The demonstration that RNAi effectively inhibits henipavirus replication in vitro, is a novel approach and may provide an effective therapy for these highly lethal, zoonotic pathogens. PMID:18687361

  17. Hepatic RNA Interference: Delivery by Synthetic Vectors

    PubMed Central

    Haynes, Matthew; Huang, Leaf

    2013-01-01

    Though the pharmaceutical industry’s infatuation with the therapeutic potential of RNA interference (RNAi) technology has finally come down from its initial lofty levels,[1] hope is by no means lost for the once-burgeoning enterprise, as recent clinical trials are beginning to show efficacy in areas ranging from amyloidosis to hypercholesterolemia to muscular dystrophy. With such resurgence comes a more informed perspective on the needs of such therapeutics: a renewed focus on true RNA drug development, and a desire for enhanced site-specific delivery.[2] In this review, we will discuss the latter with regard to hepatic targeting by synthetic vectors, covering the implications of organ and cellular physiology on conjugate structure, particle morphology, and active targeting. In presenting efficacy in a variety of disease models, we emphasize as well the extraordinary degree to which synthetic formulation improves upon and coordinates efforts with oligonucleotide development. Such advances in the understanding of and the technology behind RNAi have the potential to finally stabilize the long-term prospects RNA therapeutic development. PMID:24678447

  18. Role of RNA interference in plant improvement

    NASA Astrophysics Data System (ADS)

    Jagtap, Umesh Balkrishna; Gurav, Ranjit Gajanan; Bapat, Vishwas Anant

    2011-06-01

    Research to alter crops for their better performance involving modern technology is underway in numerous plants, and achievements in transgenic plants are impacting crop improvements in unparalleled ways. Striking progress has been made using genetic engineering technology over the past two decades in manipulating genes from diverse and exotic sources, and inserting them into crop plants for inducing desirable characteristics. RNA interference (RNAi) has recently been identified as a natural mechanism for regulation of gene expression in all higher organisms from plants to humans and promises greater accuracy and precision to plant improvement. The expression of any gene can be down-regulated in a highly explicit manner exclusive of affecting the expression of any other gene by using RNAi technologies. Additional research in this field has been focused on a number of other areas including microRNAs, hairpin RNA, and promoter methylation. Manipulating new RNAi pathways, which generate small RNA molecules to amend gene expression in crops, can produce new quality traits and having better potentiality of protection against abiotic and biotic stresses. Nutritional improvement, change in morphology, or enhanced secondary metabolite synthesis are some of the other advantages of RNAi technology. In addition to its roles in regulating gene expression, RNAi is also used as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability. Even though much advancement has been made on the field of RNAi over the preceding few years, the full prospective of RNAi for crop improvement remains to be fully realized. The intricacy of RNAi pathway, the molecular machineries, and how it relates to plant development are still to be explained.

  19. Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

    PubMed Central

    2015-01-01

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. PMID:25521794

  20. Bringing RNA Interference (RNAi) into the High School Classroom

    ERIC Educational Resources Information Center

    Sengupta, Sibani

    2013-01-01

    RNA interference (abbreviated RNAi) is a relatively new discovery in the field of mechanisms that serve to regulate gene expression (a.k.a. protein synthesis). Gene expression can be regulated at the transcriptional level (mRNA production, processing, or stability) and at the translational level (protein synthesis). RNAi acts in a gene-specific…

  1. Gene silencing in Caenorhabditis elegans by transitive RNA interference

    PubMed Central

    ALDER, MATTHEW N.; DAMES, SHALE; GAUDET, JEFFREY; MANGO, SUSAN E.

    2003-01-01

    When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5′ to 3′ synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi. PMID:12554873

  2. The Fascinating World of RNA Interference

    PubMed Central

    Naqvi, Afsar Raza; Islam, Md. Nazrul; Choudhury, Nirupam Roy; Haq., Qazi Mohd. Rizwanul

    2009-01-01

    Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways. PMID:19173032

  3. RNA interference-mediated intrinsic antiviral immunity in invertebrates.

    PubMed

    Nayak, Arabinda; Tassetto, Michel; Kunitomi, Mark; Andino, Raul

    2013-01-01

    In invertebrates such as insects and nematodes, RNA interference (RNAi) provides RNA-based protection against viruses. This form of immunity restricts viral replication and dissemination from infected cells and viruses, in turn, have evolved evasion mechanisms or RNAi suppressors to counteract host defenses. Recent advances indicate that, in addition to RNAi, other related small RNA pathways contribute to antiviral functions in invertebrates. This has led to a deeper understanding of fundamental aspects of small RNA-based antiviral immunity in invertebrates and its contribution to viral spread and pathogenesis.

  4. Silence of the strands: RNA interference in eukaryotic pathogens.

    PubMed

    Cottrell, Tricia R; Doering, Tamara L

    2003-01-01

    Double-stranded (ds) RNA interference (RNAi) is a recent technological advance that enables researchers to reduce gene expression at the post-transcriptional level. This form of RNA silencing is initiated by dsRNA, expressed in or introduced into a cell of interest, which triggers homology-dependent degradation of the corresponding mRNA. This versatile technique has remarkable promise as a tool for the study of eukaryotic pathogens. Protozoan parasites and pathogenic fungi often resist manipulation using standard molecular genetic approaches. Researchers studying these organisms need flexible molecular tools, particularly to exploit newly sequenced genomes; this review offers a practical guide to establishing RNAi in pathogenic eukaryotes.

  5. Chemical modification: the key to clinical application of RNA interference?

    PubMed Central

    Corey, David R.

    2007-01-01

    RNA interference provides a potent and specific method for controlling gene expression in human cells. To translate this potential into a broad new family of therapeutics, it is necessary to optimize the efficacy of the RNA-based drugs. As discussed in this Review, it might be possible to achieve this optimization using chemical modifications that improve their in vivo stability, cellular delivery, biodistribution, pharmacokinetics, potency, and specificity. PMID:18060019

  6. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    PubMed

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  7. RNA interference as a tool for Alzheimer's disease therapy.

    PubMed

    Orlacchio, Antonio; Bernardi, Giorgio; Orlacchio, Aldo; Martino, Sabata

    2007-11-01

    RNA interference is a biological process that controls gene silencing in all living cells. Targeting the RNA interference system represents a novel therapeutic strategy able to intercede with multiple disease-related genes and to target many neurodegenerative diseases. Recently, the design of small interfering RNA-selective compounds has become more straightforward because of the significant progress made in predictive modeling for new therapeutic approaches. Although in vivo delivery of RNA interference remains a significant obstacle, new data show that RNAi blocks gene function in vivo, suggesting a potential therapeutic approach for humans. Some groups have demonstrated the efficacy of RNAi therapy in Alzheimer's disease. Results, based on animal models, show a down-regulation of the amyloid precursor protein and a consequent reduction of the amyloid-beta peptide accumulation in the brain or the inactivation of beta-secretase (BACE1). Indeed, lentiviral vectors expressing siRNAs targeting BACE1 reduce amyloid production and the neurodegenerative and behavioural deficit in APP transgenic mice. This review highlights recent advances in RNA research and focuses on strengths and weaknesses of RNAi compounds in Alzheimer's disease. PMID:18045220

  8. RNA interference-mediated antiviral defense in insects

    PubMed Central

    Gammon, Don B.; Mello, Craig C.

    2015-01-01

    Small interfering RNA (siRNA)-mediated RNA interference (RNAi) pathways are critical for the detection and inhibition of RNA virus replication in insects. Recent work has also implicated RNAi pathways in the establishment of persistent virus infections and in the control of DNA virus replication. Accumulating evidence suggests that diverse double-stranded RNAs produced by RNA and DNA viruses can trigger RNAi responses yet many viruses have evolved mechanisms to inhibit RNAi defenses. Therefore, an evolutionary arms race exists between host RNAi pathways and invading viral pathogens. Here we review recent advances in our knowledge of how insect RNAi pathways are elicited upon infection, the strategies used by viruses to counter these defenses, and discuss recent evidence implicating Piwi-interacting RNAs in antiviral defense. PMID:26034705

  9. A system for Cre-regulated RNA interference in vivo

    PubMed Central

    Stern, Patrick; Astrof, Sophie; Erkeland, Stefan J.; Schustak, Joshua; Sharp, Phillip A.; Hynes, Richard O.

    2008-01-01

    We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker–shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. PMID:18779577

  10. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA

    PubMed Central

    Wei, Zhiyun; Batagov, Arsen O.; Carter, David R. F.; Krichevsky, Anna M.

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  11. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

    PubMed

    Wei, Zhiyun; Batagov, Arsen O; Carter, David R F; Krichevsky, Anna M

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  12. Ups and downs of RNA interference in parasitic nematodes.

    PubMed

    Britton, Collette; Samarasinghe, Buddhini; Knox, David P

    2012-09-01

    RNA interference (RNAi) is widely used in Caenorhabiditis elegans to identify essential gene function. In parasitic nematodes RNAi has been reported to result in transcript knockdown of some target genes, but not others, thus limiting its use as a potential functional genomics tool. We recently extended work in Haemonchus contortus to examine why only some genes seem to be susceptible to RNAi and to test RNAi effects in vivo. Here we review our findings, which suggest that site of gene expression influences silencing. This most likely reflects limited uptake of dsRNA from the environment, a phenomenon also observed in other free-living nematodes. We discuss new technologies to improve dsRNA delivery, such as nanoparticles being developed for therapeutic siRNA delivery, and methods to monitor RNAi effects. Alternative approaches will be important in progressing the application of RNAi to identify essential gene function in parasitic nematodes. PMID:21854774

  13. RNA interference-based nanosystems for inflammatory bowel disease therapy

    PubMed Central

    Guo, Jian; Jiang, Xiaojing; Gui, Shuangying

    2016-01-01

    Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi) precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA) and microRNA (miRNA). However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. PMID:27789943

  14. Rescue of a Dominant Mutant With RNA Interference

    PubMed Central

    Wu, Yongrui; Messing, Joachim

    2010-01-01

    Maize Mucronate1 is a dominant floury mutant based on a misfolded 16-kDa γ-zein protein. To prove its function, we applied RNA interference (RNAi) as a dominant suppressor of the mutant seed phenotype. A γ-zein RNAi transgene was able to rescue the mutation and restore normal seed phenotype. RNA interference prevents gene expression. In most cases, this is used to study gene function by creating a new phenotype. Here, we use it for the opposite purpose. We use it to reverse the creation of a mutant phenotype by restoring the normal phenotype. In the case of the maize Mucronate1 (Mc1) phenotype, interaction of a misfolded protein with other proteins is believed to be the basis for the Mc1 phenotype. If no misfolded protein is present, we can reverse the mutant to the normal phenotype. One can envision using this approach to study complex traits and in gene therapy. PMID:20876558

  15. RNA Interference Pathways in Fungi: Mechanisms and Functions

    PubMed Central

    Chang, Shwu-Shin; Zhang, Zhenyu; Liu, Yi

    2015-01-01

    RNA interference (RNAi) is a conserved eukaryotic gene regulatory mechanism that uses small non-coding RNAs to mediate post-transcriptional/transcriptional gene silencing. The fission yeast Schizosaccharomyces pombe and the filamentous fungus Neurospora crassa have served as important model systems since the beginning of RNAi studies. Studies in these two organisms and other fungi have contributed significantly to our understanding of the mechanisms and functions of RNAi in eukaryotes. In addition, surprisingly diverse RNAi-mediated processes and small RNA biogenesis pathways have been discovered in fungi. In this review, an overview is given of different fungal RNAi pathways with a focus on their mechanisms and functions. PMID:22746336

  16. Inducible RNA Interference of brlAβ in Aspergillus nidulans▿

    PubMed Central

    Barton, L. M.; Prade, R. A.

    2008-01-01

    An inducible RNA interference (RNAi) construct composed of inverted repeating alcA promoters flanking the developmental regulatory gene brlAβ was tested in Aspergillus nidulans. On inducing medium, the RNAi strains failed to sporulate and lacked brlAα and brlAβ expression. RNAi was specific for brlAβ, but not brlAα, silencing, indicating brlAα regulation by brlAβ. PMID:18757565

  17. Internal guide RNA interactions interfere with Cas9-mediated cleavage.

    PubMed

    Thyme, Summer B; Akhmetova, Laila; Montague, Tessa G; Valen, Eivind; Schier, Alexander F

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation. PMID:27282953

  18. Internal guide RNA interactions interfere with Cas9-mediated cleavage

    PubMed Central

    Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind; Schier, Alexander F.

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. PMID:27282953

  19. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  20. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

    Nikitenko, N. A.; Speiseder, T.; Lam, E.; Rubtsov, P. M.; Tonaeva, Kh. D.; Borzenok, S. A.; Dobner, T.; Prassolov, V. S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

  1. ATP requirements and small interfering RNA structure in the RNA interference pathway.

    PubMed

    Nykänen, A; Haley, B; Zamore, P D

    2001-11-01

    We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

  2. Self-assembled RNA interference microsponges for efficient siRNA delivery.

    PubMed

    Lee, Jong Bum; Hong, Jinkee; Bonner, Daniel K; Poon, Zhiyong; Hammond, Paula T

    2012-04-01

    The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell's RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

  3. Self-assembled RNA interference microsponges for efficient siRNA delivery

    NASA Astrophysics Data System (ADS)

    Lee, Jong Bum; Hong, Jinkee; Bonner, Daniel K.; Poon, Zhiyong; Hammond, Paula T.

    2012-04-01

    The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

  4. Inducing RNA interference in the arbovirus vector, Culicoides sonorensis.

    PubMed

    Mills, M K; Nayduch, D; Michel, K

    2015-02-01

    Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic haemorrhagic disease, bluetongue and most likely Schmallenberg, which cause significant economic burdens worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative small interfering RNA pathway member orthologues, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to perform RNA silencing. Injection of control double-stranded RNA targeting green fluorescent protein (dsGFP), into the haemocoel of 2-3-day-old adult female midges resulted in survival curves that support virus transmission. dsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) orthologue resulted in a 40% decrease of transcript levels and 73% shorter median survivals as compared with dsGFP-injected controls. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.

  5. Inhibition of Tulane Virus Replication in vitro with RNA Interference

    PubMed Central

    Fan, Qiang; Wei, Chao; Xia, Ming; Jiang, Xi

    2012-01-01

    RNA interference (RNAi), a conserved mechanism triggered by small interfering RNA (siRNA), has been used for suppressing gene expression through RNA degradation. The replication of caliciviruses (CVs) with RNAi was studied using the Tulane virus (TV) as a model. Five siRNAs targeting the non-structural, the major (VP1) and minor (VP2) structural genes of the TV were developed and the viruses were quantified using qPCR and TCID50 assay. Treatment of the cells with siRNA 4 hours before viral inoculation significantly reduced viral titer by up to 2.6 logs and dramatically decreased viral RNA copy numbers and viral titers 48 hours post infection in four of the five siRNAs studied. The results were confirmed by Western blot, in which the major structural protein VP1 was markedly reduced in both the cells and the culture medium. Two small protein bands of the S and P domains of the viral capsid protein were also detected in the cell lysates, although their role in viral replication remains unknown. Since the TV shares many biological properties with human noroviruses (NoVs), the successful demonstration of RNAi in TV replication would provide valuable information in control of acute gastroenteritis caused by human NoVs. PMID:23154881

  6. High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

    PubMed Central

    2011-01-01

    RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology. PMID:22018332

  7. Who Watches the Watchmen: Roles of RNA Modifications in the RNA Interference Pathway

    PubMed Central

    Xhemalce, Blerta

    2016-01-01

    RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA “epigenetic” marks. RNAs can be modified on many sites, including 5′ and 3′ ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that “write” and “erase” them as targets for therapeutic drug development. PMID:27441695

  8. Who Watches the Watchmen: Roles of RNA Modifications in the RNA Interference Pathway.

    PubMed

    Shelton, Samantha B; Reinsborough, Calder; Xhemalce, Blerta

    2016-07-01

    RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA "epigenetic" marks. RNAs can be modified on many sites, including 5' and 3' ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that "write" and "erase" them as targets for therapeutic drug development.

  9. RNA Interference in Moths: Mechanisms, Applications, and Progress

    PubMed Central

    Xu, Jin; Wang, Xia-Fei; Chen, Peng; Liu, Fang-Tao; Zheng, Shuai-Chao; Ye, Hui; Mo, Ming-He

    2016-01-01

    The vast majority of lepidopterans, about 90%, are moths. Some moths, particularly their caterpillars, are major agricultural and forestry pests in many parts of the world. However, some other members of moths, such as the silkworm Bombyx mori, are famous for their economic value. Fire et al. in 1998 initially found that exogenous double-stranded RNA (dsRNA) can silence the homolog endogenous mRNA in organisms, which is called RNA interference (RNAi). Soon after, the RNAi technique proved to be very promising not only in gene function determination but also in pest control. However, later studies demonstrate that performing RNAi in moths is not as straightforward as shown in other insect taxa. Nevertheless, since 2007, especially after 2010, an increasing number of reports have been published that describe successful RNAi experiments in different moth species either on gene function analysis or on pest management exploration. So far, more than 100 peer-reviewed papers have reported successful RNAi experiments in moths, covering 10 families and 25 species. By using classic and novel dsRNA delivery methods, these studies effectively silence the expression of various target genes and determine their function in larval development, reproduction, immunology, resistance against chemicals, and other biological processes. In addition, a number of laboratory and field trials have demonstrated that RNAi is also a potential strategy for moth pest management. In this review, therefore, we summarize and discuss the mechanisms and applications of the RNAi technique in moths by focusing on recent progresses. PMID:27775569

  10. Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

    PubMed Central

    Tomoyasu, Yoshinori

    2014-01-01

    The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting. PMID:25350485

  11. Larval RNA interference in the red flour beetle, Tribolium castaneum.

    PubMed

    Linz, David M; Clark-Hachtel, Courtney M; Borràs-Castells, Ferran; Tomoyasu, Yoshinori

    2014-10-13

    The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle's body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

  12. Abasic pivot substitution harnesses target specificity of RNA interference

    PubMed Central

    Lee, Hye-Sook; Seok, Heeyoung; Lee, Dong Ha; Ham, Juyoung; Lee, Wooje; Youm, Emilia Moonkyung; Yoo, Jin Seon; Lee, Yong-Seung; Jang, Eun-Sook; Chi, Sung Wook

    2015-01-01

    Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA–target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼80–100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications. PMID:26679372

  13. Optimizing RNA interference for application in mammalian cells.

    PubMed Central

    Medema, René H

    2004-01-01

    Over the last 2 years, the scientific community has rapidly embraced novel technologies that allow gene silencing in vertebrates. Ease of application, cost effectiveness and the possibilities for genome-wide reverse genetics have quickly turned this approach into a widely accepted, almost mandatory asset for a self-respecting laboratory in life sciences. This review discusses some of the recent technological developments that allow the application of RNAi (RNA interference) in mammalian cells. In addition, the advantages of applying RNAi to study cell cycle events and the emerging approaches to perform mutational analysis by complementation in mammalian cells are evaluated. In addition, common pitfalls and drawbacks of RNAi will be reviewed, as well as the possible ways to get around these shortcomings of gene silencing by small interfering RNA. PMID:15056071

  14. Sequence-non-specific effects of RNA interference triggers and microRNA regulators

    PubMed Central

    Olejniczak, Marta; Galka, Paulina; Krzyzosiak, Wlodzimierz J.

    2010-01-01

    RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells. PMID:19843612

  15. Biological mechanisms determining the success of RNA interference in insects.

    PubMed

    Wynant, Niels; Santos, Dulce; Vanden Broeck, Jozef

    2014-01-01

    Insects constitute the largest group of animals on this planet, having a huge impact on our environment, as well as on our quality of life. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded (ds)RNA fragments. This process not only forms the basis of a widely used reverse genetics research method in many different eukaryotes but also holds great promise to contribute to the species-specific control of agricultural pests and to combat viral infections in beneficial and disease vectoring insects. However, in many economically important insect species, such as flies, mosquitoes, and caterpillars, systemic delivery of naked dsRNA does not trigger effective gene silencing. Although many components of the RNAi pathway have initially been deciphered in the fruit fly, Drosophila melanogaster, it will be of major importance to investigate this process in a wider variety of species, including dsRNA-sensitive insects such as locusts and beetles, to elucidate the factors responsible for the remarkable variability in RNAi efficiency, as observed in different insects. In this chapter, we review the current knowledge on the RNAi pathway, as well as the most recent insights into the mechanisms that might determine successful RNAi in insects.

  16. Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

    PubMed

    Schnettler, Esther; Sterken, Mark G; Leung, Jason Y; Metz, Stefan W; Geertsema, Corinne; Goldbach, Rob W; Vlak, Just M; Kohl, Alain; Khromykh, Alexander A; Pijlman, Gorben P

    2012-12-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  17. Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

    PubMed Central

    Schnettler, Esther; Sterken, Mark G.; Leung, Jason Y.; Metz, Stefan W.; Geertsema, Corinne; Goldbach, Rob W.; Vlak, Just M.; Kohl, Alain

    2012-01-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  18. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

    PubMed

    Chung, Kwan-Ho; Hart, Christopher C; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D; Vojtek, Anne B; Turner, David L

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

  19. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    PubMed Central

    Chung, Kwan-Ho; Hart, Christopher C.; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D.; Vojtek, Anne B.; Turner, David L.

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications. PMID:16614444

  20. RNA interference targets arbovirus replication in Culicoides cells.

    PubMed

    Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

    2013-03-01

    Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

  1. RNA Interference Targets Arbovirus Replication in Culicoides Cells

    PubMed Central

    Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E.; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M.; Palmarini, Massimo

    2013-01-01

    Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses. PMID:23269795

  2. Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

    PubMed

    Palmer, Adam C; Egan, J Barry; Shearwin, Keith E

    2011-01-01

    Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

  3. RNA interference tools for the western flower thrips, Frankliniella occidentalis.

    PubMed

    Badillo-Vargas, Ismael E; Rotenberg, Dorith; Schneweis, Brandi A; Whitfield, Anna E

    2015-05-01

    The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips-tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3 days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced

  4. Suppression of prion protein in livestock by RNA interference.

    PubMed

    Golding, Michael C; Long, Charles R; Carmell, Michelle A; Hannon, Gregory J; Westhusin, Mark E

    2006-04-01

    Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After in vitro fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models.

  5. Testing the efficacy of RNA interference constructs in Aspergillus fumigatus.

    PubMed

    Henry, Christine; Mouyna, Isabelle; Latgé, Jean-Paul

    2007-04-01

    We recently developed a silencing vector in Aspergillus fumigatus which carries a hygromycin resistance marker and a transcriptional unit for hairpin RNA expression under the control of the inducible glucoamylase promoter (pGla) (Mouyna et al. in FEMS Microbiol Lett 237:317-324, 2004). We showed previously that this vector can be used for the RNA interference application of two genes ALB1 and FKS1 of which reduced mRNA levels occurred for both, with phenotypic consequences resembling disruptions of genes involved in melanin (ALB1) and beta(1-3)glucan biosynthesis (FKS1). We reported here the silencing of KRE6 and CRH1, two other genes putatively involved in cell wall biosynthesis using a similar construction under the control of the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (pgpdA). Silencing of the expression of these two genes was obtained. Further analysis of the transformants showed however that (1) a 100% loss of expression was never achieved for all genes tested (2) the vector used for RNAi is lost or modified over successive transfers resulting in an inhibition of the silencing. These disadvantages of RNAi indicate that classical gene disruption by gene replacement remains the most efficient method for a molecular analysis of gene function in A. fumigatus. PMID:17273823

  6. Emerging strategies for RNA interference (RNAi) applications in insects

    PubMed Central

    Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

    2015-01-01

    RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response. PMID:25424593

  7. Emerging strategies for RNA interference (RNAi) applications in insects.

    PubMed

    Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

    2015-01-01

    RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.

  8. Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

    PubMed Central

    Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel

    2013-01-01

    RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. PMID:23344055

  9. Nuclear Outsourcing of RNA Interference Components to Human Mitochondria

    PubMed Central

    Bandiera, Simonetta; Rüberg, Silvia; Girard, Muriel; Cagnard, Nicolas; Hanein, Sylvain; Chrétien, Dominique; Munnich, Arnold; Lyonnet, Stanislas; Henrion-Caude, Alexandra

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular

  10. A kinetic model for RNA-interference of focal adhesions

    PubMed Central

    2013-01-01

    Background Focal adhesions are integrin-based cell-matrix contacts that transduce and integrate mechanical and biochemical cues from the environment. They develop from smaller and more numerous focal complexes under the influence of mechanical force and are key elements for many physiological and disease-related processes, including wound healing and metastasis. More than 150 different proteins localize to focal adhesions and have been systematically classified in the adhesome project (http://www.adhesome.org). First RNAi-screens have been performed for focal adhesions and the effect of knockdown of many of these components on the number, size, shape and location of focal adhesions has been reported. Results We have developed a kinetic model for RNA interference of focal adhesions which represents some of its main elements: a spatially layered structure, signaling through the small GTPases Rac and Rho, and maturation from focal complexes to focal adhesions under force. The response to force is described by two complementary scenarios corresponding to slip and catch bond behavior, respectively. Using estimated and literature values for the model parameters, three time scales of the dynamics of RNAi-influenced focal adhesions are identified: a sub-minute time scale for the assembly of focal complexes, a sub-hour time scale for the maturation to focal adhesions, and a time scale of days that controls the siRNA-mediated knockdown. Our model shows bistability between states dominated by focal complexes and focal adhesions, respectively. Catch bonding strongly extends the range of stability of the state dominated by focal adhesions. A sensitivity analysis predicts that knockdown of focal adhesion components is more efficient for focal adhesions with slip bonds or if the system is in a state dominated by focal complexes. Knockdown of Rho leads to an increase of focal complexes. Conclusions The suggested model provides a kinetic description of the effect of RNA-interference

  11. Applicability of RNA interference in cancer therapy: Current status.

    PubMed

    Maduri, S

    2015-01-01

    Cancer is a manifestation of dysregulated gene function arising from a complex interplay of oncogenes and tumor suppressor genes present in our body. Cancer has been constantly chased using various therapies but all in vain as most of them are highly effective only in the early stages of cancer. Recently, RNA interference (RNAi) therapy, a comparatively new entrant is evolving as a promising player in the battle against cancer due to its post-transcriptional gene silencing ability. The most alluring feature of this non-invasive technology lies in its utility in the cancer detection and the cancer treatment at any stage. Once this technology is fully exploited it can bring a whole new era of therapeutics capable of curing cancer at any stage mainly due to its ability to target the vital processes required for cell proliferation such as response to growth factors, nutrient uptake/synthesis, and energy generation. This therapy can also be used to treat stage IV cancer, the most difficult to treat till date, by virtue of its metastasis inhibiting capability. Recent research has also proved that cancer can even be prevented by proper modulation of physiological RNAi pathways and researchers have found that many nutrients, which are a part of routine diet, can effectively modulate these pathways and prevent cancer. Even after having all these advantages the potential of RNAi therapy could not be fully tapped earlier, due to many limitations associated with the administration of RNAi based therapeutics. However, recent advancements in this direction, such as the development of small interfering RNA (siRNA) tolerant to nucleases and the development of non-viral vectors such as cationic liposomes and nanoparticles, can overcome this obstacle and facilitate the clinical use of RNAi based therapeutics in the treatment of cancer. The present review focuses on the current status of RNAi therapeutics and explores their potential as future diagnostics and therapeutics against

  12. Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera).

    PubMed

    Snell, Terry W; Shearer, Tonya L; Smith, Hilary A

    2011-04-01

    RNA interference (RNAi) is a powerful technique for functional genomics, yet no studies have reported its successful application to zooplankton. Many zooplankton, particularly microscopic metazoans of phylum Rotifera, have unique life history traits for which genetic investigation has been limited. In this paper, we report the development of RNAi methods for rotifers, with the exogenous introduction of double-stranded RNA (dsRNA) through the use of a lipofection reagent. Transfection with dsRNA for heat shock protein 90, the membrane-associated progesterone receptor, and mitogen-activated protein kinase significantly increased the proportion of non-reproductive females. Additionally, a fluorescence-based lectin binding assay confirmed the significant suppression of four of six glycosylation enzymes that were targeted with dsRNA. Suppression of mRNA transcripts was confirmed with quantitative PCR. Development of RNAi for rotifers promises to enhance the ability for assessing genetic regulation of features critical to their life history and represents a key step toward functional genomics research in zooplankton.

  13. RNA interference in nematodes and the chance that favored Sydney Brenner

    PubMed Central

    Félix, Marie-Anne

    2008-01-01

    The efficiency of RNA interference varies between different organisms, even among nematodes. A recent report of successful RNA interference in the nematode Panagrolaimus superbus in BMC Molecular Biology has implications for the comparative study of the functional genomics of nematode species, and prompts reflections on the choice of Caenorhabditis elegans as a model organism. PMID:19014674

  14. Metabolic engineering of cottonseed oil biosynthesis pathway via RNA interference.

    PubMed

    Xu, Zhongping; Li, Jingwen; Guo, Xiaoping; Jin, Shuangxia; Zhang, Xianlong

    2016-01-01

    Cottonseed oil is recognized as an important oil in food industry for its unique characters: low flavor reversion and the high level of antioxidants (VitaminE) as well as unsaturated fatty acid. However, the cottonseed oil content of cultivated cotton (Gossypium hirsutum) is only around 20%. In this study, we modified the accumulation of oils by the down-regulation of phosphoenolpyruvate carboxylase 1 (GhPEPC1) via RNA interference in transgenic cotton plants. The qRT-PCR and enzyme activity assay revealed that the transcription and expression of GhPEPC1 was dramatically down-regulated in transgenic lines. Consequently, the cottonseed oil content in several transgenic lines showed a significant (P < 0.01) increase (up to 16.7%) without obvious phenotypic changes under filed condition when compared to the control plants. In order to elucidate the molecular mechanism of GhPEPC1 in the regulation of seed oil content, we quantified the expression of the carbon metabolism related genes of transgenic GhPEPC1 RNAi lines by transcriptome analysis. This analysis revealed the decrease of GhPEPC1 expression led to the increase expression of triacylglycerol biosynthesis-related genes, which eventually contributed to the lipid biosynthesis in cotton. This result provides a valuable information for cottonseed oil biosynthesis pathway and shows the potential of creating high cottonseed oil germplasm by RNAi strategy for cotton breeding. PMID:27620452

  15. Antiviral Stratagems Against HIV-1 Using RNA Interference (RNAi) Technology

    PubMed Central

    Vlachakis, Dimitrios; Tsiliki, Georgia; Pavlopoulou, Athanasia; Roubelakis, Maria G.; Champeris Tsaniras, Spyridon; Kossida, Sophia

    2013-01-01

    The versatility of human immunodeficiency virus (HIV)-1 and its evolutionary potential to elude antiretroviral agents by mutating may be its most invincible weapon. Viruses, including HIV, in order to adapt and survive in their environment evolve at extremely fast rates. Given that conventional approaches which have been applied against HIV have failed, novel and more promising approaches must be employed. Recent studies advocate RNA interference (RNAi) as a promising therapeutic tool against HIV. In this regard, targeting multiple HIV sites in the context of a combinatorial RNAi-based approach may efficiently stop viral propagation at an early stage. Moreover, large high-throughput RNAi screens are widely used in the fields of drug development and reverse genetics. Computer-based algorithms, bioinformatics, and biostatistical approaches have been employed in traditional medicinal chemistry discovery protocols for low molecular weight compounds. However, the diversity and complexity of RNAi screens cannot be efficiently addressed by these outdated approaches. Herein, a series of novel workflows for both wet- and dry-lab strategies are presented in an effort to provide an updated review of state-of-the-art RNAi technologies, which may enable adequate progress in the fight against the HIV-1 virus. PMID:23761954

  16. Harnessing RNA interference for the treatment of viral infections.

    PubMed

    Arbuthnot, Patrick

    2010-01-01

    Exploiting the RNA interference (RNAi) pathway to inhibit viral gene expression has become an active field of research. The approach has potential for therapeutic application and several viruses are susceptible to RNAi-mediated knockdown. Differences in the characteristics of individual viruses require that viral gene silencing be tailored to specific infections. Important considerations are viral tissue tropism, acute or chronic nature of the infection and the efficiency with which antiviral sequences can be delivered to affected tissue. Both synthetic short interfering RNAs (siRNAs) and expressed RNAi activators are being developed for viral therapy. The sustained silencing of expressed antiviral sequences is useful for countering chronic viral infection. siRNAs, which may be chemically modified to improve specificity and stability, are being developed for knockdown of viruses that cause acute or chronic infections. Preventing viral escape from silencing is important and overcoming this problem using combinatorial RNAi or through silencing of host dependency factors is promising. Although improving delivery efficiency and limiting off-target effects remain obstacles, rapid progress continues to be made in the field and it is likely that the goal of achieving licensed RNAi-based viral therapies will soon be realized. PMID:20697601

  17. RNA interference targeting raptor inhibits proliferation of gastric cancer cells

    SciTech Connect

    Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

    2011-06-10

    Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G{sub 0}/G{sub 1}-phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D{sub 3} and p21{sup Waf1}, which stabilizes cyclin D/cdk4 complex for G{sub 1}-S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

  18. Metabolic engineering of cottonseed oil biosynthesis pathway via RNA interference

    PubMed Central

    Xu, Zhongping; Li, Jingwen; Guo, Xiaoping; Jin, Shuangxia; Zhang, Xianlong

    2016-01-01

    Cottonseed oil is recognized as an important oil in food industry for its unique characters: low flavor reversion and the high level of antioxidants (VitaminE) as well as unsaturated fatty acid. However, the cottonseed oil content of cultivated cotton (Gossypium hirsutum) is only around 20%. In this study, we modified the accumulation of oils by the down-regulation of phosphoenolpyruvate carboxylase 1 (GhPEPC1) via RNA interference in transgenic cotton plants. The qRT-PCR and enzyme activity assay revealed that the transcription and expression of GhPEPC1 was dramatically down-regulated in transgenic lines. Consequently, the cottonseed oil content in several transgenic lines showed a significant (P < 0.01) increase (up to 16.7%) without obvious phenotypic changes under filed condition when compared to the control plants. In order to elucidate the molecular mechanism of GhPEPC1 in the regulation of seed oil content, we quantified the expression of the carbon metabolism related genes of transgenic GhPEPC1 RNAi lines by transcriptome analysis. This analysis revealed the decrease of GhPEPC1 expression led to the increase expression of triacylglycerol biosynthesis-related genes, which eventually contributed to the lipid biosynthesis in cotton. This result provides a valuable information for cottonseed oil biosynthesis pathway and shows the potential of creating high cottonseed oil germplasm by RNAi strategy for cotton breeding. PMID:27620452

  19. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    PubMed

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  20. FOXO regulates RNA interference in Drosophila and protects from RNA virus infection

    PubMed Central

    Spellberg, Michael J.; Marr, Michael T.

