Science.gov

Sample records for genomewide rna interference

  1. Integrative analysis of genome-wide RNA interference screens.

    PubMed

    Berndt, Jason D; Biechele, Travis L; Moon, Randall T; Major, Michael B

    2009-01-01

    High-throughput genetic screens have exponentially increased the functional annotation of the genome over the past 10 years. Likewise, genome-scale efforts to map DNA methylation, chromatin state and occupancy, messenger RNA expression patterns, and disease-associated genetic polymorphisms, and proteome-wide efforts to map protein-protein interactions, have also created vast resources of data. An emerging trend involves combining multiple types of data, referred to as integrative screening. Examples include papers that report integrated data generated from large-scale RNA interference screens on the Wnt/beta-catenin pathway with either genotypic or proteomic data in colorectal cancer. These studies demonstrate the power of data integration to generate focused, validated data sets and to identify high-confidence candidate genes for follow-up experiments. We present the ongoing evolution and new strategies for the integrative screening approach with respect to understanding and treating human disease. PMID:19436058

  2. 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 ...

  3. Next-generation libraries for robust RNA interference-based genome-wide screens

    PubMed Central

    Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

    2015-01-01

    Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

  4. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

    PubMed Central

    Tomoyasu, Yoshinori; Miller, Sherry C; Tomita, Shuichiro; Schoppmeier, Michael; Grossmann, Daniela; Bucher, Gregor

    2008-01-01

    Background RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects. Results We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans. Conclusion Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches. PMID:18201385

  5. 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

  6. 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

  7. An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Ramirez, Christina N.; Radu, Constantin; Calder, Paul A.; Beauchamp, Lesslie; Farazi, T.; Landthaler, M.; Tuschi, T.; Magdaleno, Susan

    2013-01-01

    Abstract MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder–Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function. PMID:23153064

  8. The RNA interference revolution.

    PubMed

    Lenz, G

    2005-12-01

    The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing. PMID:16302089

  9. 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

  10. Editor meets silencer: crosstalk between RNA editing and RNA interference

    PubMed Central

    Nishikura, Kazuko

    2010-01-01

    The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions. PMID:17139332

  11. RNA Interference in Ticks

    PubMed Central

    Kocan, Katherine M.; Blouin, Edmour; de la Fuente, José

    2011-01-01

    Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5. RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10. RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines

  12. 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

  13. 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

  14. RNA Interference for Wheat Functional Gene Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this appr...

  15. 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

  16. 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.

  17. Redefining regulation of DNA methylation by RNA interference

    PubMed Central

    Muthusamy, Viswanathan; Bosenberg, Marcus; Wajapeyee, Narendra

    2013-01-01

    Epigenetic changes refer to heritable changes that may modulate gene expression without affecting DNA sequence. DNA methylation is one such heritable epigenetic change, which is causally associated with the transcription regulation of many genes in the mammalian genome. Altered DNA methylation has been implicated in a wide variety of human diseases including cancer. Understanding the regulation of DNA methylation is likely to improve the ability to diagnose and treat these diseases. With the advent of high-throughput RNA interference (RNAi) screens, answering epigenetic questions on a genomic scale is now possible. Two recent genome-wide RNAi screens have addressed the regulation of DNA methylation in cancer, leading to the identification of the regulators of epigenetic silencing by oncogenic RAS and how epigenetic silencing of the tumor suppressor RASSF1A is maintained. These RNAi screens have much wider applications, since similar screens can now be adapted to identify the mechanism of silencing of any human disease-associated gene that is epigenetically regulated. In this review, we discuss two recent genome-wide RNAi screens for epigenetic regulators and explore potential applications in understanding DNA methylation and gene expression regulation in mammalian cells. We also discuss some of the key unanswered questions in the field of DNA methylation and suggest genome-wide RNAi screens designed to answer them. PMID:20620207

  18. 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

  19. 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

  20. RNA Interference in Infectious Tropical Diseases

    PubMed Central

    Hong, Young S.

    2008-01-01

    Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi. PMID:18344671

  1. RNA interference Pathways in Filamentous Fungi

    PubMed Central

    Liu, Yi

    2015-01-01

    RNA interference is a conserved eukaryotic homology-dependent post-transcriptional gene silencing mechanism. The filamentous fungus Neurospora crassa is one of the first organisms used for RNAi studies. Quelling and Meiotic Silencing by Unpaired DNA (MSUD) are two RNAi related phenomena discovered in Neurospora and their characterizations have contributed significantly to our understanding of RNAi mechanisms in eukaryotes. More recently, a type of DNA damage-induced small RNA, microRNA-like small RNAs and Dicer-independent small silencing RNAs have been discovered in Neurospora crassa which can regulate gene expression. In addition, there are at least six different pathways responsible for the production of these small RNAs, indicating that this fungus is an important model system to study small RNA function and biogenesis. The RNAi studies in other filamentous fungi such as Cryphonectria paracitica and Aspergillus provide evidences that RNAi plays an important role in antiviral defense and RNAi mechanism is widely conserved in filamentous fungi, and RNAi has been commonly used as an efficient tool for studying the gene function. The discovery of the endogenous small RNAs from M. circinelloides further indicates the richness and complex of the RNAi field in eukaryotes. PMID:20680389

  2. Genome-wide characterization of maize miRNA genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in plant growth and development. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling ident...

  3. Endogenous RNA interference is driven by copy number

    PubMed Central

    Cruz, Cristina; Houseley, Jonathan

    2014-01-01

    A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001 PMID:24520161

  4. 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.

  5. Genome-Wide Approaches for RNA Structure Probing.

    PubMed

    Silverman, Ian M; Berkowitz, Nathan D; Gosai, Sager J; Gregory, Brian D

    2016-01-01

    RNA molecules of all types fold into complex secondary and tertiary structures that are important for their function and regulation. Structural and catalytic RNAs such as ribosomal RNA (rRNA) and transfer RNA (tRNA) are central players in protein synthesis, and only function through their proper folding into intricate three-dimensional structures. Studies of messenger RNA (mRNA) regulation have also revealed that structural elements embedded within these RNA species are important for the proper regulation of their total level in the transcriptome. More recently, the discovery of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) has shed light on the importance of RNA structure to genome, transcriptome, and proteome regulation. Due to the relatively small number, high conservation, and importance of structural and catalytic RNAs to all life, much early work in RNA structure analysis mapped out a detailed view of these molecules. Computational and physical methods were used in concert with enzymatic and chemical structure probing to create high-resolution models of these fundamental biological molecules. However, the recent expansion in our knowledge of the importance of RNA structure to coding and regulatory RNAs has left the field in need of faster and scalable methods for high-throughput structural analysis. To address this, nuclease and chemical RNA structure probing methodologies have been adapted for genome-wide analysis. These methods have been deployed to globally characterize thousands of RNA structures in a single experiment. Here, we review these experimental methodologies for high-throughput RNA structure determination and discuss the insights gained from each approach. PMID:27256381

  6. 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. PMID:24681887

  7. 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

  8. 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

  9. 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…

  10. Genome-wide analysis of microRNA and mRNA expression signatures in cancer

    PubMed Central

    Li, Ming-hui; Fu, Sheng-bo; Xiao, Hua-sheng

    2015-01-01

    Cancer is an extremely diverse and complex disease that results from various genetic and epigenetic changes such as DNA copy-number variations, mutations, and aberrant mRNA and/or protein expression caused by abnormal transcriptional regulation. The expression profiles of certain microRNAs (miRNAs) and messenger RNAs (mRNAs) are closely related to cancer progression stages. In the past few decades, DNA microarray and next-generation sequencing techniques have been widely applied to identify miRNA and mRNA signatures for cancers on a genome-wide scale and have provided meaningful insights into cancer diagnosis, prognosis and personalized medicine. In this review, we summarize the progress in genome-wide analysis of miRNAs and mRNAs as cancer biomarkers, highlighting their diagnostic and prognostic roles. PMID:26299954

  11. The promises and pitfalls of RNA-interference-based therapeutics

    PubMed Central

    Castanotto, Daniela; Rossi, John J.

    2009-01-01

    The discovery that gene expression can be controlled by the Watson–Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence — a process known as RNA interference — has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded. PMID:19158789

  12. 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

  13. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

    PubMed Central

    Meliopoulos, Victoria A.; Andersen, Lauren E.; Birrer, Katherine F.; Simpson, Kaylene J.; Lowenthal, John W.; Bean, Andrew G. D.; Stambas, John; Stewart, Cameron R.; Tompkins, S. Mark; van Beusechem, Victor W.; Fraser, Iain; Mhlanga, Musa; Barichievy, Samantha; Smith, Queta; Leake, Devin; Karpilow, Jon; Buck, Amy; Jona, Ghil; Tripp, Ralph A.

    2012-01-01

    Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. PMID:22247330

  14. Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells

    PubMed Central

    Balakrishnan, Ilango; Yang, Xiaodong; Brown, Joseph; Ramakrishnan, Aravind; Torok–Storb, Beverly; Kabos, Peter; Hesselberth, Jay R.; Pillai, Manoj M.

    2014-01-01

    Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. PMID:24038734

  15. 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

  16. Hairpin dsRNA does not trigger RNA interference in Candida albicans cells.

    PubMed

    Staab, Janet F; White, Theodore C; Marr, Kieren A

    2011-01-01

    RNA interference/silencing mechanisms triggered by double-stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well-conserved Argonaute and a non-canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down-regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans. PMID:20737430

  17. 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

  18. Identification of alternative splicing regulators by RNA interference in Drosophila

    PubMed Central

    Park, Jung W.; Parisky, Katherine; Celotto, Alicia M.; Reenan, Robert A.; Graveley, Brenton R.

    2004-01-01

    Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. PMID:15492211

  19. miRNA Digger: a comprehensive pipeline for genome-wide novel miRNA mining.

    PubMed

    Yu, Lan; Shao, Chaogang; Ye, Xinghuo; Meng, Yijun; Zhou, Yincong; Chen, Ming

    2016-01-01

    MicroRNAs (miRNAs) are important regulators of gene expression. The recent advances in high-throughput sequencing (HTS) technique have greatly facilitated large-scale detection of the miRNAs. However, thoroughly discovery of novel miRNAs from the available HTS data sets remains a major challenge. In this study, we observed that Dicer-mediated cleavage sites for the processing of the miRNA precursors could be mapped by using degradome sequencing data in both animals and plants. In this regard, a novel tool, miRNA Digger, was developed for systematical discovery of miRNA candidates through genome-wide screening of cleavage signals based on degradome sequencing data. To test its sensitivity and reliability, miRNA Digger was applied to discover miRNAs from four organs of Arabidopsis. The results revealed that a majority of already known mature miRNAs along with their miRNA*s expressed in these four organs were successfully recovered. Notably, a total of 30 novel miRNA-miRNA* pairs that have not been registered in miRBase were discovered by miRNA Digger. After target prediction and degradome sequencing data-based validation, eleven miRNA-target interactions involving six of the novel miRNAs were identified. Taken together, miRNA Digger could be applied for sensitive detection of novel miRNAs and it could be freely downloaded from http://www.bioinfolab.cn/miRNA_Digger/index.html. PMID:26732371

  20. Genome-Wide Mapping of Yeast RNA Polymerase II Termination

    PubMed Central

    Schaughency, Paul; Merran, Jonathan; Corden, Jeffry L.

    2014-01-01

    Yeast RNA polymerase II (Pol II) terminates transcription of coding transcripts through the polyadenylation (pA) pathway and non-coding transcripts through the non-polyadenylation (non-pA) pathway. We have used PAR-CLIP to map the position of Pol II genome-wide in living yeast cells after depletion of components of either the pA or non-pA termination complexes. We show here that Ysh1, responsible for cleavage at the pA site, is required for efficient removal of Pol II from the template. Depletion of Ysh1 from the nucleus does not, however, lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators, but in contrast to Nrd1, this readthrough is less processive, or more susceptible to pausing. The data presented here provide delineation of in vivo Pol II termination regions and highlight differences in the sequences that signal termination of different classes of non-pA transcripts. PMID:25299594

  1. 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

  2. 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

  3. Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila.

    PubMed

    Kemp, Cordula; Mueller, Stefanie; Goto, Akira; Barbier, Vincent; Paro, Simona; Bonnay, François; Dostert, Catherine; Troxler, Laurent; Hetru, Charles; Meignin, Carine; Pfeffer, Sébastien; Hoffmann, Jules A; Imler, Jean-Luc

    2013-01-15

    The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific. PMID:23255357

  4. 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

  5. RNA interference directed to CDK2 inhibits HIV-1 transcription.

    PubMed

    Ammosova, Tatyana; Berro, Reem; Kashanchi, Fatah; Nekhai, Sergei

    2005-10-25

    We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target. PMID:16085226

  6. 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

  7. 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

  8. 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.

  9. RNA Interference Prevents Autosomal-Dominant Hearing Loss.

    PubMed

    Shibata, Seiji B; Ranum, Paul T; Moteki, Hideaki; Pan, Bifeng; Goodwin, Alexander T; Goodman, Shawn S; Abbas, Paul J; Holt, Jeffrey R; Smith, Richard J H

    2016-06-01

    Hearing impairment is the most common sensory deficit. It is frequently caused by the expression of an allele carrying a single dominant missense mutation. Herein, we show that a single intracochlear injection of an artificial microRNA carried in a viral vector can slow progression of hearing loss for up to 35 weeks in the Beethoven mouse, a murine model of non-syndromic human deafness caused by a dominant gain-of-function mutation in Tmc1 (transmembrane channel-like 1). This outcome is noteworthy because it demonstrates the feasibility of RNA-interference-mediated suppression of an endogenous deafness-causing allele to slow progression of hearing loss. Given that most autosomal-dominant non-syndromic hearing loss in humans is caused by this mechanism of action, microRNA-based therapeutics might be broadly applicable as a therapy for this type of deafness. PMID:27236922

  10. 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

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

    PubMed Central

    Mills, Mary K.; Nayduch, D.; Michel, K.

    2014-01-01

    Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic hemorrhagic disease, bluetongue, and likely Schmallenberg, which cause significant economic burden 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 siRNA pathway member orthologs, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to preform RNA silencing. Injection of control dsRNA, dsGFP, into the hemocoel 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) ortholog, resulted in a 40% decrease of transcript levels and 73% shortened median survivals as compared to 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. PMID:25293805

  12. 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. PMID:25293805

  13. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

    PubMed

    Nojima, Takayuki; Gomes, Tomás; Carmo-Fonseca, Maria; Proudfoot, Nicholas J

    2016-03-01

    The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis. PMID:26844429

  14. 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

  15. MicroRNA-binding viral protein interferes with Arabidopsis development.

    PubMed

    Chellappan, Padmanabhan; Vanitharani, Ramachandran; Fauquet, Claude M

    2005-07-19

    MicroRNAs (miRNAs) are small (approximately 21 nt), noncoding RNAs that negatively regulate target mRNAs at the posttranscriptional level that are involved in development. In plants, virus-induced disease symptoms often result in developmental abnormalities resembling perturbation of miRNA-mediated function. Here, we report that expression in transgenic plants of a geminivirus-encoded AC4 protein from African cassava mosaic virus Cameroon Strain (ACMV), a suppressor of posttranscriptional gene silencing, was correlated with decreased accumulation of host miRNAs and increased development abnormalities in Arabidopsis. Down-regulation of miRNA correlated with an up-regulation of target mRNA level. In vitro binding assays revealed the ability of AC4 of ACMV (A-AC4) but not East African cassava mosaic Cameroon virus AC2 to bind single-stranded forms of miRNAs and short interfering RNAs but not double-stranded RNA forms. Normally, a labile intermediate during the miRNA biogenesis/RNA-induced silencing complex assembly, miRNA*, was below the level of detection, indicating that AC4 might interfere at a point downstream of the miRNA duplex unwinding process. The association of AC4 with miRNA was demonstrated by the association of A-AC4-GFP fusion protein, extracted from Arabidopsis protoplasts, with 2'-O-methyloligonucleotide complementary to miR159 (miR159*) and by the presence of miRNA with the A-AC4-GFP fusion protein after immunoprecipitation with antibody against GFP. In both assays, A-AC4 protein and miRNA complexes were copurified. These results provide direct evidence that AC4 is a unique virus-encoded posttranscriptional gene-silencing suppressor protein that binds to and presumably inactivates mature miRNAs and thus blocks the normal miRNA-mediated regulation of target mRNAs, resulting in developmental defects in Arabidopsis. PMID:16006510

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

    PubMed

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

  17. 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

  18. The efficiency of RNA interference in Bursaphelenchus xylophilus.

    PubMed

    Park, Jung-Eun; Lee, Kyong Yun; Lee, Se-Jin; Oh, Wan-Suk; Jeong, Pan-Young; Woo, Taeha; Kim, Chang-Bae; Paik, Young-Ki; Koo, Hyeon-Sook

    2008-07-31

    RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products. PMID:18525237

  19. 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

  20. 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

  1. 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

  2. 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

  3. Efficacy of a Novel Class of RNA Interference Therapeutic Agents

    PubMed Central

    Matsumoto, Takahiro; D'Alessandro-Gabazza, Corina N.; Gil-Bernabe, Paloma; Boveda-Ruiz, Daniel; Naito, Masahiro; Kobayashi, Tetsu; Toda, Masaaki; Mizutani, Takayuki; Taguchi, Osamu; Morser, John; Eguchi, Yutaka; Kuroda, Masahiko; Ochiya, Takahiro; Hayashi, Hirotake; Gabazza, Esteban C.; Ohgi, Tadaaki

    2012-01-01

    RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application. PMID:22916145

  4. Kinetic models of the interference of gene transcription to ncRNA and mRNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-06-01

    The experiments indicate that the transcription of genes into ncRNA can positively or negatively interfere with transcription into mRNA. We propose two kinetic models describing this effect. The first model is focused on the ncRNA-induced chromatin modification facilitating the transcription of the downstream gene into mRNA. The second model includes the competition between the transcription into ncRNA and the binding of activator to a regulatory site of the downstream gene transcribed into mRNA. Our analysis based on the mean-field kinetic equations and Monte Carlo simulations shows the likely dependences of the transcription rate on RNA polymerase concentration in situations with different rate-limiting steps. Our models can also be used to scrutinize the dependence of the transcription rate on other kinetic parameters. Our kinetic Monte Carlo simulations show that the first model predicts stochastic bursts in the mRNA formation provided that the transcription into ncRNA is slow, while the second model predicts in addition anti-phase stochastic bursts in the mRNA and ncRNA formation provided that that the protein attachment to and detachment from a regulatory site is slow.

  5. Scavenger Receptor Mediates Systemic RNA Interference in Ticks

    PubMed Central

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Xuenan, Xuan; Suzuki, Hiroshi; Linggatong Galay, Remil; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-01-01

    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks. PMID:22145043

  6. siRNA Design Software for a Target Gene-Specific RNA Interference

    PubMed Central

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  7. siRNA Design Software for a Target Gene-Specific RNA Interference.

    PubMed

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  8. An efficient RNA interference screening strategy for gene functional analysis

    PubMed Central

    2012-01-01

    Background RNA interference (RNAi) is commonly applied in genome-scale gene functional screens. However, a one-on-one RNAi analysis that targets each gene is cost-ineffective and laborious. Previous studies have indicated that siRNAs can also affect RNAs that are near-perfectly complementary, and this phenomenon has been termed an off-target effect. This phenomenon implies that it is possible to silence several genes simultaneously with a carefully designed siRNA. Results We propose a strategy that is combined with a heuristic algorithm to design suitable siRNAs that can target multiple genes and a group testing method that would reduce the number of required RNAi experiments in a large-scale RNAi analysis. To verify the efficacy of our strategy, we used the Orchid expressed sequence tag data as a case study to screen the putative transcription factors that are involved in plant disease responses. According to our computation, 94 qualified siRNAs were sufficient to examine all of the predicated 229 transcription factors. In addition, among the 94 computer-designed siRNAs, an siRNA that targets both TF15 (a previously identified transcription factor that is involved in the plant disease-response pathway) and TF21 was introduced into orchids. The experimental results showed that this siRNA can simultaneously silence TF15 and TF21, and application of our strategy successfully confirmed that TF15 is involved in plant defense responses. Interestingly, our second-round analysis, which used an siRNA specific to TF21, indicated that TF21 is a previously unidentified transcription factor that is related to plant defense responses. Conclusions Our computational results showed that it is possible to screen all genes with fewer experiments than would be required for the traditional one-on-one RNAi screening. We also verified that our strategy is capable of identifying genes that are involved in a specific phenotype. PMID:22988976

  9. 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

  10. Dissecting Wnt/beta-catenin signaling during gastrulation using RNA interference in mouse embryos.

    PubMed

    Lickert, Heiko; Cox, Brian; Wehrle, Christian; Taketo, Makoto M; Kemler, Rolf; Rossant, Janet

    2005-06-01

    Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/beta-catenin signaling, we have used genome-wide expression profiling of conditional beta-catenin mutant embryos. Known Wnt/beta-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets. PMID:15857914

  11. 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

  12. 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

  13. A small molecule enhances RNA interference and promotes microRNA processing

    PubMed Central

    Shan, Ge; Li, Yujing; Zhang, Junliang; Li, Wendi; Szulwach, Keith E; Duan, Ranhui; Faghihi, Mohammad A; Khalil, Ahmad M; Lu, Lianghua; Paroo, Zain; Chan, Anthony W S; Shi, Zhangjie; Liu, Qinghua; Wahlestedt, Claes; He, Chuan; Jin, Peng

    2010-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific post-transcriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proof-of-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics. PMID:18641635

  14. RNA interference: concept to reality in crop improvement.

    PubMed

    Saurabh, Satyajit; Vidyarthi, Ambarish S; Prasad, Dinesh

    2014-03-01

    The phenomenon of RNA interference (RNAi) is involved in sequence-specific gene regulation driven by the introduction of dsRNA resulting in inhibition of translation or transcriptional repression. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in opening a new vista for crop improvement. RNAi technology is precise, efficient, stable and better than antisense technology. It has been employed successfully to alter the gene expression in plants for better quality traits. The impact of RNAi to improve the crop plants has proved to be a novel approach in combating the biotic and abiotic stresses and the nutritional improvement in terms of bio-fortification and bio-elimination. It has been employed successfully to bring about modifications of several desired traits in different plants. These modifications include nutritional improvements, reduced content of food allergens and toxic compounds, enhanced defence against biotic and abiotic stresses, alteration in morphology, crafting male sterility, enhanced secondary metabolite synthesis and seedless plant varieties. However, crop plants developed by RNAi strategy may create biosafety risks. So, there is a need for risk assessment of GM crops in order to make RNAi a better tool to develop crops with biosafety measures. This article is an attempt to review the RNAi, its biochemistry, and the achievements attributed to the application of RNAi in crop improvement. PMID:24402564

  15. 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

  16. dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    Pan, Zhong-Hua; Gao, Kun; Hou, Cheng-Xiang; Wu, Ping; Qin, Guang-Xing; Geng, Tao; Guo, Xi-Jie

    2015-07-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA. PMID:25839934

  17. 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

  18. Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In thi...