    2015-01-01

    Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNA-binding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. Here, we show that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, we find increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. Our study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions. PMID:26553999

  1. Suppression of Bedbug's Reproduction by RNA Interference of Vitellogenin.

    PubMed

    Moriyama, Minoru; Hosokawa, Takahiro; Tanahashi, Masahiko; Nikoh, Naruo; Fukatsu, Takema

    2016-01-01

    Recent resurgence of the bedbug Cimex lectularius is a global problem on the public health. On account of the worldwide rise of insecticide-resistant bedbug populations, exploration of new approaches to the bedbug control and management is anticipated. In this context, gene silencing by RNA interference (RNAi) has been considered for its potential application to pest control and management, because RNAi enables specific suppression of target genes and thus flexible selection of target traits to be disrupted. In this study, in an attempt to develop a control strategy targeting reproduction of the bedbug, we investigated RNAi-mediated gene silencing of vitellogenin (Vg), a major yolk protein precursor essential for oogenesis. From the bedbug transcriptomes, we identified a typical Vg gene and a truncated Vg gene, which were designated as ClVg and ClVg-like, respectively. ClVg gene was highly expressed mainly in the fat body of adult females, which was more than 100 times higher than the expression level of ClVg-like gene, indicating that ClVg gene is the primary functional Vg gene in the bedbug. RNAi-mediated suppression of ClVg gene expression in adult females resulted in drastically reduced egg production, atrophied ovaries, and inflated abdomen due to hypertrophied fat bodies. These phenotypic consequences are expected not only to suppress the bedbug reproduction directly but also to deteriorate its feeding and survival indirectly via behavioral modifications. These results suggest the potential of ClVg gene as a promising target for RNAi-based population management of the bedbug. PMID:27096422

  2. RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA.

    PubMed

    Zhou, Xuguo; Wheeler, Marsha M; Oi, Faith M; Scharf, Michael E

    2008-08-01

    RNA interference (RNAi) represents a breakthrough technology for conducting functional genomics research in non-model organisms and for the highly targeted control of insect pests. This study investigated RNAi via voluntary feeding in the economically important pest termite, Reticulitermes flavipes. We used a high-dose double-stranded (ds) RNA feeding approach to silence two termite genes: one encoding an endogenous digestive cellulase enzyme and the other a caste-regulatory hexamerin storage protein. Contrary to results from previous low-dose studies that examined injection-based RNAi, high-dose silencing of either gene through dsRNA feeding led to significantly reduced group fitness and mortality. Hexamerin silencing in combination with ectopic juvenile hormone treatments additionally led to lethal molting impacts and increased differentiation of presoldier caste phenotypes (a phenotype that is not capable of feeding). These results provide the first examples of insecticidal effects from dsRNA feeding in a termite. Additionally, these results validate a high-throughput bioassay approach for use in (i) termite functional genomics research, and (ii) characterizing target sites of conventional and novel RNAi-based termiticides. PMID:18625404

  3. [The Nobel Prize in Physiology or Medicine for 2006 for the discovery of RNA interference].

    PubMed

    Bernards, R

    2006-12-30

    The Nobel Prize in Physiology or Medicine has been awarded to Andrew Fire and Craig Mello for their discovery of RNA interference, i.e. the suppression of gene activity by double-stranded RNA. Small interfering RNA molecules (siRNAs), notably the antisense strand, recognise and inhibit the corresponding mRNA, thereby silencing the appropriate gene. RNA interference can help to determine the function of genes and may assist in the development ofnew drugs. It may also lead to a better understanding of mechanisms of drug resistance. In addition, siRNAs themselves may prove to have therapeutic value as many diseases are the result of alterations in gene activity.

  4. Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

    PubMed Central

    Fulton, Amy; Peters, Sarah T.; Perkins, Gillian A.; Jarosinski, Keith W.; Damiani, Armando; Brosnahan, Margaret; Buckles, Elizabeth L.; Osterrieder, Nikolaus; Van de Walle, Gerlinde R.

    2009-01-01

    Background Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA's), delivered intranasally, could prove a valuable alternative for infection control. siRNA's against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model. Methodology/Principal Fndings siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection. Conclusions/Significance siRNA treatment has potential for both prevention and early treatment of EHV-1 infections. PMID:19122813

  5. Intergenic transcriptional interference is blocked by RNA polymerase III transcription factor TFIIIB in Saccharomyces cerevisiae.

    PubMed

    Korde, Asawari; Rosselot, Jessica M; Donze, David

    2014-02-01

    The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such "extratranscriptional" activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467-ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins.

  6. Versatile RNA Interference Nanoplatform for Systemic Delivery of RNAs

    PubMed Central

    2015-01-01

    Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells. PMID:24779637

  7. [Perspectives of RNA interference application in the therapy of diseases associated with defects in alternative RNA splicing].

    PubMed

    Wysokiński, Daniel; Błasiak, Janusz

    2012-09-18

    The primary transcript of an eukaryotic gene (pre-mRNA) is composed of coding regions--exons intervened by non-coding introns--which are removed in the RNA splicing process, leading to the formation of mature, intron-free mRNA. Alternative splicing of pre-mRNA is responsible for high complexity of the cellular proteome and expresses effective use of genetic information contained in genomic DNA. Alternative splicing plays important roles in the organism, including apoptosis regulation or development and plasticity of the nervous system. The main role of alternative splicing is differential, dependent on conditions and the cell type, splicing of mRNA, generating diverse transcripts from one gene, and, after the translation, different isoforms of a particular protein. Because of the high complexity of this mechanism, alternative splicing is particularly prone to errors. The perturbations resulting from mutations in the key sequences for splicing regulations are especially harmful. The pathogenesis of numerous diseases results from disturbed alternative RNA splicing, and those include cancers and neurodegenerative disorders. The treatment of these conditions is problematic due to their genetic background and currently RNA interference, which is a common mechanism of eukaryotic gene regulation, is being studied. Initial successes in the attempts of silencing the expression of faulty protein isoforms support the idea of using RNA interference in targeting disease related to disturbances in alternative splicing of RNA.

  8. Autoregulation of Inducible Nitric Oxide Synthase Expression by RNA Interference Provides Neuroprotection in Neonatal Rats

    PubMed Central

    Wang, Zhi; Feng, Chenzhuo; Zhao, Huijuan; Ren, Xiaoyan; Peng, Shuling; Zuo, Zhiyi

    2015-01-01

    We have shown that autoregulation of gene expression by RNA interference is achievable in cell cultures. To determine whether this novel concept could be used to produce neuroprotection under in vivo condition, postnatal day (PND) 3 rats received intracerebroventricular injection of lentivirus that carried or did not carry code for short hairpin RNA (shRNA) of inducible nitric oxide synthase (iNOS). The expression of this shRNA was controlled by an iNOS promoter (piNOS-shRNA) or cytomegalovirus promoter (pCMV-shRNA). The rats were subjected to brain hypoxia-ischemia at PND7. Ischemic brain tissues had increased iNOS expression. This increase was attenuated by virus carrying piNOS-shRNA. Virus carrying pCMV-shRNA reduced iNOS to a level that was lower than control. Brain tissue loss and functional impairment after the hypoxia-ischemia were attenuated by the virus carrying piNOS-shRNA but not by pCMV-shRNA. Our results provide proof-of-concept evidence that autoregulation of iNOS expression by RNA interference induces neuroprotection in vivo and that appropriate regulation of gene expression is important. PMID:25767617

  9. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea

    PubMed Central

    Marraffini, Luciano A.; Sontheimer, Erik J.

    2010-01-01

    Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway. PMID:20125085

  10. Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.

    PubMed

    Boutla, A; Delidakis, C; Livadaras, I; Tsagris, M; Tabler, M

    2001-11-13

    Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

  11. Delayed Newcastle disease virus replication using RNA interference to target the nucleoprotein.

    PubMed

    Hutcheson, Jessica M; Susta, Leonardo; Stice, Steven L; Afonso, Claudio L; West, Franklin D

    2015-07-01

    Each year millions of chickens die from Newcastle disease virus (NDV) worldwide leading to severe economic and food losses. Current vaccination campaigns have limitations especially in developing countries, due to elevated costs, need of trained personnel for effective vaccine administration, and functional cold chain network to maintain vaccine viability. These problems have led to heightened interest in producing new antiviral strategies, such as RNA interference (RNAi). RNAi methodology is capable of substantially decreasing viral replication at a cellular level, both in vitro and in vivo. In this study, we utilize microRNA (miRNA)-expressing constructs (a type of RNA interference) in an attempt to target and knockdown five NDV structural RNAs for nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), and large (L) protein genes. Immortalized chicken embryo fibroblast cells (DF-1) that transiently expressed miRNA targeting NP mRNA, showed increased resistance to NDV-induced cytopathic effects, as determined by cell count, relative to the same cells expressing miRNA against alternative NDV proteins. Upon infection with NDV, DF-1 cells constitutively expressing the NP miRNA construct had improved cell survival up to 48 h post infection (h.p.i) and decreased viral yield up to 24 h.p.i. These results suggest that overexpression of the NP miRNA in cells and perhaps live animal may provide resistance to NDV. PMID:26050911

  12. Inhibition of RNA interference and modulation of transposable element expression by cell death in Drosophila.

    PubMed

    Xie, Weiwu; Liang, Chengzhi; Birchler, James A

    2011-08-01

    RNA interference (RNAi) regulates gene expression by sequence-specific destruction of RNA. It acts as a defense mechanism against viruses and represses the expression of transposable elements (TEs) and some endogenous genes. We report that mutations and transgene constructs that condition cell death suppress RNA interference in adjacent cells in Drosophila melanogaster. The reversal of RNAi is effective for both the white (w) eye color gene and green fluorescent protein (GFP), indicating the generality of the inhibition. Antiapoptotic transgenes that reverse cell death will also reverse the inhibition of RNAi. Using GFP and a low level of cell death produced by a heat shock-head involution defective (hs-hid) transgene, the inhibition appears to occur by blocking the conversion of double-stranded RNA (dsRNA) to short interfering RNA (siRNA). We also demonstrate that the mus308 gene and endogenous transposable elements, which are both regularly silenced by RNAi, are increased in expression and accompanied by a reduced level of siRNA, when cell death occurs. The finding that chronic ectopic cell death affects RNAi is critical for an understanding of the application of the technique in basic and applied studies. These results also suggest that developmental perturbations, disease states, or environmental insults that cause ectopic cell death would alter transposon and gene expression patterns in the organism by the inhibition of small RNA silencing processes. PMID:21596898

  13. Satellite RNAs interfere with the function of viral RNA silencing suppressors.

    PubMed

    Shen, Wan-Xia; Au, Phil Chi Khang; Shi, Bu-Jun; Smith, Neil A; Dennis, Elizabeth S; Guo, Hui-Shan; Zhou, Chang-Yong; Wang, Ming-Bo

    2015-01-01

    Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function. PMID:25964791

  14. Powering up the molecular therapy of RNA interference by novel nanoparticles.

    PubMed

    Liao, Wenzhen; Li, Wen; Zhang, Tiantian; Kirberger, Micheal; Liu, Jun; Wang, Pei; Chen, Wei; Wang, Yong

    2016-06-21

    RNA interference technology has been widely applied in biomedical therapy in recent years. A type of small RNA molecule - siRNA could regulate the expression of disease related genes by breaking down the integrity of mRNA with high specificity. However, the low efficiency of siRNA delivery to its target seriously hampered the RNAi therapy. Compared with viral-based delivery systems, non-viral-based nanoparticles are more suitable for disease treatment due to reduced cellular toxicity, higher loading capacity, and better biocompatibility. This review article highlights several nanoparticle-based siRNA delivery systems, including liposomes, cationic solid lipid nanoparticles, reconstituted high density lipoprotein, polymeric nanoparticles, cationic cell penetrating peptides, and inorganic nanoparticles. The molecular mechanism of gene silencing, clinical examples, and the limitations of current technology related to nanomaterial sciences, are also discussed. PMID:27221980

  15. A Simple Laboratory Practical to Illustrate RNA Mediated Gene Interference Using Drosophila Cell Culture

    ERIC Educational Resources Information Center

    Buluwela, Laki; Kamalati, Tahereh; Photiou, Andy; Heathcote, Dean A.; Jones, Michael D.; Ali, Simak

    2010-01-01

    RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode "in vitro"…

  16. RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium species)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium ssp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function ...

  17. How Golden Is Silence? Teaching Undergraduates the Power and Limits of RNA Interference

    ERIC Educational Resources Information Center

    Kuldell, Natalie H.

    2006-01-01

    It is hard and getting harder to strike a satisfying balance in teaching. Time dedicated to student-generated models or ideas is often sacrificed in an effort to "get through the syllabus." I describe a series of RNA interference (RNAi) experiments for undergraduate students that simultaneously explores fundamental concepts in gene regulation,…

  18. RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive ex...

  19. Identification of giant Mimivirus protein functions using RNA interference

    PubMed Central

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases. PMID:25972846

  20. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference.

  1. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference. PMID:27142766

  2. eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference

    NASA Astrophysics Data System (ADS)

    Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak; Song, Huaidong; Papadopoulos, Evangelos; Qi, Hank H.; Jedrychowski, Mark; Güttler, Thomas; Guo, Cuicui; Luna, Rafael E.; Gygi, Steven P.; Huang, Stephen A.; Wagner, Gerhard

    2015-05-01

    MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

  3. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing.

    PubMed

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently known small RNA classes and place them in context of the reconstructed evolutionary history of the RNA interference (RNAi) protein machinery. This synthesis indicates that the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: (1) sense-antisense transcriptional products, (2) genome-encoded, imperfectly complementary hairpin sequences, and (3) larger noncoding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNAi. They were recruited alongside RNaseIII domains and RNA-dependent RNA polymerase (RdRP) domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleocytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  4. Disruption of Spodoptera exigua larval development by silencing chitin synthase gene A with RNA interference.

    PubMed

    Chen, X; Tian, H; Zou, L; Tang, B; Hu, J; Zhang, W

    2008-12-01

    RNA interference (RNAi) is a powerful tool for rapidly analyzing gene functions. However, little is known about the possible use of dsRNA/siRNA as a pest control method. Here, we demonstrate that dsRNA/siRNA can induce the silence of chitin synthase gene A (CHSA), which is an important gene for the growth and development of cuticles and trachea in beet armyworm, Spodoptera exigua. Based on the in vitro RNAi experiments in an insect cell line (Trichoplusia ni High 5), in vivo RNAi was performed by injecting synthesized dsRNA/siRNA into the 4th instar larvae of S. exigua. Significantly lower levels of CHSA transcripts were detected. In addition, the cuticle of these insects was disordered and the epithelial walls of larval trachea did not expand uniformly in injected individuals. Moreover, Injections significantly increased abnormalities relative to control larvae. These results highlighted the possibility of dsRNA/siRNA for gene function studies in lepidopteran insects and future pest control. PMID:18662430

  5. RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

    PubMed

    Goic, Bertsy; Vodovar, Nicolas; Mondotte, Juan A; Monot, Clément; Frangeul, Lionel; Blanc, Hervé; Gausson, Valérie; Vera-Otarola, Jorge; Cristofari, Gael; Saleh, Maria-Carla

    2013-04-01

    How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

  6. The Role of RNA Interference (RNAi) in Arbovirus-Vector Interactions

    PubMed Central

    Blair, Carol D.; Olson, Ken E.

    2015-01-01

    RNA interference (RNAi) was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (ds)RNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (si)RNA, micro (mi)RNA, and Piwi-interacting (pi)RNA pathways. The exogenous (exo-)siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector’s antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed. PMID:25690800

  7. The role of RNA interference (RNAi) in arbovirus-vector interactions.

    PubMed

    Blair, Carol D; Olson, Ken E

    2015-02-17

    RNA interference (RNAi) was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (ds)RNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (si)RNA, micro (mi)RNA, and Piwi-interacting (pi)RNA pathways. The exogenous (exo-)siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector's antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed.

  8. Novel siRNA-loaded Bubble Liposomes with Ultrasound Exposure for RNA Interference

    NASA Astrophysics Data System (ADS)

    Endo-Takahashi, Yoko; Negishi, Yoichi; Suzuki, Ryo; Maruyama, Kazuo; Aramaki, Yukihiko

    2011-09-01

    Recently, we have developed novel polyethyleneglycol (PEG) modified liposomes (Bubble liposomes; BLs) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. We have shown that the combination of BLs and US was also useful for the delivery of siRNA. However, for use in intravenous administration, there is room for improvement in the colocalization of BLs and siRNA in blood vessels and the stability of siRNA. In this study, we have attempted to prepare novel siRNA-loaded BLs (si-BLs) using cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). As a result, siRNA loaded onto the surface of BLs could be observed. Furthermore, siRNA-loaded BLs were stable even in the presence of serum. The specific gene silencing effect caused by transfection with si-BLs and US could be also observed. Thus, si-BLs with US-exposure may be a useful novel transfection method for siRNA delivery to a target tissue or organ via systemic injection.

  9. Applications of RNA interference in cancer therapeutics as a powerful tool for suppressing gene expression.

    PubMed

    He, Song; Zhang, Dechun; Cheng, Fang; Gong, Fanghong; Guo, Yanan

    2009-11-01

    Cancer poses a tremendous therapeutic challenge worldwide, highlighting the critical need for developing novel therapeutics. A promising cancer treatment modality is gene therapy, which is a form of molecular medicine designed to introduce into target cells genetic material with therapeutic intent. The history of RNA interference (RNAi) has only a dozen years, however, further studies have revealed that it is a potent method of gene silencing that has developed rapidly over the past few years as a result of its extensive importance in the study of genetics, molecular biology and physiology. RNAi is a natural process by which small interfering RNA (siRNA) duplex directs sequence specific post-transcriptional silencing of homologous genes by binding to its complementary mRNA and triggering its elimination. RNAi has been extensively used as a novel and effective gene silencing tool for the fundamental research of cancer therapeutics, and has displayed great potential in clinical treatment.

  10. Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference

    PubMed Central

    Wheeler, David; Darby, Brian J.; Todd, Timothy C.; Herman, Michael A.

    2012-01-01

    Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously. PMID:23483038

  11. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    PubMed

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

  12. Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

    PubMed

    Swevers, L; Huvenne, H; Menschaert, G; Kontogiannatos, D; Kourti, A; Pauchet, Y; ffrench-Constant, R; Smagghe, G

    2013-12-01

    In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double-stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA-mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi-related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi-RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi. PMID:24580832

  13. Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference

    PubMed Central

    Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

    2015-01-01

    RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells. PMID:25731667

  14. Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

    PubMed

    Swevers, L; Huvenne, H; Menschaert, G; Kontogiannatos, D; Kourti, A; Pauchet, Y; ffrench-Constant, R; Smagghe, G

    2013-12-01

    In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double-stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA-mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi-related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi-RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

  15. Tumor-targeted RNA-interference: functional non-viral nanovectors

    PubMed Central

    Pan, Xinghua; Thompson, Rachel; Meng, Xiaojie; Wu, Daocheng; Xu, Liang

    2011-01-01

    While small interfering RNA (siRNA) and microRNA (miRNA) have attracted extensive attention and showed significant promise for the study, diagnosis and treatment of human cancers, delivering siRNA or miRNA specifically and efficiently into tumor cells in vivo remains a great challenge. Delivery barriers, which arise mainly from the routes of administration associated with complex physiochemical microenvironments of the human body and the unique properties of RNAs, hinder the development of RNA-interference (RNAi)-based therapeutics in clinical practice. However, in available delivery systems, non-viral nanoparticle-based gene/RNA-delivery vectors, or nanovectors, are showing powerful delivery capacities and huge potential for improvements in functional nanomaterials, including novel fabrication approaches which would greatly enhance delivery performance. In this review, we summarize the currently recognized RNAi delivery barriers and the anti-barrier requirements related to vectors' properties. Recent efforts and achievements in the development of novel nanomaterials, nanovectors fabrication methods, and delivery approaches are discussed. We also review the outstanding needs in the areas of material synthesis and assembly, multifunction combinations, proper delivery and assisting approaches that require more intensive investigation for the comprehensive and effective delivery of RNAi by non-viral nanovectors. PMID:21572539

  16. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies.

    PubMed

    Deng, Yan; Wang, Chi Chiu; Choy, Kwong Wai; Du, Quan; Chen, Jiao; Wang, Qin; Li, Lu; Chung, Tony Kwok Hung; Tang, Tao

    2014-04-01

    During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges.

  17. Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone Aiptasia pallida.

    PubMed

    Dunn, Simon R; Phillips, Wendy S; Green, Douglas R; Weis, Virginia M

    2007-06-01

    Since the discovery of the ancient eukaryotic process of RNA-mediated gene silencing, the reverse-genetics technique RNA interference (RNAi) has increasingly been used to examine gene function in vertebrate and invertebrate systems. In this study, we report on the use of RNAi, adapted from studies on animal model systems, to manipulate gene expression in a symbiotic marine cnidarian. We describe gene knockdown of actin and of acasp--a cysteine protease, or caspase--in the symbiotic sea anemone Aiptasia pallida. Knockdown was assessed qualitatively with in situ hybridizations for both genes. Quantitative PCR and caspase activity assays were used as a quantitative measure of knockdown for acasp. PMID:17565114

  18. Gold nanoparticle interference study during the isolation, quantification, purity and integrity analysis of RNA.

    PubMed

    Sanabria, Natasha M; Vetten, Melissa; Andraos, Charlene; Boodhia, Kailen; Gulumian, Mary

    2014-01-01

    Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220-340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190-220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190-220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.

  19. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing

    PubMed Central

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently-known small RNA classes and place them in context of the reconstructed evolutionary history of the RNAi protein machinery. This synthesis indicates the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: 1) sense-antisense transcriptional products, 2) genome-encoded, imperfectly-complementary hairpin sequences, and 3) larger non-coding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNA interference. They were recruited alongside RNaseIII domains and RdRP domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleo-cytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  20. Inhibiting tumor growth by targeting liposomally encapsulated CDC20siRNA to tumor vasculature: therapeutic RNA interference.

    PubMed

    Majumder, Poulami; Bhunia, Sukanya; Bhattacharyya, Jayanta; Chaudhuri, Arabinda

    2014-04-28

    Many cancer cells over express CDC20 (Cell Division Cycle homologue 20), a key cell cycle regulator required for the completion of mitosis in organisms from yeast to human. A recent in vitro study showed that specific knockdown of CDC20 expression using CDC20siRNA can significantly inhibit growth of human pancreatic carcinoma cells. However, preclinical study aimed at demonstrating therapeutic potential of CDC20siRNA in inhibiting tumor growth has just begun. Using a syngeneic C57BL/6J mouse tumor model, herein we show that intravenous administration of a 19bp synthetic CDC20siRNA encapsulated within α5β1 integrin receptor selective liposomes of pegylated RGDK-lipopeptide inhibits melanoma tumor growth. Liposomally encapsulated CDC20siRNA was found to be efficient in silencing the expression of CDC20 in tumor and endothelial cells at both mRNA and protein levels under in vitro settings. Findings in the flow cytometric studies confirmed the presence of significantly enhanced populations of the G2/M phase in cells treated with liposomally encapsulated CDC20siRNA. Immunohistochemical staining of tumor cryosections from mice treated with liposomally encapsulated fluorescently labeled siRNAs revealed tumor vasculatures targeting capabilities of the present liposomal formulations. The colocalizations of the TUNEL and VE-cadherin positive cells in tumor cryosections are consistent with tumor growth inhibition being mediated via apoptosis of the tumor endothelial cells. In summary, the presently disclosed liposomal formulation of CDC20siRNA is a promising RNA interference tool for use in anti-angiogenic cancer therapy. PMID:24556418

  1. Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system.

    PubMed

    Bivalkar-Mehla, Shalmali; Vakharia, Janaki; Mehla, Rajeev; Abreha, Measho; Kanwar, Jagat Rakesh; Tikoo, Akshay; Chauhan, Ashok

    2011-01-01

    Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.

  2. Inhibition of acidic mammalian chitinase by RNA interference suppresses ovalbumin-sensitized allergic asthma.

    PubMed

    Yang, Ching-Jen; Liu, Yu-Kuo; Liu, Chao-Lin; Shen, Chia-Ning; Kuo, Ming-Ling; Su, Chien-Chang; Tseng, Ching-Ping; Yen, Tzu-Chen; Shen, Chia-Rui

    2009-12-01

    Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.

  3. RNA Interference Induced by the Cationic Lipid Delivery of siRNA

    NASA Astrophysics Data System (ADS)

    Bouxsein, Nathan

    2005-03-01

    Recent discoveries demonstrate that the introduction of synthetically prepared duplexes of 19-21 bp short interfering RNAs (siRNA) into mammalian cells results in the cleavage of target mRNA leading to post transcriptional gene silencing [1]. Our work focuses on the cationic-lipid (CL) mediated delivery of siRNA into mammalian cell lines in an approach similar to CL based gene delivery [2]. Co-transfection of a target and a non-target reporter plasmid followed by the CL delivery of a sequence specific siRNA allows us to probe the silencing efficiency (SE) of the target plasmid relative to non-specific silencing of both plasmids. We have created a phase diagram for SE as a function of the complex membrane charge density and as a function of the CL:siRNA charge ratio. X-ray diffraction was performed to probe the structure of the complexes at points along the phase diagram. Funding provided by NIH AI-12520, AI-20611 and GM-59288. [1] Elbashir et. al., Nature, 411 494-498 (2001) [2] Ewert et. al., Curr. Med. Chem. 11 133-149 (2004)

  4. RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects.

    PubMed

    Geldhof, P; Visser, A; Clark, D; Saunders, G; Britton, C; Gilleard, J; Berriman, M; Knox, D

    2007-05-01

    RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges. PMID:17201997

  5. Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference

    PubMed Central

    Borel, Florie; Kay, Mark A; Mueller, Christian

    2014-01-01

    RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. PMID:24352214

  6. [Silencing HSV1 gD expression in cultured cells by RNA interference].

    PubMed

    Zhu, Qin-Chang; Ren, Zhe; Zhang, Chun-Long; Zhang, Mei-Ying; Liao, Hong-Juan; Liu, Qiu-Ying; Zhang, Pei-Zhuo; Li, Jiu-Xiang; Hu, Chao-Feng; Wang, Hua-Dong; Wang, Yi-Fei

    2007-01-01

    To explore the anti-HSV-1 effect of silencing gD gene expression by RNA interference, five 21-nucleotide duplex small interfering RNAs(siRNAs) targeting the HSV1 gD sequence were designed and the gD-EGFP fusion gene expression vector was constructed, then co-transfected into Vero cell, and screened the effective siRNA through analyzing the intensity of the EGFP fluorescence. Finally, the anti-HSV1 effect was confirmed by plaque reduction assay, real-time PCR and daughter virus titration of HSV1 infected Vero cells transfected with siRNAs. The study demonstrated that siRNAs could effectively and specifically inhibit gD gene expression in HSV1-infected cells, but only had a little effect on HSV1 infection, so taking gD as the target of siRNA against HSV1 needs further study.

  7. Interference of hepatitis C virus RNA replication by short interfering RNAs

    NASA Astrophysics Data System (ADS)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  8. Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference

    PubMed Central

    Coburn, Glen A.; Cullen, Bryan R.

    2002-01-01

    Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle. PMID:12186906

  9. Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas.

    PubMed

    Huvet, Arnaud; Béguel, Jean-Philippe; Cavaleiro, Nathalia Pereira; Thomas, Yoann; Quillien, Virgile; Boudry, Pierre; Alunno-Bruscia, Marianne; Fabioux, Caroline

    2015-06-01

    Feeding strategies and digestive capacities can have important implications for variation in energetic pathways associated with ecological and economically important traits, such as growth or reproduction in bivalve species. Here, we investigated the role of amylase in the digestive processes of Crassostrea gigas, using in vivo RNA interference. This approach also allowed us to investigate the relationship between energy intake by feeding and gametogenesis in oysters. Double-stranded (ds)RNA designed to target the two α-amylase genes A and B was injected in vivo into the visceral mass of oysters at two doses. These treatments caused significant reductions in mean mRNA levels of the amylase genes: -50.7% and -59% mRNA A, and -71.9% and -70.6% mRNA B in 15 and 75 µg dsRNA-injected oysters, respectively, relative to controls. Interestingly, reproductive knock-down phenotypes were observed for both sexes at 48 days post-injection, with a significant reduction of the gonad area (-22.5% relative to controls) and germ cell under-proliferation revealed by histology. In response to the higher dose of dsRNA, we also observed reductions in amylase activity (-53%) and absorption efficiency (-5%). Based on these data, dynamic energy budget modeling showed that the limitation of energy intake by feeding that was induced by injection of amylase dsRNA was insufficient to affect gonadic development at the level observed in the present study. This finding suggests that other driving mechanisms, such as endogenous hormonal modulation, might significantly change energy allocation to reproduction, and increase the maintenance rate in oysters in response to dsRNA injection.

  10. Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy

    PubMed Central

    Bisset, Darren R.; Stepniak-Konieczna, Ewa A.; Zavaljevski, Maja; Wei, Jessica; Carter, Gregory T.; Weiss, Michael D.; Chamberlain, Joel R.

    2015-01-01

    RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3′ UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUGexp) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUGexp mRNA in the human α-skeletal muscle actin long-repeat (HSALR) mouse model of DM1. RNAi expression cassettes were delivered to HSALR mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSALR mice, including a reduction in the CUGexp mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUGexp mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSALR mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies. PMID:26082468

  11. RNA interference against interleukin-5 attenuates airway inflammation and hyperresponsiveness in an asthma model.

    PubMed

    Chen, Shao-xing; Huang, Feng-ying; Tan, Guang-hong; Wang, Cai-chun; Huang, Yong-hao; Wang, Hua; Zhou, Song-lin; Chen, Fan; Lin, Ying-ying; Liu, Jun-bao

    2009-01-01

    Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5x10(6) IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.

  12. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference.

    PubMed

    Murphy, Katherine A; Tabuloc, Christine A; Cervantes, Kevin R; Chiu, Joanna C

    2016-01-01

    RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally found growing on the surface of fruit crops, constitutes a major component of the Drosophila microbiome, and is highly attractive to Drosophila. Thus, this naturally attractive yeast biopesticide can deliver dsRNA to an insect pest without the need for genetic crop modification. We demonstrate that this biopesticide decreases larval survivorship, and reduces locomotor activity and reproductive fitness in adults, which are indicative of general health decline. To our knowledge, this is the first study to show that yeast can be used to deliver dsRNA to an insect pest. PMID:26931800

  13. Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti.

    PubMed

    Franz, Alexander W E; Sanchez-Vargas, Irma; Adelman, Zach N; Blair, Carol D; Beaty, Barry J; James, Anthony A; Olson, Ken E

    2006-03-14

    Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.

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

    PubMed

    Orii, Hidefumi; Mochii, Makoto; Watanabe, Kenji

    2003-04-01

    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.

  15. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference

    PubMed Central

    Murphy, Katherine A.; Tabuloc, Christine A.; Cervantes, Kevin R.; Chiu, Joanna C.

    2016-01-01

    RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally found growing on the surface of fruit crops, constitutes a major component of the Drosophila microbiome, and is highly attractive to Drosophila. Thus, this naturally attractive yeast biopesticide can deliver dsRNA to an insect pest without the need for genetic crop modification. We demonstrate that this biopesticide decreases larval survivorship, and reduces locomotor activity and reproductive fitness in adults, which are indicative of general health decline. To our knowledge, this is the first study to show that yeast can be used to deliver dsRNA to an insect pest. PMID:26931800

  16. Darwin's "Abominable Mystery": the role of RNA interference in the evolution of flowering plants.

    PubMed

    Cibrián-Jaramillo, A; Martienssen, R A

    2009-01-01

    Darwin was famously concerned that the sudden appearance and rapid diversification of flowering plants in the mid-Cretaceous could not have occurred by gradual change. Here, we review our attempts to resolve the relationships among the major seed plant groups, i.e., cycads, ginkgo, conifers, gnetophytes, and flowering plants, and to provide a pipeline in which these relationships can be used as a platform for identifying genes of functional importance in plant diversification. Using complete gene sets and unigenes from 16 plant species, genes with positive partitioned Bremer support at major nodes were used to identify overrepresented gene ontology (GO) terms. Posttranscriptional silencing via RNA interference (RNAi) was overrepresented at several major nodes, including between monocots and dicots during early angiosperm divergence. One of these genes, RNA-dependent RNA polymerase 6, is required for the biogenesis of trans-acting small interfering RNA (tasiRNA), confers heteroblasty and organ polarity, and restricts maternal specification of the germline. Processing of small RNA and transfer between neighboring cells underlies these roles and may have contributed to distinct mutant phenotypes in plants, and in particular in the early split of the monocots and eudicots. PMID:20508061

  17. Isolation and characterization of homologous TRBP cDNA for RNA interference in Penaeus monodon.

    PubMed

    Yang, Lishi; Li, Xiaolan; Huang, Jianhua; Zhou, Falin; Su, Tianfeng; Jiang, Shigui

    2013-02-01

    The transactivation response RNA-binding protein (TRBP) interacts with Dicer and binds to double-stranded RNA as a critical component of the RNA-induced silencing complex, which is a key complex in the RNA interference pathway. The full-length cDNA of TRBP from the tiger prawn, Penaeus monodon, (PmTRBP; 1548 bp long with a 1029 bp coding region) was isolated. The encoded polypeptide of 343 amino acids had a predicted molecular mass of 36.8 kDa. Sequence homology and phylogenetic analysis indicated that PmTRBP was evolutionarily closest to TRBP1 from Litopenaeus vannamei, with the three double-stranded RNA-binding motifs that were typical of the TRBP family. Tissue expression profile analysis by quantitative real-time reverse transcription polymerase chain reaction showed that PmTRBP1 was constitutively expressed in all the examined tissues, with a predominant expression in the lymphatic organs and with the weakest expression in the ovaries. Significantly upregulated PmTRBP1 expression was elicited by systemic injections of Staphylococcus aureus, Vibrio vulnificus, and white spot syndrome virus, thereby revealing its pathogen inducibility. Furthermore, exogenous viral nucleoside analogs (high-molecular-weight poly(I:C) dsRNAs as well as R484 single-stranded RNA) were remarkably induced PmTRBP1 transcription at 48 h and 9 h post-injection, respectively, which suggested that PmTRBP1 might function in tiger prawn antibacterial and antiviral response.

  18. Transgenic sugarcane resistant to Sorghum mosaic virus based on coat protein gene silencing by RNA interference.

    PubMed

    Guo, Jinlong; Gao, Shiwu; Lin, Qinliang; Wang, Hengbo; Que, Youxiong; Xu, Liping

    2015-01-01

    As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

  19. GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

    PubMed

    Matsumoto, Yukiko; Hattori, Makoto

    2016-03-01

    RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species. PMID:26728387

  20. Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

    PubMed

    Hatano, Akihiko; Shiraishi, Mitsuya; Terado, Nanae; Tanabe, Atsuhiro; Fukuda, Kenji

    2015-10-15

    Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

  1. Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing.

    PubMed

    Kidwell, Mary Anne; Chan, Jessica M; Doudna, Jennifer A

    2014-10-10

    The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans. PMID:25135636

  2. Evolutionarily Conserved Roles of the Dicer Helicase Domain in Regulating RNA Interference Processing*

    PubMed Central

    Kidwell, Mary Anne; Chan, Jessica M.; Doudna, Jennifer A.

    2014-01-01

    The enzyme Dicer generates 21–25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans. PMID:25135636

  3. Discovery of midgut genes for the RNA interference control of corn rootworm.

    PubMed

    Hu, Xu; Richtman, Nina M; Zhao, Jian-Zhou; Duncan, Keith E; Niu, Xiping; Procyk, Lisa A; Oneal, Meghan A; Kernodle, Bliss M; Steimel, Joseph P; Crane, Virginia C; Sandahl, Gary; Ritland, Julie L; Howard, Richard J; Presnail, James K; Lu, Albert L; Wu, Gusui

    2016-01-01

    RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality. PMID:27464714

  4. GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

    PubMed

    Matsumoto, Yukiko; Hattori, Makoto

    2016-03-01

    RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species.

  5. Discovery of midgut genes for the RNA interference control of corn rootworm

    PubMed Central

    Hu, Xu; Richtman, Nina M.; Zhao, Jian-Zhou; Duncan, Keith E.; Niu, Xiping; Procyk, Lisa A.; Oneal, Meghan A.; Kernodle, Bliss M.; Steimel, Joseph P.; Crane, Virginia C.; Sandahl, Gary; Ritland, Julie L.; Howard, Richard J.; Presnail, James K.; Lu, Albert L.; Wu, Gusui

    2016-01-01

    RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by “blebbing” of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality. PMID:27464714

  6. Statistical Methods for Analysis of High-Throughput RNA Interference Screens

    PubMed Central

    Birmingham, Amanda; Selfors, Laura M.; Forster, Thorsten; Wrobel, David; Kennedy, Caleb J.; Shanks, Emma; Santoyo-Lopez, Javier; Dunican, Dara J.; Long, Aideen; Kelleher, Dermot; Smith, Queta; Beijersbergen, Roderick L.; Ghazal, Peter; Shamu, Caroline E.