  19. Genome-Wide siRNA Screening Using Forward Transfection: Identification of Modulators of Membrane Trafficking in Mammalian Cells.

    PubMed

    Bexiga, Mariana G; Simpson, Jeremy C

    2016-01-01

    RNA interference (RNAi) has become an essential tool for molecular and cellular biologists to dissect cell function. In recent years its application has been extended to genome-wide studies, enabling the systematic identification of new cell regulation mechanisms and drug targets. In this chapter, a protocol for a genome-wide RNAi screen coupled to high-content microscopy is presented. Specifically we describe key features of assay design, plate layout, and a protocol for forward transfection of small interfering RNAs (siRNAs) in a 384-well plate format. As an example of its application in identifying modulators of membrane trafficking, we also provide a protocol to measure the efficacy of intracellular delivery of the B subunit of Shiga-like toxin to the Golgi complex. Finally we show an automated image analysis routine that can be used to extract single cell data from the screen, thereby providing a quantitative ranking of how a large panel of siRNAs affects this biological process. PMID:27581283

  20. 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.

  1. 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. PMID:25629590

  2. 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

  3. Retention and Loss of RNA Interference Pathways in Trypanosomatid Protozoans

    PubMed Central

    Murta, Silvane M. F.; Vieira, Ana Carolina; Turco, Salvatore J.; Tschudi, Christian; Ullu, Elisabetta; Beverley, Stephen M.

    2010-01-01

    RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence. PMID:21060810

  4. 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

  5. Combination of microRNA expression profiling with genome-wide SNP genotyping to construct a coronary artery disease-related miRNA-miRNA synergistic network.

    PubMed

    Hua, Lin; Xia, Hong; Zhou, Ping; Li, Dongguo; Li, Lin

    2014-12-01

    In recent years, microRNAs (miRNAs) were found to play critical roles in many important biological processes. On the other hand, the rapid development of genome-wide association studies (GWAS) help identify potential genetic variants associated with the disease phenotypic variance. Therefore, we suggested a combined analysis of microRNA expression profiling with genome-wide Single Nucleotide Polymorphism (SNP) genotyping to identify potential disease-related biomarkers. Considering functional SNPs in miRNA genes or target sites might be important signals associated with human complex diseases, we constructed a miRNA-miRNA synergistic network related to coronary artery disease (CAD) by performing a genome-wide scan for SNPs in human miRNA 3' -untranslated regions (UTRs) target sites and computed potential SNP cooperation effects contributing to disease based on potential miRNA-SNP interactions reported recently. Furthermore, we identified some potential CAD-related miRNAs by analyzing the constructed miRNAmiRNA synergistic network. As a result, the predicted miRNA-miRNA network and miRNA clusters were validated by significantly high interaction effects of CAD-related miRNAs. Accurate classification performances were obtained for all of the identified miRNA clusters, and the sensitivity and specificity were all more than 90%. The network topological analysis confirmed some novel CAD-related miRNAs identified recently by experiments. Our method might help to understand miRNA function and CAD disease, as well as to explore the novel mechanisms involved. PMID:25641175

  6. Genome-wide profiling of the microRNA-mRNA regulatory network in skeletal muscle with aging

    PubMed Central

    Kim, Ji Young; Park, Young-Kyu; Lee, Kwang-Pyo; Lee, Seung-Min; Kang, Tae-Wook; Kim, Hee-Jin; Dho, So Hee; Kim, Seon-Young; Kwon, Ki-Sun

    2014-01-01

    Skeletal muscle degenerates progressively, losing mass (sarcopenia) over time, which leads to reduced physical ability and often results in secondary diseases such as diabetes and obesity. The regulation of gene expression by microRNAs is a key event in muscle development and disease. To understand genome-wide changes in microRNAs and mRNAs during muscle aging, we sequenced microRNAs and mRNAs from mouse gastrocnemius muscles at two different ages (6 and 24 months). Thirty-four microRNAs (15 up-regulated and 19 down-regulated) were differentially expressed with age, including the microRNAs miR-206 and -434, which were differentially expressed in aged muscle in previous studies. Interestingly, eight microRNAs in a microRNA cluster at the imprinted Dlk1-Dio3 locus on chromosome 12 were coordinately down-regulated. In addition, sixteen novel microRNAs were identified. Integrative analysis of microRNA and mRNA expression revealed that microRNAs may contribute to muscle aging through the positive regulation of transcription, metabolic processes, and kinase activity. Many of the age-related microRNAs have been implicated in human muscular diseases. We suggest that genome-wide microRNA profiling will expand our knowledge of microRNA function in the muscle aging process. PMID:25063768

  7. RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

    PubMed

    Unniyampurath, Unnikrishnan; Pilankatta, Rajendra; Krishnan, Manoj N

    2016-01-01

    The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term. PMID:26927085

  8. HITS-CLIP yields genome-wide insights into brain alternative RNA processing

    NASA Astrophysics Data System (ADS)

    Licatalosi, Donny D.; Mele, Aldo; Fak, John J.; Ule, Jernej; Kayikci, Melis; Chi, Sung Wook; Clark, Tyson A.; Schweitzer, Anthony C.; Blume, John E.; Wang, Xuning; Darnell, Jennifer C.; Darnell, Robert B.

    2008-11-01

    Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

  9. Genome-wide identification of RNA editing in hepatocellular carcinoma.

    PubMed

    Kang, Lin; Liu, Xiaoqiao; Gong, Zhoulin; Zheng, Hancheng; Wang, Jun; Li, Yingrui; Yang, Huanming; Hardwick, James; Dai, Hongyue; Poon, Ronnie T P; Lee, Nikki P; Mao, Mao; Peng, Zhiyu; Chen, Ronghua

    2015-02-01

    We did whole-transcriptome sequencing and whole-genome sequencing on nine pairs of Hepatocellular carcinoma (HCC) tumors and matched adjacent tissues to identify RNA editing events. We identified mean 26,982 editing sites with mean 89.5% canonical A→G edits in each sample using an improved bioinformatics pipeline. The editing rate was significantly higher in tumors than adjacent normal tissues. Comparing the difference between tumor and normal tissues of each patient, we found 7 non-synonymous tissue specific editing events including 4 tumor-specific edits and 3 normal-specific edits in the coding region, as well as 292 edits varying in editing degree. The significant expression changes of 150 genes associated with RNA editing were found in tumors, with 3 of the 4 most significant genes being cancer related. Our results show that editing might be related to higher gene expression. These findings indicate that RNA editing modification may play an important role in the development of HCC. PMID:25462863

  10. 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

  11. RNA interference of IL-10 in leukemic B-1 cells.

    PubMed

    McCarthy, Brian A; Mansour, Amal; Lin, Yi-Chu; Kotenko, Sergei; Raveche, Elizabeth

    2004-07-23

    RNA interference, or RNAi, is designed to work by Watson-Crick base pairing and to result in a posttranscriptional block in protein synthesis. Antiapoptotic proteins are a major focus of cancer therapy and make attractive targets for RNAi. An IL-10 RNAi sequence was designed in accordance with Tuschl rules and was modeled to a hairpin configuration. In chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, the failure to undergo apoptosis may be responsible for the accumulation of malignant B-1 cells. Interleukin-10, despite controversy, has been shown to have antiapoptotic properties, and increased endogenous IL-10 production has been found in CLL by several labs. A malignant B-1 cell line, LNC, derived from an NZB mouse (a murine model for CLL) was utilized as a target for IL-10 RNAi. Our earlier studies of antisense IL-10 resulted in antiproliferative and proapoptotic effects. The cytotoxic effects of IL-10 RNAi were dose- and time-dependent, with an optimal dose 10-fold lower than that of antisense IL-10. IL-10 RNAi lowered IL-10 protein as measured by ELISA. 2 micro M IL-10 RNAi initiated a G2/M block and a decrease in the message for cdc25C, the M-phase inducer phosphatase. IL-10 RNAi efficiently induced apoptosis. Bcl7C, a member of the antiapoptotic Bcl family, was significantly down-regulated. IL-10 modulating Bcl7C expression represents a novel mechanism in the evasion of apoptosis. This approach, by itself or in conjunction with current therapies, merits consideration in similar B-cell malignancies. PMID:15270555

  12. Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

    PubMed Central

    Canella, Donatella; Praz, Viviane; Reina, Jaime H.; Cousin, Pascal; Hernandez, Nouria

    2010-01-01

    Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units. PMID:20413673

  13. Advances in RNA interference: dsRNA treatment in trees and grapevines for insect pest population suppression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is a breakthrough technology that has significantly impacted contemporary approaches to control the damage caused by insect pests. Most well-known RNAi studies continue to rely on injecting the dsRNA molecules directly into the organism; this approach is not suitable for use...

  14. Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

    PubMed Central

    Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

    2014-01-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  15. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

    PubMed

    Chan, Yujia A; Aristizabal, Maria J; Lu, Phoebe Y T; Luo, Zongli; Hamza, Akil; Kobor, Michael S; Stirling, Peter C; Hieter, Philip

    2014-04-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  16. Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

    PubMed Central

    Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

    2016-01-01

    Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

  17. Genome-wide analysis of mRNA decay patterns during early Drosophila development

    PubMed Central

    2010-01-01

    Background The modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development. Results Here we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo. Conclusions Our work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay. PMID:20858238

  18. Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes.

    PubMed

    Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

    2016-04-01

    Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with thePUK1(FGRRES_01058) pseudokinase gene important for sexual reproduction inFusarium graminearum, we found that two tandem stop codons, UA(1831)GUA(1834)G, in its kinase domain were changed to UG(1831)GUG(1834)G by RNA editing in perithecia. To confirm A-to-I editing ofPUK1transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia.PUK1was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG(1831)GUG(1834)G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites inF. graminearumoccur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69PUK1-like pseudogenes with stop codons in ORFs.PUK1orthologs and other pseudogenes also displayed stage-specific expression and editing inNeurospora crassaandF. verticillioides Furthermore,F. graminearumdiffers from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing inF. graminearumpreferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

  19. RNA interference targeting SHP-1 attenuates myocardial infarction in rats.

    PubMed

    Sugano, Masahiro; Tsuchida, Keiko; Hata, Tomoji; Makino, Naoki

    2005-12-01

    The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) plays a key role in apoptosis and decreases phosphorylation of Akt. Apoptosis of cardiomyocytes is thought to contribute to the increased area of acute myocardial infarction (AMI), and Akt activation exerts a powerful cardioprotective effect after ischemia. Thus, a therapeutic strategy designed to inhibit expression of SHP-1 would be beneficial in AMI. Here we report that siRNA targeting SHP-1 reduced infarct size in a rat model of AMI. Upon injection into the ischemic left ventricular wall, the vector-based siRNA significantly suppressed the increase in the SHP-1 mRNA and the SHP-1 protein levels. The siRNA vector also significantly reduced the SHP-1 that bound to Fas-R. The SHP-1 siRNA vector increased phospho-Akt and reduced DNA fragmentation and caspase activity compared with the scramble siRNA vector. Finally, the area of myocardial infarction was significantly smaller with the SHP-1 siRNA vector than with the scramble siRNA vector at 2 days after LCA ligation. In conclusion, SHP-1 in the heart increased from the early stage of AMI, and this increase was thought to contribute to the increased area of myocardial infarction. Suppression of SHP-1 with the SHP-1 siRNA vector markedly reduced the infarct size in AMI. PMID:16223786

  20. Genome-Wide Analysis of miRNA targets in Brachypodium and Biomass Energy Crops

    SciTech Connect

    Green, Pamela J.

    2015-08-11

    MicroRNAs (miRNAs) contribute to the control of numerous biological processes through the regulation of specific target mRNAs. Although the identities of these targets are essential to elucidate miRNA function, the targets are much more difficult to identify than the small RNAs themselves. Before this work, we pioneered the genome-wide identification of the targets of Arabidopsis miRNAs using an approach called PARE (German et al., Nature Biotech. 2008; Nature Protocols, 2009). Under this project, we applied PARE to Brachypodium distachyon (Brachypodium), a model plant in the Poaceae family, which includes the major food grain and bioenergy crops. Through in-depth global analysis and examination of specific examples, this research greatly expanded our knowledge of miRNAs and target RNAs of Brachypodium. New regulation in response to environmental stress or tissue type was found, and many new miRNAs were discovered. More than 260 targets of new and known miRNAs with PARE sequences at the precise sites of miRNA-guided cleavage were identified and characterized. Combining PARE data with the small RNA data also identified the miRNAs responsible for initiating approximately 500 phased loci, including one of the novel miRNAs. PARE analysis also revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. The project included generation of small RNA and PARE resources for bioenergy crops, to facilitate ongoing discovery of conserved miRNA-target RNA regulation. By associating specific miRNA-target RNA pairs with known physiological functions, the research provides insights about gene regulation in different tissues and in response to environmental stress. This, and release of new PARE and small RNA data sets should contribute basic knowledge to enhance breeding and may suggest new strategies for improvement of biomass energy crops.

  1. Establishing an in vivo assay system to identify components involved in environmental RNA interference in the western corn rootworm.

    PubMed

    Miyata, Keita; Ramaseshadri, Parthasarathy; Zhang, Yuanji; Segers, Gerrit; Bolognesi, Renata; Tomoyasu, Yoshinori

    2014-01-01

    The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR. PMID:25003334

  2. Joint modeling of RNase footprint sequencing profiles for genome-wide inference of RNA structure.

    PubMed

    Zou, Chenchen; Ouyang, Zhengqing

    2015-10-30

    Recent studies have revealed significant roles of RNA structure in almost every step of RNA processing, including transcription, splicing, transport and translation. RNase footprint sequencing (RNase-seq) has emerged to dissect RNA structures at the genome scale. However, it remains challenging to analyze RNase-seq data because of the issues of signal sparsity, variability and correlations among various RNases. We present a probabilistic framework, joint Poisson-gamma mixture (JPGM), for integrative modeling of multiple RNase-seq profiles. Combining JPGM with hidden Markov model allows genome-wide inference of RNA structures. We apply the joint modeling approach for inferring base pairing states on simulated data sets and RNase-seq profiles of the double-strand specific RNase V1 and single-strand specific RNase S1 in yeast. We demonstrate that joint analysis of V1 and S1 profiles outputs interpretable RNA structure states, while approaches that analyze each profile separately do not. The joint modeling approach predicts the structure states of all nucleotides in 3196 transcripts of yeast without compromising accuracy, while the simple thresholding approach misses 43% of the nucleotides. Furthermore, the posterior probabilities outputted by our model are able to resolve the structural ambiguity of ≈300 000 nucleotides with overlapping V1 and S1 cleavage sites. Our model also generates RNA accessibilities, which are associated with three-dimensional conformations. PMID:26400167

  3. 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

  4. Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays

    PubMed Central

    Honkela, Antti; Peltonen, Jaakko; Topa, Hande; Charapitsa, Iryna; Matarese, Filomena; Grote, Korbinian; Stunnenberg, Hendrik G.; Reid, George; Lawrence, Neil D.; Rattray, Magnus

    2015-01-01

    Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes. PMID:26438844

  5. Genome-Wide Profiling of RNA-Protein Interactions Using CLIP-Seq.

    PubMed

    Stork, Cheryl; Zheng, Sika

    2016-01-01

    UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein-RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein-epitope tag is then used to immunoprecipitate the protein-RNA complexes. After stringent washing and gel separation the RBP-RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP. PMID:26965263

  6. Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays.

    PubMed

    Honkela, Antti; Peltonen, Jaakko; Topa, Hande; Charapitsa, Iryna; Matarese, Filomena; Grote, Korbinian; Stunnenberg, Hendrik G; Reid, George; Lawrence, Neil D; Rattray, Magnus

    2015-10-20

    Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes. PMID:26438844

  7. Genome-wide screen for miRNA targets using the MISSION target ID library.

    PubMed

    Coussens, Matthew J; Forbes, Kevin; Kreader, Carol; Sago, Jack; Cupp, Carrie; Swarthout, John

    2012-01-01

    The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3Î"TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%). PMID:22508434

  8. [shRNAs driven by K14 promoter induce tissue-specific RNA interference].

    PubMed

    Dai, Rong; Shen, Si-Jun; Wan, Peng-Cheng; Shi, Guo-Qing; Meng, Qing-Yong; Liu, Shou-Ren

    2011-07-01

    RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA Pol II promoters can direct cell- or tissue-specific gene silencing. A eGFP-shRNA fusion construct transcribed from an RNA Pol II promoter (K14 promoter) was used to induce gene-specific shRNA silencing ofBMP4 gene expression. Recombinant vectors (pEGFP-C1-shRNA, psiCHECK-BMP4, and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-C1-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibition efficiency was tested by a luciferase assay. The results showed that all the six interference sequences inhibited the expression of BMP4 with high efficiency (>60%), and the interference sequence 5# showed the highest efficiency. For in vivo screening of JB6-C41 cells transfected with vector pEGFP-K14-shRNA, the inhibition efficiency was assayed by quantitative RT-PCR and Western blotting analyses. The results showed that the mRNA and protein products of the exogenous BMP4 gene were efficiently and specifically inhibited. The efficiency of gene silencing was greater than 60%, except for sequence 3#. The declines in mRNA and protein expression levels were significantly correlated during gene silence by the shRNA. This system may be adapted for in vivo shRNA expression and gene silencing. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in the analysis of the mechanisms of hair follicle development in sheep. PMID:22049690

  9. Genome-wide identification of microRNA-related variants associated with risk of Alzheimer's disease.

    PubMed

    Ghanbari, Mohsen; Ikram, M Arfan; de Looper, Hans W J; Hofman, Albert; Erkeland, Stefan J; Franco, Oscar H; Dehghan, Abbas

    2016-01-01

    MicroRNAs (miRNAs) serve as key post-transcriptional regulators of gene expression. Genetic variation in miRNAs and miRNA-binding sites may affect miRNA function and contribute to disease risk. Here, we investigated the extent to which variants within miRNA-related sequences could constitute a part of the functional variants involved in developing Alzheimer's disease (AD), using the largest available genome-wide association study of AD. First, among 237 variants in miRNAs, we found rs2291418 in the miR-1229 precursor to be significantly associated with AD (p-value = 6.8 × 10(-5), OR = 1.2). Our in-silico analysis and in-vitro miRNA expression experiments demonstrated that the variant's mutant allele enhances the production of miR-1229-3p. Next, we found miR-1229-3p target genes that are associated with AD and might mediate the miRNA function. We demonstrated that miR-1229-3p directly controls the expression of its top AD-associated target gene (SORL1) using luciferase reporter assays. Additionally, we showed that miR-1229-3p and SORL1 are both expressed in the human brain. Second, among 42,855 variants in miRNA-binding sites, we identified 10 variants (in the 3' UTR of 9 genes) that are significantly associated with AD, including rs6857 that increases the miR-320e-mediated regulation of PVRL2. Collectively, this study shows that miRNA-related variants are associated with AD and suggests miRNA-dependent regulation of several AD genes. PMID:27328823

  10. 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

  11. High-Throughput RNA Interference Screening: Tricks of the Trade

    PubMed Central

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

    2016-01-01

    The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality. PMID:23616418

  12. Light-activated RNA interference in human embryonic stem cells.

    PubMed

    Huang, Xiao; Hu, Qirui; Braun, Gary B; Pallaoro, Alessia; Morales, Demosthenes P; Zasadzinski, Joseph; Clegg, Dennis O; Reich, Norbert O

    2015-09-01

    We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine. PMID:26086448

  13. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference

    PubMed Central

    Naito, Yuki; Yamada, Tomoyuki; Ui-Tei, Kumiko; Morishita, Shinichi; Saigo, Kaoru

    2004-01-01

    siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing. PMID:15215364

  14. [RNA interference: biogenesis molecular mechanisms and its applications in cervical cancer].

    PubMed

    Peralta-Zaragoza, Oscar; Bermúdez-Morales, Víctor Hugo; Madrid-Marina, Vicente

    2010-01-01

    RNAi (RNA interference) is a natural process by which eukaryotic cells silence gene expression through small interference RNAs (siRNA) which are complementary to messenger RNA (mRNA). In this process, the siRNA that are 21-25 nucleotides long and are known as microRNA (miRNA), either associate with the RNA-induced silencing complex (RISC), which targets and cleaves the complementary mRNAs by the endonucleolytic pathway, or repress the translation. It is also possible to silence exogenous gene expression during viral infections by using DNA templates to transcribe siRNA with properties that are identical to those of bioactive microRNA. Persistent human papillomavirus (HPV) infection is the main etiological agent during cervical cancer development and the HPV E6 and E7 oncogenes, which induce cellular transformation and immortalization, represent strategic targets to be silenced with siRNA. In several in vitro and in vivo studies, it has been demonstrated that the introduction of siRNA directed against the E6 and E7 oncogenes in human tumoral cervical cells transformed by HPV, leads to the efficient silencing of HPV E6 and E7 oncogene expression, which induces the accumulation of the products of the p53 and pRb tumor suppressor genes and activates the mechanism of programmed cell death by apoptosis; thus, the progression of the tumoral growth process may be prevented. The goal of this review is to analyze the microRNA biogenesis process in the silencing of gene expression and to discuss the different protocols for the use of siRNA as a potential gene therapy strategy for the treatment of cervical cancer. PMID:20415061

  15. Multipurpose modular lentiviral vectors for RNA interference and transgene expression.

    PubMed

    Kesireddy, Venu; van der Ven, Peter F M; Fürst, Dieter O

    2010-07-01

    We have created a multipurpose modular lentiviral vector system for expressing both transgenes and miRNA 30-based short hairpins (shRNAmirs) for RNAi. The core of the resulting vector system, pLVmir, allows a simple two step cloning procedure for expressing shRNAmirs under the control of a Pol II promoter in both a constitutive and conditional manner. The adapted cloning method includes a PCR-free method for transferring shRNAmir based RNAi clones from a publicly available library (Open Biosystems). The addition of a Pol II promoter-driven shRNAmir cassette and broadening the choice of Pol III promoters and silencing triggers offers great flexibility to this system. The combination of several preexisting and additional modules created here caters to common needs of researchers. Our modular vector system was validated regarding functionality of promoters, inducibility and reversibility. We successfully applied the system to knockdown Xirp2 mRNA expression in H2kb-tsA58 muscle cells and determined that this had no spurious effect on the expression of a closely related protein. Finally, our set of lentiviral vectors may be used to achieve synergistic effects, for simultaneous knockdown of two genes, as a rescue plasmid and for studying mutant proteins in a physiological context. PMID:19798586

  16. 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

  17. 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

  18. Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

    PubMed Central

    Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

    2011-01-01

    Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

  19. Genome-wide analysis of mRNA polysomal profiles with spotted DNA microarrays.

    PubMed

    Melamed, Daniel; Arava, Yoav

    2007-01-01

    The sedimentation of an mRNA in sucrose gradients is highly affected by its ribosomal association. Sedimentation analysis has therefore become routine for studying changes in ribosomal association of mRNAs of interest. DNA microarray technology has been combined with sedimentation analysis to characterize changes in ribosomal association for thousands of mRNAs in parallel. Such analyses revealed mRNAs that are translationally regulated and have provided new insights into the translation process. In this chapter, we describe possible experimental designs for analyzing genome-wide changes in ribosomal association, and discuss some of their advantages and disadvantages. We then provide a detailed protocol for analysis of polysomal fractions using spotted DNA microarrays. PMID:17923236

  20. Genome-wide analysis of FOXO3 mediated transcription regulation through RNA polymerase II profiling.

    PubMed

    Eijkelenboom, Astrid; Mokry, Michal; de Wit, Elzo; Smits, Lydia M; Polderman, Paulien E; van Triest, Miranda H; van Boxtel, Ruben; Schulze, Almut; de Laat, Wouter; Cuppen, Edwin; Burgering, Boudewijn M T

    2013-01-01

    Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO-regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. PMID:23340844

  1. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

    PubMed

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. PMID:26026149

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

    PubMed Central

    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

  3. RNA interference (RNAi) patents and human health related applications of RNAi.

    PubMed

    Ebhardt, H Alexander

    2007-01-01

    The Nobel Prize in Physiology or Medicine in 2006 was shared by A.Z. Fire and C.C. Mello. The honour was given to these two principal investigators for demonstrating in the nematode Caenorhabditis elegans that double stranded RNA directs cleavage of messenger RNAs (mRNA) in a homologous manner. This process was termed RNA interference (RNAi) and was published in 1998. Since then, further research revealed that small 21-22 nts long RNAs guide an RNA-induced silencing complex (RISC) to a target mRNA causing translational inhibition or mRNA cleavage. This review will focus on RNAi patents, delivery of RNAi to combat human disease and reviewing some recent applications regarding detection and possible cure of human diseases using RNAi. PMID:19075926

  4. 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

  5. Mutational interference mapping experiment (MIME) for studying RNA structure and function.

    PubMed

    Smyth, Redmond P; Despons, Laurence; Huili, Gong; Bernacchi, Serena; Hijnen, Marcel; Mak, Johnson; Jossinet, Fabrice; Weixi, Li; Paillart, Jean-Christophe; von Kleist, Max; Marquet, Roland

    2015-09-01

    RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding. PMID:26237229

  6. 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

  7. 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...