    2009-01-01

    RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow. PMID:19644458

  7. Design and Methods of Large-Scale RNA Interference Screens in Drosophila.

    PubMed

    Zhou, Jia; Tong, Chao

    2016-01-01

    Drosophila is an ideal model system for addressing important questions in biology. The use of RNA interference (RNAi) to knockdown gene expression in fly tissues is both very effective and relatively simple. In the past few decades, genome-wide UAS-RNAi transgenic libraries and thousands of Gal4 strains have been generated and have facilitated large-scale in vivo RNAi screening. Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced. PMID:27581292

  8. Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

    PubMed Central

    2011-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice. Methods We utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development. Results The EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis. Conclusions Slug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC. PMID:21599940

  9. A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference.

    PubMed

    Takahashi, Masaki; Hohjoh, Hirohiko

    2014-11-01

    Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named "ASP-score". The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.

  10. Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

    PubMed Central

    Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

    2012-01-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

  11. Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zhang, Hui; Cao, Xudong; Qiao, Jun; Sheng, Jinliang; Guo, Fei; Chen, Chuangfu

    2011-10-01

    Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P<0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning. PMID:21698446

  12. Virus-derived gene expression and RNA interference vector for grapevine.

    PubMed

    Kurth, Elizabeth G; Peremyslov, Valera V; Prokhnevsky, Alexey I; Kasschau, Kristin D; Miller, Marilyn; Carrington, James C; Dolja, Valerian V

    2012-06-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

  13. RNA interference can be used to disrupt gene function in tardigrades

    PubMed Central

    Tenlen, Jennifer R.; McCaskill, Shaina; Goldstein, Bob

    2012-01-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We show that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions, and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments. PMID:23187800

  14. A rapid and scalable system for studying gene function in mice using conditional RNA interference

    PubMed Central

    Premsrirut, Prem K.; Dow, Lukas E.; Kim, Sang Yong; Camiolo, Matthew; Malone, Colin D.; Miething, Cornelius; Scuoppo, Claudio; Zuber, Johannes; Dickins, Ross A.; Kogan, Scott C.; Shroyer, Kenneth R.; Sordella, Raffaella; Hannon, Gregory J.; Lowe, Scott W.

    2011-01-01

    Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PMID:21458673

  15. DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

    PubMed Central

    Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

    2008-01-01

    P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

  16. RNA interference can be used to disrupt gene function in tardigrades.

    PubMed

    Tenlen, Jennifer R; McCaskill, Shaina; Goldstein, Bob

    2013-05-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.

  17. Inhibitory effects and analysis of RNA interference on thioredoxin glutathione reductase expression in Schistosoma japonicum.

    PubMed

    Han, Yanhui; Fu, Zhiqiang; Hong, Yang; Zhang, Min; Han, Hongxiao; Lu, Ke; Yang, Jianmei; Li, Xiangrui; Lin, Jiaojiao

    2014-08-01

    Schistosomes infect around 280 million people worldwide. The worms survive in the veins of the final host, where thioredoxin glutathione reductase (TGR) activity helps the parasites to survive in the aerobic environment. In the present study, we synthesized 4 small interfering RNAs (siRNA S1, S2, S3, and S4) targeting the Schistosoma japonicum (Sj) TGR gene and used them to knockdown the TGR gene. The knockdown effects of the siRNAs on SjTGR, and the thioredoxin reductase (TrxR) activity of SjTGR, were evaluated in vitro. The results of transfection with the siRNAs via the soaking method in vitro were confirmed by flow cytometry. S2 siRNA at a final concentration of 200 nM partially inhibited the expression of SjTGR at both the transcript and protein levels in vitro. TrxR-activity was lower in worms in the S2 siRNA-treated group compared with the control groups. Further analysis revealed that purified recombinant SjTGR could remove oxygen free radicals but not H(2)O(2) directly, which may explain the incomplete effects of RNA interference on SjTGR. The results of this study indicate that SjTGR may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage.

  18. RNA interference can be used to disrupt gene function in tardigrades.

    PubMed

    Tenlen, Jennifer R; McCaskill, Shaina; Goldstein, Bob

    2013-05-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments. PMID:23187800

  19. Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNA-seq, RNA interference and irradiation approach

    PubMed Central

    2012-01-01

    Background Planarian stem cells, or neoblasts, drive the almost unlimited regeneration capacities of freshwater planarians. Neoblasts are traditionally described by their morphological features and by the fact that they are the only proliferative cell type in asexual planarians. Therefore, they can be specifically eliminated by irradiation. Irradiation, however, is likely to induce transcriptome-wide changes in gene expression that are not associated with neoblast ablation. This has affected the accurate description of their specific transcriptomic profile. Results We introduce the use of Smed-histone-2B RNA interference (RNAi) for genetic ablation of neoblast cells in Schmidtea mediterranea as an alternative to irradiation. We characterize the rapid, neoblast-specific phenotype induced by Smed-histone-2B RNAi, resulting in neoblast ablation. We compare and triangulate RNA-seq data after using both irradiation and Smed-histone-2B RNAi over a time course as means of neoblast ablation. Our analyses show that Smed-histone-2B RNAi eliminates neoblast gene expression with high specificity and discrimination from gene expression in other cellular compartments. We compile a high confidence list of genes downregulated by both irradiation and Smed-histone-2B RNAi and validate their expression in neoblast cells. Lastly, we analyze the overall expression profile of neoblast cells. Conclusions Our list of neoblast genes parallels their morphological features and is highly enriched for nuclear components, chromatin remodeling factors, RNA splicing factors, RNA granule components and the machinery of cell division. Our data reveal that the regulation of planarian stem cells relies on posttranscriptional regulatory mechanisms and suggest that planarians are an ideal model for this understudied aspect of stem cell biology. PMID:22439894

  20. Drosophila Dicer-2 has an RNA interference-independent function that modulates Toll immune signaling.

    PubMed

    Wang, Zhaowei; Wu, Di; Liu, Yongxiang; Xia, Xiaoling; Gong, Wanyun; Qiu, Yang; Yang, Jie; Zheng, Ya; Li, Jingjing; Wang, Yu-Feng; Xiang, Ye; Hu, Yuanyang; Zhou, Xi

    2015-10-01

    Dicer-2 is the central player for small interfering RNA biogenesis in the Drosophila RNA interference (RNAi) pathway. Intriguingly, we found that Dicer-2 has an unconventional RNAi-independent function that positively modulates Toll immune signaling, which defends against Gram-positive bacteria, fungi, and some viruses, in both cells and adult flies. The loss of Dicer-2 expression makes fruit flies more susceptible to fungal infection. We further revealed that Dicer-2 posttranscriptionally modulates Toll signaling because Dicer-2 is required for the proper expression of Toll protein but not for Toll protein stability or Toll mRNA transcription. Moreover, Dicer-2 directly binds to the 3' untranslated region (3'UTR) of Toll mRNA via its PAZ (Piwi/Argonaute/Zwille) domain and is required for protein translation mediated by Toll 3'UTR. The loss of Toll 3'UTR binding activity makes Dicer-2 incapable of promoting Toll signaling. These data indicate that the interaction between Dicer-2 and Toll mRNA plays a pivotal role in Toll immune signaling. In addition, we found that Dicer-2 is also required for the Toll signaling induced by two different RNA viruses in Drosophila cells. Consequently, our findings uncover a novel RNAi-independent function of Dicer-2 in the posttranscriptional regulation of Toll protein expression and signaling, indicate an unexpected intersection of the RNAi pathway and the Toll pathway, and provide new insights into Toll immune signaling, Drosophila Dicer-2, and probably Dicer and Dicer-related proteins in other organisms. PMID:26601278

  1. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference

    PubMed Central

    Hochstrasser, Megan L.; Taylor, David W.; Bhat, Prashant; Guegler, Chantal K.; Sternberg, Samuel H.; Nogales, Eva; Doudna, Jennifer A.

    2014-01-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA–E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage. PMID:24748111

  2. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-01

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  3. Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

    PubMed

    Cernilogar, Filippo M; Onorati, Maria Cristina; Kothe, Greg O; Burroughs, A Maxwell; Parsi, Krishna Mohan; Breiling, Achim; Lo Sardo, Federica; Saxena, Alka; Miyoshi, Keita; Siomi, Haruhiko; Siomi, Mikiko C; Carninci, Piero; Gilmour, David S; Corona, Davide F V; Orlando, Valerio

    2011-11-06

    RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

  4. Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin.

    PubMed

    Ganesh, Shanthi; Koser, Martin L; Cyr, Wendy A; Chopda, Girish R; Tao, Junyan; Shui, Xue; Ying, Bo; Chen, Dongyu; Pandya, Purva; Chipumuro, Edmond; Siddiquee, Zakir; Craig, Kevin; Lai, Chengjung; Dudek, Henryk; Monga, Satdarshan P; Wang, Weimin; Brown, Bob D; Abrams, Marc T

    2016-09-01

    The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding β-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143-54. ©2016 AACR. PMID:27390343

  5. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    SciTech Connect

    Iida, Tetsushi; Iida, Naoko; Tsutsui, Yasuhiro; Yamao, Fumiaki; Kobayashi, Takehiko

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  6. RNA interference in plant parasitic nematodes: a summary of the current status.

    PubMed

    Lilley, C J; Davies, L J; Urwin, P E

    2012-04-01

    SUMMARYRNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants. PMID:22217302

  7. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

    PubMed

    McCaskill, Jana L; Marsh, Glenn A; Monaghan, Paul; Wang, Lin-Fa; Doran, Timothy; McMillan, Nigel A J

    2013-01-01

    Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

  8. Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

    PubMed Central

    De Arras, Lesly; Guthrie, Brandon S.; Alper, Scott

    2014-01-01

    Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production. PMID:25407484

  9. RNA interference: from an ancient mechanism to a state of the art therapeutic application?

    PubMed

    Arenz, Christoph; Schepers, Ute

    2003-08-01

    Now that the sequencing of many genomes has been completed, the basic challenges are finding the genes and predicting their functions. Up until now, a large information gap has existed between the knowledge of genome sequence and our knowledge of protein function. The assessment of gene function may be performed using the tools of reverse genetics, including knock-out mice, antisense oligomers, aptamers, and ribozymes. These approaches have been superseded by RNA interference (RNAi), which exhibits much more potency for the investigation of protein function than the techniques listed above. As already known some years ago, RNAi is based on an ancient anti-viral defense mechanism in lower eukaryotes. It is induced by double-stranded RNA and its processing to 21-23 nt small interfering RNAs (siRNAs), which cause the degradation of homologous endogenous mRNA. The way RNAi works has still to be determined, but it already serves as a first-choice approach to generate loss-of-function phenotypes among a broad variety of eukaryotic species, such as nematodes, flies, plants, fungi and mammals. RNAi also represents an extremely powerful tool, becoming a therapeutic approach to curing infectious diseases originated by viral or parasitic invasion. In this review we present the current view of how RNAi works in different eukaryotic species and its high potential for functional genomics and in rational drug design. PMID:12955224

  10. Using RNA-interference to investigate the innate immune response in mouse macrophages.

    PubMed

    De Arras, Lesly; Guthrie, Brandon S; Alper, Scott

    2014-11-03

    Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

  11. In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

    PubMed

    Wang, Gaili; He, Wenqi; Song, Deguang; Li, Jida; Bao, Yingfu; Lu, Rongguang; Bi, Jingying; Zhao, Kui; Gao, Feng

    2014-05-01

    Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

  12. Late-bolting transgenic Chinese cabbage obtained by RNA interference technique.

    PubMed

    Xia, Guang-Qing; Zhu, Jun-Yi; He, Qi-Wei; Zhao, Shuang-Yi; Wang, Cui-Hua

    2007-10-01

    LEAFY (LFY) gene plays an important role in determining plant flowering mainly by controlling the timing of phase transition. Constitutive under-expression of LFY in Arabidopsis resulted in the formation of a late-flowering and highly branching phenotype. In this paper, an RNAi approach was used in down-regulated LFY gene expression to delay Chinese cabbage (Brassica rapa L. ssp. pekinensis) bolting and flowering. The results show that transgenic plant has a later transition to the reproductive phase, and the transgenic plants have more branches, more leaves, but a lower height. Results of RQ-RT-PCR analysis show that LFY gene expression was greatly reduced in transgenic plants. These results suggest that inhibiting LFY gene expression by RNA interference can delay bolting in a cold-sensitive long-day (LD) condition. Late flowering of Chinese cabbage can be used as a good genetic resource for the breeding late-bolting Chinese cabbage.

  13. Illuminating the gateway of gene silencing: perspective of RNA interference technology in clinical therapeutics.

    PubMed

    Sindhu, Annu; Arora, Pooja; Chaudhury, Ashok

    2012-07-01

    A novel laboratory revolution for disease therapy, the RNA interference (RNAi) technology, has adopted a new era of molecular research as the next generation "Gene-targeted prophylaxis." In this review, we have focused on the chief technological challenges associated with the efforts to develop RNAi-based therapeutics that may guide the biomedical researchers. Many non-curable maladies, like neurodegenerative diseases and cancers have effectively been cured using this technology. Rapid advances are still in progress for the development of RNAi-based technologies that will be having a major impact on medical research. We have highlighted the recent discoveries associated with the phenomenon of RNAi, expression of silencing molecules in mammals along with the vector systems used for disease therapeutics.

  14. RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

    PubMed Central

    Abdurakhmonov, Ibrokhim Y.; Ayubov, Mirzakamol S.; Ubaydullaeva, Khurshida A.; Buriev, Zabardast T.; Shermatov, Shukhrat E.; Ruziboev, Haydarali S.; Shapulatov, Umid M.; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z.; Percy, Richard G.; Devor, Eric J.; Sharma, Govind C.; Sripathi, Venkateswara R.; Kumpatla, Siva P.; van der Krol, Alexander; Kater, Hake D.; Khamidov, Khakimdjan; Salikhov, Shavkat I.; Jenkins, Johnie N.; Abdukarimov, Abdusattor; Pepper, Alan E.

    2016-01-01

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization. PMID:26941765

  15. Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

    PubMed Central

    Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

    2014-01-01

    RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

  16. Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens.

    PubMed

    Warnock, Neil D; Wilson, Leonie; Canet-Perez, Juan V; Fleming, Thomas; Fleming, Colin C; Maule, Aaron G; Dalzell, Johnathan J

    2016-07-01

    Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants. PMID:27033013

  17. RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.).

    PubMed

    Abdurakhmonov, Ibrokhim Y; Ayubov, Mirzakamol S; Ubaydullaeva, Khurshida A; Buriev, Zabardast T; Shermatov, Shukhrat E; Ruziboev, Haydarali S; Shapulatov, Umid M; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z; Percy, Richard G; Devor, Eric J; Sharma, Govind C; Sripathi, Venkateswara R; Kumpatla, Siva P; van der Krol, Alexander; Kater, Hake D; Khamidov, Khakimdjan; Salikhov, Shavkat I; Jenkins, Johnie N; Abdukarimov, Abdusattor; Pepper, Alan E

    2016-01-01

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.

  18. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.

    PubMed

    Barrangou, Rodolphe; Birmingham, Amanda; Wiemann, Stefan; Beijersbergen, Roderick L; Hornung, Veit; Smith, Anja van Brabant

    2015-04-20

    The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.

  19. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

    PubMed Central

    Barrangou, Rodolphe; Birmingham, Amanda; Wiemann, Stefan; Beijersbergen, Roderick L.; Hornung, Veit; Smith, Anja van Brabant

    2015-01-01

    The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications. PMID:25800748

  20. Toward a durable anti-HIV gene therapy based on RNA interference.

    PubMed

    Berkhout, Ben

    2009-09-01

    Basic research in the field of molecular biology led to the discovery of the mechanism of RNA interference (RNAi) in Caenorhabditis elegans in 1998. RNAi is now widely appreciated as an important gene control mechanism in mammals, and several RNAi-based gene-silencing applications have already been used in clinical trials. In this review I will discuss RNAi approaches to inhibit the pathogenic human immunodeficiency virus type 1 (HIV-1), which establishes a chronic infection that would most likely require a durable gene therapy approach. Viruses, such as HIV-1, are particularly difficult targets for RNAi attack because they mutate frequently, which allows viral escape by mutation of the RNAi target sequence. Combinatorial RNAi strategies are required to prevent viral escape. PMID:19796072

  1. RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.).

    PubMed

    Abdurakhmonov, Ibrokhim Y; Ayubov, Mirzakamol S; Ubaydullaeva, Khurshida A; Buriev, Zabardast T; Shermatov, Shukhrat E; Ruziboev, Haydarali S; Shapulatov, Umid M; Saha, Sukumar; Ulloa, Mauricio; Yu, John Z; Percy, Richard G; Devor, Eric J; Sharma, Govind C; Sripathi, Venkateswara R; Kumpatla, Siva P; van der Krol, Alexander; Kater, Hake D; Khamidov, Khakimdjan; Salikhov, Shavkat I; Jenkins, Johnie N; Abdukarimov, Abdusattor; Pepper, Alan E

    2016-01-01

    RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization. PMID:26941765

  2. PsOr1, a potential target for RNA interference-based pest management.

    PubMed

    Zhao, Y Y; Liu, F; Yang, G; You, M S

    2011-02-01

    Insect pests cause billions of dollars in agricultural losses, and attempts to kill them have resulted in growing threats from insecticide resistance, dietary pesticide pollution and environmental destruction. New approaches to control refractory insect pests are therefore needed. The host-plant preferences of insect pests rely on olfaction and are mediated via a seven transmembrane-domain odorant receptor (Or) family. The present study reports the cloning and characterization of PsOr1, the first candidate member of the Or gene family from Phyllotreta striolata, a devastating beetle pest that causes damage worldwide. PsOr1 is remarkably well conserved with respect to other insect orthologues, including DmOr83b from Drosophila melanogaster. These insect orthologues form an essential non-conventional Or sub-family and may play an important and generalized role in insect olfaction. We designed double-stranded (ds) RNA directly against the PsOr1 gene and exploited RNA interference (RNAi) to control P. striolata. The chemotactic behavioural measurements showed that adult beetles were unable to sense the attractant or repellent odour stimulus after microinjection of dsRNA against PsOr1. Reverse Transcription (RT)-PCR analysis showed specific down-regulation of mRNA transcript levels for this gene. Furthermore, host-plant preference experiments confirmed that silencing PsOr1 by RNAi treatment impaired the host-plant preferences of P. striolata for cruciferous vegetables. These results demonstrate that this insect control approach of using RNAi to target PsOr1 and its orthologues might be effective in blocking host-plant-seeking behaviours in diverse insect pests. The results also support the theory that this unique receptor type plays an essential general role in insect olfaction. PMID:20854479

  3. Microfluidic platforms for RNA interference screening of virus-host interactions.

    PubMed

    Schudel, Benjamin R; Harmon, Brooke; Abhyankar, Vinay V; Pruitt, Benjamin W; Negrete, Oscar A; Singh, Anup K

    2013-03-01

    RNA interference (RNAi) is a powerful tool for functional genomics with the capacity to comprehensively analyze host-pathogen interactions. High-throughput RNAi screening is used to systematically perturb cellular pathways and discover therapeutic targets, but the method can be tedious and requires extensive capital equipment and expensive reagents. To aid in the development of an inexpensive miniaturized RNAi screening platform, we have developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry and infection. First, a multilayer polydimethylsiloxane (PDMS)-based spotting device was used to array siRNA molecules into 96 microwells targeting markers of endocytosis, along with siRNA controls. By using a PDMS-based spotting device, we remove the need for a microarray printer necessary to perform previously described small scale (e.g. cellular microarrays) and microchip-based RNAi screening, while still minimizing reagent usage tenfold compared to conventional screening. Second, the siRNA spotted array was transferred to a reversibly sealed PDMS-based screening platform containing microchannels designed to enable efficient cell loading and transfection of mammalian cells while preventing cross-contamination between experimental conditions. Validation of the screening platform was examined using Vesicular stomatitis virus and emerging pathogen Rift Valley fever virus, which demonstrated virus entry pathways of clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively. The techniques here are adaptable to other well-characterized infection pathways with a potential for large scale screening in high containment biosafety laboratories.

  4. RNA Interference of Gonadotropin-Inhibitory Hormone Gene Induces Arousal in Songbirds

    PubMed Central

    Ubuka, Takayoshi; Mukai, Motoko; Wolfe, Jordan; Beverly, Ryan; Clegg, Sarah; Wang, Ariel; Hsia, Serena; Li, Molly; Krause, Jesse S.; Mizuno, Takanobu; Fukuda, Yujiro; Tsutsui, Kazuyoshi; Bentley, George E.; Wingfield, John C.

    2012-01-01

    Gonadotropin-inhibitory hormone (GnIH) was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi) of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts. PMID:22279571

  5. Bactrocera dorsalis male sterilization by targeted RNA interference of spermatogenesis: empowering sterile insect technique programs

    PubMed Central

    Dong, Yong-Cheng; Wang, Zhi-Jian; Chen, Zhen-Zhong; Clarke, Anthony R.; Niu, Chang-Ying

    2016-01-01

    RNA interference (RNAi) is a genetic technique which has novel application for sustainable pest control. The Sterile Insect Technique (SIT) uses releases of mass-produced, sterile male insects to out-compete wild males for mates to reduce pest populations. RNAi sterilization of SIT males would have several advantages over radiation sterilization, but to achieve this appropriate target genes must first be identified and then targeted with interference technology. With this goal, eight spermatogenesis related candidate genes were cloned and tested for potential activity in Bactrocera dorsalis. The knockdown of candidate genes by oral delivery of dsRNAs did not influence the mating of male flies, but significantly affected the daily average number of eggs laid by females, and reduced egg hatching rate by 16–60%. RNAi negatively affected spermatozoa quantitatively and qualitatively. Following the mating of lola-/topi-/rac-/rho-/upd-/magu-silenced males, we recorded a significant decrease in number and length of spermatozoa in female spermatheca compared to gfp-silenced control group. In a greenhouse trial, the number of damaged oranges and B. dorsalis larvae were significantly reduced in a dsrho-treated group compared with the dsgfp group. This study provides strong evidence for the use RNAi in pest management, especially for the improvement of SIT against B. dorsalis and other species. PMID:27767174

  6. Mcam Silencing With RNA Interference Using Magnetofection has Antitumor Effect in Murine Melanoma

    PubMed Central

    Prosen, Lara; Markelc, Bostjan; Dolinsek, Tanja; Music, Branka; Cemazar, Maja; Sersa, Gregor

    2014-01-01

    The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by magnetofection. PMID:25350580

  7. RNA interference technology used for the study of aquatic virus infections.

    PubMed

    Reshi, Mohammad Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2014-09-01

    Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. Recent advances in biotechnology have had a significant impact on disease reduction in aquaculture. RNAi is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of specific genes in living systems. RNA interference technology has emerged as a powerful tool for manipulating gene expression in the laboratory. This technology represents a new therapeutic approach for treating aquatic diseases, including viral infections. RNAi technology is based on a naturally occurring post-transcriptional gene silencing process mediated by the formation of dsRNA. RNAi has been proven widely effective for gene knockdown in mammalian cultured cells, but its utility in fish remains unexplored. This review aims to highlight the RNAi technology that has made significant contributions toward the improvement of aquatic animal health and will also summarize the current status and future strategies concerning the therapeutic applications of RNAi to combat viral disease in aquacultured organisms. PMID:24945574

  8. Efficient gene silencing in mesenchymal stem cells by substrate-mediated RNA interference.

    PubMed

    Hsu, Shan-Hui; Huang, Guo-Shiang; Ho, Tung-Tso; Feng, Fuh

    2014-11-01

    We described a novel substrate-mediated RNA interference (RNAi) technology to investigate the effect of neural crest marker expression on the multipotency of human gingival fibroblasts (HGFs). HGFs showed significantly higher neural and chondrogenic differentiation potentials compared with adult bone-marrow-derived mesenchymal stem cells and stem cells from human exfoliated deciduous teeth. By sending target-specific RNAi agents with the conventional vehicle (PolyFect), we observed that the multipotency of HGFs was closely associated with the expression of neural crest marker gene Forkhead box D3 (FoxD3). Using the novel chitosan substrate-mediated method, we successfully delivered short-hairpin RNA constructs to HGFs grown on chitosan without the use of conventional vehicles. The delivery efficiency measured by flow cytometry showed a 10-fold increase for HGFs on chitosan versus those on culture dish, and the cell viability was >95%. Moreover, HGFs with FoxD3 gene knockdown did not form spheroids on chitosan. Based on this working principle, we further selected the gene-silenced population from HGFs. The nonsilenced HGFs showed much higher neural differentiation ability with the nestin expression 40-fold greater than FoxD3-silenced population after induction, suggesting the feasibility of the method to silence genes. The new substrate-mediated gene silencing platform that combines the use of substrate and RNAi can be used to clarify the functions of important genes without suffering the toxicity. PMID:24624901

  9. RNA interference screen for human genes associated with West Nile virus infection.

    PubMed

    Krishnan, Manoj N; Ng, Aylwin; Sukumaran, Bindu; Gilfoy, Felicia D; Uchil, Pradeep D; Sultana, Hameeda; Brass, Abraham L; Adametz, Rachel; Tsui, Melody; Qian, Feng; Montgomery, Ruth R; Lev, Sima; Mason, Peter W; Koski, Raymond A; Elledge, Stephen J; Xavier, Ramnik J; Agaisse, Herve; Fikrig, Erol

    2008-09-11

    West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

  10. From The Cover: Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

    NASA Astrophysics Data System (ADS)

    Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

    2004-04-01

    Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

  11. RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design.

    PubMed

    Terenius, Olle; Papanicolaou, Alexie; Garbutt, Jennie S; Eleftherianos, Ioannis; Huvenne, Hanneke; Kanginakudru, Sriramana; Albrechtsen, Merete; An, Chunju; Aymeric, Jean-Luc; Barthel, Andrea; Bebas, Piotr; Bitra, Kavita; Bravo, Alejandra; Chevalier, François; Collinge, Derek P; Crava, Cristina M; de Maagd, Ruud A; Duvic, Bernard; Erlandson, Martin; Faye, Ingrid; Felföldi, Gabriella; Fujiwara, Haruhiko; Futahashi, Ryo; Gandhe, Archana S; Gatehouse, Heather S; Gatehouse, Laurence N; Giebultowicz, Jadwiga M; Gómez, Isabel; Grimmelikhuijzen, Cornelis J P; Groot, Astrid T; Hauser, Frank; Heckel, David G; Hegedus, Dwayne D; Hrycaj, Steven; Huang, Lihua; Hull, J Joe; Iatrou, Kostas; Iga, Masatoshi; Kanost, Michael R; Kotwica, Joanna; Li, Changyou; Li, Jianghong; Liu, Jisheng; Lundmark, Magnus; Matsumoto, Shogo; Meyering-Vos, Martina; Millichap, Peter J; Monteiro, Antónia; Mrinal, Nirotpal; Niimi, Teruyuki; Nowara, Daniela; Ohnishi, Atsushi; Oostra, Vicencio; Ozaki, Katsuhisa; Papakonstantinou, Maria; Popadic, Aleksandar; Rajam, Manchikatla V; Saenko, Suzanne; Simpson, Robert M; Soberón, Mario; Strand, Michael R; Tomita, Shuichiro; Toprak, Umut; Wang, Ping; Wee, Choon Wei; Whyard, Steven; Zhang, Wenqing; Nagaraju, Javaregowda; Ffrench-Constant, Richard H; Herrero, Salvador; Gordon, Karl; Swevers, Luc; Smagghe, Guy

    2011-02-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

  12. RNA interference technology used for the study of aquatic virus infections.

    PubMed

    Reshi, Mohammad Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2014-09-01

    Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. Recent advances in biotechnology have had a significant impact on disease reduction in aquaculture. RNAi is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of specific genes in living systems. RNA interference technology has emerged as a powerful tool for manipulating gene expression in the laboratory. This technology represents a new therapeutic approach for treating aquatic diseases, including viral infections. RNAi technology is based on a naturally occurring post-transcriptional gene silencing process mediated by the formation of dsRNA. RNAi has been proven widely effective for gene knockdown in mammalian cultured cells, but its utility in fish remains unexplored. This review aims to highlight the RNAi technology that has made significant contributions toward the improvement of aquatic animal health and will also summarize the current status and future strategies concerning the therapeutic applications of RNAi to combat viral disease in aquacultured organisms.

  13. Efficient Inhibition of Human Glioma Development by RNA Interference-Mediated Silencing of PAK5

    PubMed Central

    Gu, Xuefeng; Wang, Ce; Wang, Xuefeng; Ma, Guoda; Li, You; Cui, Lili; Chen, Yanyan; Zhao, Bin; Li, Keshen

    2015-01-01

    Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment. PMID:25632266

  14. RNA interference technology to control pest sea lampreys--a proof-of-concept.

    PubMed

    Heath, George; Childs, Darcy; Docker, Margaret F; McCauley, David W; Whyard, Steven

    2014-01-01

    The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

  15. RNA Interference Technology to Control Pest Sea Lampreys - A Proof-of-Concept

    PubMed Central

    Heath, George; Childs, Darcy; Docker, Margaret F.; McCauley, David W.; Whyard, Steven

    2014-01-01

    The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0–fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species. PMID:24505485

  16. RNA interference technology to control pest sea lampreys--a proof-of-concept.

    PubMed

    Heath, George; Childs, Darcy; Docker, Margaret F; McCauley, David W; Whyard, Steven

    2014-01-01

    The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species. PMID:24505485

  17. Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts.

    PubMed

    Zhai, Zhiyang; Sooksa-nguan, Thanwalee; Vatamaniuk, Olena K

    2009-02-01

    Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) beta-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-month-old mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost- and space-efficient initial screening and selection of genes for subsequent in planta studies.

  18. RNA interference based approach to down regulate Osmoregulators of whitefly Bemisia tabaci: potential technology for the control of whitefly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) could offer potential for insect pest management. Insects feeding exclusively on plant sap depend on osmotic pressure...

  19. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways

    PubMed Central

    Clemens, James C.; Worby, Carolyn A.; Simonson-Leff, Nancy; Muda, Marco; Maehama, Tomohiko; Hemmings, Brian A.; Dixon, Jack E.

    2000-01-01

    We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project. PMID:10823906

  20. EGFP-EGF1-conjugated PLGA nanoparticles for targeted delivery of siRNA into injured brain microvascular endothelial cells for efficient RNA interference.

    PubMed

    Chen, Chen; Mei, Heng; Shi, Wei; Deng, Jun; Zhang, Bo; Guo, Tao; Wang, Huafang; Hu, Yu

    2013-01-01

    Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer.

  1. Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants.

    PubMed

    Hemmes, Hans; Kaaij, Lucas; Lohuis, Dick; Prins, Marcel; Goldbach, Rob; Schnettler, Esther

    2009-07-01

    The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at positions 173-175 are involved in the siRNA binding and suppressor activities. PMID:19282433

  2. Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea.

    PubMed

    Choi, Man-Yeon; Vander Meer, Robert K; Coy, Monique; Scharf, Michael E

    2012-08-01

    Insect neuropeptide hormones represent more than 90% of all insect hormones. The PBAN/pyrokinin family is a major group of insect neuropeptides, and they are expected to be found from all insect groups. These species-specific neuropeptides have been shown to have a variety of functions from embryo to adult. PBAN is well understood in moth species relative to sex pheromone biosynthesis, but other potential functions are yet to be determined. Recently, we focused on defining the PBAN gene and peptides in fire ants in preparation for an investigation of their function(s). RNA interference (RNAi) technology is a convenient tool to investigate unknown physiological functions in insects, and it is now an emerging method for development of novel biologically-based control agents as alternatives to insecticides. This could be a paradigm shift that will avoid many problems associated with conventional chemical insecticides. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target from two insect groups; a social insect, the fire ant (Solenopsis invicta) and a non-social insect, the corn earworm (Helicoverpa zea). Both insects are economically important pests. We report negative impacts after PBAN dsRNA treatment to suppress PBAN gene transcription during developmental and adult stages of both species, e.g. increased adult and larval mortality, delayed pupal development and decreased sex pheromone production in the moth. This is an important first step in determining the multiple functions of the PBAN gene in these two insects. This work illustrates the variety of phenotypic effects observed after RNAi silencing of the PBAN gene and suggests the possibility of novel biologically-based insect pest control methods. PMID:22705256

  3. Transgenic RNA interference (RNAi)-derived field resistance to cassava brown streak disease.

    PubMed

    Ogwok, Emmanuel; Odipio, John; Halsey, Mark; Gaitán-Solís, Eliana; Bua, Anton; Taylor, Nigel J; Fauquet, Claude M; Alicai, Titus

    2012-12-01

    Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (ΔCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ΔCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ΔCP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.

  4. Transcriptome Analysis in Cotton Boll Weevil (Anthonomus grandis) and RNA Interference in Insect Pests

    PubMed Central

    Coelho, Roberta Ramos; Antonino de Souza Jr, José Dijair; Togawa, Roberto Coiti; Silva-Junior, Orzenil Bonfim; Pappas-Jr, Georgios Joannis; da Silva, Maria Cristina Mattar; Engler, Gilbert; Grossi-de-Sa, Maria Fatima

    2013-01-01

    Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects. PMID:24386449

  5. Transcriptome analysis in cotton boll weevil (Anthonomus grandis) and RNA interference in insect pests.

    PubMed

    Firmino, Alexandre Augusto Pereira; Fonseca, Fernando Campos de Assis; de Macedo, Leonardo Lima Pepino; Coelho, Roberta Ramos; Antonino de Souza, José Dijair; Togawa, Roberto Coiti; Silva-Junior, Orzenil Bonfim; Pappas-Jr, Georgios Joannis; da Silva, Maria Cristina Mattar; Engler, Gilbert; Grossi-de-Sa, Maria Fatima

    2013-01-01

    Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families' data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.

  6. RNA interference: Applications and advances in insect toxicology and insect pest management.

    PubMed

    Kim, Young Ho; Soumaila Issa, Moustapha; Cooper, Anastasia M W; Zhu, Kun Yan

    2015-05-01

    Since its discovery, RNA interference (RNAi) has revolutionized functional genomic studies due to its sequence-specific nature of post-transcriptional gene silencing. In this paper, we provide a comprehensive review of the recent literature and summarize the current knowledge and advances in the applications of RNAi technologies in the field of insect toxicology and insect pest management. Many recent studies have focused on identification and validation of the genes encoding insecticide target proteins, such as acetylcholinesterases, ion channels, Bacillus thuringiensis receptors, and other receptors in the nervous system. RNAi technologies have also been widely applied to reveal the role of genes encoding cytochrome P450 monooxygenases, carboxylesterases, and glutathione S-transferases in insecticide detoxification and resistance. More recently, studies have focused on understanding the mechanism of insecticide-mediated up-regulation of detoxification genes in insects. As RNAi has already shown great potentials for insect pest management, many recent studies have also focused on host-induced gene silencing, in which several RNAi-based transgenic plants have been developed and tested as proof of concept for insect pest management. These studies indicate that RNAi is a valuable tool to address various fundamental questions in insect toxicology and may soon become an effective strategy for insect pest management. PMID:25987228

  7. RNA interference-based suppression of phosphoenolpyruvate carboxylase results in susceptibility of rapeseed to osmotic stress.

    PubMed

    Chen, Mei; Tang, Yunlai; Zhang, Jingmei; Yang, Mingfeng; Xu, Yinong

    2010-06-01

    The diverse functions of phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) in C(3) plants are not as well understood as in C(4) plants. To investigate the functions of PEPCase in C(3) plants, rapeseed (Brassica napus L.) PEPCase gene (referred to as BNPE15) was silenced by the RNA interference (RNAi) technique. Under normal growth conditions, no significant difference in lipid content and fatty acid composition were found between wild-type (WT) and transgenic rapeseed plants. However, when these plants were subjected to osmotic stress induced by osmoticum polyethylene glycol (PEG-6000), membrane permeability and membrane lipid peroxidization in roots and leaves of transgenic plants were higher than those of WT plants. It suggested that transgenic plants are more susceptible to osmotic stress than WT plants. Taken together, the results showed that the suppression of PEPCase by RNAi leads to susceptibility to osmotic stress in rapeseed, and PEPCase is involved in the response of C(3) plants to environmental stress.