  8. Inhibition of avian tumor viruses by vector-based RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) has been shown to reduce the replication of certain animal viruses both in cell culture and in live animals. We developed RNAi-based anti-viral strategies against two important chicken pathogens: avian leukosis virus (ALV) and Marek’s Disease virus MDV). Entry plasmids conta...

  9. Functional specialization among insect chitinase family genes revealed by RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum, were examined using gene-specific RNA interference (RNAi). One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype ...

  10. 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"…

  11. 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,…

  12. 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 ...

  13. Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

    PubMed Central

    2012-01-01

    Background RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. Results We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. Conclusions We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis. PMID:22243803

  14. Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

    PubMed Central

    Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

    2015-01-01

    Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

  15. Genome-Wide Analysis of MicroRNA Responses to the Phytohormone Abscisic Acid in Populus euphratica

    PubMed Central

    Duan, Hui; Lu, Xin; Lian, Conglong; An, Yi; Xia, Xinli; Yin, Weilun

    2016-01-01

    MicroRNA (miRNA) is a type of non-coding small RNA with a regulatory function at the posttranscriptional level in plant growth development and in response to abiotic stress. Previous studies have not reported on miRNAs responses to the phytohormone abscisic acid (ABA) at a genome-wide level in Populus euphratica, a model tree for studying abiotic stress responses in woody plants. Here we analyzed the miRNA response to ABA at a genome-wide level in P. euphratica utilizing high-throughput sequencing. To systematically perform a genome-wide analysis of ABA-responsive miRNAs in P. euphratica, nine sRNA libraries derived from three groups (control, treated with ABA for 1 day and treated with ABA for 4 days) were constructed. Each group included three libraries from three individual plantlets as biological replicate. In total, 151 unique mature sequences belonging to 75 conserved miRNA families were identified, and 94 unique sequences were determined to be novel miRNAs, including 56 miRNAs with miRNA* sequences. In all, 31 conserved miRNAs and 31 novel miRNAs response to ABA significantly differed among the groups. In addition, 4132 target genes were predicted for the conserved and novel miRNAs. Confirmed by real-time qPCR, expression changes of miRNAs were inversely correlated with the expression profiles of their putative targets. The Populus special or novel miRNA-target interactions were predicted might be involved in some biological process related stress tolerance. Our analysis provides a comprehensive view of how P. euphratica miRNA respond to ABA, and moreover, different temporal dynamics were observed in different ABA-treated libraries. PMID:27582743

  16. Genome-Wide Analysis of MicroRNA Responses to the Phytohormone Abscisic Acid in Populus euphratica.

    PubMed

    Duan, Hui; Lu, Xin; Lian, Conglong; An, Yi; Xia, Xinli; Yin, Weilun

    2016-01-01

    MicroRNA (miRNA) is a type of non-coding small RNA with a regulatory function at the posttranscriptional level in plant growth development and in response to abiotic stress. Previous studies have not reported on miRNAs responses to the phytohormone abscisic acid (ABA) at a genome-wide level in Populus euphratica, a model tree for studying abiotic stress responses in woody plants. Here we analyzed the miRNA response to ABA at a genome-wide level in P. euphratica utilizing high-throughput sequencing. To systematically perform a genome-wide analysis of ABA-responsive miRNAs in P. euphratica, nine sRNA libraries derived from three groups (control, treated with ABA for 1 day and treated with ABA for 4 days) were constructed. Each group included three libraries from three individual plantlets as biological replicate. In total, 151 unique mature sequences belonging to 75 conserved miRNA families were identified, and 94 unique sequences were determined to be novel miRNAs, including 56 miRNAs with miRNA(*) sequences. In all, 31 conserved miRNAs and 31 novel miRNAs response to ABA significantly differed among the groups. In addition, 4132 target genes were predicted for the conserved and novel miRNAs. Confirmed by real-time qPCR, expression changes of miRNAs were inversely correlated with the expression profiles of their putative targets. The Populus special or novel miRNA-target interactions were predicted might be involved in some biological process related stress tolerance. Our analysis provides a comprehensive view of how P. euphratica miRNA respond to ABA, and moreover, different temporal dynamics were observed in different ABA-treated libraries. PMID:27582743

  17. 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.

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

    PubMed Central

    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

    2015-01-01

    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-centered RNA induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis. PMID:26018492

  19. Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-α RNA interference for IBD therapy

    PubMed Central

    Xiao, Bo; Laroui, Hamed; Ayyadurai, Saravanan; Viennois, Emilie; Charania, Moiz A.; Zhang, Yuchen; Merlin, Didier

    2013-01-01

    The application of RNA interference (RNAi) for inflammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211–275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mM). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy. PMID:23820013

  20. Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-α RNA interference for IBD therapy.

    PubMed

    Xiao, Bo; Laroui, Hamed; Ayyadurai, Saravanan; Viennois, Emilie; Charania, Moiz A; Zhang, Yuchen; Merlin, Didier

    2013-10-01

    The application of RNA interference (RNAi) for inflammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy. PMID:23820013

  1. Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

    PubMed Central

    Meier, Roger; Franceschini, Andrea; Horvath, Peter; Tetard, Marilou; Mancini, Roberta; von Mering, Christian; Helenius, Ari

    2014-01-01

    ABSTRACT The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3+ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we

  2. 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

  3. 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. PMID:23435119

  4. 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

  5. Ewing's Sarcoma: Development of RNA Interference-Based Therapy for Advanced Disease

    PubMed Central

    Simmons, Olivia; Maples, Phillip B.; Senzer, Neil; Nemunaitis, John

    2012-01-01

    Ewing's sarcoma tumors are associated with chromosomal translocation between the EWS gene and the ETS transcription factor gene. These unique target sequences provide opportunity for RNA interference(i)-based therapy. A summary of RNAi mechanism and therapeutically designed products including siRNA, shRNA and bi-shRNA are described. Comparison is made between each of these approaches. Systemic RNAi-based therapy, however, requires protected delivery to the Ewing's sarcoma tumor site for activity. Delivery systems which have been most effective in preclinical and clinical testing are reviewed, followed by preclinical assessment of various silencing strategies with demonstration of effectiveness to EWS/FLI-1 target sequences. It is concluded that RNAi-based therapeutics may have testable and achievable activity in management of Ewing's sarcoma. PMID:22523703

  6. 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

  7. 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

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

    PubMed

    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

  9. Tobamovirus-resistant tobacco generated by RNA interference directed against host genes.

    PubMed

    Asano, Momoko; Satoh, Rena; Mochizuki, Atsuko; Tsuda, Shinya; Yamanaka, Takuya; Nishiguchi, Masamichi; Hirai, Katsuyuki; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

    2005-08-15

    Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants. PMID:16081069

  10. RNA Interference as A Potential Therapeutic Treatment for Inflammation Associated Lung Injury

    PubMed Central

    Lomas-Neira, Joanne; Chung, Chun-Shiang; Ayala, Alfred

    2008-01-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain important sources of morbidity for patients in the ICUs in the developed world. However, imagine having as a therapeutic tool, the ability to regulate, in a tissue specific manner, the expression of a given gene. RNA interference, as potentially such a method of selectively suppressing protein expression, has evolved as an important tool in the study of gene specific function and targeted therapeutics. Significant progress has been made in identifying potential gene targets integral to the pathways leading to the development of inflammation-associated lung injury. This review will discuss the progress, thus far, in the application of in vivo RNA interference-based gene therapy in the investigation of inflammation-associated lung injury. PMID:19079669

  11. Gold Nanoparticle Interference Study during the Isolation, Quantification, Purity and Integrity Analysis of RNA

    PubMed Central

    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. PMID:25470814

  12. Experimental study on inhibition of the growth of human adenoid cystic cancer cells by RNA interference targeting against survivingene

    PubMed Central

    Wang, Xin; Xiong, Yu; Zhang, Congji; Zhou, Jixiang; Yang, Jun; Wang, Kun; Xia, Xiyan

    2016-01-01

    Objective: To observe the influence of RNA interference targeting against survivin gene on the biological behaviors of human adenoid cystic cancer (ACC) cells and propose the action mechanism. Method: Specific siRNA (small interfering RNA) was constructed and transfected into ACC-2 cells using liposomes. The expressions of survivin and Caspase-3 in the transfected ACC-2 cells were detected by Western Blot and RT-PCR. Cell apoptosis was detected by transmission electron microscopy, TUNEL method and flow cytometry; ultrastructural changes and cell cycles were observed. Results: Recombinant siRNA interference plasmid specifically targeting against survivin gene was constructed successfully. Survivin protein expression in the transfected ACC-2 cells was downregulated significantly, while Caspase-3 protein and mRNA expressions were upregulated and cell proliferation was inhibited considerably. Conclusion: Recombinant siRNA interference plasmid inhibited survivin mRNA and protein expressions at high efficiency, thereby inhibiting the proliferation of ACC cells. PMID:27158333

  13. Investigation of RNA interference suppression of matrix metalloproteinase-9 in mouse model of atherosclerosis

    PubMed Central

    Jin, Zhe-Xiu; Xiong, Qiang; Jia, Fang; Sun, Chun-Ling; Zhu, Hong-Tao; Ke, Fu-Sheng

    2015-01-01

    Objective: To investigate the effect of RNA interference of matrix metalloproteinase (MMP)-9 on atherosclerosis on atherosclerosis in apolipoprotein E (ApoE)-/- mouse. Methods: ApoE-/- mouse strain and three cell lines (293T, NIH3T3 and Raw264.7) were used in the present study to investigate the effect of MMP-9 silencing by RNA interference. Thirty 10-week-old ApoE-/- mice were randomly assigned to a control group, lentiviruses with naked vector group and Lentiviruses-MMP-9 intervention group (n = 10). Aortic atherosclerotic plaques of the mice were stained with immunohistochemical techniques, the MMP-9 and high-sensitivity C-reactive protein levels of three groups were detected simultaneously. Expression of MMP-9 was significantly down-regulated in interference group. MMP-9 and high-sensitivity C-reactive protein levels in MMP-9 interference group were significantly lower than that of the control group. Conclusion: The expression of MMP-9 is closely related to vulnerability of atherosclerotic plaques. Silencing of MMP-9 expression acts as a positive role in maintenance of atherosclerotic plaque stability. The present study provides novel experimental insight for the treatment of vulnerable plaques in atherosclerosis. PMID:26131101

  14. 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

  15. Genome-wide siRNA screen for mediators of NF-κB activation

    PubMed Central

    Gewurz, Benjamin E.; Towfic, Fadi; Mar, Jessica C.; Shinners, Nicholas P.; Takasaki, Kaoru; Zhao, Bo; Cahir-McFarland, Ellen D.; Quackenbush, John; Xavier, Ramnik J.; Kieff, Elliott

    2012-01-01

    Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β–, and 79 for TNFα–mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β–, or TNFα–mediated canonical NFκB activation. PMID:22308454

  16. 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

  17. Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery.

    PubMed

    Miller, W Allen; Shen, Ruizhong; Staplin, William; Kanodia, Pulkit

    2016-03-01

    Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like 'sponges' that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5' to 3' degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host's RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race. PMID:26900786

  18. Reversible Suppression of Cyclooxygenase 2 (COX-2) Expression In Vivo by Inducible RNA Interference

    PubMed Central

    Zaiss, Anne K.; Zuber, Johannes; Chu, Chun; Machado, Hidevaldo B.; Jiao, Jing; Catapang, Arthur B.; Ishikawa, Tomo-o; Gil, Jose S.; Lowe, Scott W.; Herschman, Harvey R.

    2014-01-01

    Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3′untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2. PMID:24988319

  19. SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

    PubMed Central

    Boswell, Sarah A.; Cloonan, Nicole; Mullen, Thomas E.; Ling, Joseph J.; Miller, Nimrod; Kuersten, Scott; Ma, Yong-Chao; McCarroll, Steven A.; Grimmond, Sean M.; Springer, Michael

    2014-01-01

    mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment. PMID:24586954

  20. 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

  1. Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference.

    PubMed

    Ma, H B; Lu, Q; Liang, J; Zhang, X Y

    2011-01-01

    Cellulases are pathogenic substances suspected to be responsible for the development of the early symptoms of nematode disease. The pine wood nematode, Bursaphelenchus xylophilus (Parasitaphelenchidae), is the causal agent of pine wilt disease, which kills millions of pine trees. We used RNA interference (RNAi), a reverse genetic tool, to analyze the function of the endo-β-1,4-glucanase gene of B. xylophilus, which causes the most serious forest tree disease in China and the rest of eastern Asia. Silencing of this gene was detected through real-time PCR and cellulase activity assays after soaking for 24 h in dsRNA. The cellulase gene silencing effects differed among various siRNAs. The propagation and dispersal ability of these nematodes decreased when the endo-β-1,4-glucanase gene was silenced. It is important to select an effective siRNA before performing an RNAi test. PMID:21948755

  2. Gene Silencing by RNA Interference in the White Rot Fungus Phanerochaete chrysosporium▿

    PubMed Central

    Matityahu, Avi; Hadar, Yitzhak; Dosoretz, Carlos G.; Belinky, Paula A.

    2008-01-01

    The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families. PMID:18606804

  3. 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.

  4. 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

  5. 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. PMID:25883379

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

    PubMed

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

    2015-09-01

    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 (CUG(exp)) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUG(exp) mRNA in the human α-skeletal muscle actin long-repeat (HSA(LR)) mouse model of DM1. RNAi expression cassettes were delivered to HSA(LR) 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 HSA(LR) mice, including a reduction in the CUG(exp) 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 CUG(exp) mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSA(LR) 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

  7. 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

  8. 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

  9. Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.

    PubMed Central

    Johnston, R E; Tovell, D R; Brown, D T; Faulkner, P

    1975-01-01

    Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating. Images PMID:1165599

  10. Long-term effect of systemic RNA interference on circadian clock genes in hemimetabolous insects.

    PubMed

    Uryu, Outa; Kamae, Yuichi; Tomioka, Kenji; Yoshii, Taishi

    2013-04-01

    RNA interference (RNAi) strategy, which enables gene-specific knock-down of transcripts, has been spread across a wide area of insect studies for investigating gene function without regard to model and non-model insects. This technique is of particular benefit to promote molecular studies on non-model insects. However, the optimal conditions for RNAi are still not well understood because of its variable efficiency depending on the species, target genes, and experimental conditions. To apply RNAi technique to long-running experiments such as chronobiological studies, the effects of RNAi have to persist throughout the experiment. In this study, we attempted to determine the optimal concentration of double-stranded RNA (dsRNA) for systemic RNAi and its effective period in two different insect species, the cricket Gryllus bimaculatus and the firebrat Thermobia domestica. In both species, higher concentrations of dsRNA principally yielded a more efficient knock-down of mRNA levels of tested clock genes, although the effect depended on the gene and the species. Surprisingly, the effect of the RNAi reached its maximum effect 1-2 weeks and 1 month after the injection of dsRNA in the crickets and the firebrats, respectively, suggesting a slow but long-term effect of RNAi. Our study provides fundamental information for utilizing RNAi technique in any long-running experiment. PMID:23458340

  11. RNA Interference of the PBAN/Pyrokinin Gene: Impact on Ant, Solenopsis invicta, and Moth, Helicoverpa zea, Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, an emerging RNA interference (RNAi) technology has shown high potential for development of novel biologically-based control agents as alternatives to insecticides. This represents a paradigm shift that will avoid many problems associated with conventional insecticides. Insect neuropeptide ...

  12. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  13. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

    PubMed Central

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J.; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-01-01

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation. PMID:27112822

  14. Overview of Genomic Insights into Chicken Growth Traits Based on Genome-Wide Association Study and microRNA Regulation

    PubMed Central

    Xu, Zhenqiang; Nie, Qinghua; Zhang, Xiquan

    2013-01-01

    Over the two past decades, a significant number of studies have observed animal growth traits to examine animal genetic mechanisms due to their ease of measurement and high heritability. Chicken which has a significant impact on fundamental biology is a major source of protein worldwide, making it an ideal model for examining animal growth trait development. The genetic mechanisms of chicken growth traits have been studied using quantitative trait loci mapping through genome-scan and candidate gene approaches, genome-wide association studies (GWAS), comparative genomic strategies, microRNA (miRNA) regulation of growth development analysis, and epigenomic analysis. This review focuses on chicken GWAS and miRNA regulation of growth traits. Several recently published GWAS reports showed that most genome-wide significant single nucleotide polymorphisms are located on chromosomes 1 and 4 in chickens. Chicken growth, particularly skeletal muscle growth and development, is greatly regulated by miRNA. Using dwarf and normal chickens, let-7b was found to be involved in determining chicken dwarf phenotypes by regulating growth hormone receptor gene expression. PMID:24082823

  15. Impact of Delay to Cryopreservation on RNA Integrity and Genome-Wide Expression Profiles in Resected Tumor Samples

    PubMed Central

    Durand, Geoffroy; Robinot, Nivonirina; Wozniak, Magdalena B.; Galateau-Salle, Françoise; Byrnes, Graham; Hainaut, Pierre; Le Calvez-Kelm, Florence

    2013-01-01

    The quality of tissue samples and extracted mRNA is a major source of variability in tumor transcriptome analysis using genome-wide expression microarrays. During and immediately after surgical tumor resection, tissues are exposed to metabolic, biochemical and physical stresses characterized as “warm ischemia”. Current practice advocates cryopreservation of biosamples within 30 minutes of resection, but this recommendation has not been systematically validated by measurements of mRNA decay over time. Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analyzed gene expression variation in samples of 3 hepatocellular carcinomas (HCC) and 3 lung carcinomas (LC) cryopreserved at times up to 2 hours after resection. RNA Integrity Numbers (RIN) revealed no significant deterioration of mRNA up to 2 hours after resection. Genome-wide transcriptome analysis detected non-significant gene expression variations of −3.5%/hr (95% CI: −7.0%/hr to 0.1%/hr; p = 0.054). In LC, no consistent gene expression pattern was detected in relation with warm ischemia. In HCC, a signature of 6 up-regulated genes (CYP2E1, IGLL1, CABYR, CLDN2, NQO1, SCL13A5) and 6 down-regulated genes (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1) was identified (FDR <0.05). Overall, our observations support current recommendation of time to cryopreservation of up to 30 minutes and emphasize the need for identifying tissue-specific genes deregulated following resection to avoid misinterpreting expression changes induced by warm ischemia as pathologically significant changes. PMID:24278187

  16. An RNA aptamer that interferes with the DNA binding of the HSF transcription activator

    PubMed Central

    Zhao, Xiaoching; Shi, Hua; Sevilimedu, Aarti; Liachko, Nicole; Nelson, Hillary C. M.; Lis, John T.

    2006-01-01

    Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem–loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-κB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF. PMID:16893958

  17. The role of RNA interference in the developmental separation of blood and lymphatic vasculature

    PubMed Central

    2014-01-01

    Background Dicer is an RNase III enzyme that cleaves double stranded RNA and generates functional interfering RNAs that act as important regulators of gene and protein expression. Dicer plays an essential role during mouse development because the deletion of the dicer gene leads to embryonic death. In addition, dicer-dependent interfering RNAs regulate postnatal angiogenesis. However, the role of dicer is not yet fully elucidated during vascular development. Methods In order to explore the functional roles of the RNA interference in vascular biology, we developed a new constitutive Cre/loxP-mediated inactivation of dicer in tie2 expressing cells. Results We show that cell-specific inactivation of dicer in Tie2 expressing cells does not perturb early blood vessel development and patterning. Tie2-Cre; dicerfl/fl mutant embryos do not show any blood vascular defects until embryonic day (E)12.5, a time at which hemorrhages and edema appear. Then, midgestational lethality occurs at E14.5 in mutant embryos. The developing lymphatic vessels of dicer-mutant embryos are filled with circulating red blood cells, revealing an impaired separation of blood and lymphatic vasculature. Conclusion Thus, these results show that RNA interference perturbs neither vasculogenesis and developmental angiogenesis, nor lymphatic specification from venous endothelial cells but actually provides evidence for an epigenetic control of separation of blood and lymphatic vasculature. PMID:24690185

  18. Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

    PubMed

    Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

    2014-05-16

    Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast. PMID:24328131

  19. Reprogramming Leukemia Cells to Terminal Differentiation and Growth Arrest by RNA Interference of PU.1

    PubMed Central

    Papetti, Michael; Skoultchi, Arthur I.