  8. Applications of RNA interference-based gene silencing in animal agriculture.

    PubMed

    Long, Charles R; Tessanne, Kimberly J; Golding, Michael C

    2010-01-01

    Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.

  9. Targeting Th17 Cells with Small Molecules and Small Interference RNA

    PubMed Central

    Lin, Hui; Song, Pingfang; Zhao, Yi; Xue, Li-Jia; Liu, Yi; Chu, Cong-Qiu

    2015-01-01

    T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+ T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage. PMID:26792955

  10. Increased keratinocyte proliferation initiated through downregulation of desmoplakin by RNA interference

    SciTech Connect

    Wan Hong . E-mail: hong.wan@cancer.org.uk; South, Andrew P.; Hart, Ian R.

    2007-07-01

    The intercellular adhesive junction desmosomes are essential for the maintenance of tissue structure and integrity in skin. Desmoplakin (Dp) is a major obligate plaque protein which plays a fundamental role in anchoring intermediate filaments to desmosomal cadherins. Evidence from hereditary human disease caused by mutations in the gene encoding Dp, e.g. Dp haploinsufficiency, suggests that alterations in Dp expression result not only in the disruption of tissue structure and integrity but also could evoke changes in keratinocyte proliferation. We have used transient RNA interference (RNAi) to downregulate Dp specifically in HaCaT keratinocytes. We showed that this Dp downregulation also caused reduced expression of several other desmosomal proteins. Increased cell proliferation and enhanced G{sub 1}-to-S-phase entry in the cell cycle, as monitored by colonial cellular density and BrdU incorporation, were seen in Dp RNAi-treated cells. These proliferative changes were associated with elevated phospho-ERK1/2 and phospho-Akt levels. Furthermore, this increase in phospho-ERK/1/2 and phospho-Akt levels was sustained in Dp RNAi-treated cells at confluence whereas in control cells there was a significant reduction in phosphorylation of ERK1/2. This study indicates that Dp may participate in the regulation of keratinocyte cell proliferation by, in part at least, regulating cell cycle progression.

  11. T7 peptide-functionalized nanoparticles utilizing RNA interference for glioma dual targeting.

    PubMed

    Kuang, Yuyang; An, Sai; Guo, Yubo; Huang, Shixian; Shao, Kun; Liu, Yang; Li, Jianfeng; Ma, Haojun; Jiang, Chen

    2013-09-15

    Among all the malignant brain tumors, glioma is the deadliest and most common form with poor prognosis. Gene therapy is regarded as a promising way to halt the progress of the disease or even cure the tumor and RNA interference (RNAi) stands out. However, the existence of the blood-brain barrier (BBB) and blood tumor barrier (BTB) limits the delivery of these therapeutic genes. In this work, the delivery system targeting to the transferrin (Tf) receptor highly expressed on both BBB and glioma was successfully synthesized and would not compete with endogenous Tf. U87 cells stably express luciferase were employed here to simulate tumor and the RNAi experiments in vitro and in vivo validated that the gene silencing activity was 2.17-fold higher with the targeting ligand modification. The dual-targeting gene delivery system exhibits a series of advantages, such as high efficiency, low toxicity, stability and high transaction efficiency, which may provide new opportunities in RNAi therapeutics and nanomedicine of brain tumors.

  12. Targeting Th17 Cells with Small Molecules and Small Interference RNA.

    PubMed

    Lin, Hui; Song, Pingfang; Zhao, Yi; Xue, Li-Jia; Liu, Yi; Chu, Cong-Qiu

    2015-01-01

    T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4(+) T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage. PMID:26792955

  13. Promise and challenge of RNA interference-based therapy for cancer.

    PubMed

    Petrocca, Fabio; Lieberman, Judy

    2011-02-20

    Cancer therapeutics still fall far short of our goals for treating patients with locally advanced or metastatic disease. Until recently, almost all cancer drugs were crude cytotoxic agents that discriminate poorly between cancer cells and normally dividing cells. The development of targeted biologics that recognize tumor cell surface antigens and of specific inhibitors of pathways dysregulated in cancer cells or normal cellular pathways on which a cancer cell differentially depends has provided hope for converting our increasing understanding of cellular transformation into intelligently designed anticancer therapeutics. However, new drug development is painfully slow, and the pipeline of new therapeutics is thin. The discovery of RNA interference (RNAi), a ubiquitous cellular pathway of gene regulation that is dysregulated in cancer cells, provides an exciting opportunity for relatively rapid and revolutionary approaches to cancer drug design. Small RNAs that harness the RNAi machinery may become the next new class of drugs for treating a variety of diseases. Although it has only been 9 years since RNAi was shown to work in mammalian cells, about a dozen phase I to III clinical studies have already been initiated, including four for cancer. So far there has been no unexpected toxicity and suggestions of benefit in one phase II study. However, the obstacles for RNAi-based cancer therapeutics are substantial. This article will discuss how the endogenous RNAi machinery might be harnessed for cancer therapeutics, why academic researchers and biotech and pharmaceutical companies are so excited, and what the obstacles are and how they might be overcome.

  14. Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

    PubMed

    Hansen, Klavs R; Burns, Gavin; Mata, Juan; Volpe, Thomas A; Martienssen, Robert A; Bähler, Jürg; Thon, Geneviève

    2005-01-01

    Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing. PMID:15632061

  15. Modulating Drug Resistance by Targeting BCRP/ABCG2 Using Retrovirus-Mediated RNA Interference

    PubMed Central

    Yuan, Jianhui; Liu, Wenlan; Deng, Tingting; Li, Zigang; Jin, Yi; Hu, Zhangli

    2014-01-01

    Background The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications. Methodology/Principal Findings To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor. Conclusions/Significance The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors. PMID:25076217

  16. Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: evidence from Manduca sexta and Blattella germanica.

    PubMed

    Garbutt, Jennie S; Bellés, Xavier; Richards, Elaine H; Reynolds, Stuart E

    2013-02-01

    RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi. PMID:22664137

  17. Establishment and evaluation of stable cell lines inhibiting foot-and-mouth disease virus by RNA interference.

    PubMed

    Gu, Yuan-Xing; Gao, Zong-Liang; Zhou, Jian-Hua; Zhang, Jie; Liu, Yong-Sheng

    2014-01-01

    RNA interference (RNAi) has been proved to be a powerful tool for foot-and-mouth disease virus FMDV inhibition in vitro and in vivo. We established five stable baby hamster kidney 21 cell lines (BHK-21) containing five short hairpin RNAs (shRNAs) expression plasmids (p3D1shRNA, p3D2shRNA, p3D3shRNA, p3D4shRNA, and p3D5shRNA) targeting 3D gene of FMDV. Immunofluorescent assay, virus titration, and real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) were conducted to detect the effect of shRNAs on FMDV replication. After challenged with FMDV of O/CHA/99, two cell lines (p3D1shRNA and p3D4shRNA) showed a significant reduction in the synthesis of viral protein and RNA, accompanied by a sharp decrease in viral yield, and the inhibition could last for at least thirty passages. We developed an efficient procedure for the establishment and evaluation of stable cell lines for anti-FMDV research based on RNAi technology, which can be a candidate method for anti-FMDV research.

  18. The efficiency of RNA interference for conferring stable resistance to Plum Pox Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plum transformed with an intron hairpin RNA CP (ihRNA-CP) were resistant to PPV infection through the specific process of RNA silencing involving both small interfering -RNA interfering (siRNA) and a methylated virus transgene. This recognition process specifically targeted the triggered PPV genome...

  19. Breast cancer cell line MDA-MB-231 miRNA profile expression after BIK interference: BIK involvement in autophagy.

    PubMed

    Ruiz Esparza-Garrido, Ruth; Torres-Márquez, María Eugenia; Viedma-Rodríguez, Rubí; Velázquez-Wong, Ana Claudia; Salamanca-Gómez, Fabio; Rosas-Vargas, Haydeé; Velázquez-Flores, Miguel Ángel

    2016-05-01

    B-cell lymphoma 2 (BCL2)-interacting killer (apoptosis inducing) (BIK) has been proposed as a tumor suppressor in diverse types of cancers. However, BIK's overexpression in breast cancer (BC) and in non-small lung cancer cells (NSCLCs), associated with a poor prognosis, suggests its participation in tumor progression. In this study, we evaluated the global expression pattern of microRNAs (miRNAs), messenger RNA (mRNA) expression changes in autophagy, and autophagic flux after BIK interference. BIK gene expression was silenced by small interfering RNA (siRNA) in BC cell MDA-MB-231, and BIK interference efficiency was tested by real-time PCR and by Western blotting. BIK expression levels decreased by 75 ± 18 % in the presence of 600 nM siRNA, resulting in the abolishment of BIK expression by 94 ± 30 %. BIK interference resulted in the overexpression of 17 miRNAs that, according to the DIANA-miRPath v3.0 database, are mainly implied in the control of cell signaling, gene expression, and autophagy. The autophagy array revealed downregulation of transcripts which participate in autophagy, and their interactome revealed a complex network, where hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), α-synuclein (SNCA), unc-51-like autophagy activating kinase 1/2 (ULK1/2), and mitogen-activated protein kinase 3 (MAPK3) were shown to be signaling hubs. LC3-II expression-an autophagy marker-was increased by 169 ± 25 % after BIK interference, which indicates the involvement of BIK in autophagy. Altogether, our results indicate-for the first time-that BIK controls the expression of miRNAs, as well as the autophagic flux in MDA-MB-231 cells. PMID:26662110

  20. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  1. Parental RNA interference of genes involved in embryonic development of the western corn rootworm, Diabrotica virgifera virgifera LeConte.

    PubMed

    Khajuria, Chitvan; Vélez, Ana M; Rangasamy, Murugesan; Wang, Haichuan; Fishilevich, Elane; Frey, Meghan L F; Carneiro, Newton Portilho; Gandra, Premchand; Narva, Kenneth E; Siegfried, Blair D

    2015-08-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control.

  2. RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.

    PubMed

    Ortved, Kyla F; Austin, Bethany S; Scimeca, Michael S; Nixon, Alan J

    2016-01-01

    Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1β (IL-1β), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1β activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1β by RNA interference (RNAi) may drive a reduction in IL-1β. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1β (shIL-1β) would significantly decrease IL-1β expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1β in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1β knockdown led to significantly decreased expression of IL-1β, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1β using rAAV2 vector expressing a short hairpin IL-1β knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints. PMID:27073697

  3. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs.

    PubMed

    Rinaldi, Gabriel; Morales, Maria E; Alrefaei, Yousef N; Cancela, Martín; Castillo, Estela; Dalton, John P; Tort, José F; Brindley, Paul J

    2009-10-01

    Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the S. mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate l-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by >or=80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases. PMID:19463860

  4. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    PubMed

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

  5. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    PubMed

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  6. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

    PubMed Central

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang

    2016-01-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  7. Suppression of Bedbug’s Reproduction by RNA Interference of Vitellogenin

    PubMed Central

    Moriyama, Minoru; Hosokawa, Takahiro; Tanahashi, Masahiko; Nikoh, Naruo; Fukatsu, Takema

    2016-01-01

    Recent resurgence of the bedbug Cimex lectularius is a global problem on the public health. On account of the worldwide rise of insecticide-resistant bedbug populations, exploration of new approaches to the bedbug control and management is anticipated. In this context, gene silencing by RNA interference (RNAi) has been considered for its potential application to pest control and management, because RNAi enables specific suppression of target genes and thus flexible selection of target traits to be disrupted. In this study, in an attempt to develop a control strategy targeting reproduction of the bedbug, we investigated RNAi-mediated gene silencing of vitellogenin (Vg), a major yolk protein precursor essential for oogenesis. From the bedbug transcriptomes, we identified a typical Vg gene and a truncated Vg gene, which were designated as ClVg and ClVg-like, respectively. ClVg gene was highly expressed mainly in the fat body of adult females, which was more than 100 times higher than the expression level of ClVg-like gene, indicating that ClVg gene is the primary functional Vg gene in the bedbug. RNAi-mediated suppression of ClVg gene expression in adult females resulted in drastically reduced egg production, atrophied ovaries, and inflated abdomen due to hypertrophied fat bodies. These phenotypic consequences are expected not only to suppress the bedbug reproduction directly but also to deteriorate its feeding and survival indirectly via behavioral modifications. These results suggest the potential of ClVg gene as a promising target for RNAi-based population management of the bedbug. PMID:27096422

  8. [Evaluation of the binding affinity and RNA interference of low-molecular-weight chitosan/siRNA complexes using an imaging system].

    PubMed

    Kawaguchi, Yasuhisa; Okuda, Tomoyuki; Ban, Tatsunori; Danjo, Kazumi; Okamoto, Hirokazu

    2009-04-01

    Chitosan is one of the attractive non-viral carriers for gene delivery including siRNA. However, common chitosan, which has a relatively high molecular weight, is insoluble in water, which might make it difficult to apply clinically. In this study, we investigated the efficacy of low-molecular-weight chitosan (LMWC), which is soluble in water, as a carrier for siRNA delivery. To evaluate the binding affinity and RNA interference (RNAi) of LMWC/siRNA complexes, a multi-well imaging system (IVIS) was adapted. CT26 cells stably expressing firefly luciferase (CT26/Luc cells) were established to evaluate RNAi. Evaluation of RNAi using lipofectamine(TM) 2000 was carried out by employing a luminometer with cell lysis and IVIS without cell lysis. The results were closely correlated, suggesting the advantages of the multi-well imaging system regarding screening, the visualization of results, and nondestructive evaluation. Fluorescence generated by ethidium bromide intercalated in the double strand of siRNA was markedly quenched at a higher ratio of LMWC to siRNA (N/P) and lower pH. Evaluation of the particle size and zeta potential of LMWC/siRNA complexes also indicated the higher binding affinity of LMWC with siRNA. At N/P=300 and pH 6.5, which satisfied the high-level binding affinity of LMWC with siRNA, significantly lower luminescence was detected in CT26/Luc cells treated with LMWC/siRNA compared with those treated with LMWC alone, suggesting the presence of RNAi. These results suggested that LMWC may be an effective carrier for siRNA delivery, and that the multi-well imaging system may be a powerful tool to evaluate the binding affinity and RNAi.

  9. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA.

    PubMed

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E; Siegfried, Blair D

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  10. Silencing of c-kit with small interference RNA attenuates inflammation in a murine model of allergic asthma.

    PubMed

    Wu, Wei; Wang, Tao; Dong, Jia-Jia; Liao, Zeng-Lin; Wen, Fu-Qiang

    2012-07-01

    Asthma is a chronic respiratory disease characterized by the inflammation of the airways due to infiltration and activation of several inflammatory cells that produce cytokines. c-kit, a proto-oncogene that encodes a tyrosine kinase receptor, has been found to be associated with allergic inflammation. The aim of the present study was to assess whether silencing of c-kit with small interference RNA (siRNA) would attenuate inflammation in allergic asthma. A mouse model of ovalbumin (OVA)-induced allergic asthma was treated with systemic administration of anti-c-kit siRNA to inhibit the expression of the c-kit gene. siRNAs were injected through the vena caudalis. We measured inflammatory response in both anti-c-kit siRNA-treated and control mice. Systemic administration of siRNA could effectively inhibit the expression of the c-kit gene and reduce the infiltration of inflammatory cells (eosinophils and lymphocytes) into the lung tissue and bronchoalveolar lavage fluid. In addition, we found that c-kit siRNA can decrease the production of the T-helper type 2 (Th2) cytokines, interleukin 4 (IL-4) and IL-5, but has no influence on IFN-γ generation. These results show that inhibition of c-kit expression with siRNA can reduce the inflammatory response in allergic asthma.

  11. Gene silencing of 4-1BB by RNA interference inhibits acute rejection in rats with liver transplantation.

    PubMed

    Shi, Yang; Hu, Shuqun; Song, Qingwei; Yu, Shengcai; Zhou, Xiaojun; Yin, Jun; Qin, Lei; Qian, Haixin

    2013-01-01

    The 4-1BB signal pathway plays a key role in organ transplantation tolerance. In this study, we have investigated the effect of gene silencing of 4-1BB by RNA interference (RNAi) on the acute rejection in rats with liver transplantation. The recombination vector of lentivirus that contains shRNA targeting the 4-1BB gene (LV-sh4-1BB) was constructed. The liver transplantation was performed using the two-cuff technique. Brown-Norway (BN) recipient rats were infected by the recombinant LVs. The results showed that gene silencing of 4-1BB by RNAi downregulated the 4-1BB gene expression of the splenic lymphocytes in vitro, and the splenic lymphocytes isolated from the rats with liver transplantation. LV-sh4-1BB decreased the plasma levels of liver injury markers including AST, ALT, and BIL and also decreased the level of plasma IL-2 and IFN- γ in recipient rats with liver transplantation. Lentivirus-mediated delivery of shRNA targeting 4-1BB gene prolonged the survival time of recipient and alleviated the injury of liver morphology in recipient rats with liver transplantation. In conclusion, our results demonstrate that gene silencing of 4-1BB by RNA interference inhibits the acute rejection in rats with liver transplantation.

  12. RNA interference against transcription elongation factor SII does not support its role in transcription-coupled nucleotide excision repair.

    PubMed

    Mackinnon-Roy, Christine; Stubbert, Lawton J; McKay, Bruce C

    2011-01-10

    RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.

  13. RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria

    PubMed Central

    2011-01-01

    Background The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. Results We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. Conclusion This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals. PMID:21679422

  14. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  15. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA

    PubMed Central

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E.; Siegfried, Blair D.

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA’s and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA’s. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  16. Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference.

    PubMed

    Zhao, Lina; Yang, Mengmeng; Shen, Qida; Liu, Xiaojun; Shi, Zuokun; Wang, Shigui; Tang, Bin

    2016-01-01

    RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice. PMID:27328657

  17. Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference

    PubMed Central

    Zhao, Lina; Yang, Mengmeng; Shen, Qida; Liu, Xiaojun; Shi, Zuokun; Wang, Shigui; Tang, Bin

    2016-01-01

    RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice. PMID:27328657

  18. Interplay between RNA interference and heat shock response systems in Drosophila melanogaster

    PubMed Central

    Funikov, S. Yu; Kanapin, A. A.; Logacheva, M. D.; Penin, A. A.; Snezhkina, A. V.; Shilova, V. Yu.; Garbuz, D. G.; Zatsepina, O. G.

    2016-01-01

    The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. Here, we investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster. We demonstrated that pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. It was also shown that deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR. PMID:27805906

  19. Feeding-based RNA interference of a trehalose phosphate synthase gene in the brown planthopper, Nilaparvata lugens.

    PubMed

    Chen, J; Zhang, D; Yao, Q; Zhang, J; Dong, X; Tian, H; Chen, J; Zhang, W

    2010-12-01

    The brown planthopper, Nilaparvata lugens, is the most devastating rice insect pest to have given rise to an outbreak in recent years. RNA interference (RNAi) is a technological breakthrough that has been developed as a powerful tool for studying gene function and for the highly targeted control of insect pests. Here, we examined the effects of using a feeding-based RNAi technique to target the gene trehalose phosphate synthase (TPS) in N. lugens. The full-length cDNA of N. lugens TPS (NlTPS) is 3235 bp and has an open reading frame of 2424 bp, encoding a protein of 807 amino acids. NlTPS was expressed in the fat body, midgut and ovary. Quantitative real-time PCR (qRT-PCR) analysis revealed that NlTPS mRNA is expressed continuously with little change during the life of the insect. Efficient silencing of the TPS gene through double-stranded RNA (dsRNA) feeding led to rapid and significant reduction levels of TPS mRNA and enzymatic activity. Additionally, the development of N. lugens larvae that had been fed with the dsRNA was disturbed, resulting in lethality, and the cumulative survival rates dropped to 75.56, 64.44, 55.56 and 40.00% after continuous ingestion of 0.5 µg/µl dsRNA for 2, 4, 7 and 10 days, respectively. These values were significantly lower than those of the insects in the control group, suggesting that NlTPS dsRNA may be useful as a means of insect pest control. PMID:20726907

  20. Knockdown of Nogo gene by short hairpin RNA interference promotes functional recovery of spinal cord injury in a rat model.

    PubMed

    Liu, Guo-Min; Luo, Yun-Gang; Li, Juan; Xu, Kun

    2016-05-01

    The specific myelin component Nogo protein is one of the major inhibitory molecules of spinal cord axonal outgrowth following spinal cord injury. The present study aimed to investigate the effects of silencing Nogo protein with shRNA interference on the promotion of functional recovery in a rat model with spinal cord hemisection. Nogo-A short hairpin RNAs (Nogo shRNAs) were constructed and transfected into rats with spinal cord hemisection by adenovirus-mediated transfection. Reverse transcription‑polymerase chain reaction and western blotting were performed to analyze the expression of Nogo-A and Growth Associated Protein 43 (GAP-43). In addition, Basso Beattie Bresnahan (BBB) scores were used to assess the functional recovery of rats following spinal cord injury. The results demonstrated that expression of the Nogo‑A gene was observed to be downregulated following transfection and GAP‑43 expression was observed to increase. The BBB scores were increased following treatment with Nogo shRNAs, indicating functional recovery of the injured nerves. Thus, Nogo-A shRNA interference can knockdown Nogo gene expression and upregulate GAP-43 to promote the functional recovery of spinal cord injury in rats. This finding may advance progress toward assisting the regeneration of injured neurons through the use of Nogo-A shRNA. PMID:27035338

  1. RNA Interference Mitigates Motor and Neuropathological Deficits in a Cerebellar Mouse Model of Machado-Joseph Disease

    PubMed Central

    Onofre, Isabel; Albuquerque, David; Déglon, Nicole; Pereira de Almeida, Luís

    2014-01-01

    Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease. PMID:25144231

  2. Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells.

    PubMed

    Ferreira, Helena Lage; Spilki, Fernando Rosado; de Almeida, Renata Servan; Santos, Márcia M A B; Arns, Clarice Weis

    2007-04-01

    Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. In this study, we designed specific short interfering RNA (siRNA) targeting the AMPV nucleoprotein (N) and fusion (F) genes. Three days post-virus infection, virus titration, real time RT-PCR, and RT-PCR assays were performed to verify the effect of siRNA in AMPV replication. A marked decrease in virus titers from transfected CER cells treated with siRNA/N was observed. Also, the production of N, F, and G mRNAs in AMPV was decreased. Results indicate that N-specific siRNA can inhibit virus replication. In future studies, a combination of siRNAs targeting the RNA polymerase complex may be used as a tool to study AMPV replication and/or antiviral therapy.

  3. RNA interference in Colorado potato beetle: steps toward development of dsRNA as a commercial insecticide

    PubMed Central

    Palli, Subba Reddy

    2015-01-01

    Colorado potato beetle (CPB) is a notorious pest on potatoes and has a remarkable ability to detoxify plant chemicals and develop resistance against insecticides. dsRNA targeting CPB genes could be expressed in potato plants to control this pest. However, previous attempts at introducing transgenic potato plants to control CPB were not highly successful. Recent studies showed that feeding dsRNA expressed in bacteria works very well to kill CPB. To realize the potential of RNAi to control this and other economically important pests, more efficient methods for production and delivery of dsRNA need to be developed. Extensive research to determine off-target and non-target effects, environmental fate and potential for resistance development is also essential. PMID:26705514

  4. In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

    RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.

  5. Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase.

    PubMed

    Van Ekert, Evelien; Powell, Charles A; Shatters, Robert G; Borovsky, Dov

    2014-11-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pivotal role in the control of reproduction in adults and metamorphism in larval mosquitoes. This report describes an approach to control Aedes aegypti using RNAi against JH acid methyl transferase (AeaJHAMT), the ultimate enzyme in the biosynthetic pathway of JH III that converts JH acid III (JHA III) into JH III. In female A. aegypti that were injected or fed jmtA dsRNA targeting the AeaJHAMT gene (jmtA) transcript, egg development was inhibited in 50% of the treated females. In mosquito larvae that were fed transgenic Pichia pastoris cells expressing long hair pin (LHP) RNA, adult eclosion was delayed by 3 weeks causing high mortality. Northern blot analyses and qPCR studies show that jmtA dsRNA causes inhibition of jmtA transcript in adults and larvae, which is consistent with the observed inhibition of egg maturation and larval development. Taken together, these results suggest that jmtA LHP RNA expressed in heat inactivated genetically modified P. pastoris cells could be used to control mosquito populations in the marsh.

  6. Analysis of RNA Interference Lines Identifies New Functions of Maternally-Expressed Genes Involved in Embryonic Patterning in Drosophila melanogaster.

    PubMed

    Liu, Niankun; Lasko, Paul

    2015-03-31

    Embryonic patterning in Drosophila melanogaster is initially established through the activity of a number of maternally expressed genes that are expressed during oogenesis. mRNAs from some of these genes accumulate in the posterior pole plasm of the oocyte and early embryo and localize further into RNA islands, which are transient ring-like structures that form around the nuclei of future primordial germ cells (pole cells) at stage 3 of embryogenesis. As mRNAs from several genes with known functions in anterior-posterior patterning and/or germ cell specification accumulate in RNA islands, we hypothesized that some other mRNAs that localize in this manner might also function in these developmental processes. To test this, we investigated the developmental functions of 51 genes whose mRNAs accumulate in RNA islands by abrogating their activity in the female germline using RNA interference. This analysis revealed requirements for ttk, pbl, Hip14, eIF5, eIF4G, and CG9977 for progression through early oogenesis. We observed dorsal appendage defects in a proportion of eggs produced by females expressing double-stranded RNA targeting Mkrn1 or jvl, implicating these two genes in dorsal-ventral patterning. In addition, posterior patterning defects and a reduction in pole cell number were seen in the progeny of Mkrn1 females. Because the mammalian ortholog of Mkrn1 acts as an E3 ubiquitin ligase, these results suggest an additional link between protein ubiquitination and pole plasm activity.

  7. PDX-1 is a therapeutic target for pancreatic cancer, insulinoma and islet neoplasia using a novel RNA interference platform.

    PubMed

    Liu, Shi-He; Rao, Donald D; Nemunaitis, John; Senzer, Neil; Zhou, Guisheng; Dawson, David; Gingras, Marie-Claude; Wang, Zhaohui; Gibbs, Richard; Norman, Michael; Templeton, Nancy S; Demayo, Francesco J; O'Malley, Bert; Sanchez, Robbi; Fisher, William E; Brunicardi, F Charles

    2012-01-01

    Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a "drugable" target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNA(PDX-1), was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNA(humanPDX-1) lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNA(mousePDX-1) lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNA(mousePDX-1) lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.

  8. Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase.

    PubMed

    Van Ekert, Evelien; Powell, Charles A; Shatters, Robert G; Borovsky, Dov

    2014-11-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pivotal role in the control of reproduction in adults and metamorphism in larval mosquitoes. This report describes an approach to control Aedes aegypti using RNAi against JH acid methyl transferase (AeaJHAMT), the ultimate enzyme in the biosynthetic pathway of JH III that converts JH acid III (JHA III) into JH III. In female A. aegypti that were injected or fed jmtA dsRNA targeting the AeaJHAMT gene (jmtA) transcript, egg development was inhibited in 50% of the treated females. In mosquito larvae that were fed transgenic Pichia pastoris cells expressing long hair pin (LHP) RNA, adult eclosion was delayed by 3 weeks causing high mortality. Northern blot analyses and qPCR studies show that jmtA dsRNA causes inhibition of jmtA transcript in adults and larvae, which is consistent with the observed inhibition of egg maturation and larval development. Taken together, these results suggest that jmtA LHP RNA expressed in heat inactivated genetically modified P. pastoris cells could be used to control mosquito populations in the marsh. PMID:25111689

  9. Targeting MACC1 by RNA interference inhibits proliferation and invasion of bladder urothelial carcinoma in T24 cells

    PubMed Central

    Xu, Song-Tao; Ding, Xiang; Ni, Qing-Feng; Jin, Shao-Ju

    2015-01-01

    The purpose of this article is to research on whether MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma (BUC). In this study, the expression of MACC1 gene was knocked down by RNA interference (RNAi) in the T24 cell (human BUC cell). The transcription level of MACC1 was detected by RT-PCR. Activities of MACC1, caspase-3, caspase-8, Bax and Met (mesenchymal-epithelial transition factor) protein were measured by Western blot. The cell proliferation and apoptosis were detected by MTT and flow cytometry. The cell’s invasion ability was performed on Matrigel transwell assay. We also detect MMP2 (metalloproteinase-2) proteins by ELISA. The results showed that the level of MACC1 mRNA and protein was significantly reduced after RNAi. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference. Apoptosis studies also showed that MACC1 gene interference in T24 loses its anti-apoptotic effects. The expression of apoptosis proteins (Caspase-3, Caspase-8 and Bax) increased significantly due to the MACC1 RNAi. The level of Met protein was down-regulated obviously due to RNAi. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to MACC1 RNAi. Further studies showed that the secretion of MMP-2 was reduced by RNAi. It can conclude that the ability of proliferation and invasion in T24 cells can be inhibited by RNAi-targeting MACC1. As a result, MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma. PMID:26339359

  10. A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.

    PubMed

    Port, Fillip; Hausmann, George; Basler, Konrad

    2011-11-01

    Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export. PMID:21886182

  11. RNA interference for CFTR attenuates lung fluid absorption at birth in rats

    PubMed Central

    Li, Tianbo; Koshy, Shyny; Folkesson, Hans G

    2008-01-01

    Background Small interfering RNA (siRNA) against αENaC (α-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs. Methods Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality. Results αENaC and CFTR mRNA and protein decreased by ~80% and ~85%, respectively, following αENaC and CFTR silencing. Extravascular lung water and mortality increased after αENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells αENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced αENaC mRNA and protein. αENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. Conclusion Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth. PMID:18652671

  12. Decreased expression of RNA interference machinery, Dicer and Drosha, is associated with poor outcome in ovarian cancer patients

    SciTech Connect

    Merritt, William M.; Lin, Yvonne G.; Han, Liz Y.; Kamat, Aparna A.; Spannuth, Whitney A.; Schmandt, Rosemarie; Urbauer, Diana; Pennacchio, Len A.; Cheng, Jan-Fang; Zeidan, Alexandra; Wang, Hua; Mueller, Peter; Lenburg, Marc E.; Gray, Joe W.; Mok, Samuel; Birrer, Michael J.; Lopez-Berestein, Gabriel; Coleman, Robert L.; Bar-Eli, Menashe; Sood, Anil K.

    2008-05-06

    The clinical and functional significance of RNA interference (RNAi) machinery, Dicer and Drosha, in ovarian cancer is not known and was examined. Dicer and Drosha expression was measured in ovarian cancer cell lines (n=8) and invasive epithelial ovarian cancer specimens (n=111) and correlated with clinical outcome. Validation was performed with previously published cohorts of ovarian, breast, and lung cancer patients. Anti-Galectin-3 siRNA and shRNA transfections were used for in vitro functional studies. Dicer and Drosha mRNA and protein levels were decreased in 37% to 63% of ovarian cancer cell lines and in 60% and 51% of human ovarian cancer specimens, respectively. Low Dicer was significantly associated with advanced tumor stage (p=0.007), and low Drosha with suboptimal surgical cytoreduction (p=0.02). Tumors with both high Dicer and Drosha were associated with increased median patient survival (>11 years vs. 2.66 years for other groups; p<0.001). In multivariate analysis, high Dicer (HR=0.48; p=0.02), high-grade histology (HR=2.46; p=0.03), and poor chemoresponse (HR=3.95; p<0.001) were identified as independent predictors of disease-specific survival. Findings of poor clinical outcome with low Dicer expression were validated in separate cohorts of cancer patients. Galectin-3 silencing with siRNA transfection was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% compared to non-targeting sequences), and similar in cell lines with high Dicer. Our findings demonstrate the clinical and functional impact of RNAi machinery alterations in ovarian carcinoma and support the use of siRNA constructs that do not require endogenous Dicer and Drosha for therapeutic applications.

  13. Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations.

    PubMed

    Lawrence, Richard J; Pikaard, Craig S

    2003-10-01

    Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs. PMID:12974816

  14. In Vitro Gene Silencing of the Fish Microsporidian Heterosporis saurida by RNA Interference

    PubMed Central

    Kumar, Gokhlesh; Abdel-Baki, Abdel-Azeem; Dkhil, Mohamed A.; El-Matbouli, Mansour; Al-Quraishy, Saleh

    2016-01-01

    Heterosporis saurida, a microsporidian parasite of lizardfish, Saurida undosquamis, causes severe economic losses in marine aquaculture. Among the novel approaches being explored for treatment of parasitic infections in aquaculture is small interfering RNA molecules. The aim of the present study was to investigate the efficiency of using siRNA to knock down expression of specific genes of H. saurida in vitro. For this purpose, siRNAs specific for ATP/ADP antiporter 1 and methionine aminopeptidase II genes were designed and tested using a previously developed in vitro cultivation model. Silencing of H. saurida target genes was assessed and the efficacy of using siRNA for inhibition of gene expression was measured by quantitative real-time polymerase chain reaction (PCR). Silencing of ATP/ADP antiporter 1 or methionine aminopeptidase II by siRNA reduced H. saurida infection levels in EK-1 cells 40% and 60%, respectively, as measured by qRT-PCR and spore counts. Combined siRNA treatment of both ATP/ADP antiporter 1 and methionine aminopeptidase II siRNAs was more effective against H. saurida infection as seen by the 16S rRNA level and spore counts. Our study concluded that siRNA could be used to advance development of novel approaches to inhibit H. saurida and provide an alternative approach to combat microsporidia. PMID:27228357

  15. RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly.

    PubMed

    Raza, Amir; Malik, Hassan Jamil; Shafiq, Muhammad; Amin, Imran; Scheffler, Jodi A; Scheffler, Brian E; Mansoor, Shahid

    2016-01-01

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA) against both aquaporin (AQP) and a sucrase gene, alpha glucosidase (AGLU). These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR) was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci) and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops. PMID:27105353

  16. Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA interference-based bone anabolic strategy.

    PubMed

    Liang, Chao; Guo, Baosheng; Wu, Heng; Shao, Ningsheng; Li, Defang; Liu, Jin; Dang, Lei; Wang, Cheng; Li, Hui; Li, Shaohua; Lau, Wing Ki; Cao, Yu; Yang, Zhijun; Lu, Cheng; He, Xiaojuan; Au, D W T; Pan, Xiaohua; Zhang, Bao-Ting; Lu, Changwei; Zhang, Hongqi; Yue, Kinman; Qian, Airong; Shang, Peng; Xu, Jiake; Xiao, Lianbo; Bian, Zhaoxiang; Tan, Weihong; Liang, Zicai; He, Fuchu; Zhang, Lingqiang; Lu, Aiping; Zhang, Ge

    2015-03-01

    Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer-functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.

  17. Differential nanotoxicological and neuroinflammatory liabilities of non-viral vectors for RNA interference in the central nervous system.

    PubMed

    Godinho, Bruno M D C; McCarthy, David J; Torres-Fuentes, Cristina; Beltrán, Caroll J; McCarthy, Joanna; Quinlan, Aoife; Ogier, Julien R; Darcy, Raphael; O'Driscoll, Caitriona M; Cryan, John F

    2014-01-01

    Progression of RNA interference-based gene silencing technologies for the treatment of disorders of the central nervous system (CNS) depends on the availability of efficient non-toxic nanocarriers. Despite advances in the field of nanotechnology undesired and non-specific interactions with different brain-cell types occur and are poorly investigated. To this end, we studied the cytotoxic and neuroinflammatory effects of widely-used transfection reagents and modified amphiphilic β-cyclodextrins (CDs). All non-viral vectors formed positively charged nanoparticles with distinctive physicochemical properties. Differential and significant cytotoxic effects were observed among commercially available cationic vectors, whereas CDs induced limited disruptions of cellular membrane integrity and mitochondrial dehydrogenase activity. Interestingly, murine derived BV2 microglia cells and a rat striatal in vitro model of Huntington's disease (ST14A-HTT120Q) were more susceptible to toxicity than human U87 astroglioma cells. BV2 microglia presented significant increases in cytokine, toll-like receptor 2 and cyclooxygenase-2 gene expression after transfection with selected commercial vectors but not with CD.siRNA nanoparticles. Non-viral siRNA nanoparticles formulated with G6 polyamidoamine (PAMAM) also significantly increased cytokine gene expression in the brain following injections into the mouse striatum. Together our data identify modified CDs as nanosystems that enable siRNA delivery to the brain with low levels of cytotoxicity and immunological activation. PMID:24138827

  18. Delivery Systems for the Direct Application of siRNAs to Induce RNA Interference (RNAi) In Vivo

    PubMed Central

    Aigner, Achim

    2006-01-01

    RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described. PMID:17057369

  19. RNA Interference based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly

    PubMed Central

    Raza, Amir; Malik, Hassan Jamil; Shafiq, Muhammad; Amin, Imran; Scheffler, Jodi A.; Scheffler, Brian E.; Mansoor, Shahid

    2016-01-01

    Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) that could offer great potential for insect pest management. The diet of insects feeding exclusively on phloem sieves contains water and sugars as main components, and the uptake of the liquid food greatly depends on the osmotic pressure within the insect body. Based on this physiological mechanism, transgenic plants of Nicotiana tabacum were generated expressing double stranded RNA (dsRNA) against both aquaporin (AQP) and a sucrase gene, alpha glucosidase (AGLU). These two genes are involved in osmotic pressure maintenance particularly in sap sucking insects, and the aim was to disrupt osmoregulation within the insect ultimately leading to mortality. Real time quantitative PCR (RT-qPCR) was performed to assess the suppression of gene expression in Bemisia tabaci (B. tabaci) and mortality was recorded during transgenic tobacco feeding bioassays. Feeding of insects on plants expressing dsRNA significantly reduced the transcript level of the target genes in B. tabaci after six days of feeding and more than 70% mortality was observed in B. tabaci fed on transgenic plants compared to the control plants. Our data shows that down-regulation of genes related to osmoregulation may find practical applications for the control of this important pest in cotton and other crops. PMID:27105353

  20. Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

    PubMed

    D'Agostino, Vito Giuseppe; Lal, Preet; Mantelli, Barbara; Tiedje, Christopher; Zucal, Chiara; Thongon, Natthakan; Gaestel, Matthias; Latorre, Elisa; Marinelli, Luciana; Seneci, Pierfausto; Amadio, Marialaura; Provenzani, Alessandro

    2015-11-10

    Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

  1. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

    PubMed

    Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

    2015-01-01

    Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the "PICKY" software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients.

  2. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    PubMed Central

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jose R.

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Delivered RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1, and OPR) in the hemi-biotrophic fungus F. oxysporum f. sp. conglutinans. Expression of double stranded RNA (dsRNA) molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75, 83, and 72% reduction for FOW2, FRP1, and OPR, respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30–50% survival and OPR between 45 and 70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants. PMID:25654075

  3. Advances in RNA interference technology and its impact on nutritional improvement, disease and insect control in plants.

    PubMed

    Katoch, Rajan; Thakur, Neelam

    2013-03-01

    This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

  4. Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels.

    PubMed

    French, Andrew S; Meisner, Shannon; Liu, Hongxia; Weckström, Matti; Torkkeli, Päivi H

    2015-01-01

    Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100-1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596-708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction. PMID:26257659

  5. Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

    PubMed Central

    French, Andrew S.; Meisner, Shannon; Liu, Hongxia; Weckström, Matti; Torkkeli, Päivi H.

    2015-01-01

    Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100–1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596–708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction. PMID:26257659

  6. Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

    PubMed Central

    Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

    2013-01-01

    RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

  7. Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

    PubMed Central

    Spenkuch, Felix; Hinze, Gerald; Kellner, Stefanie; Kreutz, Christoph; Micura, Ronald; Basché, Thomas; Helm, Mark

    2014-01-01

    The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases. PMID:25300485

  8. Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor.

    PubMed

    Spenkuch, Felix; Hinze, Gerald; Kellner, Stefanie; Kreutz, Christoph; Micura, Ronald; Basché, Thomas; Helm, Mark

    2014-11-10

    The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.

  9. Inhibition of influenza A virus matrix and nonstructural gene expression using RNA interference.

    PubMed

    McMillen, Cynthia M; Beezhold, Donald H; Blachere, Francoise M; Othumpangat, Sreekumar; Kashon, Michael L; Noti, John D

    2016-10-01

    Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influenza virus therapy.

  10. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells.

    PubMed

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  11. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    NASA Astrophysics Data System (ADS)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  12. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

    PubMed

    Dykxhoorn, Derek M; Schlehuber, Lisa D; London, Irving M; Lieberman, Judy

    2006-04-11

    A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

  13. Adenoviral-mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma.

    PubMed

    Fan, Rui; Li, Xiaohua; Du, Wenqi; Zou, Xue; Du, Rui; Zhao, Lina; Luo, Guanhong; Mo, Ping; Xia, Lin; Pan, Yanglin; Shi, Yongquan; Lian, Zhaorui; Feitelson, Mark A; Nie, Yongzhan; Liu, Jie; Fan, Daiming

    2011-06-15

    Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5-year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1-S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl-2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl-2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad-URG11-siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad-URG11-siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.

  14. Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

    PubMed Central

    Nebane, N. Miranda; Coric, Tatjana; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

    2016-01-01

    The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens. PMID:26663785

  15. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    NASA Astrophysics Data System (ADS)

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  16. Inhibition of Newcastle disease virus replication by RNA interference targeting the matrix protein gene in chicken embryo fibroblasts.

    PubMed

    Yin, Renfu; Ding, Zhuang; Liu, Xinxin; Mu, Lianzhi; Cong, Yanlong; Stoeger, Tobias

    2010-07-01

    Newcastle disease (ND) is an infectious viral disease of birds caused by the Newcastle disease virus (NDV), also known as avian paramyxovirus type 1 (AMPV-1), which leads to severe economic losses in the poultry industry worldwide. In this study, the application of RNA interference (RNAi) for inhibiting the replication of NDV in cell culture by targeting the viral matrix protein gene (M) is described. Two M-specific shRNA-expressing plasmid constructs, named pS(M641) and pS(M827), were evaluated for antiviral activity against the NDV strain NA-1 by cytopathic effects (CPE), virus titration and real-time RT-PCR. After 36h of infection, both pS(M641) and pS(M827) reduced virus titers by 79.4- and 31.6-fold, respectively, and they down-regulated mRNA expression levels of the matrix protein gene M by 94.6% and 84.8%, respectively, in chicken embryo fibroblast (CEF) cells, while only pS(M641) significantly decreased CPE, compared to the control group. These results indicated that the M gene 641 and 827 sites represent potential antiviral therapy targets, and RNAi targeting of the M gene could not only represent an effective treatment in Newcastle disease but also aid as a method for studying the replication of NDV.

  17. PRDM16 is associated with evasion of apoptosis by prostatic cancer cells according to RNA interference screening.

    PubMed

    Zhu, Shaoxing; Xu, Yipeng; Song, Mei; Chen, Guiping; Wang, Hua; Zhao, Yang; Wang, Zongping; Li, Fangyin

    2016-10-01

    Histone methylation, which is regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs), has been indicated to be involved in a variety of diseases, particularly in cancer, including androgen‑independent prostate cancer (PCa). However, the functions of HMTs and HDTs in cancer have largely remained elusive. The present study, utilized an RNA interference screening using a lentiviral small hairpin (sh)RNA library to systematically elucidate the function of HMTs and HDTs in PCa cell growth and viability. Nine HMTs and HDTs, namely FBXO11, PRDM10, JMJD8, MLL, SETD4, JMJD7, PRMT2, MEN1 and PRDM16, were identified to affect DU145 cell viability, as indicated by an MTS assay subsequent to knockdown of the specific genes using shRNA pools. Furthermore, flow cytometric analysis and western blot analysis of apoptosis‑associated proteins indicated that PRDM16 has an anti‑apoptotic role in PCa cells. In addition, the spliced form, sPRDM16/MEL1S, was detected to be overexpressed in PCa cell lines. In conclusion, the present study indicated an important oncogenic role of sPRDM16/MEL1S in PCa and suggested that PRDM16 may represent a novel therapeutic target. PMID:27511603

  18. PRDM16 is associated with evasion of apoptosis by prostatic cancer cells according to RNA interference screening.

    PubMed

    Zhu, Shaoxing; Xu, Yipeng; Song, Mei; Chen, Guiping; Wang, Hua; Zhao, Yang; Wang, Zongping; Li, Fangyin

    2016-10-01

    Histone methylation, which is regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs), has been indicated to be involved in a variety of diseases, particularly in cancer, including androgen‑independent prostate cancer (PCa). However, the functions of HMTs and HDTs in cancer have largely remained elusive. The present study, utilized an RNA interference screening using a lentiviral small hairpin (sh)RNA library to systematically elucidate the function of HMTs and HDTs in PCa cell growth and viability. Nine HMTs and HDTs, namely FBXO11, PRDM10, JMJD8, MLL, SETD4, JMJD7, PRMT2, MEN1 and PRDM16, were identified to affect DU145 cell viability, as indicated by an MTS assay subsequent to knockdown of the specific genes using shRNA pools. Furthermore, flow cytometric analysis and western blot analysis of apoptosis‑associated proteins indicated that PRDM16 has an anti‑apoptotic role in PCa cells. In addition, the spliced form, sPRDM16/MEL1S, was detected to be overexpressed in PCa cell lines. In conclusion, the present study indicated an important oncogenic role of sPRDM16/MEL1S in PCa and suggested that PRDM16 may represent a novel therapeutic target.

  19. Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes

    PubMed Central

    Matsa, Elena; Dixon, James E.; Medway, Christopher; Georgiou, Orestis; Patel, Minal J.; Morgan, Kevin; Kemp, Paul J.; Staniforth, Andrew; Mellor, Ian; Denning, Chris

    2014-01-01

    Aims Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. Methods and results We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K+ currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). Conclusions These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart. PMID:23470493

  20. RNA interference targeting tNOX attenuates cell migration via a mechanism that involves membrane association of Rac

    SciTech Connect

    Liu, S.-C.; Yang, J.-J.; Shao, K.-N.; Chueh, P.J.

    2008-01-25

    tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.

  1. Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection

    PubMed Central

    Ayllón, Nieves; Naranjo, Victoria; Hajdušek, Ondrej; Villar, Margarita; Galindo, Ruth C.; Kocan, Katherine M.; Alberdi, Pilar; Šíma, Radek; Cabezas-Cruz, Alejandro; Rückert, Claudia; Bell-Sakyi, Lesley; Kazimírová, Mária; Havlíková, Sabína; Klempa, Boris; Kopáček, Petr; de la Fuente, José

    2015-01-01

    Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found. PMID:26186700

  2. Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection.

    PubMed

    Ayllón, Nieves; Naranjo, Victoria; Hajdušek, Ondrej; Villar, Margarita; Galindo, Ruth C; Kocan, Katherine M; Alberdi, Pilar; Šíma, Radek; Cabezas-Cruz, Alejandro; Rückert, Claudia; Bell-Sakyi, Lesley; Kazimírová, Mária; Havlíková, Sabína; Klempa, Boris; Kopáček, Petr; de la Fuente, José

    2015-01-01

    Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

  3. RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

    PubMed

    Ouyang, Jie-Xiu; Luo, Tao; Sun, Hui-Yun; Huang, Jian; Tang, Dan-Feng; Wu, Lei; Zheng, Yue-Hui; Zheng, Li-Ping

    2013-08-01

    Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

  4. Helicobacter pylori interferes with an embryonic stem cell micro RNA cluster to block cell cycle progression

    PubMed Central

    2011-01-01

    Background MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections. PMID:22027184

  5. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug.

    PubMed

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed. PMID:27144586

  6. RNA interference (RNAI) as a tool to engineer high nutritional value in chicory (Chicorium intybus).

    PubMed

    Asad, M

    2006-01-01

    The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.

  7. Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

    PubMed Central

    Bansal, Raman; Mittapelly, Priyanka; Chen, Yuting; Mamidala, Praveen; Zhao, Chaoyang; Michel, Andy

    2016-01-01

    The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed. PMID:27144586

  8. Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model

    PubMed Central

    Bric, Anka; Miething, Cornelius; Bialucha, Carl Uli; Scuoppo, Claudio; Zender, Lars; Krasnitz, Alexander; Xuan, Zhenyu; Zuber, Johannes; Wigler, Michael; Hicks, James; McCombie, Richard W.; Hemann, Michael T.; Hannon, Gregory J.; Powers, Scott; Lowe, Scott W.

    2009-01-01

    SUMMARY Short hairpin RNAs (shRNAs) capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor suppressor gene loss in mice. By selecting for shRNAs capable of accelerating lymphomagenesis in a well-characterized mouse lymphoma model, we identified over ten candidate tumor suppressors, including Sfrp1, Numb, Mek1, and Angiopoietin 2. Several components of the DNA damage response machinery were also identified, including Rad17, which acts as a haploinsufficient tumor suppressor that responds to oncogenic stress and whose loss is associated with poor prognosis in human patients. Our results emphasize the utility of in vivo RNAi screens, identify and validate a diverse set of tumor suppressors, and have therapeutic implications. PMID:19800577

  9. Enhancing Cellulase Production in Thermophilic Fungus Myceliophthora thermophila ATCC42464 by RNA Interference of cre1 Gene Expression.

    PubMed

    Yang, Fan; Gong, Yanfen; Liu, Gang; Zhao, Shengming; Wang, Juan

    2015-07-01

    The role of CRE1 in a thermophilic fungus, Myceliophthora thermophila ATCC42464, was studied using RNA interference. In the cre1-silenced strain C88, the filter paper hydrolyzing activity and β-1,4-endoglucanase activity were 3.76-, and 1.31-fold higher, respectively, than those in the parental strain when the strains were cultured in inducing medium for 6 days. The activities of β-1,4-exoglucanase and cellobiase were 2.64-, and 5.59-fold higher, respectively, than those in the parental strain when the strains were cultured for 5 days. Quantitative reverse-transcription polymerase chain reaction showed that the gene expression of egl3, cbh1, and cbh2 was significantly increased in transformant C88 compared with the wild-type strain. Therefore, our findings suggest the feasibility of improving cellulase production by modifying the regulator expression, and an attractive approach to increasing the total cellulase productivity in thermophilic fungi.

  10. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

    PubMed Central

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  11. Single-target RNA interference for the blockade of multiple interacting proinflammatory and profibrotic pathways in cardiac fibroblasts.

    PubMed

    Tank, Juliane; Lindner, Diana; Wang, Xiaomin; Stroux, Andrea; Gilke, Leona; Gast, Martina; Zietsch, Christin; Skurk, Carsten; Scheibenbogen, Carmen; Klingel, Karin; Lassner, Dirk; Kühl, Uwe; Schultheiss, Heinz-Peter; Westermann, Dirk; Poller, Wolfgang

    2014-01-01

    Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals. PMID:24239602

  12. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

    PubMed

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  13. HAWC Response to Lighting Activity

    NASA Astrophysics Data System (ADS)

    Lara, A.

    2014-12-01

    The High Altitude Water Cherenkov (HAWC) observatory is being constructed at the Sierra Negra volcano (4100 m a.s.l.) in Mexico. HAWC's primary purpose is the study of both: galactic and extra-galactic sources of high energy gamma rays. HAWC will consist of 300 large water Cherenkov detectors (WCD), each instrumented with 4 photo-multipliers (PMTs). The Data taking has already started while construction continues, with the completion projected for late 2014. The HAWC scaler system records the rates of individual PMTs giving the opportunity of study relatively low energy transients as solar energetic particles and the solar modulation of galactic cosmic rays. In this work, we present the observations of scaler rate enhancements associated with lightning activity observed close to HAWC (i. e. at high altitude). In particular, we present the time and space coincidence of the lighting strikes and the scaler enhancements and our preliminary speculations on the origin of the detector response to the lighting activity.

  14. [Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene].

    PubMed

    Zheng, Chuanyi; Chen, Zhenggang; Bai, Enqi; Li, Zhengzheng; Yang, Kun

    2016-05-01

    目的:筛选出人GLUT3基因有效的RNA干扰(RNA interference,RNAi)序列,并构建出慢病毒RNAi载体。方法:根据GLUT3基因mRNA序列设计合成siRNA片段4个,分别定向克隆至pLV-shRNA载体上,并将构建的质粒转染HeLa细胞,运用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测GLUT3mRNA的表达量验证其干扰效果。筛选出其中有效的质粒与病毒包装质粒共转染293T细胞进行包装,收获并浓缩重组慢病毒颗粒,测定病毒颗粒滴度后,将病毒感染U251胶质瘤细胞以测定感染慢病毒干扰载体后胶质瘤细胞内GLUT3的表达情况。结果:重组RNAi质粒pLV-shRNA-GLUT3-1,pLV-shRNA-GLUT3-2,pLV-shRNA-GLUT3-3,pLV-shRNA- GLUT3-4经测序证实质粒载体构建成功;4个干扰质粒在HeLa细胞中均可以明显抑制GLUT3-mRNA的表达。pLV-shRNA-GLUT3可以在293T细胞中成功包装。收集293T细胞分泌的病毒上清浓缩后测定病毒颗粒LV-GLUT3滴度为1.5×109 TU/mL。与感染阴性对照慢病毒颗粒(0.3641±0.044)相比,胶质瘤U251细胞感染慢病毒颗粒LV- GLUT3后,GLUT3蛋白相对表达明显降低(0.108±0.016,t=16.267,P<0.001)。结论:成功构建了人GLUT3基因有效的慢病毒RNAi载体,为进一步研究GLUT3的生物学功能奠定了基础。.

  15. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    PubMed

    Beard, Philippa M; Griffiths, Samantha J; Gonzalez, Orland; Haga, Ismar R; Pechenick Jowers, Tali; Reynolds, Danielle K; Wildenhain, Jan; Tekotte, Hille; Auer, Manfred; Tyers, Mike; Ghazal, Peter; Zimmer, Ralf; Haas, Jürgen

    2014-01-01

    Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  16. Reversible suppression of an essential gene in adult mice using transgenic RNA interference

    PubMed Central

    McJunkin, Katherine; Mazurek, Anthony; Premsrirut, Prem K.; Zuber, Johannes; Dow, Lukas E.; Simon, Janelle; Stillman, Bruce; Lowe, Scott W.

    2011-01-01

    RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8–11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system. PMID:21482754

  17. Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.

    PubMed

    Khandelia, Piyush; Yap, Karen; Makeyev, Eugene V

    2011-08-01

    Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest. PMID:21768390

  18. Use of recombinant tobacco mosaic virus to achieve RNA interference in plants against the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae).

    PubMed

    Khan, Arif Muhammad; Ashfaq, Muhammad; Kiss, Zsofia; Khan, Azhar Abbas; Mansoor, Shahid; Falk, Bryce W

    2013-01-01

    The citrus mealybug, Planococcus citri, is an important plant pest with a very broad plant host range. P. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms induced on a range of host plants, but P. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. Better strategies for managing this pest are greatly needed. RNA interference (RNAi) is an emerging tool for functional genomics studies and is being investigated as a practical tool for highly targeted insect control. Here we investigated whether RNAi effects can be induced in P. citri and whether candidate mRNAs could be identified as possible targets for RNAi-based P. citri control. RNAi effects were induced in P. citri, as demonstrated by specific target reductions of P. citri actin, chitin synthase 1 and V-ATPase mRNAs after injection of the corresponding specific double-stranded RNA inducers. We also used recombinant Tobacco mosaic virus (TMV) to express these RNAi effectors in Nicotiana benthamiana plants. We found that P. citri showed lower fecundity and pronounced death of crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi against P. citri, and that recombinant TMV is an effective tool for evaluating candidate RNAi effectors in plants.

  19. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

    PubMed

    Woerner, Andreas C; Frottin, Frédéric; Hornburg, Daniel; Feng, Li R; Meissner, Felix; Patra, Maria; Tatzelt, Jörg; Mann, Matthias; Winklhofer, Konstanze F; Hartl, F Ulrich; Hipp, Mark S

    2016-01-01

    Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. PMID:26634439

  20. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    PubMed

    Yamanaka, Tomoyuki; Wong, Hon Kit; Tosaki, Asako; Bauer, Peter O; Wada, Koji; Kurosawa, Masaru; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2014-01-01

    In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms. PMID:24705917

  1. Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation

    PubMed Central

    Tosaki, Asako; Bauer, Peter O.; Wada, Koji; Kurosawa, Masaru; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2014-01-01

    In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms. PMID:24705917

  2. Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle.

    PubMed

    Lü, Feng-Gong; Fu, Kai-Yun; Guo, Wen-Chao; Li, Guo-Qing

    2015-10-10

    In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata, and were provisionally designated LdJHEH1 and LdJHEH2. Both mRNAs were detectable in the thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2, and a mixture of dsJHEH1-1+dsJHEH2-1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 (LdKr-h1) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2, or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle. PMID:26079572

  3. RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.

    PubMed

    Trzcińska-Daneluti, Agata M; Chen, Anthony; Nguyen, Leo; Murchie, Ryan; Jiang, Chong; Moffat, Jason; Pelletier, Lawrence; Rotin, Daniela

    2015-06-01

    Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.

  4. Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle.

    PubMed

    Lü, Feng-Gong; Fu, Kai-Yun; Guo, Wen-Chao; Li, Guo-Qing

    2015-10-10

    In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata, and were provisionally designated LdJHEH1 and LdJHEH2. Both mRNAs were detectable in the thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2, and a mixture of dsJHEH1-1+dsJHEH2-1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 (LdKr-h1) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2, or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle.

  5. Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

    PubMed

    Wu, Hong-Xia; Wang, Hua-Lei; Guo, Xiao-Feng; Yang, Yu-Jiao; Ma, Jin-Zhu; Wang, Tie-Cheng; Gao, Yu-Wei; Zhao, Yong-Kun; Yang, Song-Tao; Xia, Xian-Zhu

    2013-10-01

    To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

  6. Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

    PubMed

    Chen, Weizao; Liu, Mingqiu; Jiao, Ye; Yan, Weiyao; Wei, Xuefeng; Chen, Jiulian; Fei, Liang; Liu, Yang; Zuo, Xiaoping; Yang, Fugui; Lu, Yonggan; Zheng, Zhaoxin

    2006-04-01

    Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD. PMID:16537624

  7. Cytotype regulation by telomeric P elements in Drosophila melanogaster: evidence for involvement of an RNA interference gene.

    PubMed

    Simmons, Michael J; Ryzek, Don-Felix; Lamour, Cecile; Goodman, Joseph W; Kummer, Nicole E; Merriman, Peter J

    2007-08-01

    P elements inserted at the left telomere of the X chromosome evoke the P cytotype, a maternally inherited condition that regulates the P-element family in the Drosophila germline. This regulation is completely disrupted in stocks heterozygous for mutations in aubergine, a gene whose protein product is involved in RNA interference. However, cytotype is not disrupted in stocks heterozygous for mutations in two other RNAi genes, piwi and homeless (spindle-E), or in a stock heterozygous for a mutation in the chromatin protein gene Enhancer of zeste. aubergine mutations exert their effects in the female germline, where the P cytotype is normally established and through which it is maintained. These effects are transmitted maternally to offspring of both sexes independently of the mutations themselves. Lines derived from mutant aubergine stocks reestablish the P cytotype quickly, unlike lines derived from stocks heterozygous for a mutation in Suppressor of variegation 205, the gene that encodes the telomere-capping protein HP1. Cytotype regulation by telomeric P elements may be tied to a system that uses RNAi to regulate the activities of telomeric retrotransposons in Drosophila. PMID:17603126

  8. Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

    PubMed

    Xie, Xi; Liu, Zhiye; Liu, Mingxin; Tao, Tian; Shen, Xiquan; Zhu, Dongfa

    2016-09-01

    Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal. PMID:27267122

  9. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    PubMed

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera. PMID:26659592

  10. RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits.

    PubMed

    Schijlen, Elio G W M; de Vos, C H Ric; Martens, Stefan; Jonker, Harry H; Rosin, Faye M; Molthoff, Jos W; Tikunov, Yury M; Angenent, Gerco C; van Tunen, Arjen J; Bovy, Arnaud G

    2007-07-01

    Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed. PMID:17478633

  11. Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts

    PubMed Central

    Grainger, Deborah L.; Kutzler, Lydia; Rannels, Sharon L.; Kimball, Scot R.

    2016-01-01

    REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 -/- mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin. PMID:27335637

  12. Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

    PubMed Central

    Chen, Si-Le; Cai, Shi-Rong; Zhang, Xin-Hua; Li, Wen-Feng; Zhai, Er-Tao; Peng, Jian-Jun; Wu, Hui; Chen, Chuang-Qi; Ma, Jin-Ping; Wang, Zhao; He, Yu-Long

    2016-01-01

    The aim of the present study was to investigate the expression level of collapsin response mediator protein 4 (CRMP-4) in human colorectal cancer (CRC) tissue and to evauluate its impact on SW480 cell proliferation, in addition to tumor growth in a mouse xenograft model. Clinical CRC tissue samples were collected to detect the CRMP-4 protein expression levels using western blot and immunohistochemistry analyses. A specific small interfering RNA sequence targeting the CRMP-4 gene (DPYSL3) was constructed and transfected into an SW480 cell line using a lentivirus vector to obtain a stable cell line with low expression of CRMP-4. The effectiveness of the interference was evaluated using western blot and reverse transcription-quantitative polymerase chain reaction, and the cell proliferation was determined using MTT and BrdU colorimetric methods. Tumor growth was assessed by subcutaneously inoculating the constructed cells into BALB/c nude mice. The protein expression levels of CRMP-4 were markedly increased in colon tumor tissue of the human samples. The proliferation of SW480 cells and the tumor growth rate in nude mice of the si-CPMR-4 group were evidently depressed compared with the si-scramble group. Thus, the present results suggest that CRMP-4 may be involved in the pathogenesis of CRC. PMID:27698685

  13. Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

    PubMed Central

    Chen, Si-Le; Cai, Shi-Rong; Zhang, Xin-Hua; Li, Wen-Feng; Zhai, Er-Tao; Peng, Jian-Jun; Wu, Hui; Chen, Chuang-Qi; Ma, Jin-Ping; Wang, Zhao; He, Yu-Long

    2016-01-01

    The aim of the present study was to investigate the expression level of collapsin response mediator protein 4 (CRMP-4) in human colorectal cancer (CRC) tissue and to evauluate its impact on SW480 cell proliferation, in addition to tumor growth in a mouse xenograft model. Clinical CRC tissue samples were collected to detect the CRMP-4 protein expression levels using western blot and immunohistochemistry analyses. A specific small interfering RNA sequence targeting the CRMP-4 gene (DPYSL3) was constructed and transfected into an SW480 cell line using a lentivirus vector to obtain a stable cell line with low expression of CRMP-4. The effectiveness of the interference was evaluated using western blot and reverse transcription-quantitative polymerase chain reaction, and the cell proliferation was determined using MTT and BrdU colorimetric methods. Tumor growth was assessed by subcutaneously inoculating the constructed cells into BALB/c nude mice. The protein expression levels of CRMP-4 were markedly increased in colon tumor tissue of the human samples. The proliferation of SW480 cells and the tumor growth rate in nude mice of the si-CPMR-4 group were evidently depressed compared with the si-scramble group. Thus, the present results suggest that CRMP-4 may be involved in the pathogenesis of CRC.

  14. Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support.

    PubMed

    Boutter, Jeannette; Huang, Yun; Marovca, Blerim; Vonderheit, Andreas; Grotzer, Michael A; Eckert, Cornelia; Cario, Gunnar; Wollscheid, Bernd; Horvath, Peter; Bornhauser, Beat C; Bourquin, Jean-Pierre

    2014-11-30

    Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc- when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc-. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment. PMID:25415224

  15. Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support

    PubMed Central

    Marovca, Blerim; Vonderheit, Andreas; Grotzer, Michael A.; Eckert, Cornelia; Cario, Gunnar; Wollscheid, Bernd; Horvath, Peter

    2014-01-01

    Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc− when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc−. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment. PMID:25415224

  16. Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

    PubMed

    Xie, Xi; Liu, Zhiye; Liu, Mingxin; Tao, Tian; Shen, Xiquan; Zhu, Dongfa

    2016-09-01

    Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal.

  17. Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

    PubMed

    Wang, Meng; Wang, Diandong; Zhang, Xi; Wang, Xu; Liu, Wencui; Hou, Xiaomeng; Huang, Xiaoyin; Xie, Bingyan; Cheng, Xinyue

    2016-05-01

    RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode

  18. The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response

    PubMed Central

    Mehrabadi, Mohammad; Hussain, Mazhar; Matindoost, Leila

    2015-01-01

    ABSTRACT RNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response in Spodoptera frugiperda cells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viral p35 gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by a p35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis. IMPORTANCE Our findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a

  19. Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes.

    PubMed

    Shi, Ji-Feng; Xu, Qing-Yu; Sun, Qiang-Kun; Meng, Qing-Wei; Mu, Li-Li; Guo, Wen-Chao; Li, Guo-Qing

    2016-10-01

    Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae. PMID:27524277

  20. Enhancing chemosensitivity in oral squamous cell carcinoma by lentivirus vector-mediated RNA interference targeting EGFR and MRP2

    PubMed Central

    Chen, Ying-Ju; Chen, Shiuan-Yin; Lovel, Ronald; Ku, Yi-Chu; Lai, Yi-Hui; Hung, Chiao-Ling; Li, Yu-Fen; Lu, Yin-Che; Tai, Chien-Kuo

    2016-01-01

    Oral cancer is the eighth most common type of cancer among men worldwide, with an age-standardized rate of 6.3 per 100,000, and is the fourth leading cause of cancer-associated mortality among men in Taiwan. Cisplatin and 5-fluorouracil (5-FU) are two of the most frequently utilized chemotherapy drugs for the treatment of oral cancer. Although oral cancer patients initially benefit from chemotherapy with these drugs, they may develop resistance to them, which worsens their prognosis and reduces survival rates. It has been reported that increased levels of epidermal growth factor receptor (EGFR) and multidrug resistance-associated protein 2 (MRP2) induce drug resistance in numerous types of human cancer. Therefore, the present study employed lentivirus vector-mediated RNA interference (RNAi) in order to target the genes encoding EGFR and MRP2 in the oral squamous cell carcinoma cell line OC2. It was observed that RNAi-mediated downregulation of EGFR or MRP2 increased the sensitivity to 5-FU and cisplatin in OC2 cells. Downregulation of EGFR resulted in significant suppression of OC2 tumor growth following 5-FU administration. However, simultaneous downregulation of the two genes did not further suppress the tumor growth, indicating that MRP2 does not have a significant role in the chemosensitivity of EGFR-downregulated cells to 5-FU. In contrast, downregulation of MRP2 was demonstrated to significantly enhance the therapeutic effects of cisplatin in EGFR-downregulated OC2 tumors. The observation that the expression of MRP2 was positively correlated with the level of cisplatin resistance in cells suggests that RNAi-mediated downregulation of MRP2 may be applicable as a therapeutic approach toward reversing MRP2-dependent cisplatin resistance in oral cancer. PMID:27602148

  1. Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

    SciTech Connect

    Carbonell, Alberto; Martinez de Alba, Angel-Emilio Gago, Selma

    2008-02-05

    Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.

  2. RNA binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of RNA interference and promotes b2 dimerization.

    PubMed

    Qi, Nan; Cai, Dawei; Qiu, Yang; Xie, Jiazheng; Wang, Zhaowei; Si, Jie; Zhang, Jiamin; Zhou, Xi; Hu, Yuanyang

    2011-09-01

    Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family with a bipartite genome of positive-sense RNAs. A nonstructural protein encoded by subgenomic RNA3 of nodaviruses, B2, serves as a potent RNA silencing suppressor (RSS) by sequestering RNA duplexes. We have previously demonstrated that WhNV B2 blocks RNA silencing in cultured Drosophila cells. However, the molecular mechanism by which WhNV B2 functions remains unknown. Here, we successfully established an RNA silencing system in cells derived from Pieris rapae, a natural host of WhNV, by introducing into these cells double-stranded RNA (dsRNA)-expressing plasmids or chemically synthesized small interfering RNAs (siRNAs). Using this system, we revealed that the WhNV B2 protein inhibited Dicer-mediated dsRNA cleavage and the incorporation of siRNA into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. Based on the modeled B2 3-dimensional structure, serial single alanine replacement mutations and N-terminal deletion analyses showed that the RNA-binding domain of B2 is formed by its helices α2 and α3, while helix α1 mediates B2 dimerization. Furthermore, positive feedback between RNA binding and B2 dimerization was uncovered by gel shift assay and far-Western blotting, revealing that B2 dimerization is required for its binding to RNA, whereas RNA binding to B2 in turn promotes its dimerization. All together, our findings uncovered a novel RNA-binding mode of WhNV B2 and provided evidence that the promotion effect of RNA binding on dimerization exists in a viral RSS protein. PMID:21734038

  3. RNA Binding by a Novel Helical Fold of B2 Protein from Wuhan Nodavirus Mediates the Suppression of RNA Interference and Promotes B2 Dimerization ▿

    PubMed Central

    Qi, Nan; Cai, Dawei; Qiu, Yang; Xie, Jiazheng; Wang, Zhaowei; Si, Jie; Zhang, Jiamin; Zhou, Xi; Hu, Yuanyang

    2011-01-01

    Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family with a bipartite genome of positive-sense RNAs. A nonstructural protein encoded by subgenomic RNA3 of nodaviruses, B2, serves as a potent RNA silencing suppressor (RSS) by sequestering RNA duplexes. We have previously demonstrated that WhNV B2 blocks RNA silencing in cultured Drosophila cells. However, the molecular mechanism by which WhNV B2 functions remains unknown. Here, we successfully established an RNA silencing system in cells derived from Pieris rapae, a natural host of WhNV, by introducing into these cells double-stranded RNA (dsRNA)-expressing plasmids or chemically synthesized small interfering RNAs (siRNAs). Using this system, we revealed that the WhNV B2 protein inhibited Dicer-mediated dsRNA cleavage and the incorporation of siRNA into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. Based on the modeled B2 3-dimensional structure, serial single alanine replacement mutations and N-terminal deletion analyses showed that the RNA-binding domain of B2 is formed by its helices α2 and α3, while helix α1 mediates B2 dimerization. Furthermore, positive feedback between RNA binding and B2 dimerization was uncovered by gel shift assay and far-Western blotting, revealing that B2 dimerization is required for its binding to RNA, whereas RNA binding to B2 in turn promotes its dimerization. All together, our findings uncovered a novel RNA-binding mode of WhNV B2 and provided evidence that the promotion effect of RNA binding on dimerization exists in a viral RSS protein. PMID:21734038

  4. A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

    PubMed Central

    Schuster, Susan; Zirkel, Florian; Kurth, Andreas; van Cleef, Koen W. R.; Drosten, Christian

    2014-01-01

    ABSTRACT Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Côte d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nucleotides (nt) (RNA 1) and 1,801 nt (RNA 2). Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative, that of the Pariacoto virus (family Nodaviridae). Unexpectedly, for the putative capsid protein, maximal pairwise identity of 16% to Lake Sinai virus 2, an unclassified virus with a nonsegmented RNA genome, was found. Moreover, MoNV virions are nonenveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of ∼56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNA interference (RNAi) that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long double-stranded RNA (dsRNA) and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into small interfering RNAs (siRNAs). Phylogenetic analyses indicate that MoNV is a novel member of the family Nodaviridae that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level. IMPORTANCE The identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus with a distant relationship to nodaviruses. Another remarkable feature of Mo

  5. RNA interference-mediated targeting of DKK1 gene expression in Ishikawa endometrial carcinoma cells causes increased tumor cell invasion and migration

    PubMed Central

    YI, NUO; LIAO, QIN-PING; LI, ZHEN-HUA; XIE, BAO-JIANG; HU, YU-HONG; YI, WEI; LIU, MIN

    2013-01-01

    The Wnt signaling pathway plays an essential role in tumor invasion and migration. DKK1 functions as an important inhibitor of the pathway and represents a promising target for cancer therapy. The aim of the present study was to determine the role of DKK1 in endometrial carcinoma (EC) cell invasion and migration using RNA interference (RNAi) technology. Ishikawa EC cells were transfected at high efficiency with specific DKK1 siRNA. RT-PCR and western blot analysis were used to determine the mRNA and protein levels of DKK1, β-catenin and metalloproteinase 14 (MMP14) in siRNA-treated and -untreated cells. In addition, the invasion and migration of the EC cells were detected by invasion and migration assays. Transient transfection of DKK1 siRNA significantly inhibited the mRNA and protein levels of DKK1. Markedly increased cell invasion and migration was observed following treatment with DKK1 siRNA when compared with the negative control siRNA-treated and siRNA-untreated cells. The knockdown of DKK1 also elevated the mRNA and protein levels of β-catenin and MMP14 involved in the Wnt signaling pathway, indicating that targeting this gene may promote intracellular Wnt signal transduction and thus, accelerate EC cell invasion and migration in vitro. The RNAi-mediated targeting of DKK1 gene expression in Ishikawa EC cells resulted in increased tumor cell invasion and migration. DKK1 was identified as an inhibitor of EC cell invasion and migration via its novel role in the Wnt signaling pathway. Targeting DKK1 may therefore represent an effective anti-invasion and -migration strategy for the treatment of EC. PMID:24137406

  6. Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

    PubMed Central

    2010-01-01

    Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the expression profiles of members

  7. Dicer-2- and Piwi-Mediated RNA Interference in Rift Valley Fever Virus-Infected Mosquito Cells

    PubMed Central

    Léger, P.; Lara, E.; Jagla, B.; Sismeiro, O.; Mansuroglu, Z.; Coppée, J. Y.; Bonnefoy, E.