    2011-01-01

    Malignant transformation often leads to both loss of normal proliferation control and inhibition of cell differentiation. Some tumor cells can be stimulated to reenter their differentiation program and to undergo terminal growth arrest. The in vitro differentiation of mouse erythroleukemia (MEL) cells is an important example of tumor cell reprogramming. MEL cells are malignant erythroblasts that are blocked from differentiating into mature RBC due to dysregulated expression of the transcription factor PU.1, which binds to and represses GATA-1, the major transcriptional regulator of erythropoiesis. We used RNA interference to ask whether inhibiting PU.1 synthesis was sufficient to cause MEL cells to lose their malignant properties. We report here that transfection of MEL cells with a PU.1-specific short interfering RNA oligonucleotide causes the cells to resume erythroid differentiation, accumulate hemoglobin, and undergo terminal growth arrest. RNA interference directed at specific, aberrantly expressed transcription factors may hold promise for the development of potent antitumor therapies in other hematologic malignancies. PMID:17951405

  20. Long-term expression of miRNA for RNA interference using a novel vector system based on a negative-strand RNA virus.

    PubMed

    Honda, Tomoyuki; Yamamoto, Yusuke; Daito, Takuji; Matsumoto, Yusuke; Makino, Akiko; Tomonaga, Keizo

    2016-01-01

    RNA interference (RNAi) has emerged as a promising technique for gene therapy. However, the safe and long-term expression of small RNA molecules is a major concern for the application of RNAi therapies in vivo. Borna disease virus (BDV), a non-segmented, negative-strand RNA virus, establishes a persistent infection without obvious cytopathic effects. Unique among animal non-retroviral RNA viruses, BDV persistently establishes a long-lasting persistent infection in the nucleus. These features make BDV ideal for RNA virus vector persistently expressing small RNAs. Here, we demonstrated that the recombinant BDV (rBDV) containing the miR-155 precursor, rBDV-miR-155, persistently expressed miR-155 and efficiently silenced its target gene. The stem region of the miR-155 precursor in rBDV-miR-155 was replaceable by any miRNA sequences of interest and that such rBDVs efficiently silence the expression of target genes. Collectively, BDV vector would be a novel RNA virus vector enabling the long-term expression of miRNAs for RNAi therapies. PMID:27189575

  1. Long-term expression of miRNA for RNA interference using a novel vector system based on a negative-strand RNA virus

    PubMed Central

    Honda, Tomoyuki; Yamamoto, Yusuke; Daito, Takuji; Matsumoto, Yusuke; Makino, Akiko; Tomonaga, Keizo

    2016-01-01

    RNA interference (RNAi) has emerged as a promising technique for gene therapy. However, the safe and long-term expression of small RNA molecules is a major concern for the application of RNAi therapies in vivo. Borna disease virus (BDV), a non-segmented, negative-strand RNA virus, establishes a persistent infection without obvious cytopathic effects. Unique among animal non-retroviral RNA viruses, BDV persistently establishes a long-lasting persistent infection in the nucleus. These features make BDV ideal for RNA virus vector persistently expressing small RNAs. Here, we demonstrated that the recombinant BDV (rBDV) containing the miR-155 precursor, rBDV-miR-155, persistently expressed miR-155 and efficiently silenced its target gene. The stem region of the miR-155 precursor in rBDV-miR-155 was replaceable by any miRNA sequences of interest and that such rBDVs efficiently silence the expression of target genes. Collectively, BDV vector would be a novel RNA virus vector enabling the long-term expression of miRNAs for RNAi therapies. PMID:27189575

  2. 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. PMID:26404411

  3. 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

  4. An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.

    PubMed

    Shi, Huafang; Tschudi, Christian; Ullu, Elisabetta

    2006-12-01

    RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer. PMID:17053086

  5. 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

  6. 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

  7. Effects of RNA interference-mediated NRP-1 silencing on the proliferation and apoptosis of breast cancer cells.

    PubMed

    Han, Zhengxiang; Jiang, Guan; Zhang, Yingying; Xu, Jie; Chen, Chong; Zhang, Lansheng; Xu, Zhenyuan; Du, Xiuping

    2015-07-01

    Lentiviral expression vectors carrying human NRP-1 short hairpin RNA (shRNA) were constructed and selected to present highly efficient NRP-1/shRNA interference sequences, in order to investigate the effects of RNA interference (RNAi)-mediated NRP-1 silencing on the biological activities of breast cancer cells. Three pairs of human NRP-1 targeted specific interference sequences and one pair of non-specific control sequences were designed, synthesized and subcloned into pLB lentiviral vectors, which were further identified by polymerase chain reaction (PCR) and sequencing. Recombinant and lentiviral packaging plasmids were co-transfected into 293FT cell lines in order to produce lentiviral particles and to infect breast cancer cells with high NRP-1 expression. Flow cytometry was used to sort green fluorescent protein-positive cells. Fluorescence quantitative-reverse transcription-PCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. A cell counting kit-8 assay and an Annexin V-propidium iodide method in combination with flow cytometry were used to examine the effects of RNA interference-mediated NRP-1 gene silencing on cell proliferation, apoptosis and sensitivity to chemotherapy. The recombinant lentiviral plasmid pLB-NRP-1/shRNA was constructed successfully, as confirmed by PCR and sequencing. After the infection of recombinant lentiviral plasmids, the expression profiles of NRP-1 mRNA, and proteins of MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) were significantly lower than that of the control group (P<0.05). Compared with the control group, the MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) showed lower optical density values and higher apoptotic rates at 48, 72 and 96 h; these differences were statistically significant (P<0.05). EPI administration resulted in increased apoptosis in the MCF-7 and SK-BR-3 cell-specific interference

  8. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages

    PubMed Central

    Kishore, Shivendra; Jaskiewicz, Lukasz; Hall, Jonathan; Günthard, Huldrych F.; Beerenwinkel, Niko; Metzner, Karin J.

    2015-01-01

    Background MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM). Methods The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. Results and Conclusion PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages. PMID:26226348

  9. 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

  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. gespeR: a statistical model for deconvoluting off-target-confounded RNA interference screens.

    PubMed

    Schmich, Fabian; Szczurek, Ewa; Kreibich, Saskia; Dilling, Sabrina; Andritschke, Daniel; Casanova, Alain; Low, Shyan Huey; Eicher, Simone; Muntwiler, Simone; Emmenlauer, Mario; Rämö, Pauli; Conde-Alvarez, Raquel; von Mering, Christian; Hardt, Wolf-Dietrich; Dehio, Christoph; Beerenwinkel, Niko

    2015-01-01

    Small interfering RNAs (siRNAs) exhibit strong off-target effects, which confound the gene-level interpretation of RNA interference screens and thus limit their utility for functional genomics studies. Here, we present gespeR, a statistical model for reconstructing individual, gene-specific phenotypes. Using 115,878 siRNAs, single and pooled, from three companies in three pathogen infection screens, we demonstrate that deconvolution of image-based phenotypes substantially improves the reproducibility between independent siRNA sets targeting the same genes. Genes selected and prioritized by gespeR are validated and shown to constitute biologically relevant components of pathogen entry mechanisms and TGF-β signaling. gespeR is available as a Bioconductor R-package. PMID:26445817

  12. 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

  13. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways.

    PubMed

    Weiberg, Arne; Wang, Ming; Lin, Feng-Mao; Zhao, Hongwei; Zhang, Zhihong; Kaloshian, Isgouhi; Huang, Hsien-Da; Jin, Hailing

    2013-10-01

    Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism. PMID:24092744

  14. Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus.

    PubMed

    Marr, Edward J; Sargison, Neil D; Nisbet, Alasdair J; Burgess, Stewart T G

    2015-12-01

    This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. PMID:26212476

  15. Expression and RNA Interference of Salivary Polygalacturonase Genes in the Tarnished Plant Bug, Lygus lineolaris

    PubMed Central

    Walker, William B.; Allen, Margaret L.

    2010-01-01

    Three genes encoding polygalacturonase (PG) have been identified in Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). Earlier studies showed that the three PG gene transcripts are exclusively expressed in the feeding stages of L. lineolaris. In this report, it is shown that all three transcripts are specifically expressed in salivary glands indicating that PGs are salivary enzymes. Transcriptional profiles of the three PGs were evaluated with respect to diet, comparing live cotton plant material to artificial diet. PG2 transcript levels were consistently lower in cotton-fed insects than those reared on artificial diet. RNA interference was used to knock down expression of PG1 mRNA in adult salivary glands providing the first demonstration of the use of this method in the non-model insect, L. lineolaris. PMID:21062205

  16. Genome-Wide Approaches to Dissect the Roles of RNA Binding Proteins in Translational Control: Implications for Neurological Diseases

    PubMed Central

    Kapeli, Katannya; Yeo, Gene W.

    2012-01-01

    Translational control of messenger RNAs (mRNAs) is a key aspect of neurobiology, defects of which can lead to neurological diseases. In response to stimuli, local translation of mRNAs is activated at synapses to facilitate long-lasting forms of synaptic plasticity, the cellular basis for learning, and memory formation. Translation, as well as all other aspects of RNA metabolism, is controlled in part by RNA binding proteins (RBPs) that directly interact with mRNAs to form mRNA-protein complexes. Disruption of RBP function is becoming widely recognized as a major cause of neurological diseases. Thus understanding the mechanisms that govern the interplay between translation control and RBP regulation in both normal and diseased neurons will provide new opportunities for novel diagnostics and therapeutic intervention. As a means of studying translational control, genome-wide methods are emerging as powerful tools that have already begun to unveil mechanisms that are missed by single-gene studies. Here, we describe the roles of RBPs in translational control, review genome-wide approaches to examine translational control, and discuss how the application of these approaches may provide mechanistic insight into the pathogenic underpinnings of RBPs in neurological diseases. PMID:23060744

  17. ‘MicroRNA Targets’, a new AthaMap web-tool for genome-wide identification of miRNA targets in Arabidopsis thaliana

    PubMed Central

    2012-01-01

    Background The AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation. Methods To identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome. Results Putative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, ‘MicroRNA Targets’, was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new ‘Small RNA Targets’ web-tool. Conclusions The integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/ PMID:22800758

  18. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells.

    PubMed

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2016-03-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  19. Genome-wide identification and analysis of mRNA expression in fibroblasts, ES cells, and iPS cells

    PubMed Central

    Hirai, Hiroyuki; Kikyo, Nobuaki

    2015-01-01

    Genome-wide expression patterns of mRNA were compared between mouse embryonic fibroblasts (MEFs), embryonic stem cells (ESCs), and various types of induced pluripotent stem cells (iPSCs). iPSCs were established and maintained using modified Oct4 with or without exogenous leukemia inhibitory factor (LIF) and used to identify mRNAs that were potentially involved in the LIF-independence. The data have been deposited in the NCBI's Gene Expression Omnibus (GEO) database with the accession number GSE65563. PMID:26981399

  20. Studying membrane trafficking in the worm C. elegans by RNA interference.

    PubMed

    Balklava, Zita; Sztul, Elizabeth

    2013-01-01

    A powerful approach to gain understanding of molecular machinery responsible for membrane trafficking is through inactivation of gene function by RNA interference (RNAi). RNAi-mediated gene silencing occurs when a double-stranded RNA is introduced into cells and targets a complementary mRNA for degradation. The subsequent lack of mRNA prevents the synthesis of the corresponding protein and ultimately causes depletion of a particular gene product from the cell. The effects of such depletion can then by analyzed by functional, morphological, and biochemical assays. RNAi-mediated knockdowns of numerous gene products in cultured cells of mammalian and other species origins have provided significant new insight into traffic regulation and represent standard approaches in current cell biology. However, RNAi in the multicellular nematode Caenorhabditis elegans model allows RNAi studies within the context of a whole organism, and thus provides an unprecedented opportunity to explore effects of specific trafficking regulators within the context of distinct developmental stages and diverse cell types. In addition, various transgenic C. elegans strains have been developed that express marker proteins tagged with fluorescent proteins to facilitate the analysis of trafficking within the secretory and endocytic pathways. This chapter provides a detailed description of a basic RNAi approach that can be used to analyze the function of any gene of interest in secretory and endosomal trafficking in C. elegans. PMID:24295300

  1. Flagellum ontogeny in trypanosomes studied via an inherited and regulated RNA interference system.

    PubMed

    Bastin, P; Ellis, K; Kohl, L; Gull, K

    2000-09-01

    The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread applicability in studies of gene function and cellular processes in parasites. PMID:10954429

  2. 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

  3. Targeting Marek's disease virus by RNA interference delivered from a herpesvirus vaccine.

    PubMed

    Lambeth, Luke S; Zhao, Yuguang; Smith, Lorraine P; Kgosana, Lydia; Nair, Venugopal

    2009-01-01

    Live attenuated herpesvirus vaccines such as herpesvirus of turkey (HVT) have been used since 1970 for the control of Marek's disease (MD), a highly infectious lymphoproliferative disease of poultry. Despite the success of these vaccines in reducing losses from the disease, Marek's disease virus (MDV) strains have shown a continuing increase in virulence, presumably due to the inability of the current vaccines in preventing MDV replication. The highly specific and effective nature of RNA interference (RNAi) makes this technology particularly attractive for new antiviral strategies. In order to exploit the power of RNAi-mediated suppression of MDV replication in vivo delivered through existing vaccines, we engineered recombinant HVT expressing short hairpin RNA (shRNA) against MDV genes gB and UL29. The levels of protection induced by the RNAi-expressing HVT against virulent virus challenge were similar to the parent pHVT3 virus. However, chickens vaccinated with recombinant HVT expressing shRNA showed moderate reduction of challenge virus replication in blood and feather samples. Delivery of RNAi-based gene silencing through live attenuated vaccines for reducing replication of pathogenic viruses is a novel approach for the control of infectious diseases. PMID:18977264

  4. 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

  5. RNA Interference of Myocyte Enhancer Factor 2A Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice

    PubMed Central

    Zhao, Yu-xia; Liu, Gang-qiong; Zhang, Jin-ying

    2015-01-01

    Objective Myocyte enhancer factor-2A (MEF 2A) has been shown to be involved in atherosclerotic lesion development, but its role in preexisting lesions is still unclear. In the present study we aim to assess the role of MEF 2A in the progression of pre-existing atherosclerosis. Methods Eighty apolipoprotein E-deficient mice (APOE KO) were randomly allocated to control, scramble and MEF 2A RNA interference (RNAi) groups, and constrictive collars were used to induce plaque formation. Six weeks after surgery, lentiviral shRNA construct was used to silence the expression of MEF 2A. Carotid plaques were harvested for analysis 4 weeks after viral vector transduction. Inflammatory gene expression in the plasma and carotid plaques was determined by using ELISAs and real-time RT-PCR. Results The expression level of MEF 2A was significantly reduced in plasma and plaque in the RNAi group, compared to the control and NC groups, whereas the expression level of pro-inflammatory cytokines was markedly increased. Silencing MEF 2A using lentiviral shRNA significantly reduced the plaque collagen content and fibrous cap thickness, as well as increased plaque area. However, silencing MEF 2A had no obvious effect on plaque lipid content. Conclusions Lentivirus-mediated MEF 2A shRNA accelerates inflammation and atherosclerosis in APOE KO mice, but has no effect on lipoprotein levels in plasma. PMID:25793529

  6. RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway.

    PubMed

    Fusaro, Adriana F; Matthew, Louisa; Smith, Neil A; Curtin, Shaun J; Dedic-Hagan, Jasmina; Ellacott, Geoff A; Watson, John M; Wang, Ming-Bo; Brosnan, Chris; Carroll, Bernard J; Waterhouse, Peter M

    2006-11-01

    RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway. PMID:17039251

  7. 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

  8. A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

    PubMed Central

    Uckun, Fatih M.; Qazi, Sanjive; Ma, Hong; Yin, Lichen; Cheng, Jianjun

    2014-01-01

    The purposes of the present study were to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide unprecedented experimental evidence for a previously unrecognized causal link between CD22ΔE12 and aggressive biology of BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary BPL cells is associated with a marked inhibition of their clonogenicity. These findings provide the preclinical proof-of-concept that siRNA-mediated depletion of CD22ΔE12 may help develop effective treatments for high-risk and relapsed BPL patients who are in urgent need for therapeutic innovations. We also describe a unique polypeptide-based nanoparticle formulation of CD22ΔE12-siRNA as an RNAi therapeutic candidate targeting CD22ΔE12 that is capable of delivering its siRNA cargo into the cytoplasm of leukemia cells causing effective CD22ΔE12 depletion and marked inhibition of leukemic cell growth. Further development and optimization of this nanoparticle or other nanoformulation platforms for CD22ΔE12-siRNA may facilitate the development of an effective therapeutic RNAi strategy against a paradigm shift in therapy of aggressive or chemotherapy-resistant B-lineage lymphoid malignancies. PMID:25599086

  9. 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

  10. RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

    PubMed Central

    Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

    2014-01-01

    RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties. PMID:25332689

  11. Mathematical model of plant-virus interactions mediated by RNA interference.

    PubMed

    Neofytou, G; Kyrychko, Y N; Blyuss, K B

    2016-08-21

    Cross-protection, which refers to a process whereby artificially inoculating a plant with a mild strain provides protection against a more aggressive isolate of the virus, is known to be an effective tool of disease control in plants. In this paper we derive and analyse a new mathematical model of the interactions between two competing viruses with particular account for RNA interference. Our results show that co-infection of the host can either increase or decrease the potency of individual infections depending on the levels of cross-protection or cross-enhancement between different viruses. Analytical and numerical bifurcation analyses are employed to investigate the stability of all steady states of the model in order to identify parameter regions where the system exhibits synergistic or antagonistic behaviour between viral strains, as well as different types of host recovery. We show that not only viral attributes but also the propagating component of RNA-interference in plants can play an important role in determining the dynamics. PMID:27188250

  12. A genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis

    PubMed Central

    Goh, Wee-Siong Sho; Seah, Jun Wen Eugene; Harrison, Emily J.; Chen, Caifu; Hammell, Christopher M.; Hannon, Gregory J.

    2014-01-01

    In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ∼26-nucleotide capped precursors. However, nothing is known of how the expression of precursors is controlled or how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class. PMID:24696458

  13. Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells.

    PubMed

    Ge, Linhu; Deng, Zhansheng; Zhang, Yangde; Shao, Wenlong; Qiu, Yuan; Cui, Dong; Huang, Donghai

    2011-12-01

    In the present study, a plasmid-mediated siRNA interference vector targeting the hTERT gene was constructed and stably transfected into H1299 lung cancer cells. Using real-time quantitative fluorescent PCR technology, western blotting and flow cytometry-based cell cycle profiling, the silencing effect of this vector and its inhibitory effect on proliferation in lung cancer cells were explored. Based upon the results of our previous study, a pair of siRNA sequences was selected, and a DNA template primer was designed and synthesized. After cloning of the template primer into the promoter of the pGenesil-1.1 expression vector, the constructed interference vector was validated using enzyme digestion and gene sequencing. The recombinant interference vector and empty vector were separately transfected into H1299 lung cancer cells with cationic liposomes, and stable monoclonally transfected cells were obtained after selection with G418. After stable transfection, hTERT mRNA and protein expression levels were detected using real-time RT-PCR technology and western blotting. Using the MTT method and a colony formation assay, the growth and proliferation of the stably transfected lung cancer cells were determined. Changes in the cell cycle profile of the stably transfected lung cancer cells were detected using flow cytometry. An interference vector targeting the hTERT gene (pGenesil.1-hTERT) was successfully constructed. Enzyme digestion and gene sequencing confirmed that the sequence insertion met the criteria of the design. After transfection of H1299 cells with pGenesil.1-hTERT or an empty vector, the stably transfected monoclonal cell lines H1299-pGenesil.1-hTERT and H1299-pGenesil.1 were obtained. Compared to the control cells transfected with the empty vector, the H1299-pGenesil.1-hTERT cells had significantly lower mRNA expression of hTERT (93.97±0.83% inhibition, with P<0.001). The protein expression of hTERT in H1299-pGenesil.1-hTERT cells was significantly lower

  14. Genome-wide analysis of YB-1-RNA interactions reveals a novel role of YB-1 in miRNA processing in glioblastoma multiforme

    PubMed Central

    Wu, Shuai-Lai; Fu, Xing; Huang, Jinyan; Jia, Ting-Ting; Zong, Feng-Yang; Mu, Shi-Rong; Zhu, Hong; Yan, Yong; Qiu, Shuwei; Wu, Qun; Yan, Wei; Peng, Ying; Chen, Juxiang; Hui, Jingyi

    2015-01-01

    Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis. PMID:26240386

  15. Specific interference shRNA-expressing plasmids inhibit Hantaan virus infection in vitro and in vivo

    PubMed Central

    Liu, Yuan-yuan; Chen, Liang-jun; Zhong, Yan; Shen, Meng-xin; Ma, Nian; Liu, Bing-yu; Luo, Fan; Hou, Wei; Yang, Zhan-qiu; Xiong, Hai-rong

    2016-01-01

    Aim: To investigate the antiviral effects of vectors expressing specific short hairpin RNAs (shRNAs) against Hantaan virus (HTNV) infection in vitro and in vivo. Methods: Based on the effects of 4 shRNAs targeting different regions of HTNV genomic RNA on viral replication, the most effective RNA interference fragments of the S and M genes were constructed in pSilencer-3.0-H1 vectors, and designated pSilencer-S and pSilencer-M, respectively. The antiviral effect of pSilencer-S/M against HTNV was evaluated in both HTNV-infected Vero-E6 cells and mice. Results: In HTNV-infected Vero-E6 cells, pSilencer-S and pSilencer-M targeted the viral nucleocapsid proteins and envelope glycoproteins, respectively, as revealed in the immunofluorescence assay. Transfection with pSilencer-S or pSilencer-M (1, 2, 4 μg) markedly inhibited the viral antigen expression in dose- and time-dependent manners. Transfection with either plasmid (2 μg) significantly decreased HTNV-RNA level at 3 day postinfectin (dpi) and the progeny virus titer at 5 dpi. In mice infected with lethal doses of HTNV, intraperitoneal injection of pSilencer-S or pSilencer-M (30 μg) considerably increased the survival rates and mean time to death, and significantly reduced the mean virus yields and viral RNA level, and alleviated virus-induced pathological lesions in lungs, brains and kidneys. Conclusion: Plasmid-based shRNAs potently inhibit HTNV replication in vitro and in vivo. Our results provide a basis for development of shRNA as therapeutics for HTNV infections in humans. PMID:26972493

  16. Vezf1 protein binding sites genome-wide are associated with pausing of elongating RNA polymerase II

    PubMed Central

    Gowher, Humaira; Brick, Kevin; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2012-01-01

    The protein Vezf1 plays multiple roles important for embryonic development. In Vezf1−/− mouse embryonic stem (mES) cells, our earlier data showed widespread changes in gene-expression profiles, including decreased expression of the full-length active isoform of Dnmt3b methyltransferase and concomitant genome-wide reduction in DNA methylation. Here we show that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1−/− mES cells. Depending upon genomic location, Vezf1-mediated Pol II pausing can have different regulatory roles in transcription and splicing. We find examples of genes in which Vezf1 binding sites are located near cassette exons, and in which loss of Vezf1 leads to a change in the relative abundance of alternatively spliced messages. We further show that Vezf1 interacts with Mrg15/Mrgbp, a protein that recognizes H3K36 trimethylation, consistent with the role of histone modifications at alternatively spliced sites. PMID:22308494

  17. 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.