    2013-01-01

    Rift Valley fever virus (RVFV) is a Phlebovirus (Bunyaviridae family) transmitted by mosquitoes. It infects humans and ruminants, causing dramatic epidemics and epizootics in Africa, Yemen, and Saudi Arabia. While recent studies demonstrated the importance of the nonstructural protein NSs as a major component of virulence in vertebrates, little is known about infection of mosquito vectors. Here we studied RVFV infection in three different mosquito cell lines, Aag2 cells from Aedes aegypti and U4.4 and C6/36 cells from Aedes albopictus. In contrast with mammalian cells, where NSs forms nuclear filaments, U4.4 and Aag2 cells downregulated NSs expression such that NSs filaments were never formed in nuclei of U4.4 cells and disappeared at an early time postinfection in the case of Aag2 cells. On the contrary, in C6/36 cells, NSs nuclear filaments were visible during the entire time course of infection. Analysis of virus-derived small interfering RNAs (viRNAs) by deep sequencing indicated that production of viRNAs was very low in C6/36 cells, which are known to be Dicer-2 deficient but expressed some viRNAs presenting a Piwi signature. In contrast, Aag2 and U4.4 cells produced large amounts of viRNAs predominantly matching the S segment and displaying Dicer-2 and Piwi signatures. Whereas 21-nucleotide (nt) Dicer-2 viRNAs were prominent during early infection, the population of 24- to 27-nt Piwi RNAs (piRNAs) increased progressively and became predominant later during the acute infection and during persistence. In Aag2 and U4.4 cells, the combined actions of the Dicer-2 and Piwi pathways triggered an efficient antiviral response permitting, among other actions, suppression of NSs filament formation and allowing establishment of persistence. In C6/36 cells, Piwi-mediated RNA interference (RNAi) appeared to be sufficient to mount an antiviral response against a secondary infection with a superinfecting virus. This study provides new insights into the role of Dicer and Piwi

  8. Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

    SciTech Connect

    Hu Xiaoqu; Su Fengxi; Qin Li; Jia Weijuan; Gong Chang; Yu Fengyan; Guo Jujiang; Song Erwei . E-mail: songerwei02@yahoo.com.cn

    2006-08-04

    Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.

  9. Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in Subtype I-C/Dvulg CRISPR-Cas system

    PubMed Central

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong

    2012-01-01

    SUMMARY Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several Type I CRISPR-Cas systems. Here we report the molecular function of Subtype I-C/Dvulg Cas5d from B. halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3′ single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116 and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-subunit interference complex similar to E. coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among Type I CRISPR subtypes. PMID:22841292

  10. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    SciTech Connect

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  11. Silencing of Gonad-Inhibiting Hormone Transcripts in Litopenaeus vannamei Females by use of the RNA Interference Technology.

    PubMed

    Feijó, Rubens G; Braga, André L; Lanes, Carlos F C; Figueiredo, Márcio A; Romano, Luis A; Klosterhoff, Marta C; Nery, Luis E M; Maggioni, Rodrigo; Wasielesky, Wilson; Marins, Luis F

    2016-02-01

    The method usually employed to stimulate gonadal maturation and spawning of captive shrimp involves unilateral eyestalk ablation, which results in the removal of the endocrine complex responsible for gonad-inhibiting hormone (GIH) synthesis and release. In the present study, RNAi technology was used to inhibit transcripts of GIH in Litopenaeus vannamei females. The effect of gene silencing on gonad development was assessed by analyzing the expression of GIH and vitellogenin, respectively, in the eyestalk and ovaries of L. vannamei females, following ablation or injection with dsRNA-GIH, dsRNA-IGSF4D (non-related dsRNA), or saline solution. Histological analyses were performed to determine the stage of gonadal development and to assess the diameter of oocytes throughout the experimental procedure. Only oocytes at pre-vitellogenesis and primary vitellogenesis stages were identified in females injected with dsRNA-GIH, dsRNA-IGSF4D, or saline solution. Oocytes at all developmental stages were observed in eyestalk-ablated females, with predominance of later stages, such as secondary vitellogenesis and mature oocytes. Despite achieving 64, 73, and 71% knockdown of eyestalk GIH mRNA levels by 15, 30, and 37 days post-injection (dpi), respectively, in dsRNA-GIH-injected females, the expected increase in ovary vitellogenin mRNA expression was only observed on the 37th dpi. This is the first report of the use of RNAi technology to develop an alternative method to eyestalk ablation in captive L. vannamei shrimps.

  12. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

    PubMed

    Chin, V K; Atika Aziz, Nur A; Hudu, Shuaibu A; Harmal, Nabil S; Syahrilnizam, A; Jalilian, Farid A; Zamberi, S

    2016-10-01

    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection. PMID:27432115

  13. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

    PubMed

    Chin, V K; Atika Aziz, Nur A; Hudu, Shuaibu A; Harmal, Nabil S; Syahrilnizam, A; Jalilian, Farid A; Zamberi, S

    2016-10-01

    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection.

  14. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    PubMed Central

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-01-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates. PMID:27405089

  15. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    NASA Astrophysics Data System (ADS)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  16. Artemether Combined with shRNA Interference of Vascular Cell Adhesion Molecule-1 Significantly Inhibited the Malignant Biological Behavior of Human Glioma Cells

    PubMed Central

    Wang, Ping; Xue, Yi-Xue; Yao, Yi-Long; Yu, Bo; Liu, Yun-Hui

    2013-01-01

    Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma cells were treated with artemether at various concentrations and shRNA interfering technology was employed to silence the expression of VCAM-1. Cell viability, migration, invasiveness and apoptosis were assessed with MTT, wound healing, Transwell and Annexin V-FITC/PI staining. The expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylated Akt (p-Akt) was checked by Western blot assay. Results showed that artemether and shRNA-VCAM-1 not only significantly inhibited the migration, invasiveness and expression of MMP-2/9 and p-Akt, but also promoted the apoptosis of U87 cells. Combined treatment of both displayed the maximum inhibitory effects on the malignant biological behavior of glioma cells. Our work revealed the potential therapeutic effects of artemether and antiVCAM-1 in the treatments of gliomas. PMID:23593320

  17. RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells.

    PubMed

    Beck, Markus; Strand, Michael R

    2003-09-30

    The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes.

  18. Dissecting systemic RNA interference in the red flour beetle Tribolium castaneum: parameters affecting the efficiency of RNAi.

    PubMed

    Miller, Sherry C; Miyata, Keita; Brown, Susan J; Tomoyasu, Yoshinori

    2012-01-01

    The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects. PMID:23133513

  19. Lentiviral-mediated RNA interference targeting stathmin1 gene in human gastric cancer cells inhibits proliferation in vitro and tumor growth in vivo

    PubMed Central

    2013-01-01

    Background Gastric cancer is highly aggressive disease. Despite advances in diagnosis and therapy, the prognosis is still poor. Various genetic and molecular alterations are found in gastric cancer that underlies the malignant transformation of gastric mucosa during the multistep process of gastric cancer pathogenesis. The detailed mechanism of the gastric cancer development remains uncertain. In present study we investigated the potential role of stathmin1 gene in gastric cancer tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting stathmin1 as a form of gastric cancer treatment. Methods A lentiviral vector encoding a short hairpin RNA (shRNA) targeted against stathmin1 was constructed and transfected into the packaging cells HEK 293 T and the viral supernatant was collected to transfect MKN-45 cells. The transwell chemotaxis assay and the CCK-8 assay were used to measure migration and proliferation of tumor cells, respectively. Quantitative real-time PCR and western blotting were used to detect the expression levels of stathmin1. Results Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. Significant decreases in stathmin1 mRNA and protein expression were detected in gastric cells carrying lentiviral stathmin-shRNA vector and also significantly inhibited the proliferation, migration in gastric cancer cells and tumorigenicity in Xenograft Animal Models. Conclusions Our findings suggest that stathmin1 overexpression is common in gastric cancer and may play a role in its pathogenesis. Lentivirus mediated RNAi effectively reduced stathmin1 expression in gastric cells. In summary, shRNA targeting of stathmin1 can effectively inhibits human gastric cancer cell growth in vivo and may be a potential therapeutic strategy for gastric cancer. PMID:24040910

  20. Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference.

    PubMed

    Ni, Wei; Qiao, Jun; Ma, Qiman; Wang, Jiangde; Wang, Dawei; Zhao, Xinxia; Cao, Yang; Li, Qifeng; Hu, Shengwei; Chen, Chuangfu

    2015-06-15

    Bovine viral diarrhea virus (BVDV) should be a ubiquitous viral pathogen to the cattle and sheep industry. This pathogen is responsible for severe economic losses. We previously showed that plasmid-mediated dual short hairpin RNA (shRNA) efficiently inhibit BVDV replication in bovine kidney epithelial (MDBK) cells. In this study, we delivered the dual shRNA system to sheep fibroblasts and generated transgenic cell colonies. These transgenic fibroblasts were further used for somatic cell nuclear transfer (SCNT). Three lambs were born at full term, but perished soon after birth. Integration of shRNA into the genome of cloned sheep was confirmed by PCR and expression of shRNA in transgenic sheep was confirmed by real-time PCR. Kidney epithelial cells were isolated from transgenic sheep and challenged with multiple BVDV subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). The dual shRNA expressed in transgenic kidney epithelial cells significantly inhibited BVDV replication in a cross-resistance manner. Our results showed that transgenic RNAi might be a useful tool for preparation of transgenic animals with increased resistance to BVDV.

  1. Silencing MRP1-4 genes by RNA interference enhances sensitivity of human hepatoma cells to chemotherapy

    PubMed Central

    Su, Zheng; Liu, Gaojie; Fang, Tingfeng; Wang, Yang; Zhang, Huayao; Yang, Shanglin; Wei, Jinxing; Lv, Zejian; Tan, Langping; Liu, Jianping

    2016-01-01

    Aim: Besides surgical treatment, systematic chemotherapy plays a crucial role in HCC treatment, especially for patients with advanced HCC. However, none of the single-drug-treatment strategies have shown significant survival benefit due to a high incidence rate of chemoresistance. This study was designed to observe the effect of small interfering of RNA (SiRNA) targeting multidrug resistance-related protein 1-4 (MRP1, MRP2, MRP3, and MRP4) in modulating drug resistance of HepG2/ADM and SMMC7721/ADM cells. Methods: HepG2/Adriamycin (ADM) and SMMC7721/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity and half inhibitory concentration (IC50) of drugs was calculated. Flow cytometry was employed to analyze cell cycle distribution. MRP1-4 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression of proteins was analyzed by Western blot. The growth curve was draw and the cell apoptosis was also observed. Animal experiment was used to compare the cell growth. Results: MTT assay showed that the values of IC50 and RI of HepG2/ADM and SMMC7721/ADM decreased after siRNA treatment in HepG2/ADM cells and SMMC7721/ADM cells. QRT-PCR analysis demonstrated the MRP1-4 mRNA expression decreased significantly in HepG2/ADM cells and SMMC7721/ADM cells after siRNA transfection. In addition, compared with parental cells, MRP1-4 protein expressions apparently decreased in SMMC7721/ADM and HepG2/ADM cells. Flow cytometry showed significantly elevated apoptosis rate following MRP1-4 siRNA transfection. Animal experiment suggested that silencing MRP1-4 gene in vivo inhibited tumor growth. Conclusion: Inhibition of MRP1-4 by small interfering RNA enhanced and selectively restored sensitivity of hepatoma cells to drugs. MRP1-4 siRNA might represent a new therapeutic option for HCC. PMID:27398162

  2. Light-activated Reassembly of Split GFP

    PubMed Central

    Kent, Kevin P.; Boxer, Steven G.

    2011-01-01

    Truncated Green Fluorescent Protein (GFP) with the 11th β-strand removed is potentially interesting for bioconjugation, imaging, and the preparation of semi-synthetic proteins with novel spectroscopic or functional properties. Surprisingly, the truncated GFP generated by removing the 11th strand, once refolded, does not reassemble with a synthetic peptide corresponding to strand 11, but does reassemble following light activation. The mechanism of this process has been studied in detail by absorption, fluorescence and Raman spectroscopy. The chromophore in this refolded truncated GFP is found to be in the trans configuration. Upon exposure to light a photostationary state is formed between the trans and cis conformations of the chromophore, and only truncated GFP with the cis configuration of the chromophore binds the peptide. A kinetic model describing the light activated reassembly of this split GFP is discussed. This unique light-driven reassembly is potentially useful for controlling protein-protein interactions. PMID:21351768

  3. Light-activated communication in synthetic tissues.

    PubMed

    Booth, Michael J; Schild, Vanessa Restrepo; Graham, Alexander D; Olof, Sam N; Bayley, Hagan

    2016-04-01

    We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional electrical communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way. PMID:27051884

  4. Light-activated communication in synthetic tissues

    PubMed Central

    Booth, Michael J.; Schild, Vanessa Restrepo; Graham, Alexander D.; Olof, Sam N.; Bayley, Hagan

    2016-01-01

    We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional electrical communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way. PMID:27051884

  5. Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

    PubMed

    Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

    2013-01-01

    Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

  6. Genome-wide screening using RNAi (RNA interference) to study host factors in viral replication and pathogenesis

    PubMed Central

    Houzet, Laurent; Jeang, Kuan-Teh

    2012-01-01

    With the recent development of siRNA and shRNA expression libraries, RNAi technology has been extensively employed to identify genes involved in diverse cellular processes, such as signal transduction, cell cycle, cancer biology and host-pathogen interactions. In the field of viral infection, this approach has already identified hundreds of new genes not previously known to be important for various virus lifecycles. In this brief review, we focus on recent studies performed using genome-wide RNAi-based screens in mammalian cells for the identification of essential host factors for viral infection and pathogenesis. PMID:21727185

  7. Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

    PubMed Central

    Qiu, Xiu-Wen; Wu, Xiao-Qin; Huang, Lin; Ye, Jian-Ren

    2016-01-01

    As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD. PMID:26797602

  8. Silence of MACC1 expression by RNA interference inhibits proliferation, invasion and metastasis, and promotes apoptosis in U251 human malignant glioma cells

    PubMed Central

    SUN, LONGFENG; LI, GANG; DAI, BING; TAN, WEI; ZHAO, HONGWEN; LI, XIAOFEI; WANG, AIPING

    2015-01-01

    The overexpression of metastasis-associated in colon cancer 1 (MACC1) has been demonstrated not only in colon cancer, but also in various other types of cancer. Gliomas are the most common type of intracranial tumors, and recent studies have reported MACC1 to be involved in human glioma progression. The present study aimed to investigate the effects of MACC1 expression silencing in glioma cells using RNA interference, in order to determine the underlying biological mechanisms of glioma progression, including proliferation, apoptosis, invasion and metastasis. The expression levels of MACC1 were determined in various types of U251 glioma cells using western blot analyses. MACC1-specific short hairpin RNA (shRNA) was used to silence the expression of MACC1 in the U251 cells. The results obtained following MACC1 silencing demonstrated a significant inhibition of cell proliferation, invasion and migration, as well as a marked enhancement of apoptosis. MACC1 shRNA-induced inhibition of cell proliferation was observed by colony forming and MTT assays, and cell apoptosis was measured using flow cytometry and Hoechst staining. In addition, inhibition of cell invasion and migration was assessed using wound healing and transwell assays. Western blotting and fluorescence-activated cell sorting (FACS) revealed a G0/G1 phase cell cycle arrest regulated by cyclins D1 and E; cell apoptosis regulated by caspase-3; and cell invasion and migration regulated by matrix metalloproteinases 2 and 9, respectively. The present study demonstrated that the expression levels of MACC1 were significantly correlated with the biological processes underlying glioma cell proliferation, invasion and metastasis. Therefore, MACC1 may serve as a promising novel therapeutic target in human glioma. Notably, the inhibition of MACC1 expression by shRNA may prove to be an effective genetic therapeutic strategy for glioma treatment. PMID:26043756

  9. RNA Interference: A New Mechanism by Which FMRP Acts in the Normal Brain? What Can Drosophila Teach Us?

    ERIC Educational Resources Information Center

    Siomi, Haruhiko; Ishizuka, Akira; Siomi, Mikiko C.

    2004-01-01

    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…

  10. RNA interference targeting extracellular matrix metalloproteinase inducer (CD147) inhibits growth and increases chemosensitivity in human cervical cancer cells.

    PubMed

    Zhang, F; Zeng, Y L; Zhang, X G; Chen, W J; Yang, R; Li, S J

    2013-01-01

    Overexpression of extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN CD147) has been implicated in the growth and survival of malignant cells. However, its presence and role in cervical cancer cells has not been well-studied. In the present study, small interfering RNA (siRNA) was designed and synthesized to breakdown the expression of CD147. The present data demonstrated that 24 and 48 hours after transfecting CD147 siRNA, both the CD147 mRNA and protein expression were significantly inhibited as determined by quantitative real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Meanwhile, simultaneous silencing of CD147 resulted in distinctly increasing MMP-9, VEGF, and MDR-1. Further studies demonstrated decreased CD147 expression, resulted in G1/S phase transition with flow cytometry analysis, as well as the resistance of the cells to 5-FU. These findings provide further evidence that CD147 may become a promising therapeutic target for human cervical cancer and a potential chemotherapy-sensitizing agent.

  11. RNA interference-mediated knockdown of brain-derived neurotrophic factor (BDNF) promotes cell cycle arrest and apoptosis in B-cell lymphoma cells.

    PubMed

    Xia, D; Li, W; Zhang, L; Qian, H; Yao, S; Qi, X

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily that has been reported to be involved in a number of neurological and psychological situations. Recently, high expression level of BDNF is observed in diverse human malignancies, delineating a role of BDNF in tumorigenesis. Nevertheless, its effect on B-cell lymphoma remains unclear. In this study, RNA interference technology mediated by short hairpin RNA (shRNA) was performed to inhibit endogenous BDNF expression in B-cell lymphoma cells. Results showed that knockdown of BDNF reduced cell growth and proliferation of Raji and Ramos cells. Furthermore, down-regulation of BDNF induced a cell cycle arrest at G0/G1 phase in Raji cells, and consequently led to cell apoptosis in vitro. Meanwhile, down-regulation of Bcl-2 and up-regulation of Bax, activated caspase-3 and caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP) were observed in Raji cells when endogenous BDNF was inhibited. Besides, we also found that suppression of BDNF in Raji cells increased their sensitivity to chemotherapeutic drug, 5-Fluorouracil (5-FU). Our research provides a promising therapeutic strategy for human B-cell lymphoma by targeting BDNF.

  12. RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

    PubMed Central

    2005-01-01

    The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos. PMID:15951844

  13. PEI-g-PEG-RGD/Small Interference RNA Polyplex-Mediated Silencing of Vascular Endothelial Growth Factor Receptor and Its Potential as an Anti-Angiogenic Tumor Therapeutic Strategy

    PubMed Central

    Kim, Jihoon; Kim, Sung Wan

    2011-01-01

    Tumor angiogenesis appears to be achieved by the expression of vascular endothelial growth factor (VEGF) within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. VEGF's angiogenic actions are mediated through its high-affinity binding to 2 endothelium-specific receptor tyrosine kinase, Flt-1 (VEGFR1), and Flk-1/KDR (VEGFR2). RNA interference-mediated knockdown of protein expression at the messenger RNA level provides a new therapeutic strategy to overcome various diseases. To achieve high efficacy in RNA interference-mediated therapy, it is critical to develop an efficient delivering system to deliver small interference RNA (siRNA) into tissues or cells site-specifically. We previously reported an angiogenic endothelial cell-targeted polymeric gene carrier, PEI-g-PEG-RGD. This targeted carrier was developed by the conjugation of the ανβ3/ανβ5 integrin-binding RGD peptide (ACDCRGDCFC) to the cationic polymer, branched polyethylenimine, with a hydrophilic polyethylene glycol (PEG) spacer. In this study, we used PEI-g-PEG-RGD to deliver siRNA against VEGFR1 into tumor site. The physicochemical properties of PEI-g-PEG-RGD/siRNA complexes was evaluated. Further, tumor growth profile was also investigated after systemic administration of PEI-g-PEG-RGD/siRNA complexes. PMID:21375397

  14. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  15. RNA interference targeting CD147 inhibits the invasion of human cervical squamous carcinoma cells by downregulating MMP-9.

    PubMed

    Fan, Xiaobin; Wu, Weiguang; Shi, Haixia; Han, Jianqiu

    2013-07-01

    Cervical squamous carcinoma is a highly invasive tumour that has a great capacity to metastasise. Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin superfamily, is a widely distributed cell surface glycoprotein. It is highly expressed on malignant tumour cell surfaces, including human cervical squamous carcinoma. It also plays a critical role in the invasive and metastatic activity of malignant cells by stimulating the expression of matrix metalloproteinases (MMPs). The anti-invasive effect of small interfering RNA (siRNA) against CD147 on human cervical squamous carcinoma cells and its possible pathways has been investigated. The downregulation of CD147 by transfection with siRNA resulted in MMP-9 expression and decreased activity in the cervical squamous carcinoma cell line SiHa. In vitro analysis showed that the invasive capacity of SiHa cells decreased. Thus CD147 inhibition and subsequent MMP-9 deletion may have anti-tumour effects by inhibiting the invasiveness of human cervical squamous carcinoma cells.

  16. A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth, Plutella xylostella.

    PubMed

    Gu, Xiaojun; Kumar, Sunil; Kim, Eunjin; Kim, Yonggyun

    2015-09-01

    Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI=5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.

  17. Mechanics of light-activated network polymers

    NASA Astrophysics Data System (ADS)

    Long, Kevin Nicholas

    Mechanically responsive, environmentally activated polymers can undergo large, complex deformation in response to external stimuli such as thermal, luminous, and chemical changes to the environment. Light as a stimulus provides unique application potential because it allows for remote, rapid, and isothermal activation of the material with precise spatial control via existing optical technologies. While certain systems have received considerable attention, the state of the art of most light-activated polymers is limited to basic characterization and demonstrations. To make such materials available to the engineering and scientific communities, physically based theoretical and computational tools are required to guide experimental and design efforts that capitalize on their complex photo-mechanical couplings. The central objective of this thesis is to develop a multi-physics constitutive modeling framework to simulate the continuum scale, photo mechanical behavior of light-activated polymers and implement it into a finite element analysis setting. This framework is independent of specific underlying photo-stimulation mechanisms and is discussed in the context of photo-activated shape memory polymers and network rearranging polymers. Next, the framework is applied to the light-activated network rearranging polymer system, which is relaxed of stress upon irradiation with UV light, and a suite of characterization and application oriented experiments are carried out to calibrate and validate the model's predictive capabilities. The calibrated model is used to investigate several applications such as photo-activated stress relaxation of notched specimens, bending actuation, creep, the buckling of equi-biaxially deformed and irradiated films, and photomechanically formed 1D channels and ridges. Modeling creep involves additional complexity through simultaneous deformation and irradiation, and so the model framework is extended to cover such scenarios. Experiments, finite

  18. RNA interference-mediated knockdown of translationally controlled tumor protein induces apoptosis, and inhibits growth and invasion in glioma cells

    PubMed Central

    JIN, HUA; ZHANG, XUEXIN; SU, JUN; TENG, YUEQIU; REN, HUAN; YANG, LIZHUANG

    2015-01-01

    Translationally controlled tumor protein (TCTP) is a highly conserved, growth-associated and small molecule protein, which is highly expressed in various types of tumor cell. TCTP can promote the growth and suppress apoptosis of tumor cels. However, few studies have reported the effects of TCTP in gliomas. In the present study, a glioma cell line was established, which was stably transfected with TCTP short hairpin ribonucleic acid (shRNA), to investigate the impact of downregulated expression of TCTP on the proliferation, apoptosis and invasion of glioma cells. Western blot and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that TCTP shRNA effectively reduced the expression of TCTP in the U251 glioma cell line. MTT and colony formation assays revealed that downregulated expression of TCTP significantly inhibited glioma cell proliferation. Cell cycle analysis using flow cytometry revealed that the cells in the pRNA-H1.1-TCTP group were arrested in the G0/G1 phase of the cell cycle. Western blot analysis detected downregulated expression levels of cyclins, including Cyclin D1, Cyclin E and Cyclin B. Annexin V-fluorescein isothiocyanate/propidium iodide and Hoechst staining demonstrated that the apoptotic rate of the cells in the pRNA-H1.1-TCTP group was significantly higher than that of the cells in the pRNA-H1.1-control group, with upregulated expression levels of B-cell-associated X protein and cleaved-caspase-3 and downregulated expression of B-cell lmyphoma-2 in the apoptotic process. Wound healing and Transwell assays revealed that downregulated expression of TCTP significantly inhibited the migration and invasiveness of the glioma cells; and the expression levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were also significantly affected. In conclusion, the present study demonstrated that downregulated expression of TCTP significantly inhibited proliferation and invasion, and induced apoptosis in the glioma

  19. Silencing in Apolygus lucorum of the olfactory coreceptor Orco gene by RNA interference induces EAG response declining to two putative semiochemicals.

    PubMed

    Zhou, Yan-Le; Zhu, Xiao-Qiang; Gu, Shao-Hua; Cui, Huan-huan; Guo, Yu-Yuan; Zhou, Jing-Jiang; Zhang, Yong-Jun

    2014-01-01

    Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is an agronomically important pest that causes severe economic damage to the cotton, fruit, and vegetable industries. Similar to other insects, A. lucorum can perceive and discriminate olfactory cues. A highly conserved and broadly expressed olfactory coreceptor (Orco) is crucial for insect olfaction, and Orco orthologs have been identified in several insect species. In this study, a homology-based polymerase chain reaction (PCR) method was utilized to identify AlucOrco, an Orco ortholog essential for olfaction in A. lucorum. AlucOrco shares significant sequence homology with known Orco proteins in other insects. Quantitative real-time PCR (qRT-PCR) analysis revealed that AlucOrco was abundantly expressed in adult A. lucorum. AlucOrco expression level was the highest in the antennae; by contrast, AlucOrco showed negligible expression level in other tissues. We injected AlucOrco siRNA into the conjunctivum between the prothorax and mesothorax of A. lucorum and evaluated its expression 36 h after RNA interference. The results of qRT-PCR demonstrated that the level of mRNA expression was significantly reduced (>90%) in AlucOrco siRNA-treated A. lucorum than in water-injected and non-injected controls. The electroantennogram responses of A. lucorum to two putative semiochemicals, trans-2-hexenal and trans-2-hexenyl butyrate, were also reduced significantly (∼80%) in RNAi-treated A. lucorum than in the controls. These results suggest that AlucOrco is crucial in mediating odorant perception of A. lucorum, especially in perceiving trans-2-hexenal and trans-2-hexenyl butyrate. PMID:24216470

  20. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    PubMed

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2014-01-01

    Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs. PMID:24489765

  1. Functions of nuclear receptor HR3 during larval-pupal molting in Leptinotarsa decemlineata (Say) revealed by in vivo RNA interference.

    PubMed

    Guo, Wen-Chao; Liu, Xin-Ping; Fu, Kai-Yun; Shi, Ji-Feng; Lü, Feng-Gong; Li, Guo-Qing

    2015-08-01

    Our previous results revealed that RNA interference-aided knockdown of Leptinotarsa decemlineata FTZ-F1 (LdFTZ-F1) reduced 20E titer, and impaired pupation. In this study, we characterized a putative LdHR3 gene, an early-late 20E-response gene upstream of LdFTZ-F1. Within the first, second and third larval instars, three expression peaks of LdHR3 occurred just before the molt. In the fourth (final) larval instar 80 h after ecdysis and prepupal stage 3 days after burying into soil, two LdHR3 peaks occurred. The LdHR3 expression peaks coincide with the peaks of circulating 20E level. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdHR3 expression in the final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene Ldshd repressed the expression. Moreover, Hal rescued the transcript levels in the Ldshd-silenced larvae. Thus, 20E peaks activate the expression of LdHR3. Furthermore, ingesting dsRNA against LdHR3 successfully knocked down the target gene, and impaired pupation. Finally, knockdown of LdHR3 upregulated the transcription of three ecdysteroidogenesis genes (Ldphm, Lddib and Ldshd), increased 20E titer, and activated the expression of two 20E-response genes (LdEcR and LdFTZ-F1). Thus, LdHR3 functions in regulation of pupation in the Colorado potato beetle. PMID:26005119

  2. Involvement of doublesex and mab-3-related transcription factors in human female germ cell development demonstrated by xenograft and interference RNA strategies.

    PubMed

    Poulain, Marine; Frydman, Nelly; Tourpin, Sophie; Muczynski, Vincent; Mucsynski, Vincent; Souquet, Benoit; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie; Livera, Gabriel

    2014-10-01

    We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development.

  3. Involvement of doublesex and mab-3-related transcription factors in human female germ cell development demonstrated by xenograft and interference RNA strategies.

    PubMed

    Poulain, Marine; Frydman, Nelly; Tourpin, Sophie; Muczynski, Vincent; Mucsynski, Vincent; Souquet, Benoit; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie; Livera, Gabriel

    2014-10-01

    We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development. PMID:25082981

  4. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    PubMed

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2014-01-01

    Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

  5. RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus.

    PubMed

    Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

    2015-04-01

    Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus. PMID:24282064

  6. RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus.

    PubMed

    Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

    2015-04-01

    Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.

  7. Light-activated self-propelled colloids

    PubMed Central

    Palacci, J.; Sacanna, S.; Kim, S.-H.; Yi, G.-R.; Pine, D. J.; Chaikin, P. M.

    2014-01-01

    Light-activated self-propelled colloids are synthesized and their active motion is studied using optical microscopy. We propose a versatile route using different photoactive materials, and demonstrate a multiwavelength activation and propulsion. Thanks to the photoelectrochemical properties of two semiconductor materials (α-Fe2O3 and TiO2), a light with an energy higher than the bandgap triggers the reaction of decomposition of hydrogen peroxide and produces a chemical cloud around the particle. It induces a phoretic attraction with neighbouring colloids as well as an osmotic self-propulsion of the particle on the substrate. We use these mechanisms to form colloidal cargos as well as self-propelled particles where the light-activated component is embedded into a dielectric sphere. The particles are self-propelled along a direction otherwise randomized by thermal fluctuations, and exhibit a persistent random walk. For sufficient surface density, the particles spontaneously form ‘living crystals’ which are mobile, break apart and reform. Steering the particle with an external magnetic field, we show that the formation of the dense phase results from the collisions heads-on of the particles. This effect is intrinsically non-equilibrium and a novel principle of organization for systems without detailed balance. Engineering families of particles self-propelled by different wavelength demonstrate a good understanding of both the physics and the chemistry behind the system and points to a general route for designing new families of self-propelled particles. PMID:25332383

  8. Micro RNA-98 interferes with expression interleukin-10 in peripheral B cells of patients with lung cancer

    NASA Astrophysics Data System (ADS)

    Li, Yun; Rong, Jian; Qin, Jie; He, Jin-Yuan; Chen, Hui-Guo; Huang, Shao-Hong

    2016-09-01

    Interleukin (IL)-10-producing B cells (B10 cells) plays an important role in the tumor tolerance. High frequency of peripheral B10 cell was reported in patients with lung cancer recently. Micro RNA (miR) regulates some gene expression. This study test a hypothesis that miR-98 suppresses the expression of IL-10 in B cells of subjects with lung cancer. The results showed that the levels of miR-98 were significantly less in peripheral B cells of patients with lung cancer than that in healthy subjects. IL-10 mRNA levels in peripheral B cells were significantly higher in lung cancer patients as compared with healthy controls. A negative correlation was identified between miR-98 and IL-10 in peripheral B cells. Serum IL-13 was higher in lung cancer patients than that in healthy controls. The levels of IL-13 were also negatively correlated with IL-10 in B cells. Exposure B10 cells to IL-13 in the culture or over expression of miR-98 reduced the expression of IL-10 in B cells. Administration with miR-98-laden liposomes inhibited the lung cancer growth in a mouse model. In conclusion, up regulation of miR-98 inhibits the expression of IL-10 in B cells, which may contribute to inhibit the lung cancer tolerance in the body.

  9. RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

    PubMed

    Gruber, Jens; Boese, Guido; Tuschl, Thomas; Osborn, Mary; Weber, Klaus

    2004-07-01

    The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

  10. Evolutionary constraints acting on DDX3X protein potentially interferes with Rev-mediated nuclear export of HIV-1 RNA.

    PubMed

    Sharma, Deepak; Bhattacharya, Jayanta

    2010-01-01

    Differential host-pathogen interactions direct viral replication in infected cells. In HIV-1 infected cells, nuclear export of viral RNA transcripts into cellular cytoplasm is governed by interaction of HIV-1 Rev, Exportin-1 (CRM-1) and DDX3X. Knock down of DDX3X has been shown to drastically impair HIV replication. Here we show that evolutionary forces are responsible for demarking previously unidentified critical functionally important residues on the surface of DDX3X. Using computational approaches, we show that these functional residues, depending on their location, are capable of regulating ATPase and RNA helicase functions of DDX3X. The potential of these residues in designing better blockers against HIV-1 replication was also assessed. Also, using stepwise docking simulations, we could identify DDX3X-CRM-1 interface and its critical functional residues. Our data would help explain the role of DDX3X in HIV-1 Rev function with potential to design new intervention strategies against HIV-1 replication.

  11. Micro RNA-98 interferes with expression interleukin-10 in peripheral B cells of patients with lung cancer.

    PubMed

    Li, Yun; Rong, Jian; Qin, Jie; He, Jin-Yuan; Chen, Hui-Guo; Huang, Shao-Hong

    2016-01-01

    Interleukin (IL)-10-producing B cells (B10 cells) plays an important role in the tumor tolerance. High frequency of peripheral B10 cell was reported in patients with lung cancer recently. Micro RNA (miR) regulates some gene expression. This study test a hypothesis that miR-98 suppresses the expression of IL-10 in B cells of subjects with lung cancer. The results showed that the levels of miR-98 were significantly less in peripheral B cells of patients with lung cancer than that in healthy subjects. IL-10 mRNA levels in peripheral B cells were significantly higher in lung cancer patients as compared with healthy controls. A negative correlation was identified between miR-98 and IL-10 in peripheral B cells. Serum IL-13 was higher in lung cancer patients than that in healthy controls. The levels of IL-13 were also negatively correlated with IL-10 in B cells. Exposure B10 cells to IL-13 in the culture or over expression of miR-98 reduced the expression of IL-10 in B cells. Administration with miR-98-laden liposomes inhibited the lung cancer growth in a mouse model. In conclusion, up regulation of miR-98 inhibits the expression of IL-10 in B cells, which may contribute to inhibit the lung cancer tolerance in the body. PMID:27605397

  12. Silencing stromal interaction molecule 1 by RNA interference inhibits the proliferation and migration of endothelial progenitor cells

    SciTech Connect

    Kuang, Chun-yan; Yu, Yang; Guo, Rui-wei; Qian, De-hui; Wang, Kui; Den, Meng-yang; Shi, Yan-kun; Huang, Lan

    2010-07-23

    Research highlights: {yields} STIM1 and TRPC1 are expressed in EPCs. {yields} Knockdown of STIM1 inhibits the proliferation, migration and SOCE of EPCs. {yields} TRPC1-SOC cooperates with STIM1 to mediate the SOCE of EPCs. -- Abstract: Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.

  13. Micro RNA-98 interferes with expression interleukin-10 in peripheral B cells of patients with lung cancer

    PubMed Central

    Li, Yun; Rong, Jian; Qin, Jie; He, Jin-yuan; Chen, Hui-guo; Huang, Shao-hong

    2016-01-01

    Interleukin (IL)-10-producing B cells (B10 cells) plays an important role in the tumor tolerance. High frequency of peripheral B10 cell was reported in patients with lung cancer recently. Micro RNA (miR) regulates some gene expression. This study test a hypothesis that miR-98 suppresses the expression of IL-10 in B cells of subjects with lung cancer. The results showed that the levels of miR-98 were significantly less in peripheral B cells of patients with lung cancer than that in healthy subjects. IL-10 mRNA levels in peripheral B cells were significantly higher in lung cancer patients as compared with healthy controls. A negative correlation was identified between miR-98 and IL-10 in peripheral B cells. Serum IL-13 was higher in lung cancer patients than that in healthy controls. The levels of IL-13 were also negatively correlated with IL-10 in B cells. Exposure B10 cells to IL-13 in the culture or over expression of miR-98 reduced the expression of IL-10 in B cells. Administration with miR-98-laden liposomes inhibited the lung cancer growth in a mouse model. In conclusion, up regulation of miR-98 inhibits the expression of IL-10 in B cells, which may contribute to inhibit the lung cancer tolerance in the body. PMID:27605397

  14. Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

    PubMed

    Xiao, Su; Chen, Yu Chi; Buehler, Eugen; Mandal, Swati; Mandal, Ajeet; Betenbaugh, Michael; Park, Myung Hee; Martin, Scott; Shiloach, Joseph

    2016-11-01

    For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. A 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport, and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1-was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Biotechnol. Bioeng. 2016;113: 2403-2415. © 2016 Wiley Periodicals, Inc. PMID:27215166

  15. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

    PubMed

    Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

    2016-07-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27417115

  16. Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

    PubMed Central

    Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

    2015-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

  17. Development of an RNA Interference Tool, Characterization of Its Target, and an Ecological Test of Caste Differentiation in the Eusocial Wasp Polistes

    PubMed Central

    Havukainen, Heli; Henshaw, Michael T.; Amdam, Gro V.

    2011-01-01

    Recent advancements in genomics provide new tools for evolutionary ecological research. The paper wasp genus Polistes is a model for social insect evolution and behavioral ecology. We developed RNA interference (RNAi)-mediated gene silencing to explore proposed connections between expression of hexameric storage proteins and worker vs. gyne (potential future foundress) castes in naturally-founded colonies of P. metricus. We extended four fragments of putative hexamerin-encoding P. metricus transcripts acquired from a previous study and fully sequenced a gene that encodes Hexamerin 2, one of two proposed hexameric storage proteins of P. metricus. MALDI-TOF/TOF, LC-MSMS, deglycosylation, and detection of phosphorylation assays showed that the two putative hexamerins diverge in peptide sequence and biochemistry. We targeted the hexamerin 2 gene in 5th (last)-instar larvae by feeding RNAi-inducing double-stranded hexamerin 2 RNA directly to larvae in naturally-founded colonies in the field. Larval development and adult traits were not significantly altered in hexamerin 2 knockdowns, but there were suggestive trends toward increased developmental time and less developed ovaries, which are gyne characteristics. By demonstrating how data acquisition from 454/Roche pyrosequencing can be combined with biochemical and proteomics assays and how RNAi can be deployed successfully in field experiments on Polistes, our results pave the way for functional genomic research that can contribute significantly to learning the interactions of environment, development, and the roles they play in paper wasp evolution and behavioral ecology. PMID:22069460

  18. The chitin biosynthesis pathway in Entamoeba and the role of glucosamine-6-P isomerase by RNA interference.

    PubMed

    Samanta, Sintu Kumar; Ghosh, Sudip K

    2012-11-01

    Entamoeba histolytica, the causative agent of amoebiasis, infects through its cyst form. A thick chitin wall protects the cyst from the harsh environment outside of the body. It is known that chitin is synthesized only during encystation, but the chitin synthesis pathway (CSP) of Entamoeba is not well characterized. In this report, we have identified the genes involved in chitin biosynthesis from the Entamoeba genome database and verified their expression profile at the transcriptional level in encysting Entamoeba invadens. Semi-quantitative RT-PCR (sqRT-PCR) analysis showed that all the chitin pathway genes are entirely absent or transcribed at low levels in trophozoites. The mRNA expression of most of the CSP genes reached their maximum level between 9 and 12h after the in vitro initiation of encystation. Double-stranded RNA-mediated silencing of glucosamine-6-P isomerase (Gln6Pi) reduced chitin synthesis to 62-64%, which indicates that Gln6Pi might be a key enzyme for regulating chitin synthesis in Entamoeba. The study of different enzymes involved in glycogen metabolism revealed that stored glycogen is converted to glucose during encystation. It is clear from the sqRT-PCR analysis that the rate of glycolysis decreases as encystation proceeds. Encystation up-regulates the expression of glycogen phosphorylase, which is responsible for glycogen degradation. The significant decrease in chitin synthesis in encysting cells treated with a specific inhibitor of glycogen phosphorylase indicates that the glucose obtained from the degradation of stored glycogen in trophozoites might be one of the major sources of glucose for chitin synthesis.

  19. Silencing PP2A Inhibitor by Lenti-shRNA Interference Ameliorates Neuropathologies and Memory Deficits in tg2576 Mice

    PubMed Central

    Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Shi, Hai-Rong; Liu, Xing-Hua; Chai, Gao-Shang; Yao, Xiu-Qing; Zhang, Jia-Yu; Peng, Cai-Xia; Hu, Juan; Li, Xia-Chun; Wang, Qun; Wang, Jian-Zhi

    2013-01-01

    Deficits of protein phosphatase-2A (PP2A) play a crucial role in tau hyperphosphorylation, amyloid overproduction, and synaptic suppression of Alzheimer's disease (AD), in which PP2A is inactivated by the endogenously increased inhibitory protein, namely inhibitor-2 of PP2A (I2PP2A). Therefore, in vivo silencing I2PP2A may rescue PP2A and mitigate AD neurodegeneration. By infusion of lentivirus-shRNA targeting I2PP2A (LV-siI2PP2A) into hippocampus and frontal cortex of 11-month-old tg2576 mice, we demonstrated that expression of LV-siI2PP2A decreased remarkably the elevated I2PP2A in both mRNA and protein levels. Simultaneously, the PP2A activity was restored with the mechanisms involving reduction of the inhibitory binding of I2PP2A to PP2A catalytic subunit (PP2AC), repression of the inhibitory Leu309-demethylation and elevation of PP2AC. Silencing I2PP2A induced a long-lasting attenuation of amyloidogenesis in tg2576 mice with inhibition of amyloid precursor protein hyperphosphorylation and β-secretase activity, whereas simultaneous inhibition of PP2A abolished the antiamyloidogenic effects of I2PP2A silencing. Finally, silencing I2PP2A could improve learning and memory of tg2576 mice with preservation of several memory-associated components. Our data reveal that targeting I2PP2A can efficiently rescue Aβ toxicities and improve the memory deficits in tg2576 mice, suggesting that I2PP2A could be a promising target for potential AD therapies. PMID:23922015

  20. Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance.

    PubMed

    Rodríguez-Hernández, Ana M; Gosalvez, Blanca; Sempere, Raquel N; Burgos, Lorenzo; Aranda, Miguel A; Truniger, Verónica

    2012-09-01

    Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

  1. RNA interference-mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease.

    PubMed

    Ma, Junqing; Chen, Wei; Zhang, Lijie; Tucker, Byron; Zhu, Guochun; Sasaki, Hajime; Hao, Liang; Wang, Lin; Ci, Hongliang; Jiang, Hongbing; Stashenko, Philip; Li, Yi-Ping

    2013-04-01

    Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

  2. Light-Activated Content Release from Liposomes

    PubMed Central

    Leung, Sarah J.; Romanowski, Marek

    2012-01-01

    Successful integration of diagnostic and therapeutic actions at the level of individual cells requires new materials that combine biological compatibility with functional versatility. This review focuses on the development of liposome-based functional materials, where payload release is activated by light. Methods of sensitizing liposomes to light have progressed from the use of organic molecular moieties to the use of metallic plasmon resonant structures. This development has facilitated application of near infrared light for activation, which is preferred for its deep penetration and low phototoxicity in biological tissues. Presented mechanisms of light-activated liposomal content release enable precise in vitro manipulation of minute amounts of reagents, but their use in clinical diagnostic and therapeutic applications will require demonstration of safety and efficacy. PMID:23139729

  3. Visible-Light-Activated Molecular Switches.

    PubMed

    Bléger, David; Hecht, Stefan

    2015-09-21

    The ability to influence key properties of molecular systems by using light holds much promise for the fields of materials science and life sciences. The cornerstone of such systems is molecules that are able to reversibly photoisomerize between two states, commonly referred to as photoswitches. One serious restriction to the development of functional photodynamic systems is the necessity to trigger switching in at least one direction by UV light, which is often damaging and penetrates only partially through most media. This review provides a summary of the different conceptual strategies for addressing molecular switches in the visible and near-infrared regions of the optical spectrum. Such visible-light-activated molecular switches tremendously extend the scope of photoswitchable systems for future applications and technologies.

  4. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.

  5. Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

    PubMed

    Carissimo, Guillaume; Pondeville, Emilie; McFarlane, Melanie; Dietrich, Isabelle; Mitri, Christian; Bischoff, Emmanuel; Antoniewski, Christophe; Bourgouin, Catherine; Failloux, Anna-Bella; Kohl, Alain; Vernick, Kenneth D

    2015-01-13

    Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

  6. RNA interference of broad gene expression mimics antimetamorphic effect of pyriproxyfen on the beet armyworm, Spodoptera exigua.

    PubMed

    Kim, Jiwan; Kim, Yonggyun

    2012-12-01

    A larva-to-pupa metamorphosis is induced by a low or undetectable level of juvenile hormone (JH) during last instar in holometabolous insects. An exogenous application of JH agonist, pyriproxyfen (PYR), inhibited pupal metamorphosis of the beet armyworm, Spodoptera exigua. Last instar larvae of S. exigua exhibited increase of body size at first 3 days along with active feeding behavior. Also, at this period, prothoracic gland increased in size, while corpora allata remained little change. Storage proteins were accumulated in hemolymph plasma from penultimate to last instars, during which two storage protein genes (SeHex and SeSP1) were actively expressed. A Broad-Complex 1 (BRC1) gene of S. exigua (SeBRC1) was partially cloned and showed a specific expression at the last instar in all tested tissues including hemocytes, fat body, epidermis, gut, nerve, and salivary gland. Knockdown of SeBRC1 expression by its specific double-strand RNA mimicked the antimetamorphic effect induced by PYR treatment. PYR treatment at early last instar inhibited expression of SeBRC1, but did not that of other nuclear receptor, βFTZ-F1. These results indicate that a transcriptional factor, SeBRC1, plays a crucial role in pupal metamorphosis of S. exigua.

  7. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. PMID:27106120

  8. Inhibition of the expression of aquaporin‑1 by RNA interference in pulmonary epithelial cells and its effects on water transport.

    PubMed

    Zhang, Qiuyue; Fu, Jianhua; Xue, Xindong

    2016-01-01

    In the present study, the effect of aquaporin‑1 (AQP1) on fluid transportation in pulmonary epithelial cells, and the role of AQP1 in alveolar fluid clearance were investigated to provide an experimental foundation to elucidate the pathogenesis of hyperoxic lung edema. An siRNA transfection technique was used to silence AQP1 in the A549 cell line. The transfected cells were randomized into a hyperoxia exposure and an air control group, with a negative control group set for each group. Cell volume was determined using flow cytometry, and Pf values were used to determine osmotic water permeability. Cell volume was found to be reduced in the AQP1‑silenced A549 cells, compared with the negative control group 72 h following air exposure. In addition, cell volume was reduced in the AQP1‑silenced A549 cells, compared with the negative control group 48 and 72 h following hyperoxia exposure. The osmotic water permeability of the AQP1‑silenced cells was reduced in the air control and hyperoxia exposure groups, compared with the negative control group 48 and 72 h following exposure. The volume and cell membrane osmotic water permeability of the A549 cells were reduced, compared with those in the control group following AQP1‑silencing, which indicated that the downregulation of AQP1 impedes extracellular to intracellular fluid transportation. Therefore, the disturbance in alveolar fluid clearance resulting from the downregulation of AQP1 following hyperoxia exposure may be one of the key mechanisms responsible for hyperoxic lung edema. PMID:26549133

  9. Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota

    PubMed Central

    Carissimo, Guillaume; Pondeville, Emilie; McFarlane, Melanie; Dietrich, Isabelle; Mitri, Christian; Bischoff, Emmanuel; Antoniewski, Christophe; Bourgouin, Catherine; Failloux, Anna-Bella; Kohl, Alain; Vernick, Kenneth D.

    2015-01-01

    Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o’nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens. PMID:25548172

  10. Inhibition of the expression of aquaporin‑1 by RNA interference in pulmonary epithelial cells and its effects on water transport.

    PubMed

    Zhang, Qiuyue; Fu, Jianhua; Xue, Xindong

    2016-01-01

    In the present study, the effect of aquaporin‑1 (AQP1) on fluid transportation in pulmonary epithelial cells, and the role of AQP1 in alveolar fluid clearance were investigated to provide an experimental foundation to elucidate the pathogenesis of hyperoxic lung edema. An siRNA transfection technique was used to silence AQP1 in the A549 cell line. The transfected cells were randomized into a hyperoxia exposure and an air control group, with a negative control group set for each group. Cell volume was determined using flow cytometry, and Pf values were used to determine osmotic water permeability. Cell volume was found to be reduced in the AQP1‑silenced A549 cells, compared with the negative control group 72 h following air exposure. In addition, cell volume was reduced in the AQP1‑silenced A549 cells, compared with the negative control group 48 and 72 h following hyperoxia exposure. The osmotic water permeability of the AQP1‑silenced cells was reduced in the air control and hyperoxia exposure groups, compared with the negative control group 48 and 72 h following exposure. The volume and cell membrane osmotic water permeability of the A549 cells were reduced, compared with those in the control group following AQP1‑silencing, which indicated that the downregulation of AQP1 impedes extracellular to intracellular fluid transportation. Therefore, the disturbance in alveolar fluid clearance resulting from the downregulation of AQP1 following hyperoxia exposure may be one of the key mechanisms responsible for hyperoxic lung edema.

  11. Fundamental Study of Detection of Muscle Hypertrophy-Oriented Gene Doping by Myostatin Knock Down Using RNA Interference

    PubMed Central

    Takemasa, Tohru; Yakushiji, Naohisa; Kikuchi, Dale Manjiro; Deocaris, Custer; Widodo; Machida, Masanao; Kiyosawa, Hidenori

    2012-01-01

    To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray. PMID:24149203

  12. Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma

    PubMed Central

    Xu, Chang; Wang, Pei; Liu, Yan; Zhang, Yuzheng; Fan, Wenhong; Upton, Melissa P.; Lohavanichbutr, Pawadee; Houck, John R.; Doody, David R.; Futran, Neal D.; Zhao, Lue Ping; Schwartz, Stephen M.; Chen, Chu; Méndez, Eduardo

    2013-01-01

    In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC–specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03–11.11) and 3.45 (95% CI: 1.84–6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA–mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials. PMID:23341773

  13. Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation.

    PubMed

    Bai, Hongkun; Qiao, Hui; Li, Fajun; Fu, Hongtuo; Sun, Shengming; Zhang, Wenyi; Jin, Shubo; Gong, Yongsheng; Jiang, Sufei; Xiong, Yiwei

    2015-05-10

    Vitellogenin (Vg) is the precursor of yolk protein, which functions as a nutritive resource that is important for embryonic growth and gonad development. In this study, the cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag (EST) analysis and the rapid amplification of cDNA ends (RACE) approach. The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa. Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary, hemocytes, and hepatopancreas. As ovaries developed, the expression level of Mn-Vg increased in both the hepatopancreas and ovary. In the hepatopancreas, the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary. The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development. Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages. Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary. In adult females, Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary, and two injection treatment dramatically delayed ovary maturation. Vg RNA interference down-regulated the vitellogenin receptor (VgR) expression level in the ovary, which illustrates the close relationship between Vg and VgR in the process of vitellogenesis. PMID:25499697

  14. Bright light activates a trigeminal nociceptive pathway

    PubMed Central

    Okamoto, Keiichiro; Tashiro, Akimasa; Chang, Zheng; Bereiter, David A.

    2010-01-01

    Bright light can cause ocular discomfort and/or pain; however, the mechanism linking luminance to trigeminal nerve activity is not known. In this study we identify a novel reflex circuit necessary for bright light to excite nociceptive neurons in superficial laminae of trigeminal subnucleus caudalis (Vc/C1). Vc/C1 neurons encoded light intensity and displayed a long delay (>10 s) for activation. Microinjection of lidocaine into the eye or trigeminal root ganglion (TRG) inhibited light responses completely, whereas topical application onto the ocular surface had no effect. These findings indicated that light-evoked Vc/C1 activity was mediated by an intraocular mechanism and transmission through the TRG. Disrupting local vasomotor activity by intraocular microinjection of the vasoconstrictive agents, norepinephrine or phenylephrine, blocked light-evoked neural activity, whereas ocular surface or intra-TRG microinjection of norepinephrine had no effect. Pupillary muscle activity did not contribute since light-evoked responses were not altered by atropine. Microinjection of lidocaine into the superior salivatory nucleus diminished light-evoked Vc/C1 activity and lacrimation suggesting that increased parasympathetic outflow was critical for light-evoked responses. The reflex circuit also required input through accessory visual pathways since both Vc/C1 activity and lacrimation were prevented by local blockade of the olivary pretectal nucleus. These findings support the hypothesis that bright light activates trigeminal nerve activity through an intraocular mechanism driven by a luminance-responsive circuit and increased parasympathetic outflow to the eye. PMID:20206444

  15. A high-throughput RNA interference (RNAi)-based approach using hairy roots for the study of plant-rhizobia interactions.

    PubMed

    Sinharoy, Senjuti; Pislariu, Catalina I; Udvardi, Michael K

    2015-01-01

    Legumes are major contributors to sustainable agriculture; their key feature is their ability to fix atmospheric nitrogen through symbiotic nitrogen fixation. Legumes are often recalcitrant to regeneration and transformation by Agrobacterium tumefaciens; however, A. rhizogenes-mediated root transformation and composite plant generation are rapid and convenient alternatives to study root biology, including root nodule symbiosis. RNA interference (RNAi), coupled with A. rhizogenes-mediated root transformation, has been very successfully used for analyses of gene function by reverse genetics. Besides being applied to model legumes (Medicago truncatula and Lotus japonicus), this method has been adopted for several other legumes due to the ease and relative speed with which transgenic roots can be generated. Several protocols for hairy root transformation have been published. Here we describe an improved hairy root transformation protocol and the methods to study nodulation in Medicago. We also highlight the major differences between our protocol and others, and key steps that need to be adjusted in order to translate this method to other legumes.

  16. RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

    PubMed

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

    2014-06-19

    The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  17. Novel Genes Participating in the Formation of Prismatic and Nacreous Layers in the Pearl Oyster as Revealed by Their Tissue Distribution and RNA Interference Knockdown

    PubMed Central

    Koyama, Hiroki; Mizutani, Saeri; Ota, Ayaka; Osakabe, Yuki; Nagai, Kiyohito; Maeyama, Kaoru; Okamoto, Kikuhiko; Kanoh, Satoshi; Asakawa, Shuichi; Watabe, Shugo

    2014-01-01

    In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers. PMID:24454739

  18. RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

    PubMed

    Van Ekert, E; Wang, M; Miao, Y-G; Brent, C S; Hull, J J

    2016-10-01

    Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus. PMID:27189651

  19. Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins.

    PubMed

    Shigemitsu, Takanari; Ozaki, Shinji; Saito, Yuhi; Kuroda, Masaharu; Morita, Shigeto; Satoh, Shigeru; Masumura, Takehiro

    2012-03-01

    Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.

  20. Cyclooxygenase-2 Silencing for the Treatment of Colitis: A Combined In Vivo Strategy Based on RNA Interference and Engineered Escherichia Coli

    PubMed Central

    Spisni, Enzo; Valerii, Maria C; De Fazio, Luigia; Cavazza, Elena; Borsetti, Francesca; Sgromo, Annamaria; Candela, Marco; Centanni, Manuela; Rizello, Fernando; Strillacci, Antonio

    2015-01-01

    Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColishCOX2), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColishCOX2 in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColishCOX2-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColishCOX2 strategy as a promising tool for molecular therapy in intestinal inflammatory diseases. PMID:25393372

  1. RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

    PubMed

    Van Ekert, E; Wang, M; Miao, Y-G; Brent, C S; Hull, J J

    2016-10-01

    Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.

  2. A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila.

    PubMed

    Gobert, Vanessa; Osman, Dani; Bras, Stéphanie; Augé, Benoit; Boube, Muriel; Bourbon, Henri-Marc; Horn, Thomas; Boutros, Michael; Haenlin, Marc; Waltzer, Lucas

    2010-06-01

    Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory "CDK8 module," composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals. PMID:20368357

  3. RNA Interference in Chicken Embryos

    NASA Astrophysics Data System (ADS)

    van Hateren, Nick J.; Jones, Rachel S.; Wilson, Stuart A.

    The chicken has played an important role in biological discoveries since the 17th century (Stern, 2005). Many investigations into vertebrate development have utilized the chicken due to the accessibility of the chick embryo and its ease of manipulation (Brown et al., 2003). However, the lack of genetic resources has often handicapped these studies and so the chick is frequently overlooked as a model organism for the analysis of vertebrate gene function in favor of mice or zebrafish. In the past six years this situation has altered dramatically with the generation of over half a million expressed sequence tags and >20,000 fully sequenced chicken cDNAs (Boardman et al. 2002; Caldwell et al., 2005; Hubbard et al., 2005) together with a 6X coverage genome sequence (Hillier et al., 2004). These resources have created a comprehensive catalogue of chicken genes with readily accessible cDNA and EST resources available via ARK-GENOMICS (www.ark-genomics.org) for the functional analysis of vertebrate gene function.

  4. Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba.

    PubMed

    Serikawa, Masayuki; Miwa, Kumiko; Kondo, Takao; Oyama, Tokitaka

    2008-04-01

    Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis. PMID:18281417

  5. A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection

    PubMed Central

    Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.; Hatch, Anson V.; Rasley, Amy

    2016-01-01

    ABSTRACT Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses. PMID:27226375

  6. RNA Interference-Mediated Change in Protein Body Morphology and Seed Opacity through Loss of Different Zein Proteins1[C][W][OA

    PubMed Central

    Wu, Yongrui; Messing, Joachim

    2010-01-01

    Opaque or nonvitreous phenotypes relate to the seed architecture of maize (Zea mays) and are linked to loci that control the accumulation and proper deposition of storage proteins, called zeins, into specialized organelles in the endosperm, called protein bodies. However, in the absence of null mutants of each type of zein (i.e. α, β, γ, and δ), the molecular contribution of these proteins to seed architecture remains unclear. Here, a double null mutant for the δ-zeins, the 22-kD α-zein, the β-zein, and the γ-zein RNA interference (RNAi; designated as z1CRNAi, βRNAi, and γRNAi, respectively) and their combinations have been examined. While the δ-zein double null mutant had negligible effects on protein body formation, the βRNAi and γRNAi alone only cause slight changes. Substantial loss of the 22-kD α-zeins by z1CRNAi resulted in protein body budding structures, indicating that a sufficient amount of the 22-kD zeins is necessary for maintenance of a normal protein body shape. Among different mutant combinations, only the combined βRNAi and γRNAi resulted in drastic morphological changes, while other combinations did not. Overexpression of α-kafirins, the homologues of the maize 22-kD α-zeins in sorghum (Sorghum bicolor), in the β/γRNAi mutant failed to offset the morphological alterations, indicating that β- and γ-zeins have redundant and unique functions in the stabilization of protein bodies. Indeed, opacity of the β/γRNAi mutant was caused by incomplete embedding of the starch granules rather than by reducing the vitreous zone. PMID:20237020

  7. A genome-wide RNA interference screen identifies a role for Wnt/β-catenin signaling during Rift Valley Fever Virus infection

    DOE PAGESBeta

    Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.; Hatch, Anson V.; Rasley, Amy; Negrete, Oscar A.

    2016-05-25

    Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses Lamore » Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. Lastly, these studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.« less

  8. RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture1[OPEN

    PubMed Central

    Kir, Gokhan; Ye, Huaxun; Nelissen, Hilde; Neelakandan, Anjanasree K.; Kusnandar, Andree S.; Luo, Anding; Inzé, Dirk; Sylvester, Anne W.; Yin, Yanhai; Becraft, Philip W.

    2015-01-01

    Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling. PMID:26162429

  9. Teaching dental anatomy with light-activated resins.

    PubMed

    Chalkley, Y; Denehy, G E; Schulein, T M

    1984-04-01

    A method has been described in which light-activated resins are incorporated into the dental anatomy laboratory. This procedure is a valuable addition to the anatomy course because students (1) work with a restorative material appropriate for anterior teeth, (2) learn the unique properties of the light-activated resins, and (3) apply the principles of dental anatomy to a clinically relevant task.

  10. Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Colomer, Ramon; Lupu, Ruth

    2005-05-20

    combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.

  11. The effects of RNA interference targeting Bactrocera dorsalis ds-Bdrpl19 on the gene expression of rpl19 in non-target insects.

    PubMed

    Chen, Aie; Zheng, Weiwei; Zheng, Wenping; Zhang, Hongyu

    2015-04-01

    Double-stranded RNA (dsRNA) designed to target pest genes emerges as a promising strategy for improving pest control. Therefore, it is necessary to assess the effects of dsRNA on non-target insects, such as native enemies and beneficial insects, to determine the environmental safety of such treatments. In this paper, we investigated the effects of dsRNA targeting rpl19 from Bactrocera dorsalis on non-target insects in citrus ecological systems by feeding the dsRNA to Bactrocera minax, Apis mellifera and Diachasmimorpha longicaudata. The results showed that when B. dorsalis were fed rpl19 CDS dsRNA or 3'UTR dsRNA, the expression of rpl19 was dramatically decreased. Feeding the Bdrpl19 CDS dsRNA to adult B. minax and D. longicaudata caused their respective rpl19 genes to be knocked down over 50-70 and 40%, respectively, but it had no effect on the expression of the rpl19 gene in A. mellifera. The Bdrpl19 3'UTR dsRNA did not have any silencing effects on the expression levels of rpl19 in non-target insects. This study provides evidence that dsRNA can impact non-target organisms, but the 3'UTR dsRNA may not have effects in non-target organisms.

  12. Direct observation of frequency modulated transcription in single cells using light activation

    PubMed Central

    Larson, Daniel R; Fritzsch, Christoph; Sun, Liang; Meng, Xiuhau; Lawrence, David S; Singer, Robert H

    2013-01-01

    Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI: http://dx.doi.org/10.7554/eLife.00750.001 PMID:24069527

  13. Selenium bond decreases ON resistance of light-activated switch

    NASA Technical Reports Server (NTRS)

    1965-01-01

    Vitrified amorphous selenium bond decreases the ON resistance of a gallium arsenide-silicon light-activated, low-level switch. The switch is used under a pulse condition to prolong switch life and minimize errors due to heating, devitrification, and overdrawing.

  14. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    SciTech Connect

    Doi, Nobutaka; Ogawa, Ryohei; Cui, Zheng-Guo; Morii, Akihiro; Watanabe, Akihiko; Kanayama, Shinji; Yoneda, Yuko; Kondo, Takashi

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  15. Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells

    PubMed Central

    Li, Xiao C.; Zhuo, Jia L.

    2008-01-01

    Receptor-mediated endocytosis of extracellular ANG II has been suggested to play an important role in the regulation of proximal tubule cell (PTC) function. Using immortalized rabbit PTCs as an in vitro cell culture model, we tested the hypothesis that extracellular ANG II is taken up by PTCs through angiotensin type 1 receptor (AT1; or AT1a) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT1 receptor small-interfering RNA (siRNA; AT1R siRNA) or endocytotic inhibitors exerts a physiological effect on total and apical sodium and hydrogen exchanger isoform 3 (NHE-3) protein abundance. Western blots and live cell imaging with FITC-labeled ANG II confirmed that transfection of PTCs with a human specific AT1R siRNA for 48 h selectively knocked down AT1 receptor protein by 76 ± 5% (P < 0.01), whereas transfection with a scrambled siRNA had little effect. In nontransfected PTCs, exposure to extracellular ANG II (1 nM) for 60 min at 37°C increased intracellular ANG II accumulation by 67% (control: 566 ± 55 vs. ANG II: 943 ± 160 pg/mg protein, P < 0.05) and induced mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (163 ± 15% of control, P < 0.01). AT1R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT1 receptors (557 ± 37 pg/mg protein, P < 0.05 vs. ANG II), or to colchicine, which disrupts cytoskeleton microtubules (613 ± 12 pg/mg protein, P < 0.05 vs. ANG II). AT1R siRNA, losartan, and colchicine all attenuated ANG II-induced ERK1/2 activation and total cell lysate and apical membrane NHE-3 abundance. The scrambled siRNA had no effect on ANG II endocytosis, ERK1/2 activation, or NHE-3 expression. These results suggest that AT1 receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins. PMID:17428839

  16. Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

    PubMed

    Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

    2014-12-01

    Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

  17. Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

    SciTech Connect

    Rana, Ritu; Jagadish, Nirmala; Garg, Manoj; Mishra, Deepshikha; Dahiya, Neetu; Chaurasiya, Dipak; Suri, Anil . E-mail: anil@nii.res.in

    2006-02-03

    Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

  18. RNA interference-aided knockdown of a putative saccharopine dehydrogenase leads to abnormal ecdysis in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

    PubMed

    Wan, P-J; Yang, L; Yuan, S-Y; Tang, Y-H; Fu, Q; Li, G-Q

    2015-08-01

    The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control. PMID:25908053

  19. Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells.

    PubMed

    Tan, Shinta; Hougardy, Brigitte M T; Meersma, Gert J; Schaap, Bessel; de Vries, Elisabeth G E; van der Zee, Ate G J; de Jong, Steven

    2012-05-01

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells. PMID:22328720

  20. Measurement of action spectra of light-activated processes

    NASA Astrophysics Data System (ADS)

    Ross, Justin; Zvyagin, Andrei V.; Heckenberg, Norman R.; Upcroft, Jacqui; Upcroft, Peter; Rubinsztein-Dunlop, Halina H.

    2006-01-01

    We report on a new experimental technique suitable for measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength. We demonstrate the efficiency of this new technique in determination of the action spectrum of the light mediated transport of rhodamine 123 into the parasitic protozoan Giardia duodenalis.

  1. RNA interference-mediated knockdown of long-form phosphodiesterase-4D (PDE4D) enzyme reverses amyloid-β42-induced memory deficits in mice.

    PubMed

    Zhang, Cong; Cheng, Yufang; Wang, Haitao; Wang, Chuang; Wilson, Steven P; Xu, Jiangping; Zhang, Han-Ting

    2014-01-01

    Phosphodiesterase-4 (PDE4) inhibitors enhance memory, increase hippocampal neurogenesis, and reverse amyloid-β (Aβ)-induced memory deficits. Here, we examined whether long-form PDE4D knockdown by lentiviral RNA construct containing a specific microRNA/miRNA-mir hairpin structure (4DmiRNA) reversed memory impairment caused by amyloid-β1-42 (Aβ42) in mice using the Morris water maze (MWM) and novelty object recognition tests. Western blotting analysis was used to assess protein levels of cAMP response element-binding protein (CREB, unphosphorylated and phosphorylated [pCREB]), brain-derived neurotrophic factor (BDNF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nuclear factor-κB (NF-κB) to explore the neurochemical mechanisms. Aggregated Aβ42 (0.5 μg/side) bilaterally infused in dentate gyrus decreased cAMP levels (p < 0.01) and produced memory deficits in the MWM (p < 0.01) and object recognition tests (p < 0.01). Microinfusions of lentiviruses resulted in downregulated expression of PDE4D4 and 4D5 proteins and reversed Aβ42-induced cAMP decline (p < 0.05) and memory deficits. Treatment also concomitantly increased pCREB (p < 0.05) and BDNF (p < 0.01) and reduced IL-1β (p < 0.05), TNF-α (p < 0.01), and NF-κB (p65) (p < 0.05) in the hippocampus of Aβ42-challenged mice. These results suggest that long-form PDE4D knockdown may offer a promising treatment for memory loss associated with Alzheimer's disease.

  2. Ran Involved in the Development and Reproduction Is a Potential Target for RNA-Interference-Based Pest Management in Nilaparvata lugens

    PubMed Central

    Wang, Wei-Xia; Lai, Feng-Xiang; Fu, Qiang

    2015-01-01

    Ran (RanGTPase) in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1) and vitellogenin, are involved. A putative Ran gene (NlRan) was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1) Extended body form, and failed to molt; (2) The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control. PMID:26554926

  3. The effect of light-activation sources on tooth bleaching

    PubMed Central

    Baroudi, Kusai; Hassan, Nadia Aly

    2014-01-01

    Vital bleaching is one of the most requested cosmetic dental procedures asked by patients who seek a more pleasing smile. This procedure consists of carbamide or hydrogen peroxide gel applications that can be applied in-office or by the patient (at-home/overnight bleaching system). Some in-office treatments utilise whitening light with the objective of speeding up the whitening process. The objective of this article is to review and summarise the current literature with regard to the effect of light-activation sources on in-office tooth bleaching. A literature search was conducted using Medline, accessed via the National Library of Medicine Pub Med from 2003 to 2013 searching for articles relating to effectiveness of light activation sources on in-office tooth bleaching. This study found conflicting evidence on whether light truly improve tooth whitening. Other factors such as, type of stain, initial tooth colour and subject age which can influence tooth bleaching outcome were discussed. Conclusions: The use of light activator sources with in-office bleaching treatment of vital teeth did not increase the efficacy of bleaching or accelerate the bleaching. PMID:25298598

  4. Genomic Characterization of Variable Surface Antigens Reveals a Telomere Position Effect as a Prerequisite for RNA Interference-Mediated Silencing in Paramecium tetraurelia

    PubMed Central

    Baranasic, Damir; Oppermann, Timo; Cheaib, Miriam; Cullum, John; Schmidt, Helmut

    2014-01-01

    ABSTRACT Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes. PMID:25389173

  5. Underbarrier interference

    SciTech Connect

    Ivlev, B.