  18. Genome-Wide Occupancy Profile of the RNA Polymerase III Machinery in Saccharomyces cerevisiae Reveals Loci with Incomplete Transcription Complexes†

    PubMed Central

    Moqtaderi, Zarmik; Struhl, Kevin

    2004-01-01

    We used chromatin immunoprecipitation, followed by microarray hybridization, to determine the genome-wide distribution of the RNA polymerase (Pol) III transcription apparatus in the yeast Saccharomyces cerevisiae. The Pol III transcriptome includes all tRNA genes, previously identified non-tRNA Pol III genes, and SNR52, which encodes a small nucleolar RNA. Unexpectedly, we identify eight ETC loci that are occupied by TFIIIC but not by other components of the Pol III machinery. Some ETC loci contain stretches of DNA that are highly conserved among closely related yeast species, suggesting that they may encode functional RNAs. ETC6 is located upstream of the gene encoding the τ 91 subunit of TFIIIC, suggesting the possibility of Pol III-regulated expression of a critical Pol III factor. We also identify the ZOD1 locus, which is bound by all components of the Pol III machinery and yet does not appear to express an RNA conserved among closely related yeast species. The B block motifs and several flanking nucleotides of the ZOD1 and ETC loci are very similar to each other and are highly conserved across the yeast species. Furthermore, the unusual profile of Pol III factor association with ZOD1 and the ETC loci is perfectly preserved in a different Saccharomyces species, indicating that these loci represent novel functional entities. PMID:15121834

  19. 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

  20. Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

    PubMed

    Gan, Defang; Ding, Fei; Zhuang, Dan; Jiang, Haiyang; Jiang, Tong; Zhu, Suwen; Cheng, Beijiu

    2014-08-01

    Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach. PMID:25189224

  1. 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. PMID:23691205

  2. Requirement for CRIF1 in RNA interference and Dicer-2 stability

    PubMed Central

    Lim, Su Jun; Scott, Anthony; Xiong, Xiao-Peng; Vahidpour, Shabnam; Karijolich, John; Guo, Dongdong; Pei, Shanshan; Yu, Yi-Tao; Zhou, Rui; Li, Willis X

    2014-01-01

    RNA interference (RNAi) is a eukaryotic gene-silencing system. Although the biochemistry of RNAi is relatively well defined, how this pathway is regulated remains incompletely understood. To identify genes involved in regulating the RNAi pathway, we screened for genetic mutations in Drosophila that alter the efficiency of RNAi. We identified the Drosophila homolog of the mammalian CR6-interacting factor 1 (CRIF1), also known as growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1), as a potential new regulator of the RNAi pathway. Loss-of-function mutants of Drosophila CRIF1 (dCRIF) are deficient in RNAi-mediated target gene knock-down, in the biogenesis of small interfering RNA (siRNA) molecules, and in antiviral immunity. Moreover, we show that dCRIF may function by interacting with, and stabilizing, the RNase III enzyme Dicer-2. Our results suggest that dCRIF may play an important role in regulating the RNAi pathway. PMID:25483042

  3. Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease

    PubMed Central

    Ghanbari, Mohsen; Ikram, M. Arfan; de Looper, Hans W. J.; Hofman, Albert; Erkeland, Stefan J.; Franco, Oscar H.; Dehghan, Abbas

    2016-01-01

    MicroRNAs (miRNAs) serve as key post-transcriptional regulators of gene expression. Genetic variation in miRNAs and miRNA-binding sites may affect miRNA function and contribute to disease risk. Here, we investigated the extent to which variants within miRNA-related sequences could constitute a part of the functional variants involved in developing Alzheimer’s disease (AD), using the largest available genome-wide association study of AD. First, among 237 variants in miRNAs, we found rs2291418 in the miR-1229 precursor to be significantly associated with AD (p-value = 6.8 × 10−5, OR = 1.2). Our in-silico analysis and in-vitro miRNA expression experiments demonstrated that the variant’s mutant allele enhances the production of miR-1229-3p. Next, we found miR-1229-3p target genes that are associated with AD and might mediate the miRNA function. We demonstrated that miR-1229-3p directly controls the expression of its top AD-associated target gene (SORL1) using luciferase reporter assays. Additionally, we showed that miR-1229-3p and SORL1 are both expressed in the human brain. Second, among 42,855 variants in miRNA-binding sites, we identified 10 variants (in the 3′ UTR of 9 genes) that are significantly associated with AD, including rs6857 that increases the miR-320e-mediated regulation of PVRL2. Collectively, this study shows that miRNA-related variants are associated with AD and suggests miRNA-dependent regulation of several AD genes. PMID:27328823

  4. Genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA transcriptomes in oxidative and glycolytic skeletal muscles

    PubMed Central

    Shen, Linyuan; Du, Jingjing; Xia, Yudong; Tan, Zhendong; Fu, Yuhua; Yang, Qiong; Li, Xuewei; Tang, Guoqing; Jiang, Yanzhi; Wang, Jinyong; Li, Mingzhou; Zhang, Shunhua; Zhu, Li

    2016-01-01

    The physiological, biochemical and functional differences between oxidative and glycolytic muscles play important roles in human metabolic health and in animal meat quality. To explore these differences, we determined the genome-wide landscape of DNA methylomes and their relationship with the mRNA and miRNA transcriptomes of the oxidative muscle psoas major (PMM) and the glycolytic muscle longissimus dorsi (LDM). We observed the hypo-methylation of sub-telomeric regions. A high mitochondrial content contributed to fast replicative senescence in PMM. The differentially methylated regions (DMRs) in promoters (478) and gene bodies (5,718) were mainly enriched in GTPase regulator activity and signaling cascade-mediated pathways. Integration analysis revealed that the methylation status within gene promoters (or gene bodies) and miRNA promoters was negatively correlated with mRNA and miRNA expression, respectively. Numerous genes were closely related to distinct phenotypic traits between LDM and PMM. For example, the hyper-methylation and down-regulation of HK-2 and PFKFB4 were related to decrease glycolytic potential in PMM. In addition, promoter hypo-methylation and the up-regulation of miR-378 silenced the expression of the target genes and promoted capillary biosynthesis in PMM. Together, these results improve understanding of muscle metabolism and development from genomic and epigenetic perspectives. PMID:27561200

  5. Genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA transcriptomes in oxidative and glycolytic skeletal muscles.

    PubMed

    Shen, Linyuan; Du, Jingjing; Xia, Yudong; Tan, Zhendong; Fu, Yuhua; Yang, Qiong; Li, Xuewei; Tang, Guoqing; Jiang, Yanzhi; Wang, Jinyong; Li, Mingzhou; Zhang, Shunhua; Zhu, Li

    2016-01-01

    The physiological, biochemical and functional differences between oxidative and glycolytic muscles play important roles in human metabolic health and in animal meat quality. To explore these differences, we determined the genome-wide landscape of DNA methylomes and their relationship with the mRNA and miRNA transcriptomes of the oxidative muscle psoas major (PMM) and the glycolytic muscle longissimus dorsi (LDM). We observed the hypo-methylation of sub-telomeric regions. A high mitochondrial content contributed to fast replicative senescence in PMM. The differentially methylated regions (DMRs) in promoters (478) and gene bodies (5,718) were mainly enriched in GTPase regulator activity and signaling cascade-mediated pathways. Integration analysis revealed that the methylation status within gene promoters (or gene bodies) and miRNA promoters was negatively correlated with mRNA and miRNA expression, respectively. Numerous genes were closely related to distinct phenotypic traits between LDM and PMM. For example, the hyper-methylation and down-regulation of HK-2 and PFKFB4 were related to decrease glycolytic potential in PMM. In addition, promoter hypo-methylation and the up-regulation of miR-378 silenced the expression of the target genes and promoted capillary biosynthesis in PMM. Together, these results improve understanding of muscle metabolism and development from genomic and epigenetic perspectives. PMID:27561200

  6. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo.

    PubMed

    Rouskin, Silvi; Zubradt, Meghan; Washietl, Stefan; Kellis, Manolis; Weissman, Jonathan S

    2014-01-30

    RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo. PMID:24336214

  7. Genome-Wide Uncovering of STAT3-Mediated miRNA Expression Profiles in Colorectal Cancer Cell Lines

    PubMed Central

    Zhang, Jufeng; Luo, Xia; Li, Huiming; Deng, Ling

    2014-01-01

    Colorectal cancer (CRC) is one of the most common malignancies resulting in high mortality worldwide. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor which is frequently activated and aberrantly expressed in CRC. MicroRNAs (miRNAs) are a class of small noncoding RNAs which play important roles in many cancers. However, little is known about the global miRNA profiles mediated by STAT3 in CRC cells. In the present study, we applied RNA interference to inhibit STAT3 expression and profiled the miRNA expression levels regulated by STAT3 in CRC cell lines with deep sequencing. We found that 26 and 21 known miRNAs were significantly overexpressed and downexpressed, respectively, in the STAT3-knockdown CRC cell line SW480 (SW480/STAT3-siRNA) compared to SW480 transfected with scrambled siRNAs (SW480/siRNA-control). The miRNA expression profiling was then validated by quantitative real-time PCR for selected known miRNAs. We further predicted the putative target genes for the dysregulated miRNAs and carried out functional annotation including GO enrichment and KEGG pathway analysis for selected miRNA targets. This study directly depicts STAT3-mediated miRNA profiles in CRC cells, which provides a possible way to discover biomarkers for CRC therapy. PMID:25126546

  8. 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

  9. 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

  10. 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

  11. 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

  12. Quantitative high-throughput analysis of synthetic genetic interactions in Caenorhabditis elegans by RNA interference

    PubMed Central

    Fortunato, Angelo

    2009-01-01

    Biological processes are highly dynamic but the current representation of molecular networks is static and largely qualitative. To investigate the dynamic property of genetic networks, a novel quantitative high-throughput method based on RNA interference and capable of calculating the relevance of each interaction, was developed. With this approach, it will be possible to identify not only the components of a network, but also to investigate quantitatively how network and biological processes react to perturbations. As a first application of this method, the genetic interactions of a weak loss-of-function mutation in the gene efl-1/E2F with all the genes of chromosome III were investigated during embryonic development of Caenorhabditis elegans. Fifteen synthetic genetic interactions of efl-1/E2F with the genes of chromosome III were detected, measured and ranked by statistical relevance. PMID:19059334

  13. 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

  14. 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

  15. Establishment of Functional Genomics Pipeline in Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

    PubMed

    Takata, Nozomu; Sakakura, Eriko; Kasukawa, Takeya; Sakuma, Tetsushi; Yamamoto, Takashi; Sasai, Yoshiki

    2016-06-01

    The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics. PMID:26839115

  16. Effects of chitosan nanoparticle-mediated BRAF siRNA interference on invasion and metastasis of gastric cancer cells.

    PubMed

    Huo, Jian

    2016-08-01

    To observe the changes in invasion capacity of gastric cancer BGC823 cells after being treated with chitosan-encapsulated BRAF siRNA nanoparticles, and to evaluate the effects of the nanoparticle-mediated BRAF siRNA interference on cell invasion and metastasis, BRAF siRNA was encapsulated with chitosan into nanoparticles sized 350 nm to treat gastric cancer cells. Silencing of BRAF was detected by Western blot and PCR, and cell invasion was observed by the Transwell assay. The nanoparticles significantly downregulated BRAF expression in BGC823 cells (P < 0.01) and inhibited their invasion (P < 0.001). Chitosan nanoparticle-mediated BRAF siRNA interference evidently reduced the invasion capacity of gastric cancers. PMID:25794798

  17. A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.

    PubMed Central

    Segal, Gregorio; Song, Rentao; Messing, Joachim

    2003-01-01

    In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants. PMID:14504244

  18. Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

    PubMed Central

    Sierant, Malgorzata; Paduszynska, Alina; Kazmierczak-Baranska, Julia; Nacmias, Benedetta; Sorbi, Sandro; Bagnoli, Silvia; Sochacka, Elzbieta; Nawrot, Barbara

    2011-01-01

    RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide. PMID:21559198

  19. 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

  20. RNA Interference Using c-Myc-Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models.

    PubMed

    Tangudu, Naveen K; Verma, Vinod K; Clemons, Tristan D; Beevi, Syed S; Hay, Trevor; Mahidhara, Ganesh; Raja, Meera; Nair, Rekha A; Alexander, Liza E; Patel, Anant B; Jose, Jedy; Smith, Nicole M; Zdyrko, Bogdan; Bourdoncle, Anne; Luzinov, Igor; Iyer, K Swaminathan; Clarke, Alan R; Dinesh Kumar, Lekha

    2015-05-01

    In this article, we report the development and preclinical validation of combinatorial therapy for treatment of cancers using RNA interference (RNAi). RNAi technology is an attractive approach to silence genes responsible for disease onset and progression. Currently, the critical challenge facing the clinical success of RNAi technology is in the difficulty of delivery of RNAi inducers, due to low transfection efficiency, difficulties of integration into host DNA and unstable expression. Using the macromolecule polyglycidal methacrylate (PGMA) as a platform to graft multiple polyethyleneimine (PEI) chains, we demonstrate effective delivery of small oligos (anti-miRs and mimics) and larger DNAs (encoding shRNAs) in a wide variety of cancer cell lines by successful silencing/activation of their respective target genes. Furthermore, the effectiveness of this therapy was validated for in vivo tumor suppression using two transgenic mouse models; first, tumor growth arrest and increased animal survival was seen in mice bearing Brca2/p53-mutant mammary tumors following daily intratumoral treatment with nanoparticles conjugated to c-Myc shRNA. Second, oral delivery of the conjugate to an Apc-deficient crypt progenitor colon cancer model increased animal survival and returned intestinal tissue to a non-wnt-deregulated state. This study demonstrates, through careful design of nonviral nanoparticles and appropriate selection of therapeutic gene targets, that RNAi technology can be made an affordable and amenable therapy for cancer. PMID:25695957

  1. Applications of RNA Interference in Schistosomiasis: Gene Function Identification and Development of New Therapies

    PubMed Central

    Pereira, Tiago Campos; Evangelista, Cláudia Carolina Silva; Borges, Gustavo; Zanotti-Magalhães, Eliana Maria; Magalhães, Luiz Augusto; Lopes-Cendes, Iscia

    2013-01-01

    The study of Schistosoma species has undergone a dramatic change in recent years mainly due to transcriptome, proteome, and genome analyses. In order to better understand the biology of the parasite and to develop new and more efficient/specific drugs, scientists have now the task to translate genetic information into functional data. The present paper aims to review the use of RNA interference (RNAi), a versatile technique used in gene silencing, for the dissection of the cellular/molecular biology of Schistosoma spp. In addition, we will review information on the recent development of a new generation of RNA-based drugs. Examples of specific experimental approaches will be presented and discussed, such as identification of gene function, development of therapies by targeting eggs, miracidia (as a strategy for environmental use), sporocysts (for infestation control in the intermediate host), and schistosomula/adult worms (as a treatment strategy). Furthermore, some of the main advantages, drawbacks, and future directions of these new applications and techniques will also be discussed. PMID:27335847

  2. 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

  3. 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

  4. 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

  5. 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

  6. The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts.

    PubMed

    Lu, Jian; Wei, Caihong; Zhang, Xiaoning; Xu, Lingyang; Zhang, Shifang; Liu, Jiasen; Cao, Jiaxue; Zhao, Fuping; Zhang, Li; Li, Bichun; Du, Lixin

    2013-06-01

    Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may promote muscle growth, we used RNA interference mediated by a lentiviral vector to knockdown myostatin in goat fetal fibroblast cells. We also investigated the expression changes in relevant myogenic regulatory factors (MRFs) and adipogenic regulatory factors in the absence of myostatin in goat fetal fibroblasts. Quantitative RT-PCR revealed that myostatin transcripts were significantly reduced by 75 % (P < 0.01). Western blot showed that myostatin protein expression was reduced by 95 % (P < 0.01). We also found that the mRNA expression of activin receptor IIB (ACVR2B) significantly increased by 350 % (P < 0.01), and p21 increased 172 % (P < 0.01). Furthermore, myostatin inhibition decreased Myf5 and increased MEF2C mRNA expression in goat fetal fibroblasts, suggesting that myostatin regulates MRFs differently in fibroblasts compared to muscle. In addition, the expression of adipocyte marker genes peroxisome proliferator-activated receptor (PPAR) γ and leptin, but not CCAAT/enhance-binding protein (C/EBP) α and C/EBPβ, were upregulated at the transcript level after myostatin silencing. These results suggest that we have generated a novel way to block myostatin in vitro, which could be used to improve livestock meat production and gene therapy of musculoskeletal diseases. This also suggests that myostatin plays a negative role in regulating the expression of adipogenesis related genes in goat fetal fibroblasts. PMID:23604693

  7. Does the mutant CAG expansion in huntingtin mRNA interfere with exonucleolytic cleavage of its first exon?

    PubMed Central

    Liu, Wanzhao; Pfister, Edith L.; Kennington, Lori A.; Chase, Kathryn O.; Mueller, Christian; DiFiglia, Marian; Aronin, Neil

    2016-01-01

    Background Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington’s disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments. Objectives We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA. Methods Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection. Results Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites. Conclusions Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage. PMID:27003665

  8. RNA interference as a method for target-site screening in the Western Corn Rootworm, Diabrotica virgifera virgifera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) is one of the most powerful and extraordinarily-specific means by which to silence genes. The ability of RNAi to silence genes makes it possible to ascertain function from genomic data, thereby making it an excellent choice for target-site screening. To test the efficacy of...

  9. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

    PubMed Central

    Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin; Hartono, Stella R; Chédin, Frédéric

    2015-01-01

    Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE. DOI: http://dx.doi.org/10.7554/eLife.08007.001 PMID:26182405

  10. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

    PubMed Central

    Foo, Chwan Hong; Rootes, Christina L.; Gould, Cathryn M.; Grusovin, Julian; Monaghan, Paul; Lo, Michael K.; Tompkins, S. Mark; Adams, Timothy E.; Lowenthal, John W.; Simpson, Kaylene J.; Stewart, Cameron R.; Bean, Andrew G. D.; Wang, Lin-Fa

    2016-01-01

    Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections. PMID:27010548

  11. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

    PubMed

    Deffrasnes, Celine; Marsh, Glenn A; Foo, Chwan Hong; Rootes, Christina L; Gould, Cathryn M; Grusovin, Julian; Monaghan, Paul; Lo, Michael K; Tompkins, S Mark; Adams, Timothy E; Lowenthal, John W; Simpson, Kaylene J; Stewart, Cameron R; Bean, Andrew G D; Wang, Lin-Fa

    2016-03-01

    Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections. PMID:27010548

  12. Broad Meloidogyne Resistance in Potato Based on RNA Interference of Effector Gene 16D10.

    PubMed

    Dinh, Phuong T Y; Zhang, Linhai; Mojtahedi, Hassan; Brown, Charles R; Elling, Axel A

    2015-03-01

    Root-knot nematodes (Meloidogyne spp.) are a significant problem in potato (Solanum tuberosum) production. There is no potato cultivar with Meloidogyne resistance, even though resistance genes have been identified in wild potato species and were introgressed into breeding lines. The objectives of this study were to generate stable transgenic potato lines in a cv. Russet Burbank background that carry an RNA interference (RNAi) transgene capable of silencing the 16D10 Meloidogyne effector gene, and test for resistance against some of the most important root-knot nematode species affecting potato, i.e., M. arenaria, M. chitwoodi, M. hapla, M. incognita, and M. javanica. At 35 days after inoculation (DAI), the number of egg masses per plant was significantly reduced by 65% to 97% (P < 0.05) in the RNAi line compared to wild type and empty vector controls. The largest reduction was observed in M. hapla, whereas the smallest reduction occurred in M. javanica. Likewise, the number of eggs per plant was significantly reduced by 66% to 87% in M. arenaria and M. hapla, respectively, compared to wild type and empty vector controls (P < 0.05). Plant-mediated RNAi silencing of the 16D10 effector gene resulted in significant resistance against all of the root-knot nematode species tested, whereas R Mc1(blb) , the only known Meloidogyne resistance gene in potato, did not have a broad resistance effect. Silencing of 16D10 did not interfere with the attraction of M. incognita second-stage juveniles to roots, nor did it reduce root invasion. PMID:25861119

  13. 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

  14. Antiviral RNA Interference against Orsay Virus Is neither Systemic nor Transgenerational in Caenorhabditis elegans

    PubMed Central

    Sarkies, Peter; Le Pen, Jérémie; Tanguy, Mélanie

    2015-01-01

    ABSTRACT Antiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. In Caenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection of C. elegans from viral infection. We studied the Orsay virus, a positive-strand RNA virus related to Nodaviridae and the first and only virus known to infect C. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections. IMPORTANCE Since its discovery in Caenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. In C. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected

  15. EGFP-EGF1-Conjugated PLGA Nanoparticles for Targeted Delivery of siRNA into Injured Brain Microvascular Endothelial Cells for Efficient RNA Interference

    PubMed Central

    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. PMID:23593330

  16. The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies.

    PubMed

    Nabhan, Joseph F; El-Shehabi, Fouad; Patocka, Nicholas; Ribeiro, Paula

    2007-11-01

    The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. Here, we used a bioinformatics approach to identify the proteasomal constituents of the parasitic trematode Schistosoma mansoni. A detailed search of the S. mansoni genome database identified a total of 31 putative proteasomal subunits, including 17 subunits of the regulatory (19S) complex and 14 predicted catalytic (20S) subunits. A quantitative real-time RT-PCR analysis of subunit expression levels revealed that the S. mansoni proteasome components are differentially expressed among cercaria, schistosomula, and adult worms. In particular, the data suggest that the proteasome may be downregulated during the early stages of schistosomula development and is subsequently upregulated as the parasite matures to the adult stage. To test for biological relevance, we developed a transfection-based RNA interference method to knockdown the expression of the proteasome subunit, SmRPN11/POH1. Transfection of in vitro transformed S. mansoni schistosomula with specific short-interfering RNAs (siRNAs) diminished SmRPN11/POH1 expression nearly 80%, as determined by quantitative RT-PCR analysis, and also decreased parasite viability 78%, whereas no significant effect could be seen after treatment with the same amount of an irrelevant siRNA. These results indicate that the subunit SmRPN11/POH1 is an essential gene in schistosomes and further suggest an important role for the proteasome in parasite development and survival. PMID:17892869

  17. An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis

    PubMed Central

    2010-01-01

    Background In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines. Results By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped. Conclusions In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA

  18. 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. PMID:22845735

  19. 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

  20. 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

  1. Genome-Wide Comparative In Silico Analysis of the RNA Helicase Gene Family in Zea mays and Glycine max: A Comparison with Arabidopsis and Oryza sativa

    PubMed Central

    Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  2. An RNA Interference Phenotypic Screen Identifies a Role for FGF Signals in Colon Cancer Progression

    PubMed Central

    Leushacke, Marc; Spörle, Ralf; Bernemann, Christof; Brouwer-Lehmitz, Antje; Fritzmann, Johannes; Theis, Mirko; Buchholz, Frank; Herrmann, Bernhard G.; Morkel, Markus

    2011-01-01

    In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression. PMID:21853123

  3. 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

  4. 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

  5. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.