    2011-04-15

    Research Highlights: > In tunneling a momentum, tangent to a border of the prebarrier region, is important. > A tangent momentum, transferred under the barrier, is real in contrast to normal one. > Real momenta lead to caustics points under the barrier where new branches are formed. > Resulting eigenstate can be not small after the barrier. > This results in a possibility of penetration through an almost classical barrier. - Abstract: Quantum tunneling through a two-dimensional static barrier becomes unusual when a momentum of an electron has a tangent component with respect to a border of the prebarrier region. If the barrier is not homogeneous in the direction perpendicular to tunneling a fraction of the electron state is waves propagating away from the barrier. When the tangent momentum is zero a mutual interference of the waves results in an exponentially small outgoing flux. The finite tangent momentum destroys the interference due to formation of caustics by the waves. As a result, a significant fraction of the prebarrier density is carried away from the barrier providing a not exponentially small penetration even through an almost classical barrier. The total electron energy is well below the barrier.

  6. Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms.

    PubMed

    Steiner, Jessica Kathryne; Tasaki, Junichi; Rouhana, Labib

    2016-05-01

    Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova. PMID:27149082

  7. Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms

    PubMed Central

    Steiner, Jessica Kathryne; Tasaki, Junichi; Rouhana, Labib

    2016-01-01

    Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova. PMID:27149082

  8. RNA interference-mediated knockdown of astrocyte elevated gene-1 inhibits growth, induces apoptosis, and increases the chemosensitivity to 5-fluorouracil in renal cancer Caki-1 cells.

    PubMed

    Wang, Peng; Yin, Bo; Shan, Liping; Zhang, Hui; Cui, Jun; Zhang, Mo; Song, Yongsheng

    2014-12-31

    Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

  9. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

    SciTech Connect

    Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

    2011-10-22

    Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

  10. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model.

    PubMed

    Andey, Terrick; Marepally, Srujan; Patel, Apurva; Jackson, Tanise; Sarkar, Shubhashish; O'Connell, Malaney; Reddy, Rakesh C; Chellappan, Srikumar; Singh, Pomila; Singh, Mandip

    2014-06-28

    The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h resulting in a 6

  11. RNA interference-mediated knockdown of three putative aminopeptidases N affects susceptibility of Spodoptera exigua larvae to Bacillus thuringiensis Cry1Ca.

    PubMed

    Ren, Xiang-Liang; Ma, Yan; Cui, Jin-Jie; Li, Guo-Qing

    2014-08-01

    Aminopeptidase N (APN) isoforms in insects have been documented to be involved in the mode of action of insecticidal crystal proteins (Cry) from Bacillus thuringiensis. Here we cloned two novel Seapns from the larval midgut of Spodoptera exigua, a major pest of many crops of economic importance in China. According to a phylogenetic analysis, these two novel SeAPNs, along with the four SeAPN isoforms already described, belong to six different clades. All the six SeAPNs share similar structural features. From N- to C-terminus a signal peptide, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif, and a glycosylphosphatidylinositol-anchor sequence are located. The six Seapn genes were highly expressed at the larval stage, especially in the larval gut. Ingestion during four consecutive days of double-stranded RNAs (dsRNAs) targeting Seapn1, Seapn2, Seapn3, Seapn4, Seapn5 and Seapn6 significantly reduced corresponding mRNA levels by 55.6%, 45.5%, 43.2%, 56.8%, 45.4%, and 46.0% respectively, compared with those recorded in control larvae fed on non-specific dsRNA (dsegfp). When the larvae that previously ingested phosphate buffered saline (PBS)-, dsegfp-, or six dsSeapns-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities were 71.2%, 69.3%, 52.0%, 77.2%, 43.3%, 62.0%, 65.4% and 53.8% respectively recorded after 6days. ANOVA analysis revealed that the larvae previously fed on dsSeapn1-, dsSeapn3-, and dsSeapn6-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeapn2-, dsSeapn4- and dsSeapn5-overlaid diets. Thus, these results suggest that SeAPN1, SeAPN3 and SeAPN6 may be candidate receptors for Cry1Ca in S. exigua. PMID:24932922

  12. Blue light-activated hypocrellin B damages ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Jiang, Y.; Leung, A. W. N.; Xiang, J. Y.; Xu, C. S.

    2011-10-01

    In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hyppcrellin B concentration was kept at 2.5 μM and light doses from 0.5-4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

  13. Salmonella VNP20009-mediated RNA interference of ABCB5 moderated chemoresistance of melanoma stem cell and suppressed tumor growth more potently

    PubMed Central

    Zhang, Xiaoxin; Cheng, Xiawei; Lai, Yueyang; Zhou, Yuqiang; Cao, Wenmin; Hua, Zi-Chun

    2016-01-01

    Drug resistance remains an obstacle hindering the success of chemotherapy. Cancer stem cells (CSCs) have been recently found to confer resistance to chemotherapy. Therefore functional markers of CSCs should be discovered and specific therapies targeting these cells should be developed. In our investigation, a small population of B16F10 cells which was positive for ATP-binding cassette sub-family B member 5 (ABCB5) was isolated. This population displayed characteristics similar to those of CSCs and ABCB5 was identified to confer tumor growth and drug resistance in B16F10 cell line. Although targeting ABCB5 by small short interfering RNA delivered by VNP20009 failed to inhibit tumor growth, the combined treatment of VNP-shABCB5 and chemotherapy can act synergistically to delay tumor growth and enhance survival time in a primary B16F10 mice model. Results suggest that the combined treatment of VNP-shABCB5 and chemotherapy can improve the efficacy of chemotherapeutic drugs. Therefore, this combination therapy is of potential significance for melanoma treatment. PMID:26910836

  14. RNA interference of a trehalose-6-phosphate synthase gene reveals its roles during larval-pupal metamorphosis in Bactrocera minax (Diptera: Tephritidae).

    PubMed

    Xiong, Ke-Cai; Wang, Jia; Li, Jia-Hao; Deng, Yu-Qing; Pu, Po; Fan, Huan; Liu, Ying-Hong

    2016-01-01

    Trehalose is the major blood sugar in insects, which plays a crucial role as an instant source of energy and the starting substrate for chitin biosynthesis. In insects, trehalose is synthesized by catalysis of an important enzyme, trehalose-6-phosphate synthase (TPS). In the present study, a trehalose-6-phosphate synthase gene from Bactrocera minax (BmTPS) was cloned and characterized. BmTPS contained an open reading frame of 2445 nucleotides encoding a protein of 814 amino acids with a predicted molecular weight of 92.05kDa. BmTPS was detectable in all developmental stages of Bactrocera minax and expressed higher in the final- (third-) instar larvae. Tissue-specific expression patterns of BmTPS showed that it was mainly expressed in the fat body. The 20-hydroxyecdysone (20E) induced the expression of BmTPS and three genes in the chitin biosynthesis pathway. Moreover, injection of double-stranded RNA into third-instar larvae successfully silenced the transcription of BmTPS in B. minax, and thereby decreased the activity of TPS and trehalose content. Additionally, silencing of BmTPS inhibited the expression of three key genes in the chitin biosynthesis pathway and exhibited 52% death and abnormal phenotypes. The findings demonstrate that BmTPS is indispensable for larval-pupal metamorphosis. Besides, the establishment of RNAi experimental system in B. minax would lay a solid foundation for further investigation of molecular biology and physiology of this pest. PMID:27405007

  15. RNA interference of endochitinases in the sugarcane endophyte Trichoderma virens 223 reduces its fitness as a biocontrol agent of pineapple disease.

    PubMed

    Romão-Dumaresq, Aline S; de Araújo, Welington Luiz; Talbot, Nicholas J; Thornton, Christopher R

    2012-01-01

    The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

  16. Impairment of acquisition of intravenous cocaine self-administration by RNA-interference of dopamine D1-receptors in the nucleus accumbens shell.

    PubMed

    Pisanu, Augusta; Lecca, Daniele; Valentini, Valentina; Bahi, Amine; Dreyer, Jean-Luc; Cacciapaglia, Fabio; Scifo, Andrea; Piras, Giovanna; Cadoni, Cristina; Di Chiara, Gaetano

    2015-02-01

    Microdialysis during i.v. drug self-administration (SA) have implicated nucleus accumbens (NAc) shell DA in cocaine and heroin reinforcement. However, this correlative evidence has not been yet substantiated by experimental evidence obtained by studying the effect of selective manipulation of NAc shell DA transmission on cocaine and heroin SA. In order to investigate this issue, DA D1a receptor (D1aR) expression was impaired in the NAc shell and core by locally infusing lentiviral vectors (LV) expressing specific D1aR-siRNAs (LV-siRNAs). Control rats were infused in the same areas with LV expressing GFP. Fifteen days later, rats were trained to acquire i.v. cocaine or heroin self-administration (SA). At the end of behavioral experiments, in order to evaluate the effect of LV-siRNA on D1aR expression, rats were challenged with amphetamine and the brains were processed for immunohistochemical detection of c-Fos and D1aR. Control rats acquired i.v. cocaine and heroin SA. Infusion of LV-siRNAs in the medial NAc shell reduced D1aR density and the number of c-Fos positive nuclei in the NAc shell, while sparing the core, and prevented the acquisition of cocaine, but not heroin SA. In turn, LV-siRNAs infusion in the core reduced D1aR density and the number of c-Fos positive nuclei in the same area, while sparing the shell, and failed to affect acquisition of cocaine. The differential effect of LV impairment of NAc shell D1aR on cocaine and heroin SA indicates that NAc shell DA acting on D1aR specifically mediates cocaine reinforcement. PMID:25446574

  17. Roles of NlAKTIP in the Growth and Eclosion of the Rice Brown Planthopper, Nilaparvata lugens Stål, as Revealed by RNA Interference

    PubMed Central

    Hao, Peiying; Lu, Chaofeng; Ma, Yan; Xu, Lingbo; Zhu, Jiajun; Yu, Xiaoping

    2015-01-01

    AKT-interacting protein (AKTIP) interacts with serine/threonine protein kinase B (PKB)/AKT. AKTIP modulates AKT’s activity by enhancing the phosphorylation of the regulatory site and plays a crucial role in multiple biological processes. In this study, the full length cDNA of NlAKTIP, a novel AKTIP gene in the brown planthopper (BPH) Nilaparvata lugens, was cloned. The reverse transcription quantitive PCR (RT-qPCR) results showed that the NlAKTIP gene was strongly expressed in gravid female adults, but was relatively weakly expressed in nymphs and male adult BPH. In female BPH, treatment with dsAKTIP resulted in the efficient silencing of NlAKTIP, leading to a significant reduction of mRNA levels, about 50% of those of the untreated control group at day 7 of the study. BPH fed with dsAKTIP had reduced growth with lower body weights and smaller sizes, and the body weight of BPH treated with dsAKTIP at day 7 decreased to about 30% of that of the untreated control. Treatment of dsAKTIP significantly delayed the eclosion for over 7 days relative to the control group and restricted ovarian development to Grade I (transparent stage), whereas the controls developed to Grade IV (matured stage). These results indicated that NlAKTIP is crucial to the growth and development of female BPH. This study provided a valuable clue of a potential target NlAKTIP for inhibiting the BPH, and also provided a new point of view on the interaction between BPH and resistant rice. PMID:26402675

  18. Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor

    PubMed Central

    Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

    2015-01-01

    Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships. PMID:26136688

  19. RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae1

    PubMed Central

    Subramanian, Senthil; Graham, Madge Y.; Yu, Oliver; Graham, Terrence L.

    2005-01-01

    Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing. PMID:15778457

  20. Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor.

    PubMed

    Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

    2015-01-01

    Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships. PMID:26136688

  1. RNA interference-mediated knockdown of Aurora-B alters the metastatic behavior of A549 cells via modulation of the phosphoinositide 3-kinase/Akt signaling pathway

    PubMed Central

    ZHOU, LONG DIAN; XIONG, XU; LONG, XIN HUA; LIU, ZHI LI; HUANG, SHAN HU; ZHANG, WEI

    2014-01-01

    Accumulating evidence has revealed that an elevated expression level of Aurora-B is associated with metastasis in various types of malignant tumor. However, it is currently unclear whether this molecule is involved in non-small lung cancer (NSCLC) metastasis, and the molecular mechanisms associated with Aurora-B and metastasis remain unknown. In the present study, in order to investigate whether Aurora-B is involved in the development and metastasis of NSCLC, the Aurora-B protein expression in NSCLC tissues was detected by immunohistochemistry and its association with metastasis was analyzed. The results revealed that the expression levels of the Aurora-B protein in tissues obtained from NSCLC patients with lymph node metastasis were significantly higher than those without metastatic disease. Furthermore, the effect of Aurora-B inhibition on A549 cell migration and invasion, as well as the activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was evaluated. Aurora-B was inhibited in the A549 cells using short hairpin RNA, and the cell migration and invasion rates were investigated using wound healing and Transwell invasion assays. In addition, the expression of the main proteins in the PI3K/Akt/nuclear factor-κB (NF-κB) signaling pathway, and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. The results demonstrated that cell migration and invasion were decreased as a result of silencing Aurora-B. Furthermore, the activity of the PI3K/Akt/NF-κB signaling pathway and the expression of MMP-2 and -9 protein were suppressed by silencing Aurora-B. The results of the present study indicate that the knockdown of Aurora-B suppresses A549 cell invasion and migration via the inhibition of the PI3K/Akt signaling pathway in vitro and thus, targeting Aurora-B may present a potential treatment strategy for NSCLC. PMID:25295091

  2. The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

    PubMed

    Fan, Guanglei; Bo, Jingli; Wan, Renming; Peng, Mingya; Luan, Yufen; Deng, Minbin; Xu, Longbao

    2015-05-01

    Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for

  3. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics*

    PubMed Central

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-01-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  4. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics.

    PubMed

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-09-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  5. Herschel's Interference Demonstration.

    ERIC Educational Resources Information Center

    Perkalskis, Benjamin S.; Freeman, J. Reuben

    2000-01-01

    Describes Herschel's demonstration of interference arising from many coherent rays. Presents a method for students to reproduce this demonstration and obtain beautiful multiple-beam interference patterns. (CCM)

  6. Temperature rise during experimental light-activated bleaching.

    PubMed

    Klaric, Eva; Rakic, Mario; Sever, Ivan; Tarle, Zrinka

    2015-02-01

    The purpose of this study was to evaluate the surface and intrapulpal temperatures after treatments with different bleaching gels subjected to different types of light activation. A K-type thermocouple and infrared thermometer were used to measure the temperature increase during the 15- or 30-min treatment period. Light-emitting diode with a center wavelength of 405 nm (LED405), organic light-emitting diode (OLED), and femtosecond laser were tested and compared to ZOOM2. The tooth surface was treated with five bleaching agents and Vaseline which served as a control.The generalized estimating equation (GEE) model was applied for testing the differences in temperature increase. The ZOOM2 light source led to the largest increase in mean pulpal and tooth surface temperatures of 21.1 and 22.8 °C, followed by focused femtosecond laser which increased the pulpal and surface temperatures by up to 15.7 and 16.8 °C. Treatments with unfocused femtosecond laser, LED405, and OLED induced significantly lower mean temperature increases (p < 0.001 for each comparison with ZOOM2 and focused femtosecond laser), both in the pulp chamber (up to 2.7, 2.5, and 1.4 °C) and at the tooth surface (up to 3.2, 3.4, and 1.8 °C). Significant differences between pulp chamber and tooth surface measurements were obtained for all types of bleaching gel, during treatments with ZOOM2 (p < 0.001), LED405 (p < 0.001), and unfocused (p < 0.001) and focused femtosecond laser (p ≤ 0.002). Different bleaching agents or Vaseline can serve as an isolating layer. Focused femtosecond laser and ZOOM2 produced large temperature increases in the pulp chamber and at the tooth surface. Caution is advised when using these types of light activation, while LED405, OLED, and unfocused femtosecond laser could be safely used.

  7. Preventing bee mortality with RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present a real world example of the successful use of an RNAi product for disease control. RNAi increased bee health in the presence of the bee viral pathogen, IAPV. The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsib...

  8. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    PubMed

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

  9. Alternative chromophores for use in light-activated surgical adhesives

    NASA Astrophysics Data System (ADS)

    Byrd, Brian D.; Heintzelman, Douglas L.; McNally-Heintzelman, Karen M.

    2003-06-01

    A study was conducted to determine the feasibility of using alternative chromophores in light-activated surgical adhesives. Two commonly used chromophores, indocyanine green (ICG), and methylene blue (MB) were investigated, as well as three different food colorings: red #40, blue #1, and green food coloring consisting of yellow #5 and blue #1. The study consisted of three components. First, the absorption profiles of the five chromophores, both diluted in deionized water and bound to protein, were recorded with a UV-Vis-NIR spectrophotometer. Second, the effect of accumulated thermal dosages on the stability of the absorption profiles was investigated. Third, the stability of the absorption profiles of the chromophore solutions when exposed to ambient light for an extended period of time was investigated. The peak absorption wavelengths of ICG, MB, red #40, and blue #1, were found to be 780 nm, 665 nm, 500 nm, and 630 nm respectively. The green food coloring had two absorption peaks at 417 nm and 630 nm, corresponding to the two dye components comprising this color. The peak absorption wavelength of the ICG shifted to 805 nm when bound to protein. ICG and MB showed a significant decrease in absorbance units with increased time and temperature when heated to temperatures up to 100 degrees C. Negligible change in absorption with accumulated thermal dose was observed for any of the three food colorings investigated. Photobleaching was observed in both ICG and MB solutions with exposure to a white light source. An 88% decrease in absorption was seen in ICG deionized water solution after 7 days of exposure with a corresponding 33% decrease in absorption seen in the MB deionized water solution. A negligible drop in absorption was observed from exposure to ambient light for a 12-week period with the three food colorings investigated.

  10. Multifunctional RNA Nanoparticles

    PubMed Central

    2015-01-01

    Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA–DNA hybrids aimed to conditionally activate multiple split functionalities inside cells. PMID:25267559

  11. Tuning RNA Interference by Enhancing siRNA/PAZ Recognition.

    PubMed

    Gaglione, Maria; Potenza, Nicoletta; Di Fabio, Giovanni; Romanucci, Valeria; Mosca, Nicola; Russo, Aniello; Novellino, Ettore; Cosconati, Sandro; Messere, Anna

    2013-01-10

    Chemically modified siRNAs were synthesized to enhance the corresponding silencing activities. The introduced modifications endowed siRNAs with high silencing effect, long RNAi persistence, and better serum resistance. Theoretical data allowed us to correlate the observed siRNAs interfering performance with the peculiar interactions with PAZ.

  12. Short-interference RNAs: becoming medicines

    PubMed Central

    Martínez, Tamara; Jiménez, Ana Isabel; Pañeda, Covadonga

    2015-01-01

    RNA interference is a cellular mechanism by which small molecules of double stranded RNA modulate gene expression acting on the concentration and/or availability of a given messenger RNA. Almost 10 years after Fire and Mello received the Nobel Prize for the discovery of this mechanism in flat worms, RNA interference is on the edge of becoming a new class of therapeutics. With various phase III studies underway, the following years will determine whether RNAi-therapeutics can rise up to the challenge and become mainstream medicines. The present review gives a thorough overview of the current status of this technology focusing on the path to the clinic of this new class of compounds. PMID:26648823

  13. Short-interference RNAs: becoming medicines.

    PubMed

    Martínez, Tamara; Jiménez, Ana Isabel; Pañeda, Covadonga

    2015-01-01

    RNA interference is a cellular mechanism by which small molecules of double stranded RNA modulate gene expression acting on the concentration and/or availability of a given messenger RNA. Almost 10 years after Fire and Mello received the Nobel Prize for the discovery of this mechanism in flat worms, RNA interference is on the edge of becoming a new class of therapeutics. With various phase III studies underway, the following years will determine whether RNAi-therapeutics can rise up to the challenge and become mainstream medicines. The present review gives a thorough overview of the current status of this technology focusing on the path to the clinic of this new class of compounds. PMID:26648823

  14. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial

    PubMed Central

    Fitzgerald, Kevin; Frank-Kamenetsky, Maria; Shulga-Morskaya, Svetlana; Liebow, Abigail; Bettencourt, Brian R; Sutherland, Jessica E; Hutabarat, Renta M; Clausen, Valerie A; Karsten, Verena; Cehelsky, Jeffrey; Nochur, Saraswathy V; Kotelianski, Victor; Horton, Jay; Mant, Timothy; Chiesa, Joseph; Ritter, James; Munisamy, Malathy; Vaishnaw, Akshay K; Gollob, Jared A; Simon, Amy

    2015-01-01

    baseline relative to placebo (p<0·0001). Interpretation Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. Funding Alnylam Pharmaceuticals. PMID:24094767

  15. Role of double-stranded RNA-binding proteins in RNA silencing and antiviral defense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In plants as well as in animals, the intracellular presence of double-stranded RNA (dsRNA) triggers a signal transduction pathway that uses the sequence information of dsRNA to direct silencing of homologous genes. This process, designated RNA silencing or RNA interference (RNAi), relies on a family...

  16. Light-Activated Rapid-Response Polyvinylidene-Fluoride-Based Flexible Films.

    PubMed

    Tai, Yanlong; Lubineau, Gilles; Yang, Zhenguo

    2016-06-01

    The design strategy and mechanical response mechanism of light-activated, rapid-response, flexible films are presented. Practical applications as a microrobot and a smart spring are demonstrated. PMID:27061392

  17. Light-Triggered RNA Annealing by an RNA Chaperone.

    PubMed

    Panja, Subrata; Paul, Rakesh; Greenberg, Marc M; Woodson, Sarah A

    2015-06-15

    Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.

  18. Thick Film Interference.

    ERIC Educational Resources Information Center

    Trefil, James

    1983-01-01

    Discusses why interference effects cannot be seen with a thick film, starting with a review of the origin of interference patterns in thin films. Considers properties of materials in films, properties of the light source, and the nature of light. (JN)

  19. Interference laser processing

    NASA Astrophysics Data System (ADS)

    Nakata, Yoshiki

    2016-02-01

    The most important component of quantum optics is laser interference. Interference patterns are formed by splitting a coherent beam into multiple beams and correlating them. This study introduces a variety of beam correlators and discusses their characteristics. Beam correlator basics such as interference region in terms of pulse width, group delay dispersion effects on pulse width, optical delay adjustment, and interference pattern simulation are explained. A discussion of the history of interference processing begins with the method in 1967 and continues through the advancement of shorter wavelengths and pulse widths. The recent techniques of solid-liquid-solid for 3D nanofabrication, duplicated structures with laser-induced periodic surface structure, processing inside transparent materials, and 2D and 3D periodic structures fabricated by photo-sensitization are also presented.

  20. A Mg2+-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding.

    PubMed

    Schaak, Janell E; Yakhnin, Helen; Bevilacqua, Philip C; Babitzke, Paul

    2003-09-19

    Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg(2+)-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5'-p-azidophenacyl-containing transcripts revealed a Mg(2+)-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg(2+) inhibits TRAP binding, which may be partially due to the effect of Mg(2+) on RNA tertiary structure formation. Results from expression studies using trpE'-'lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg(2+)-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE. PMID:12963367

  1. Fluorinated TiO₂ as an ambient light-activated virucidal surface coating material for the control of human norovirus.

    PubMed

    Park, Geun Woo; Cho, Min; Cates, Ezra L; Lee, David; Oh, Byung-Taek; Vinjé, Jan; Kim, Jae-Hong

    2014-11-01

    We evaluated the virucidal efficacy of light-activated fluorinated TiO₂ surface coatings on human norovirus and several surrogates (bacteriophage MS2, feline calcivirus (FCV), and murine norovirus (MNV)). Inactivation of viruses on surfaces exposed to a common fluorescent lamp was monitored and the effects of UVA intensity, temperature, and fluoride content were assessed. Destruction of RNA and capsid oxidation were evaluated for human norovirus inocula on the F-TiO₂ surfaces, while contact with the F-TiO₂ surface and exposure to residual UVA radiation of 10 μW cm(-2) for 60 min resulted in infectivity reductions for the norovirus surrogates of 2-3 log₁₀. Infectivity reductions on pristine TiO₂ surfaces in identical conditions were over 2 orders of magnitude lower. Under realistic room lighting conditions, MS2 infectivity declined below the lower detection limit after 12h. Reductions in RNA were generally low, with the exception of GII.4, while capsid protein oxidation likely played a larger role in infectivity loss. Inactivation of norovirus surrogates occurred significantly faster on F-TiO₂ compared to pristine TiO₂ surfaces. The material demonstrated antiviral action against human norovirus surrogates and was shown to effectively inhibit MS2 when exposed to residual UVA present in fluorescent room lighting conditions in a laboratory setting.

  2. Electromagnetic interference analysis for CSRH

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Chen, Zhi-Jun; Liu, Yu-Ying; Yan, Yi-Hua; Ji, Guo-Shu

    2006-06-01

    The electromagnetic interference tolerance is discussed in this paper, the radio spectrum assignment and the sensitivity of interference measurement equipment are described. Interference to CSRH and forecast from synchronous orbit satellite, and interference due to low and middle orbit satellite are analysed. Transmission formula of interference from mobile base and conservative estimation from pulse radar are presented.

  3. RNA Interference of Interferon Regulatory Factor-1 Gene Expression in THP-1 Cell Line Leads to Toll-Like Receptor-4 Overexpression/Activation As Well As Up-modulation of Annexin-II1

    PubMed Central

    Maratheftis, Christos I; Giannouli, Stavroula; Spachidou, Maria P; Panayotou, George; Voulgarelis, Michael

    2007-01-01

    Interferon regulatory factor-1 (IRF-1) is a candidate transcription factor for the regulation of the Toll-like receptor-4 (TLR-4) gene. Using a small interfering RNA-based (siRNA) process to silence IRF-1 gene expression in the leukemic monocytic cell line THP-1, we investigated whether such a modulation would alter TLR-4 expression and activation status in these cells. The siIRF-1 cells expressed elevated levels of TLR-4 mRNA and protein compared to controls by 90% and 77%, respectively. ICAM.1 protein expression and apoptosis levels were increased by 8.35- and 4.25-fold, respectively. The siIRF-1 cells overexpressed Bax mRNA compared to controls. Proteomic analysis revealed upmodulation of the Annexin-II protein in siIRF-1 THP-1 cells. Myelodysplastic syndrome (MDS) patients with an absence of full-length IRF-1 mRNA also overexpressed Annexin-II. It is plausible that this overexpression may lead to the activation of TLR-4 contributing to the increased apoptosis characterizing MDS. PMID:18084608

  4. Dealing with LOFAR interference

    NASA Astrophysics Data System (ADS)

    Offringa, André

    2011-07-01

    LOFAR is a new radio telescope built in and around the Netherlands that will probe the universe between 10 and 200 MHz. Most of LOFAR's hardware has been installed and the next step is writing the required algorithms to process LOFAR's data. One such algorithm is the detection of interference. Since LOFAR is built in a populated environment, care has to be taken to deal with interference from terrestrial origin. A detection pipeline was written that removes interference in an automated way. This pipeline is now in use and the radio environment around LOFAR is being analyzed. Results show a relatively benign radio environment with a loss of data of a few per cent due to interference.

  5. Involvement of {alpha}ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis.

    PubMed

    Li, Tianbo; Koshy, Shyny; Folkesson, Hans G

    2007-10-01

    To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against alphaENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. alphaENaC mRNA and protein were specifically reduced by approximately 65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by approximately 60% after pSi-N1 injection. Interestingly, alphaENaC and betaENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after alphaENaC silencing and reduced after Nedd4-2 silencing. alphaENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo alphaENaC or Nedd4-2 silencing and measured alphaENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated alphaENaC or Nedd4-2 mRNA and protein, thus demonstrating that alphaENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth. PMID:17693485

  6. Evaluation of Light-Activated Provisional Resin Materials for Periodontal Soft Tissue Management

    PubMed Central

    Jun, Soo-Kyung; Lee, Hae-Hyoung

    2016-01-01

    The purpose of this study was to determine mechanical properties using a compressive test with cylinder specimen (h = 6 mm and ϕ = 4 mm) as well as cytotoxicity using elutes from disk specimen (ϕ = 10 mm and h = 2 mm) against human gingival fibroblasts and oral keratinocytes with light-activated provisional resin materials (Revotek LC and Luxatemp Solar) compared to chemically activated counterpart (Snap, Trim II, and Jet). Significantly increased compressive strength (210~280 MPa) was detected in light-activated products compared to chemically activated ones (20~65 MPa, P < 0.05) and similar compressive modulus was detected in both types (0.8~1.5 and 0.5~1.3 GPa). Simultaneously, the light-activated products showed less adverse effects on the periodontal soft tissue cells in any polymerization stage compared to the chemically activated products. Particularly, chemically activated products had significantly greater adverse effects during the “polymerizing” phase compared to those that were “already set” (P < 0.05), as shown in confocal microscopic images of live and dead cells. In conclusion, light-activated provisional resin materials have better mechanical properties as well as biocompatibility against two tested types of oral cells compared to the chemically activated counterpart, which are considered as more beneficial choice for periodontal soft tissue management. PMID:27672651

  7. GAS PHASE SELECTIVE PHOTOXIDATION OF ALCOHOLS USING LIGHT-ACTIVATED TITANIUM DIOXIDE AND MOLECULAR OXYGEN

    EPA Science Inventory

    Gas Phase Selective Oxidation of Alcohols Using Light-Activated Titanium Dioxide and Molecular Oxygen

    Gas phase selective oxidations of various primary and secondary alcohols are studied in an indigenously built stainless steel up-flow photochemical reactor using ultravi...

  8. Evaluation of Light-Activated Provisional Resin Materials for Periodontal Soft Tissue Management

    PubMed Central

    Jun, Soo-Kyung; Lee, Hae-Hyoung

    2016-01-01

    The purpose of this study was to determine mechanical properties using a compressive test with cylinder specimen (h = 6 mm and ϕ = 4 mm) as well as cytotoxicity using elutes from disk specimen (ϕ = 10 mm and h = 2 mm) against human gingival fibroblasts and oral keratinocytes with light-activated provisional resin materials (Revotek LC and Luxatemp Solar) compared to chemically activated counterpart (Snap, Trim II, and Jet). Significantly increased compressive strength (210~280 MPa) was detected in light-activated products compared to chemically activated ones (20~65 MPa, P < 0.05) and similar compressive modulus was detected in both types (0.8~1.5 and 0.5~1.3 GPa). Simultaneously, the light-activated products showed less adverse effects on the periodontal soft tissue cells in any polymerization stage compared to the chemically activated products. Particularly, chemically activated products had significantly greater adverse effects during the “polymerizing” phase compared to those that were “already set” (P < 0.05), as shown in confocal microscopic images of live and dead cells. In conclusion, light-activated provisional resin materials have better mechanical properties as well as biocompatibility against two tested types of oral cells compared to the chemically activated counterpart, which are considered as more beneficial choice for periodontal soft tissue management.

  9. Enhanced anti-tumor effects of combined MDR1 RNA interference and human sodium/iodide symporter (NIS) radioiodine gene therapy using an adenoviral system in a colon cancer model

    PubMed Central

    Ahn, S J; Jeon, Y H; Lee, Y J; Lee, Y L; Lee, S-W; Ahn, B-C; Ha, J-H; Lee, J

    2010-01-01

    Using an adenoviral system as a delivery mediator of therapeutic gene, we investigated the therapeutic effects of the use of combined MDR1 shRNA and human NIS (hNIS) radioiodine gene therapy in a mouse colon xenograft model. In vitro uptake of Tc-99m sestamibi was increased approximately two-fold in cells infected with an adenovirus vector that expressed MDR1 shRNA (Ad-shMDR1) and I-125 uptake was 25-fold higher in cells infected with an adenovirus vector that expressed human NIS (Ad-hNIS) as compared with control cells. As compared with doxorubicin or I-131 treatment alone, the combination of doxorubicin and I-131 resulted in enhanced cytotoxicity for both Ad-shMDR1- and Ad-hNIS-infected cells, but not for control cells. In vivo uptake of Tc-99m sestamibi and Tc-99m pertechnetate was twofold and 10-fold higher for Ad-shMDR1 and Ad-hNIS-infected tumors as compared with tumors infected with a control adenovirus construct that expressed β-galactrosidase (Ad-LacZ), respectively. In mice treated with either doxorubicin or I-131 alone, there was a slight delay in tumor growth as compared to mice treated with Ad-LacZ. However, combination therapy with doxorubicin and I-131 induced further significant inhibition of tumor growth as compared with mice treated with Ad-LacZ. We have shown successful therapeutic efficacy of combined MDR shRNA and hNIS radioiodine gene therapy using an adenoviral vector system in a mouse colon cancer model. Adenovirus-mediated cancer gene therapy using MDR1 shRNA and hNIS would be a useful tool for the treatment of cancer cells expressing multi-drug resistant genes. PMID:20186172

  10. Generalized Multiphoton Quantum Interference

    NASA Astrophysics Data System (ADS)

    Tillmann, Max; Tan, Si-Hui; Stoeckl, Sarah E.; Sanders, Barry C.; de Guise, Hubert; Heilmann, René; Nolte, Stefan; Szameit, Alexander; Walther, Philip

    2015-10-01

    Nonclassical interference of photons lies at the heart of optical quantum information processing. Here, we exploit tunable distinguishability to reveal the full spectrum of multiphoton nonclassical interference. We investigate this in theory and experiment by controlling the delay times of three photons injected into an integrated interferometric network. We derive the entire coincidence landscape and identify transition matrix immanants as ideally suited functions to describe the generalized case of input photons with arbitrary distinguishability. We introduce a compact description by utilizing a natural basis that decouples the input state from the interferometric network, thereby providing a useful tool for even larger photon numbers.

  11. Understanding ghost interference

    NASA Astrophysics Data System (ADS)

    Qureshi, Tabish; Chingangbam, Pravabati; Shafaq, Sheeba

    2016-08-01

    The ghost interference observed for entangled photons is theoretically analyzed using wave-packet dynamics. It is shown that ghost interference is a combined effect of virtual double-slit creation due to entanglement, and quantum erasure of which-path information for the interfering photon. For the case where the two photons are of different color, it is shown that fringe width of the interfering photon depends not only on its own wavelength, but also on the wavelength of the other photon which it is entangled with.

  12. RNA in evolution.

    PubMed

    Lehman, Niles

    2010-01-01

    RNA has played a variety of roles in the evolutionary history of life on the Earth. While this molecule was once considered a poor cousin of the more influential polymers in the cell, namely DNA and proteins, a string of important discoveries over the last 50 years has revealed that RNA may in fact be the cornerstone of biological function. In particular, the finding that RNA can be catalytic, and thus possess both a genotype and a phenotype, has forced us to consider the possibility that life's origins began with RNA, and that the subsequent diversification of life is aptly described as a string of innovations by RNA to adapt to a changing environment. Some of these adaptations include riboswitches, ribonucleoproteins (RNPs), RNA editing, and RNA interference (RNAi). Although many of these functions may seem at first glance to be recent evolutionary developments, it may be the case that all of their catalytic activities trace their roots back to a primordial 'RNA World' some four billion years ago, and that RNA's diversity has a continuous thread that pervades life from its very origins. PMID:21935885

  13. The Crosslink formation of 2'-OMe oligonucleotide containing 2-amino-6-vinylpurine protects mRNA from miRNA-mediated silencing.

    PubMed

    Hagihara, Shinya; Lin, Wei-Chen; Kusano, Shuhei; Chao, Xiao-Guang; Hori, Tsuneaki; Imoto, Shuhei; Nagatsugi, Fumi

    2013-08-19

    Masking the miRNA binding site: Crosslink-forming oligonucleotide (CFO) was used for target gene-specific inhibition of microRNA (miRNA) functions. This method can interfere with specific miRNA-mRNA interactions by recognizing sequences unique to the 3'-UTR that are inherent in each mRNA.

  14. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    PubMed

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  15. <