    PubMed

    Barbie, David A; Tamayo, Pablo; Boehm, Jesse S; Kim, So Young; Moody, Susan E; Dunn, Ian F; Schinzel, Anna C; Sandy, Peter; Meylan, Etienne; Scholl, Claudia; Fröhling, Stefan; Chan, Edmond M; Sos, Martin L; Michel, Kathrin; Mermel, Craig; Silver, Serena J; Weir, Barbara A; Reiling, Jan H; Sheng, Qing; Gupta, Piyush B; Wadlow, Raymond C; Le, Hanh; Hoersch, Sebastian; Wittner, Ben S; Ramaswamy, Sridhar; Livingston, David M; Sabatini, David M; Meyerson, Matthew; Thomas, Roman K; Lander, Eric S; Mesirov, Jill P; Root, David E; Gilliland, D Gary; Jacks, Tyler; Hahn, William C

    2009-11-01

    The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer. PMID:19847166

  6. Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference

    PubMed Central

    Badaut, Jérôme; Ashwal, Stephen; Adami, Arash; Tone, Beatriz; Recker, Rebecca; Spagnoli, David; Ternon, Béatrice; Obenaus, Andre

    2011-01-01

    Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels. PMID:20877385

  7. Functionally associated targets in mantle cell lymphoma as defined by DNA microarrays and RNA interference.

    PubMed

    Ortega-Paino, Eva; Fransson, Johan; Ek, Sara; Borrebaeck, Carl A K

    2008-02-01

    Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with poor prognosis. Its hallmark is the translocation t(11:14)q (13;32), leading to overexpression of cyclin D1, a positive regulator of the cell cycle. As cyclin D1 up-regulation is not sufficient for inducing malignant transformation, we combined DNA microarray and RNA interference (RNAi) approaches to identify novel deregulated genes involved in the progression of MCL. DNA microarray analysis identified 46 genes specifically up-regulated in MCL compared with normal B cells; 20 of these were chosen for further studies based on their cellular functions, such as growth and proliferation. The Granta 519 cell line was selected as an MCL in vitro model, to set up the RNAi protocol. To confirm the functionality of overexpression of the 20 disease-associated genes, they were knocked down using small interfering RNAs (siRNAs). In particular, knockdown of 3 genes, encoding the hepatoma-derived growth factor related protein 3 (HDGFRP3), the frizzled homolog 2 (FZD2), and the dual specificity phosphatase 5 (DUSP5), induced proliferative arrest in Granta 519 MCL cells. These genes emerged as functionally associated in MCL, in relation to growth and survival, and interfering with their function would increase insight into lymphoma growth regulation, potentially leading to novel clinical intervention modalities. PMID:18024791

  8. RNA interference inhibits DUX4-induced muscle toxicity in vivo: implications for a targeted FSHD therapy.

    PubMed

    Wallace, Lindsay M; Liu, Jian; Domire, Jacqueline S; Garwick-Coppens, Sara E; Guckes, Susan M; Mendell, Jerry R; Flanigan, Kevin M; Harper, Scott Q

    2012-07-01

    No treatment exists for facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle diseases. Although FSHD can be debilitating, little effort has been made to develop targeted therapies. This lack of focus on targeted FSHD therapy perpetuated because the genes and pathways involved in the disorder were not understood. Now, more than 2 decades after efforts to decipher the root cause of FSHD began, this barrier to translation is finally lowering. Specifically, several recent studies support an FSHD pathogenesis model involving overexpression of the myopathic DUX4 gene. DUX4 inhibition has therefore emerged as a promising therapeutic strategy for FSHD. In this study, we tested a preclinical RNA interference (RNAi)-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of-principle for RNAi therapy of FSHD through DUX4 inhibition. PMID:22508491

  9. RNA interference improves myopathic phenotypes in mice over-expressing FSHD region gene 1 (FRG1).

    PubMed

    Wallace, Lindsay M; Garwick-Coppens, Sara E; Tupler, Rossella; Harper, Scott Q

    2011-11-01

    Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders. PMID:21730972

  10. 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

  11. Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

    PubMed

    Dare, Andrew P; Tomes, Sumathi; Jones, Midori; McGhie, Tony K; Stevenson, David E; Johnson, Ross A; Greenwood, David R; Hellens, Roger P

    2013-05-01

    We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. PMID:23398045

  12. RNA Interference Improves Myopathic Phenotypes in Mice Over-expressing FSHD Region Gene 1 (FRG1)

    PubMed Central

    Wallace, Lindsay M; Garwick-Coppens, Sara E; Tupler, Rossella; Harper, Scott Q

    2011-01-01

    Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1−high mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1−high mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders. PMID:21730972

  13. Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference.

    PubMed

    Wang, Geliang; Xu, Yinong

    2008-07-01

    An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics. PMID:18347801

  14. 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.

  15. A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

    PubMed

    Andritschke, Daniel; Dilling, Sabrina; Emmenlauer, Mario; Welz, Tobias; Schmich, Fabian; Misselwitz, Benjamin; Rämö, Pauli; Rottner, Klemens; Kerkhoff, Eugen; Wada, Teiji; Penninger, Josef M; Beerenwinkel, Niko; Horvath, Peter; Dehio, Christoph; Hardt, Wolf-Dietrich

    2016-01-01

    Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect. PMID:27627128

  16. A Genome-Wide siRNA Screen to Identify Modulators of Insulin Sensitivity and Gluconeogenesis

    PubMed Central

    Yang, Ruojing; Lacson, Raul G.; Castriota, Gino; Zhang, Xiaohua D.; Liu, Yaping; Zhao, Wenqing; Einstein, Monica; Camargo, Luiz Miguel; Qureshi, Sajjad; Wong, Kenny K.; Zhang, Bei B.; Ferrer, Marc; Berger, Joel P.

    2012-01-01

    Background Hepatic insulin resistance impairs insulin’s ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. Methodology/Principal Findings To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. Conclusions/Significance These results support the proposition that the proteins encoded by the genes identified in our cell

  17. RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

    PubMed Central

    Horn, Martin; Braschi, Simon; Sojka, Daniel; Ruelas, Debbie S.; Suzuki, Brian; Lim, Kee-Chong; Hopkins, Stephanie D.; McKerrow, James H.; Caffrey, Conor R.

    2010-01-01

    Background The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. Methodology/Principal Findings We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript

  18. 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

  19. Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach

    PubMed Central

    Zhang, Peng; Chen, Xiao; Wu, Jun; Xu, Ling; Luo, Xue-Qun; Ke, Zhi-Yong; Zhou, Hui; Qu, Liang-Hu; Chen, Yue-Qin

    2009-01-01

    Background MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis. Methodology/Principal Findings To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO “biological process” classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development. Conclusion/Significance The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse. PMID:19724645

  20. The future of genetics in psychology and psychiatry: Microarrays, genome-wide association, and non-coding RNA

    PubMed Central

    Plomin, Robert; Davis, Oliver S. P.

    2010-01-01

    Background Much of what we thought we knew about genetics needs to be modified in light of recent discoveries. What are the implications of these advances for identifying genes responsible for the high heritability of many behavioural disorders and dimensions in childhood? Methods Although quantitative genetics such as twin studies will continue to yield important findings, nothing will advance the field as much as identifying the specific genes responsible for heritability. Advances in molecular genetics have been driven by technology, especially DNA microarrays the size of a postage stamp that can genotype a million DNA markers simultaneously. DNA microarrays have led to a dramatic shift in research towards genome-wide association (GWA) studies. The ultimate goal of GWA is to sequence each individual’s entire genome, which has begun to happen. Results GWA studies suggest that for most complex traits and common disorders genetic effects are much smaller than previously considered: The largest effects account for only 1% of the variance of quantitative traits. This finding implies that hundreds of genes are responsible for the heritability of behavioural problems in childhood, and that it will be difficult to identify reliably these genes of small effect. Another discovery with far-reaching implications for future genetic research is the importance of non-coding RNA (DNA transcribed into RNA but not translated into amino acid sequences) which redefines what the word gene means. Non-coding RNA underlines the need for a genome-wide approach that is not limited to the 2% of DNA responsible for traditional genes that are translated into amino acid sequences. Conclusions The only safe prediction is that the fast pace of genetic discoveries will continue and will increasingly affect research in child psychology and psychiatry. DNA microarrays will make it possible to use hundreds of genes to predict genetic risk and to use these sets of genes in top-down behavioural

  1. A Genome-Wide Analysis of RNA Pseudoknots That Stimulate Efficient −1 Ribosomal Frameshifting or Readthrough in Animal Viruses

    PubMed Central

    Cheng, Qiang; Du, Zhihua

    2013-01-01

    Programmed −1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at the gag-pol frameshift junction of HIV1 harbors potential elaborated pseudoknots encompassing the frameshift site. A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event. PMID:24298557

  2. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

    PubMed Central

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  3. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates.

    PubMed

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David; Lagarrigue, Sandrine; Pitel, Frédérique

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  4. Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein

    PubMed Central

    Jia, Dongsheng; Chen, Hongyan; Zheng, Ailing; Chen, Qian; Liu, Qifei; Xie, Lianhui

    2012-01-01

    An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins. PMID:22398296

  5. Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis

    PubMed Central

    Bersani, Cinzia; Huss, Mikael; Giacomello, Stefania; Xu, Li-Di; Bianchi, Julie; Eriksson, Sofi; Jerhammar, Fredrik; Alexeyenko, Andrey; Vilborg, Anna; Lundeberg, Joakim; Lui, Weng-Onn; Wiman, Klas G.

    2016-01-01

    RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3′UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression. PMID:26672765

  6. Genome-wide analysis of TIAR RNA ligands in mouse macrophages before and after LPS stimulation

    PubMed Central

    Kharraz, Yacine; Lefort, Anne; Libert, Frédérick; Mann, Christopher J.; Gueydan, Cyril; Kruys, Véronique

    2016-01-01

    TIA-1 related protein (TIAR) is a RNA-binding protein involved in several steps of gene expression such as RNA splicing Aznarez et al. (2008) [1] and translation Piecyk et al. (2000) [2]. TIAR contains three RNA recognition motifs (RRMs) allowing its interaction with specific sequences localized in the untranslated regions (UTRs) of several mRNAs. In myeloid cells, TIAR has been shown to bind and regulate the translation and stability of various mRNA-encoding proteins important for the inflammatory response, such as TNFα Piecyk et al. (2000), Gueydan et al. (1999) [2], [3], Cox-2 Cok et al. (2003) [4] or IL-8 Suswam et al. (2005) [5]. Here, we generated two macrophage-like RAW 264.7 cell lines expressing either a tagged full-length TIAR protein or a RRM2-truncated mutant unable to bind RNA with high affinity Dember et al. (1996), Kim et al. (2013) . By a combination of RNA-IP and microarray analysis (RIP-chip), we identified mRNAs specifically bound by the full-length protein both in basal conditions and in response to LPS (GSE77577). PMID:26981431

  7. 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...

  8. 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

  9. Genome-wide DNA methylation profiles and their relationships with mRNA and the microRNA transcriptome in bovine muscle tissue (Bos taurine).

    PubMed

    Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-01-01

    DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

  10. Flow-dependent regulation of genome-wide mRNA and microRNA expression in endothelial cells in vivo

    PubMed Central

    Kumar, Sandeep; Kim, Chan Woo; Son, Dong Ju; Ni, Chih Wen; Jo, Hanjoong

    2014-01-01

    Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), in part, due to alterations in gene expression in the endothelium. While numerous in vitro studies have shown how anti-atherogenic flow and pro-atherogenic flow differently regulate gene expression of cultured endothelial cells, similar in vivo studies have been scarce. Recently, we developed a mouse model of atherosclerosis that rapidly develops robust atherosclerosis by partially ligating the left carotid artery (LCA) branches, while using the contralateral right carotid (RCA) as control. We also developed a novel method to collect endothelial-enriched RNAs from the carotids of these animals, which enabled us to perform genome-wide expression analyses of mRNAs and miRNAs in the arterial endothelium exposed to either d-flow or s-flow. These microarray results were used to identify novel mechanosensitive genes such as DNA methyltransferase-1 and miR-712 that play key roles in atherosclerosis. Here, we report these endothelial mRNA and miRNA expression profiles with in-depth information on experimental procedures along with an example of usage of these data. PMID:25977794

  11. Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)

    PubMed Central

    Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E.; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-01-01

    DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

  12. Experimental Genome-Wide Determination of RNA Polyadenylation in Chlamydomonas reinhardtii

    PubMed Central

    Bell, Stephen A.; Shen, Chi; Brown, Alishea; Hunt, Arthur G.

    2016-01-01

    The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types—Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth. PMID:26730730

  13. A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

    PubMed Central

    Papageorgiou, Angela; Rapley, Joseph; Mesirov, Jill P.; Tamayo, Pablo; Avruch, Joseph

    2015-01-01

    mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms. PMID:25790369

  14. Genome-wide identification of miRNA targets by PAR-CLIP

    PubMed Central

    Hafner, Markus; Lianoglou, Steve; Tuschl, Thomas; Betel, Doron

    2012-01-01

    MiRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable tools for the characterization of posttranscriptional gene regulation. Here we present a step-by-step protocol and guidelines for the computational analysis for the large-scale identification of miRNA target sites in cultured cells by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) of AGO proteins. PMID:22926237

  15. Study of RNA Interference Targeting NET-1 Combination with Sorafenib for Hepatocellular Carcinoma Therapy In Vitro and In Vivo

    PubMed Central

    He, Song; Wei, Ying-ze; Wang, Gui-lan; Xu, Yu-yin; Zhou, Jia-ming; Zhang, Yi-xin; Chen, Li

    2013-01-01

    The aim of this study is to explore the inhibitory effects of RNA interference (RNAi) targeting NET-1 or combined with sorafenib on HCC in vitro and in vivo and the possible underlying mechanisms. The expressions of NET-1 mRNA and protein were detected by RT-QPCR and western blot. The ability of proliferation was determined by CCK-8 assay. Apoptosis was examined by flow cytometry (FCM). Abilities of migration and invasion were measured by scratch-wound assay and transwell assay. MHCC97H cells with stable transfection of NET-1shRNA were injected subcutaneously to prepare nude mice model of HCC and Caspase-3, Caspase-8, and Caspase-9 mRNAs of tumor tissues in different groups were examined. NET-1 mRNA and protein were reduced sharply in MHCC97H cells transfected with NET-1shRNA. The abilities of proliferation and migration were inhibited and apoptosis was promoted in either NET-1shRNA or sorafenib as compared with untreated cells in vitro and in vivo (P < 0.05). The mRNA levels of caspase-3, caspase-8, and caspase-9 of tumor tissues were reduced in different treatment groups compared with untreated group, particularly in combination group. (P < 0.05). The combination NET-1shRNA with sorafenib dramatically enhanced the effects of sorafenib antitumor ,which may involve in blocking ras signaling pathway and stimulating apoptotic pathways simultaneously. PMID:24307893

  16. 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. PMID:26005118

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

    PubMed Central

    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

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

    PubMed Central

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

    2009-01-01

    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 Schistosoma 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 SmLAP 1 and 2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP 2 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 ≥ 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

  19. 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

  20. 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

  1. 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

  2. RNA interference revealed the roles of two carboxylesterase genes in insecticide detoxification in Locusta migratoria.

    PubMed

    Zhang, Jianqin; Li, Daqi; Ge, Pingting; Yang, Meiling; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2013-10-01

    Carboxylesterases (CarEs) play key roles in metabolism of specific hormones and detoxification of dietary and environmental xenobiotics in insects. We sequenced and characterized CarE cDNAs putatively derived from two different genes named LmCesA1 and LmCesA2 from the migratory locust, Locusta migratoria, one of the most important agricultural pests in the world. The full-length cDNAs of LmCesA1 (1892 bp) and LmCesA2 (1643 bp) encode 543 and 501 amino acid residues, respectively. The two deduced CarEs share a characteristic α/β-hydrolase structure, including a catalytic triad composed of Ser-Glu (Asp)-His and a consensus sequence GQSAG, which suggests that both CarEs are biologically active. Phylogenetic analysis grouped both LmCesA1 and LmCesA2 into clade A which has been suggested to be involved in dietary detoxification. Both transcripts were highly expressed in all the nymphal and adult stages, but only slightly expressed in eggs. Analyses of tissue-dependent expression and in situ hybridization revealed that both transcripts were primarily expressed in gastric caeca. RNA interference (RNAi) of LmCesA1 and LmCesA2 followed by a topical application of carbaryl or deltamethrin did not lead to a significantly increased mortality with either insecticide. However, RNAi of LmCesA1 and LmCesA2 increased insect mortalities by 20.9% and 14.5%, respectively, when chlorpyrifos was applied. These results suggest that these genes might not play a significant role in detoxification of carbaryl and deltamethrin but are most likely to be involved in detoxification of chlorpyrifos in L. migratoria. PMID:23899922

  3. The Genome-Wide Analysis of Carcinoembryonic Antigen Signaling by Colorectal Cancer Cells Using RNA Sequencing.

    PubMed

    Bajenova, Olga; Gorbunova, Anna; Evsyukov, Igor; Rayko, Michael; Gapon, Svetlana; Bozhokina, Ekaterina; Shishkin, Alexander; O'Brien, Stephen J

    2016-01-01

    Сarcinoembryonic antigen (CEA, CEACAM5, CD66) is a promoter of metastasis in epithelial cancers that is widely used as a prognostic clinical marker of metastasis. The aim of this study is to identify the network of genes that are associated with CEA-induced colorectal cancer liver metastasis. We compared the genome-wide transcriptomic profiles of CEA positive (MIP101 clone 8) and CEA negative (MIP 101) colorectal cancer cell lines with different metastatic potential in vivo. The CEA-producing cells displayed quantitative changes in the level of expression for 100 genes (over-expressed or down-regulated). They were confirmed by quantitative RT-PCR. The KEGG pathway analysis identified 4 significantly enriched pathways: cytokine-cytokine receptor interaction, MAPK signaling pathway, TGF-beta signaling pathway and pyrimidine metabolism. Our results suggest that CEA production by colorectal cancer cells triggers colorectal cancer progression by inducing the epithelial- mesenchymal transition, increasing tumor cell invasiveness into the surrounding tissues and suppressing stress and apoptotic signaling. The novel gene expression distinctions establish the relationships between the existing cancer markers and implicate new potential biomarkers for colorectal cancer hepatic metastasis. PMID:27583792

  4. Characterizing protein interactions employing a genome-wide siRNA cellular phenotyping screen.

    PubMed

    Suratanee, Apichat; Schaefer, Martin H; Betts, Matthew J; Soons, Zita; Mannsperger, Heiko; Harder, Nathalie; Oswald, Marcus; Gipp, Markus; Ramminger, Ellen; Marcus, Guillermo; Männer, Reinhard; Rohr, Karl; Wanker, Erich; Russell, Robert B; Andrade-Navarro, Miguel A; Eils, Roland; König, Rainer

    2014-09-01

    Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest. PMID:25255318

  5. Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis.

    PubMed

    Wu, Xiao-Meng; Kou, Shu-Jun; Liu, Yuan-Long; Fang, Yan-Ni; Xu, Qiang; Guo, Wen-Wu

    2015-04-01

    Somatic embryogenesis (SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of miRNAs/siRNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing (HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived, 94 other and 235 phased small interfering RNAs (siRNAs), as well as 203 target genes. The majority of the abundantly expressed miRNAs/siRNAs exhibit lower expression levels in embryogenic callus (EC) or during SE process than in nonembryogenic callus (NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors (TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, miRNA- and siRNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved miRNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE. PMID:25615015

  6. Prevention of neointimal hyperplasia in balloon-injured rat carotid artery via small interference RNA mediated downregulation of osteopontin gene.

    PubMed

    Xu, Jian; Sun, Yingxian; Wang, Tairan; Liu, Guinan

    2013-05-01

    The aim of the present study was to take osteopontin (OPN) as molecular target to study its effects on injured intima model of carotid artery in rat using perivascular transfer of OPN-small interference RNA (siRNA). OPN mRNA in cultured VSMCs was quantified by real-time RT-PCR, and OPN-siRNA-002 was determined as the most sensitive sequence and used as transfected siRNA in the subsequent animal experiments. We established rat carotid arterial intima-injured model with balloon-injured method, and then perivascularly transfected OPN-siRNA-002 to study the role of OPN-siRNA in regulating several related genes including proliferating cell nuclear antigen (PCNA), transforming growth factor β1(TGF-β1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-14 (MMP-14), as well as its role in neointimal formation. OPN mRNA and protein decreased about 50 % with corresponding decrease in intima thickness after transfecting with specific OPN-siRNA-002 compared with Pluronic control group and OPN-SCR-siRNA group on each time point (n = 6, p < 0.001), and this inhibiting effects persisted up to 14 days after balloon injury. PCNA, TGF-β1, MMP-2, and MMP-14 mRNA and protein correlated directly with the respective levels of OPN, suggesting its functions via regulating these downstream factors (n = 6, p < 0.001). OPN may be a potential target gene in reducing the risk for arterial restenosis after vascular intervention. PMID:23467880

  7. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response

    PubMed Central

    Hattori, Hiroyoshi; Janky, Rekin’s; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4–24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients. PMID:25486198

  8. A genome-wide map of hyper-edited RNA reveals numerous new sites.

    PubMed

    Porath, Hagit T; Carmi, Shai; Levanon, Erez Y

    2014-01-01

    Adenosine-to-inosine editing is one of the most frequent post-transcriptional modifications, manifested as A-to-G mismatches when comparing RNA sequences with their source DNA. Recently, a number of RNA-seq data sets have been screened for the presence of A-to-G editing, and hundreds of thousands of editing sites identified. Here we show that existing screens missed the majority of sites by ignoring reads with excessive ('hyper') editing that do not easily align to the genome. We show that careful alignment and examination of the unmapped reads in RNA-seq studies reveal numerous new sites, usually many more than originally discovered, and in precisely those regions that are most heavily edited. Specifically, we discover 327,096 new editing sites in the heavily studied Illumina Human BodyMap data and more than double the number of detected sites in several published screens. We also identify thousands of new sites in mouse, rat, opossum and fly. Our results establish that hyper-editing events account for the majority of editing sites. PMID:25158696

  9. Suppression of pancreatic cancer growth and metastasis by HMP19 identified through genome-wide shRNA screen.

    PubMed

    Kurahara, Hiroshi; Bohl, Christopher; Natsugoe, Shoji; Nishizono, Yuka; Harihar, Sitaram; Sharma, Rahul; Iwakuma, Tomoo; Welch, Danny R

    2016-08-01

    Therapeutic effectiveness against metastatic or even locally advanced pancreatic ductal adenocarcinoma (PDAC) is dismal, with 5-year survival less than 5%. Even in patients who undergo potentially curative resection, most patients' tumors recur in the liver. Improving therapies targeting or preventing liver metastases is crucial for improving prognosis. To identify genes suppressing metastasis, a genome-wide shRNA screen was done using the human non-metastatic PDAC cell line, S2-028. After identification of candidates, functional validation was done using intrasplenic and orthotopic injections in athymic mice. HMP19 strongly inhibited metastasis but also partially attenuated tumor growth in the pancreas. Knockdown of HMP19 increased localization of activated ERK1/2 in the nucleus, corresponding to facilitated cell proliferation, decreased p27(Kip1) and increased cyclin E1. Over-expression of HMP19 exerted the opposite effects. Using a tissue microarray of 84 human PDAC, patients with low expression of HMP19 showed significantly higher incidence of liver metastasis (p = 0.0175) and worse prognosis (p = 0.018) after surgery. HMP19, a new metastasis/tumor suppressor in PDAC, appears to alter signaling that leads to cell proliferation and appears to offer prognostic value in human PDAC. PMID:27012470

  10. Genome-wide mRNA sequencing of a single canine cerebellar cortical degeneration case leads to the identification of a disease associated SPTBN2 mutation

    PubMed Central

    2012-01-01

    Background Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq) of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation. Results A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq) of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the β-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that β-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle. Conclusions An 8 bp deletion in the SPTBN2 gene encoding β-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs. This study shows that mRNA

  11. 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

  12. Gene Silencing of 4-1BB by RNA Interference Inhibits Acute Rejection in Rats with Liver Transplantation

    PubMed Central

    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. PMID:23484089

  13. Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast

    PubMed Central

    Ard, Ryan; Tong, Pin; Allshire, Robin C.

    2014-01-01

    Most long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the nc-tgp1 lncRNA regulates drug tolerance by repressing the adjacent phosphate-responsive permease gene transporter for glycerophosphodiester 1 (tgp1+). We demonstrate that the act of transcribing nc-tgp1 over the tgp1+ promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1+ without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1+ is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1+ even in repressive conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast. PMID:25428589

  14. Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species

    PubMed Central

    González, Nicolas; Heeb, Stephan; Valverde, Claudio; Kay, Elisabeth; Reimmann, Cornelia; Junier, Thomas; Haas, Dieter

    2008-01-01

    Background Small RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family. Results A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression. Conclusion The discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are

  15. A genome-wide miRNA screen revealed miR-603 as a MGMT-regulating miRNA in glioblastomas

    PubMed Central

    Ng, Kimberly; Steed, Tyler; Nguyen, Thien; Futalan, Diahnn; Akers, Johnny C.; Sarkaria, Jann; Jiang, Tao; Chowdhury, Dipanjan; Carter, Bob S.; Chen, Clark C.

    2014-01-01

    MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs and characterized the top candidate, miR-603. Transfection of miR-603 suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. Moreover, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents. PMID:24994119

  16. 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

  17. 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

  18. RNA interference-mediated silencing of mitotic kinesin KIF14 disrupts cell cycle progression and induces cytokinesis failure.

    PubMed

    Carleton, Michael; Mao, Mao; Biery, Matthew; Warrener, Paul; Kim, Sammy; Buser, Carolyn; Marshall, C Gary; Fernandes, Christine; Annis, James; Linsley, Peter S

    2006-05-01

    KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression. PMID:16648480

  19. RNA Interference-Mediated Silencing of Mitotic Kinesin KIF14 Disrupts Cell Cycle Progression and Induces Cytokinesis Failure†

    PubMed Central

    Carleton, Michael; Mao, Mao; Biery, Matthew; Warrener, Paul; Kim, Sammy; Buser, Carolyn; Marshall, C. Gary; Fernandes, Christine; Annis, James; Linsley, Peter S.

    2006-01-01

    KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression. PMID:16648480

  20. Inhibition of avian leukosis virus replication by vector-based RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNAi has recently emerged as a promising antiviral technique in vertebrates. To date, most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, though vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are...

  1. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells

    PubMed Central

    HUANG, HUI-PING; LIU, WEN-JUN; GUO, QU-LIAN; BAI, YONG-QI

    2016-01-01

    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF-PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (P<0.05). The distribution of cells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (P<0.05). In

  2. A rapid and sensitive nonradioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

    PubMed Central

    Wu, Jingyan; Huang, Hsiao-Yun; Hopper, Anita K.

    2013-01-01

    The conventional small RNA isolation and detection methods for yeast cells have been designed for a small number of samples. In order to conduct a genome-wide assessment of how each gene product impacts upon small non-coding RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive nonradioactive Northern method for RNA detection. The RNA isolation procedure is highly reproducible and requires only 4 hours for processing 96 samples, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-level small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analysis of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe, and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking, and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. PMID:23417998

  3. 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

  4. 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

  5. MR-02A GENOME-WIDE miRNA SCREEN REVEALED MIR-603 AS A MGMT-REGULATING miRNA IN GLIOBLASTOMAS

    PubMed Central

    Kushwaha, Deepa; Ramakrishnan, Valya; Ng, Kimberly; Steed, Tyler; Nguyen, Thien; Futalan, Diahnn; Akers, Johnny; Tao, Jiang; Chowdhury, Dipanjan; Carter, Bob; Chen, Clark

    2014-01-01

    MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs. Comparison of these candidates to those predicted computational algorithms, including DIANA micro, Targetscan, miRanda, and microcosm showed poor agreement (3-22%), suggesting the need for empiric validation of in silico predictions. Transfection of miR-603, the top scoring candidate, suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. miR-603 cooperates with miR-181d to bind to the 3'UTR of MGMT to suppress MGMT expression. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. However, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Our results further suggest that these miRNA may regulate MGMT by direct binding of MGMT 3'UTR or through modulation of proteins that regulate MGMT stability/degradation. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents.

  6. A Genome-Wide Association Study for Diabetic Retinopathy in a Japanese Population: Potential Association with a Long Intergenic Non-Coding RNA

    PubMed Central

    Awata, Takuya; Yamashita, Hisakuni; Kurihara, Susumu; Morita-Ohkubo, Tomoko; Miyashita, Yumi; Katayama, Shigehiro; Mori, Keisuke; Yoneya, Shin; Kohda, Masakazu; Okazaki, Yasushi; Maruyama, Taro; Shimada, Akira; Yasuda, Kazuki; Nishida, Nao; Tokunaga, Katsushi; Koike, Asako

    2014-01-01

    Elucidation of the genetic susceptibility factors for diabetic retinopathy (DR) is important to gain insight into the pathogenesis of DR, and may help to define genetic risk factors for this condition. In the present study, we conducted a three-stage genome-wide association study (GWAS) to identify DR susceptibility loci in Japanese patients, which comprised a total of 837 type 2 diabetes patients with DR (cases) and 1,149 without DR (controls). From the stage 1 genome-wide scan of 446 subjects (205 cases and 241 controls) on 614,216 SNPs, 249 SNPs were selected for the stage 2 replication in 623 subjects (335 cases and 288 controls). Eight SNPs were further followed up in a stage 3 study of 297 cases and 620 controls. The top signal from the present association analysis was rs9362054 in an intron of RP1-90L14.1 showing borderline genome-wide significance (Pmet = 1.4×10−7, meta-analysis of stage 1 and stage 2, allele model). RP1-90L14.1 is a long intergenic non-coding RNA (lincRNA) adjacent to KIAA1009/QN1/CEP162 gene; CEP162 plays a critical role in ciliary transition zone formation before ciliogenesis. The present study raises the possibility that the dysregulation of ciliary-associated genes plays a role in susceptibility to DR. PMID:25364816

  7. 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

  8. 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

  9. Genome-Wide Screening of mRNA Expression in Leprosy Patients

    PubMed Central

    Belone, Andrea de Faria F.; Rosa, Patrícia S.; Trombone, Ana P. F.; Fachin, Luciana R. V.; Guidella, Cássio C.; Ura, Somei; Barreto, Jaison A.; Pinilla, Mabel G.; de Carvalho, Alex F.; Carraro, Dirce M.; Soares, Fernando A.; Soares, Cleverson T.

    2015-01-01

    Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy. PMID:26635870

  10. 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

  11. Effect of CLN3 silencing by RNA interference on the proliferation and apoptosis of human colorectal cancer cells.

    PubMed

    Zhu, Xinguo; Huang, Zhilong; Chen, Yan; Zhou, Jian; Hu, Shuiqing; Zhi, Qiaoming; Song, Shiduo; Wang, Yanan; Wan, Daiwei; Gu, Wen; Zhou, Hao; Zhang, Bo; Cao, Wei; He, Songbing

    2014-04-01

    Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten disease protein, CLN3, is antiapoptotic gene in NT2 neuronal precursor cells and a few types of cancers. However, in colorectal cancer, whether CLN3 also play its antiapoptotic role and the effect of targeted controlling CLN3 on the biological behavior of human colorectal cancer cell is unknown. We employed the sequence-specific siRNA silencing the CLN3 gene and investigated its effects on growth and apoptosis of colorectal cancer HCT116 cells, which has highest elevation of CLN3 expression among four colorectal cancer cell lines. After CLN3 specific siRNA transfection, mRNA and protein expression levels of CLN3 in HCT116 cells were noticeably decreased. Moreover, CLN3-siRNA inhibited the proliferation of colorectal cancer cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. Our current study demonstrated that CLN3 was expressed in colorectal cancer cells at a high frequency. Moreover, CLN3 down-regulation with RNA interference can inhibit proliferation, apoptosis, and cell cycle progression of colorectal cancer cells. Our study represented a potential new approach to understanding the role of CLN3 in cancer and provides a potential novel strategy colorectal cancer therapy. PMID:24556023

  12. Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9

    PubMed Central

    Park, Jeongbin; Kim, Jin-Soo; Bae, Sangsu

    2016-01-01

    Motivation: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. Results: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. Availability and implementation: Free access at http://www.rgenome.net/cas-database/. Contact: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153724

  13. Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC.

    PubMed

    Nagel, Remco; Stigter-van Walsum, Marijke; Buijze, Marijke; van den Berg, Jaap; van der Meulen, Ida H; Hodzic, Jasmina; Piersma, Sander R; Pham, Thang V; Jiménez, Connie R; van Beusechem, Victor W; Brakenhoff, Ruud H

    2015-06-01

    Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non-small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle-related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2-M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. PMID:25808837

  14. Genome-wide circulating microRNA expression profiling indicates biomarkers for epilepsy

    PubMed Central

    Wang, Jun; Yu, Jin-Tai; Tan, Lin; Tian, Yan; Ma, Jing; Tan, Chen-Chen; Wang, Hui-Fu; Liu, Ying; Tan, Meng-Shan; Jiang, Teng; Tan, Lan

    2015-01-01

    MicroRNAs (miRNAs) have been proposed as biomarkers for cancer and other diseases due to their stability in serum. In epilepsy, miRNAs have almost been studied in brain tissues and in animals' circulation, but not in circulation of human. To date, a major challenge is to develop biomarkers to improve the current diagnosis of epilepsy. The aim of this study was to evaluate whether circulating miRNAs can be used as biomarkers for epilepsy. We measured the differences in serum miRNA levels between 30 epilepsy patients and 30 healthy controls in discovery and training phases using Illumina HiSeq2000 sequencing followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. The selected miRNAs were then validated in 117 epilepsy patients and 112 healthy controls by qRT-PCR. Let-7d-5p, miR-106b-5p, -130a-3p and -146a-5p were found up-regulated, whereas miR-15a-5p and -194-5p were down-regulated in epilepsy patients compared to controls (P < 0.0001). Among these miRNAs, miR-106b-5p had the best diagnostic value for epilepsy with 80.3% sensitivity and 81.2% specificity. Circulating miRNAs were differentially regulated in epilepsy patients as compared with controls. MiR-106b-5p may serve as a novel, noninvasive biomarker to improve the current diagnosis of epilepsy. PMID:25825351

  15. 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

  16. 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.

  17. DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

    PubMed Central

    Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

    2015-01-01

    Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM) with substrate inhibition kinetics (Ki=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong

  18. 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

  19. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq).

    PubMed

    Mahat, Dig Bijay; Kwak, Hojoong; Booth, Gregory T; Jonkers, Iris H; Danko, Charles G; Patel, Ravi K; Waters, Colin T; Munson, Katie; Core, Leighton J; Lis, John T

    2016-08-01

    We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast. PMID:27442863

  20. 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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26553968

  2. 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

  3. 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

  4. 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

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

    PubMed Central

    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. PMID:26482836

  6. The Intestinal Microbiota Interferes with the microRNA Response upon Oral Listeria Infection

    PubMed Central

    Archambaud, Cristel; Sismeiro, Odile; Toedling, Joern; Soubigou, Guillaume; Bécavin, Christophe; Lechat, Pierre; Lebreton, Alice; Ciaudo, Constance; Cossart, Pascale

    2013-01-01

    ABSTRACT The intestinal tract is the largest reservoir of microbes in the human body. The intestinal microbiota is thought to be able to modulate alterations of the gut induced by enteropathogens, thereby maintaining homeostasis. Listeria monocytogenes is the agent of listeriosis, an infection transmitted to humans upon ingestion of contaminated food. Crossing of the intestinal barrier is a critical step of the infection before dissemination into deeper organs. Here, we investigated the role of the intestinal microbiota in the regulation of host protein-coding genes and microRNA (miRNA or miR) expression during Listeria infection. We first established the intestinal miRNA signatures corresponding to the 10 most highly expressed miRNAs in the murine ileum of conventional and germfree mice, noninfected and infected with Listeria. Next, we identified 6 miRNAs whose expression decreased upon Listeria infection in conventional mice. Strikingly, five of these miRNA expression variations (in miR-143, miR-148a, miR-200b, miR-200c, and miR-378) were dependent on the presence of the microbiota. In addition, as is already known, protein-coding genes were highly affected by infection in both conventional and germfree mice. By crossing bioinformatically the predicted targets of the miRNAs to our whole-genome transcriptomic data, we revealed an miRNA-mRNA network that suggested miRNA-mediated global regulation during intestinal infection. Other recent studies have revealed an miRNA response to either bacterial pathogens or commensal bacteria. In contrast, our work provides an unprecedented insight into the impact of the intestinal microbiota on host transcriptional reprogramming during infection by a human pathogen. PMID:24327339

  7. cyp51A gene silencing using RNA interference in azole-resistant Aspergillus fumigatus.

    PubMed

    Mousavi, Bita; Hedayati, Mohammad T; Teimoori-Toolabi, Ladan; Guillot, Jacques; Alizadeh, Ahad; Badali, Hamid

    2015-12-01

    An increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole-resistant A. fumigatus isolates. A 21-nucleotide small-interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole-resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT-PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 μg/ml. PMID:26448519

  8. 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

  9. 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

  10. 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. PMID:23322250

  11. A genome-wide search for eigenetically regulated genes in zebra finch using MethylCap-seq and RNA-seq

    PubMed Central

    Steyaert, Sandra; Diddens, Jolien; Galle, Jeroen; De Meester, Ellen; De Keulenaer, Sarah; Bakker, Antje; Sohnius-Wilhelmi, Nina; Frankl-Vilches, Carolina; Van der Linden, Annemie; Van Criekinge, Wim; Vanden Berghe, Wim; De Meyer, Tim

    2016-01-01

    Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2′-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control. PMID:26864856

  12. RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies.

    PubMed

    Anantharam, Arun; Lewis, Anthony; Panaghie, Gianina; Gordon, Earl; McCrossan, Zoe A; Lerner, Daniel J; Abbott, Geoffrey W

    2003-04-01

    The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function. PMID:12529362

  13. 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.

  14. Targeting Six1 by lentivirus-mediated RNA interference inhibits colorectal cancer cell growth and invasion

    PubMed Central

    Li, Zhaoming; Tian, Tian; Hu, Xiaopeng; Zhang, Xudong; Li, Lifeng; Nan, Feifei; Chang, Yu; Wang, Xinhua; Sun, Zhenchang; Lv, Feng; Zhang, Mingzhi

    2014-01-01

    The Six1 homeodomain protein is a developmental transcription factor that has been implicated in tumor onset and progression. Recently, it’s reported that overexpression of Six1 is sufficient to induce epithelial-to-mesenchymal transition (EMT) and metastasis of colorectal cancer. Moreover, its expression is significantly associated with poorer overall survival probability in advanced-stage colorectal cancer. To address whether Six1 could serve as a therapeutic target for human colorectal cancer, we used a lentivirus-mediated short hairpin RNA (shRNA) gene knockdown method to suppress the expression of Six1 in colorectal cancer cells. We showed that lentivirusmediated shRNA targeted to Six1 gene efficiently reduced its expression in colorectal cancer cells at both mRNA and protein levels. In vitro functional assays revealed that knockdown of Six1 significantly suppressed cell proliferation, and inhibited cell migration and invasion of colorectal cancer cells. Furthermore, tumor xenograft model demonstrated that downregulation of Six1 dramatically inhibited colorectal cancer growth in vivo. In conclusion, these findings suggest that lentivirus-mediated Six1 inhibition may represent a novel therapeutic approach for treatment of colorectal cancer. PMID:24551283

  15. 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. PMID:20171246

  16. 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.

  17. 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.

  18. The HIV-1 Nef Protein Binds Argonaute-2 and Functions as a Viral Suppressor of RNA Interference

    PubMed Central

    Aqil, Madeeha; Naqvi, Afsar Raza; Bano, Aalia Shahr; Jameel, Shahid

    2013-01-01

    The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR). PMID:24023945

  19. 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

  20. In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

    PubMed Central

    Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

    2013-01-01

    SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

  1. 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

  2. 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

  3. Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

    PubMed Central

    Lee, Minho; Shin, Eunkyoung; Jeon, Che Ok; Cha, Chang-Jun; Han, Seung Hyun; Kim, Su-Jin; Lee, Sang-Won; Lee, Younghoon; Ha, Nam-Chul

    2011-01-01

    Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5′ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects. PMID:22186084

  4. A Whole-Genome RNA Interference Screen for Human Cell Factors Affecting Myxoma Virus Replication

    PubMed Central

    Teferi, Wondimagegnehu M.; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole

    2013-01-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes (“hits”) and nonsignificant genes (“nonhits”) of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G1, or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G1/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-d-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy. PMID

  5. 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

  6. 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

  7. Identification and RNA Interference of the Pheromone Biosynthesis Activating Neuropeptide (PBAN) in the Common Cutworm Moth Spodoptera litura (Lepidoptera: Noctuidae).

    PubMed

    Lu, Qin; Huang, Ling-Yan; Chen, Peng; Yu, Jin-Feng; Xu, Jin; Deng, Jian-Yu; Ye, Hui

    2015-06-01

    Spodoptera litura F. is one of the most destructive insect pests of many agricultural crops and notorious for developing insecticide resistance. Developing environmental friendly control methods such as novel pheromone and RNAi-related control strategies is imperative to control this pest. In the present study, the full-length cDNA encoding the diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) was identified and characterized in S. litura. This 809-bp transcript contains a 573-nucleotide ORF encoding a 191-amino acid protein, from which five putative neuropeptides, including PBAN, DH, and α-, β-, and γ-subesophageal ganglion neuropeptides, were derived. Phylogenetic analysis showed that both the whole protein and each of the five neuropeptides have high similarities to those of DH-PBANs from other insect orders particularly Lepidoptera. Females treated with TKYFSPRLamide (the active core fragment of PBAN) produced significantly more four types of pheromone compounds (A; B; C; D) than controls. RNA interference by injection of PBAN dsRNA significantly reduced the relative expression levels of this gene in adult females (approximately reduced by 60%). As a consequence, females treated with PBAN dsRNA produced significantly less four types of pheromone compounds (A; B; C; D) than controls. These results suggest that PBAN function in activating sex pheromone biosynthesis and the RNAi of DH-PBAN gene can be induced by the injection of dsRNA into the body cavity in S. litura. This study suggests the possibility of novel pheromone-related pest control strategies based on RNAi techniques. PMID:26470263

  8. 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

  9. Pseudoscorpion mitochondria show rearranged genes and genome-wide reductions of RNA gene sizes and inferred structures, yet typical nucleotide composition bias

    PubMed Central

    2012-01-01

    Background Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes. Results We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic analyses of all 13

  10. A Genome-wide Small Interfering RNA (siRNA) Screen Reveals Nuclear Factor-κB (NF-κB)-independent Regulators of NOD2-induced Interleukin-8 (IL-8) Secretion*

    PubMed Central

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-01-01

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  11. A genome-wide small interfering RNA (siRNA) screen reveals nuclear factor-κB (NF-κB)-independent regulators of NOD2-induced interleukin-8 (IL-8) secretion.

    PubMed

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-10-10

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  12. 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

  13. 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

  14. A Re-Examination of Global Suppression of RNA Interference by HIV-1

    PubMed Central

    Sanghvi, Viraj R.; Steel, Laura F.

    2011-01-01

    The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

  15. Diphencyprone Treatment of Alopecia Areata: Postulated Mechanism of Action and Prospects for Therapeutic Synergy with RNA Interference.

    PubMed

    Bulock, Karen G; Cardia, James P; Pavco, Pamela A; Levis, William R

    2015-11-01

    Diphencyprone (DPCP) is a potent topical sensitizing agent that has been used since the late 1970s by physicians for the treatment of alopecia areata (AA), viral warts (human papillomavirus) and cutaneous metastases of melanoma. Although to date the compound is not approved as a drug by the FDA or EMA, physicians have continued to use DPCP because of its proven effects in these dermatological conditions. The use of the drug has been highly variable because of differences in compounding, and as a result, the literature reports vary widely in the concentrations used for sensitization and challenge treatment with DPCP. The efficacy of DPCP has generally been ascribed to immunological reactions by the host. Inducing inflammation with a contact sensitizer is counterintuitive to treating AA, an autoimmune disorder. We have hypothesized that the body's attempt to downregulate the inflammation caused by the contact sensitizer may also ameliorate AA. Studies using microarray and miRNA profiling may provide information about how DPCP induces inflammation in human skin at different times. Gene targets and microRNAs identified through these data may be modulated by an RNA interference approach to enhance DPCP efficacy and response rates. In addition, this approach may result in the discovery and development of drugs that are more potent and selective for the treatment of AA. PMID:26551938

  16. Genome-wide association study implicates a novel RNA gene, the lincRNA AC068718.1, as a risk factor for post-traumatic stress disorder in women

    PubMed Central

    Guffanti, Guia; Galea, Sandro; Yan, Lulu; Roberts, Andrea L.; Solovieff, Nadia; Aiello, Allison E.; Smoller, Jordan W.; De Vivo, Immaculata; Ranu, Hardeep; Uddin, Monica; Wildman, Derek E.; Purcell, Shaun; Koenen, Karestan C.

    2013-01-01

    Posttraumatic stress disorder (PTSD) is a common and debilitating mental disorder with a particularly high burden for women. Emerging evidence suggests PTSD may be more heritable among women and evidence from animal models and human correlational studies suggest connections between sex-linked biology and PTSD vulnerability, which may extend to the disorder’s genetic architecture. We conducted a genome-wide association study (GWAS) of PTSD in a primarily African American sample of women from the Detroit Neighborhood Health Study (DNHS) and tested for replication in an independent cohort of primarily European American women from the Nurses Health Study II (NHSII). We genotyped 413 DNHS women - 94 PTSD cases and 319 controls exposed to at least one traumatic event - on the Illumina HumanOmniExpress BeadChip for > 700,000 markers and tested 578 PTSD cases and 1963 controls from NHSII for replication. We performed a network-based analysis integrating data from GWAS-derived independent regions of association and the Reactome database of functional interactions. We found genome-wide significant association for one marker mapping to a novel RNA gene, lincRNA AC068718.1, for which we found suggestive evidence of replication in NHSII. Our network-based analysis indicates that our top GWAS results were enriched for pathways related to telomere maintenance and immune function. Our findings implicate a novel RNA gene, lincRNA AC068718.1, as risk factor for PTSD in women and add to emerging evidence that non-coding RNA genes may play a crucial role in shaping the landscape of gene regulation with putative pathological effects that lead to phenotypic differences. PMID:24080187

  17. Genome-wide association study implicates a novel RNA gene, the lincRNA AC068718.1, as a risk factor for post-traumatic stress disorder in women.

    PubMed

    Guffanti, Guia; Galea, Sandro; Yan, Lulu; Roberts, Andrea L; Solovieff, Nadia; Aiello, Allison E; Smoller, Jordan W; De Vivo, Immaculata; Ranu, Hardeep; Uddin, Monica; Wildman, Derek E; Purcell, Shaun; Koenen, Karestan C

    2013-12-01

    Posttraumatic stress disorder (PTSD) is a common and debilitating mental disorder with a particularly high burden for women. Emerging evidence suggests PTSD may be more heritable among women and evidence from animal models and human correlational studies suggest connections between sex-linked biology and PTSD vulnerability, which may extend to the disorder's genetic architecture. We conducted a genome-wide association study (GWAS) of PTSD in a primarily African American sample of women from the Detroit Neighborhood Health Study (DNHS) and tested for replication in an independent cohort of primarily European American women from the Nurses Health Study II (NHSII). We genotyped 413 DNHS women - 94 PTSD cases and 319 controls exposed to at least one traumatic event - on the Illumina HumanOmniExpress BeadChip for >700,000 markers and tested 578 PTSD cases and 1963 controls from NHSII for replication. We performed a network-based analysis integrating data from GWAS-derived independent regions of association and the Reactome database of functional interactions. We found genome-wide significant association for one marker mapping to a novel RNA gene, lincRNA AC068718.1, for which we found suggestive evidence of replication in NHSII. Our network-based analysis indicates that our top GWAS results were enriched for pathways related to telomere maintenance and immune function. Our findings implicate a novel RNA gene, lincRNA AC068718.1, as risk factor for PTSD in women and add to emerging evidence that non-coding RNA genes may play a crucial role in shaping the landscape of gene regulation with putative pathological effects that lead to phenotypic differences. PMID:24080187

  18. A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila

    PubMed Central

    Muerdter, Felix; Guzzardo, Paloma M.; Gillis, Jesse; Luo, Yicheng; Yu, Yang; Chen, Caifu; Fekete, Richard; Hannon, Gregory J.

    2013-01-01

    Summary A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels. PMID:23665228

  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. Targeting SPARC by lentivirus-mediated RNA interference inhibits cervical cancer cell growth and metastasis

    PubMed Central

    2012-01-01

    Background Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progressions of some cancers. However, no information has been available to date regarding the function of SPARC in cervical cancer cell growth and metastasis. Methods In this study, we isolated and established high invasive subclones and low invasive subclones from human cervical cancer cell lines HeLa and SiHa by the limited dilution method. Real-time q-RT-PCR, Western Blot and ICC were performed to investigate SPARC mRNA and protein expressions in high invasive subclones and low invasive subclones. Then lentivirus vector with SPARC shRNA was constructed and infected the highly invasive subclones. Real-time q-RT-PCR, Western Blot and ICC were also performed to investigate the changes of SPARC expression after viral infection. In functional assays, effects of SPARC knockdown on the biological behaviors of cervical cancer cells were investigated. The mechanisms of SPARC in cervical cancer proliferation, apoptosis and invasion were also researched. Results SPARC was over-expressed in the highly invasive subclones compared with the low invasive subclones. Knockdown of SPARC significantly suppressed cervical cancer cell proliferation, and induced cell cycle arrest at the G1/G0 phase through the p53/p21 pathway, also caused cell apoptosis accompanied by the decreased ratio of Bcl-2/Bax, and inhibited cell invasion and metastasis accompanied by down-regulated MMP2 and MMP9 expressions and up-regulated E-cadherin expression. Conclusion SPARC is related to the invasive phenotype of cervical cancer cells. Knockdown of SPARC significantly suppresses cervical cancer cell proliferation, induces cell apoptosis and inhibits cell invasion and metastasis. SPARC as a promoter improves cervical cancer cell growth and metastasis. PMID:23050783

  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. 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

  3. Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene.

    PubMed

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

    2011-01-01

    Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of β-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, β-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites. PMID:20699100

  4. Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression

    PubMed Central

    Boström, Adrian E.; Mwinyi, Jessica; Schiöth, Helgi B.

    2016-01-01

    Abstract Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data. PMID:27310475

  5. Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

    PubMed

    Nilsson, Emil K; Boström, Adrian E; Mwinyi, Jessica; Schiöth, Helgi B

    2016-06-01

    Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data. PMID:27310475

  6. RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats

    PubMed Central

    Luo, Zheng; Liu, Huimin; Sun, Xiaomeng; Guo, Rong; Cui, Ruibing; Ma, Xiangxing; Yan, Ming

    2013-01-01

    Discoidin domain receptor 2 (DDR2) is involved in fibrotic disease. However, the exact pathogenic implications of the receptor in early alcoholic liver disease are still controversial. We constructed plasmid vectors encoding short-hairpin RNA against DDR2 to investigate its role in alcoholic liver disease in an immortalized rat hepatic stellate cell line, HSC-T6, and in rats by MTT, RT-PCR and western blot analyses; immunohistochemistry and electron microscopy. Alcohol-induced upregulation of DDR2 was associated with the expression of matrix metalloproteinase 2, the transforming growth factor β1 signaling pathway and tissue inhibitor of metalloproteinase 1; collagen deposition; and extracellular matrix remodeling. Inhibition of DDR2 decreased HSC-T6 cell proliferation and liver injury in rats with 10-week-induced alcoholic liver disease. DDR2 may have an important role in the pathogenesis of early-stage alcoholic liver disease. Silencing DDR2 may be effective in preventing early-stage alcoholic liver disease. PMID:23409069

  7. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms

    PubMed Central

    Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    Characteristics of DIRS-1 Mediated Knock-Downs We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. Advantages of the DIRS-1–RNAi System The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5’-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes. PMID:26110905

  8. RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

    PubMed Central

    Trzcińska-Daneluti, Agata M.; Chen, Anthony; Nguyen, Leo; Murchie, Ryan; Jiang, Chong; Moffat, Jason; Pelletier, Lawrence; Rotin, Daniela

    2015-01-01

    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. PMID:25825526

  9. 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

  10. Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis

    PubMed Central

    Rafehi, Haloom; Ververis, Katherine; Balcerczyk, Aneta; Ziemann, Mark; Ooi, Jenny; Hu, Sean; Kwa, Faith A. A.; Loveridge, Shanon J.; Georgiadis, George T.; El-Osta, Assam; Karagiannis, Tom C.

    2012-01-01

    The accumulating evidence of the beneficial effects of cinnamon (Cinnamomum burmanni) in type-2 diabetes, a chronic age-associated disease, has prompted the commercialisation of various supplemental forms of the spice. One such supplement, Cinnulin PF®, represents the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavanoids. The overall aim of this study was to utilize genome-wide mRNA-Seq analysis to characterise the changes in gene expression caused by Cinnulin PF in immortalised human keratinocytes and microvascular endothelial cells, which are relevant with respect to diabetic complications. In summary, our findings provide insights into the mechanisms of action of Cinnulin PF in diabetes and diabetic complications. More generally, we identify relevant candidate genes which could provide the basis for further investigation. PMID:22953038

  11. Investigation of the biological properties of Cinnulin PF in the context of diabetes: mechanistic insights by genome-wide mRNA-Seq analysis.

    PubMed

    Rafehi, Haloom; Ververis, Katherine; Balcerczyk, Aneta; Ziemann, Mark; Ooi, Jenny; Hu, Sean; Kwa, Faith A A; Loveridge, Shanon J; Georgiadis, George T; El-Osta, Assam; Karagiannis, Tom C

    2012-01-01

    The accumulating evidence of the beneficial effects of cinnamon (Cinnamomum burmanni) in type-2 diabetes, a chronic age-associated disease, has prompted the commercialisation of various supplemental forms of the spice. One such supplement, Cinnulin PF(®), represents the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavanoids. The overall aim of this study was to utilize genome-wide mRNA-Seq analysis to characterise the changes in gene expression caused by Cinnulin PF in immortalised human keratinocytes and microvascular endothelial cells, which are relevant with respect to diabetic complications. In summary, our findings provide insights into the mechanisms of action of Cinnulin PF in diabetes and diabetic complications. More generally, we identify relevant candidate genes which could provide the basis for further investigation. PMID:22953038

  12. High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans.

    PubMed

    O'Reilly, Linda P; Knoerdel, Ryan R; Silverman, Gary A; Pak, Stephen C

    2016-01-01

    RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules mediate the inhibition of gene expression. RNAi in C. elegans can be achieved by simply feeding animals with bacteria expressing dsRNA against the gene of interest. This "feeding" method has made it possible to conduct genome-wide RNAi experiments for the systematic knockdown and subsequent investigation of almost every single gene in the genome. Historically, these genome-scale RNAi screens have been labor and time intensive. However, recent advances in automated, high-throughput methodologies have allowed the development of more rapid and efficient screening protocols. In this report, we describe a fast and efficient, liquid-based method for genome-wide RNAi screening. PMID:27581291

  13. Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells.

    PubMed

    Workenhe, S T; Ketela, T; Moffat, J; Cuddington, B P; Mossman, K L

    2016-05-12

    Oncolytic human herpes simplex virus type 1 (HSV-1) shows promising treatment efficacy in late-stage clinical trials. The anticancer activity of oncolytic viruses relies on deregulated pathways in cancer cells, which make them permissive to oncolysis. To identify pathways that restrict HSV-1 KM100-mediated oncolysis, this study used a pooled genome-wide short hairpin RNA library and found that depletion of the splicing factor arginine-rich splicing factor 2 (SRSF2) leads to enhanced cytotoxicity of breast cancer cells by KM100. Serine/arginine-rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing. Further characterization showed that KM100 infection of HS578T cells under conditions of low SRSF2 leads to pronounced apoptosis without a corresponding increase in virus replication. As DNA topoisomerase I inhibitors can limit the phosphorylation of SRSF2, we combined a topoisomerase I inhibitor chemotherapeutic with KM100 and observed synergistic anticancer effect in vitro and prolonged survival of tumor-bearing mice in vivo. PMID:26257065

  14. Genome-wide RNA-seq and ChIP-seq reveal Linc-YY1 function in regulating YY1/PRC2 activity during skeletal myogenesis

    PubMed Central

    Sun, Kun; Zhou, Liang; Zhao, Yu; Wang, Huating; Sun, Hao

    2016-01-01

    Little is known how lincRNAs are involved in skeletal myogenesis. Here we describe the discovery and functional annotation of Linc-YY1, a novel lincRNA originating from the promoter of the transcription factor (TF) Yin Yang 1 (YY1). Starting from whole transcriptome shotgun sequencing (a.k.a. RNA-seq) data from muscle C2C12 cells, a series of bioinformatics analysis was applied towards the identification of hundreds of high-confidence novel lincRNAs. Genome-wide approaches were then employed to demonstrate that Linc-YY1 functions to promote myogenesis through associating with YY1 and regulating YY1/PRC2 transcriptional activity in trans. Here we describe the details of the ChIP-seq, RNA-seq experiments, and data analysis procedures associated with the study published by Zhou and colleagues in the Nature Communications Journal in 2015 Zhou et al. (2015) [1]. The data was deposited on NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE74049. PMID:26981420

  15. RNA interference suppression of the receptor tyrosine kinase Torso gene impaired pupation and adult emergence in Leptinotarsa decemlineata.

    PubMed

    Zhu, Tao-Tao; Meng, Qing-Wei; Guo, Wen-Chao; Li, Guo-Qing

    2015-12-01

    In Drosophila melanogaster prothoracic gland (PG) cells, Torso mediates prothoracicotropic hormone (PTTH)-triggered mitogen activated protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK and ERK) to stimulate ecdysteroidogenesis. In this study, LdTorso, LdRas, LdRaf and LdERK were cloned in Leptinotarsa decemlineata. The four genes were highly or moderately expressed in the larval prothoracic glands. At the first- to third-instar stages, their expression levels were higher just before and right after the molt, and were lower in the mid instars. At the fourth-instar stage, their transcript levels were higher before prepupal stage. RNA interference-mediated knockdown of LdTorso delayed larval development, increased pupal weight, and impaired pupation and adult emergence. Moreover, knockdown of LdTorso decreased the mRNA levels of LdRas, LdRaf and LdERK, repressed the transcription of two ecdysteroidogenesis genes (LdPHM and LdDIB), lowered 20E titer, and downregulated the expression of several 20E-response genes (LdEcR, LdUSP, LdHR3 and LdFTZ-F1). Furthermore, silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT, increased JH titer, and activated the transcription of a JH early-inducible gene LdKr-h1. Thus, our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect. PMID:26518287

  16. Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication.

    PubMed

    Wang, Yi-Ping; Huang, Li-Ping; Du, Wen-Juan; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-08-01

    RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors. PMID:27387827

  17. Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism[C][W][OA

    PubMed Central

    Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M.; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima

    2012-01-01

    Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection. PMID:22822208

  18. 16S rRNA survey revealed complex bacterial communities and evidence of bacterial interference on human adenoids.

    PubMed

    Ren, Tiantian; Glatt, Dominique Ulrike; Nguyen, Tam Nhu; Allen, Emma Kaitlynn; Early, Stephen V; Sale, Michele; Winther, Birgit; Wu, Martin

    2013-02-01

    Adenoid microbiota plays an important role in the development of various infectious and non-infectious diseases of the upper airways, such as otitis media, adenotonsillitis, rhinosinusitis and adenoid hypertrophy. Studies have suggested that adenoids could act as a potential reservoir of opportunistic pathogens. However, previous bacterial surveys of adenoids were mainly culture based and therefore might only provide an incomplete and potentially biased assessment of the microbial diversity. To develop an in-depth and comprehensive understanding of the adenoid microbial communities and test the 'pathogen reservoir hypothesis', we carried out a 16S rRNA based, culture-independent survey of bacterial communities on 67 human adenoids removed by surgery. Our survey revealed highly diverse adenoid bacterial communities distinct from those of other body habitats. Despite large interpersonal variations, adenoid microbiota shared a core set of taxa and can be classified into at least five major types based on its bacterial species composition. Our results support the 'pathogen reservoir hypothesis' as we found common pathogens of otitis media to be both prevalent and abundant. Co-occurrence analyses revealed evidence consistent with the bacterial interference theory in that multiple common pathogens showed 'non-coexistence' relationships with non-pathogenic members of the commensal microflora. PMID:23113966

  19. 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

  20. RNA interference against MDM2 suppresses tumor growth and metastasis in pancreatic carcinoma SW1990HM cells.

    PubMed

    Shi, Weidong; Meng, Zhiqiang; Chen, Zhen; Hua, Yongqiang; Gao, Huifeng; Wang, Peng; Lin, Junhua; Zhou, Zhenhua; Luo, Jianmin; Liu, Luming

    2014-02-01

    In our previous study, the mouse double minute 2 (MDM2) was identified as one of the leading genes that promote the metastasis of pancreatic cancer (PC). However, the mechanism by which MDM2 promotes metastasis of PC is not understood. In this study, we show that down-regulation of MDM2 through lentivirus-mediated RNA interference could also suppress in vitro proliferation and in vivo tumor growth, and led to an obvious inhibition of both in vitro invasion and in vivo live metastases of SW1990HM cells which had an over-expression of MDM2 and a higher metastatic potential. Moreover, we also show that the down-regulation of MDM2 induced a significant decrease in MMP9, Ki-67 and increase in P53, E-Cadherin expression, and results in an altered expression of genes involved in metastasis, apoptosis, and cell proliferation. Our results suggest that MDM2 plays an important role in metastasis as well as tumor growth of PC. MDM2 could be a hopeful target for the control of PC. PMID:22200978

  1. Functional analysis of a chitinase gene during the larval-nymph transition in Panonychus citri by RNA interference.

    PubMed

    Xia, Wen-Kai; Shen, Xiao-Min; Ding, Tian-Bo; Niu, Jin-Zhi; Zhong, Rui; Liao, Chong-Yu; Feng, Ying-Cai; Dou, Wei; Wang, Jin-Jun

    2016-09-01

    Chitinases are hydrolytic enzymes that are required for chitin degradation and reconstruction in arthropods. In this study, we report a cDNA sequence encoding a putative chitinase (PcCht1) from the citrus red mite, Panonychus citri. The PcCht1 (564 aa) possessed a signal peptide, a conserver domain, and a chitin-binding domain. Structural and phylogenetic analyses found that PcCht1 had high sequence similarity to chitinases in Tetranychus urticae. Real-time quantitative PCR analyses showed that the transcript levels of PcCht1 peaked periodically in larval and nymph stages. Moreover, significant increase of PcCht1 transcript level in the larvae was observed upon the exposure of diflubenzuron. In contrast, exposures of the larvae to diflubenzuron resulted in the decreased chitin content. Furthermore, through a feeding-based RNA interference approach, we were able to reduce the PcCht1 transcript level by 59.7 % in the larvae, and consequently the treated larvae showed a very low molting rate compared with the control. Our results expanded the understanding of the important role of PcCht1 in the growth and development of P. citri. PMID:27388447

  2. RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification

    PubMed Central

    Ćwiek, Paulina; Leni, Zaira; Salm, Fabiana; Dimitrova, Valeriya; Styp-Rekowska, Beata; Chiriano, Gianpaolo; Carroll, Michael; Höland, Katrin; Djonov, Valentin; Scapozza, Leonardo; Guiry, Patrick; Arcaro, Alexandre

    2015-01-01

    Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification. PMID:25402633

  3. 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

  4. Targeting S100P Inhibits Colon Cancer Growth and Metastasis by Lentivirus-Mediated RNA Interference and Proteomic Analysis

    PubMed Central

    Jiang, Lei; Lai, Yiu-Kay; Zhang, Jinfang; Wang, Hua; Lin, Marie CM; He, Ming-liang; Kung, Hsiang-fu

    2011-01-01

    S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and β-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis. PMID:21327297

  5. 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

  6. Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein Function

    NASA Astrophysics Data System (ADS)

    Stout, Jane R.; Rizk, Rania S.; Walczak, Claire E.

    A major goal in cell biology is to understand the molecular mechanisms of the biological process under study, which requires functional information about the roles of individual proteins in the cell. For many non-genetic model organisms researchers have relied on the use of inhibitory reagents, such as antibodies that can be microinjected into cells. More recently, the advent of RNA-mediated interference (RNAi) has allowed scientists to knockdown individual proteins and to examine the consequences of the knockdown. In this chapter we present a comparison between microinjection of inhibitory reagents and RNAi for the analysis of protein function in mammalian tissue culture cells, providing both a description of the techniques as well as a discussion of the benefits and drawbacks of each approach. In addition, we present a strategy to employ RNAi for organisms without a sequenced genome. While the focus of our research is on the organization of the mitotic spindle during cell division and thus the examples utilized are from that system, the approaches described here should be readily applicable to multiple experimental models.

  7. RNA interference of Marlin-1/Jakmip1 results in abnormal morphogenesis and migration of cortical pyramidal neurons.

    PubMed

    Vidal, René L; Fuentes, Patricio; Valenzuela, José Ignacio; Alvarado-Diaz, Carlos P; Ramírez, Omar A; Kukuljan, Manuel; Couve, Andrés

    2012-08-01

    The formation of the nervous systems requires processes that coordinate proliferation, differentiation and migration of neuronal cells, which extend axons, generate dendritic branching and establish synaptic connections during development. The structural organization and dynamic remodeling of the cytoskeleton and its association to the secretory pathway are critical determinants of cell morphogenesis and migration. Marlin-1 (Jakmip1) is a microtubule-associated protein predominantly expressed in neurons and lymphoid cells. Marlin-1 participates in polarized secretion in lymphocytes, but its functional association with the neuronal cytoskeleton and its contribution to brain development have not been explored. Combining in vitro and in vivo approaches we show that Marlin-1 contributes to the establishment of neuronal morphology. Marlin-1 associates to the cytoskeleton in neurites, is required for the maintenance of an intact Golgi apparatus and its depletion produces the down-regulation of kinesin-1, a plus-end directed molecular motor with a central function in morphogenesis and migration. RNA interference of Marlin-1 in vivo results in abnormal migration of newborn pyramidal neurons during the formation of the cortex. Our results support the involvement of Marlin-1 in the acquisition of the complex architecture and migration of pyramidal neurons, two fundamental processes for the laminar layering of the cortex. PMID:22828129

  8. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  9. RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders.

    PubMed

    Petriv, Oleh I; Pilgrim, David B; Rachubinski, Richard A; Titorenko, Vladimir I

    2002-08-14

    RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders. PMID:12181365

  10. Genome-wide whole blood microRNAome and transcriptome analyses reveal miRNA-mRNA regulated host response to foodborne pathogen Salmonella infection in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miRNAs) are important regulators of gene expression and play key roles in several biological processes. However, little is known about the role of miRNAs in regulating genes involved in host response to bacterial infection. Here, we present a systematic study of miRNA and mRNA profiles fr...

  11. Genome-wide analysis for discovery of new rice miRNA reveals natural antisense miRNA (nat-miRNAs)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (21-24nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently ~240 miRNAs have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, w...

  12. Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus

    PubMed Central

    Chiba, Sotaro; Suzuki, Nobuhiro

    2015-01-01

    Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfection with the prototypic member of the genus Mycoreovirus (MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels of dcl2 and agl2 transcripts were elevated by transgenic expression of a hairpin construct of an endogenous C. parasitica gene. Disruption of dcl2 completely abolished the interference, whereas that of agl2 did not always lead to its abolishment, suggesting more crucial roles of dcl2 in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing in C. parasitica activated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing. PMID:26283371

  13. Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus.

    PubMed

    Chiba, Sotaro; Suzuki, Nobuhiro

    2015-09-01

    Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfection with the prototypic member of the genus Mycoreovirus (MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels of dcl2 and agl2 transcripts were elevated by transgenic expression of a hairpin construct of an endogenous C. parasitica gene. Disruption of dcl2 completely abolished the interference, whereas that of agl2 did not always lead to its abolishment, suggesting more crucial roles of dcl2 in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing in C. parasitica activated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing. PMID:26283371

  14. A Genome-Wide siRNA Screen Reveals Positive and Negative Regulators of the NOD2 and NF-κB Signaling Pathways

    PubMed Central

    Warner, Neil; Burberry, Aaron; Franchi, Luigi; Kim, Yun-Gi; McDonald, Christine; Sartor, Maureen A.; Núñez, Gabriel

    2013-01-01

    The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways that lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn’s disease and Blau syndrome. We used a genome-wide, small interfering RNA (siRNA) screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn’s disease risk were identified in the screen, supporting a role for NOD2 and nuclear factor κB (NF-κB) pathways in the pathogenesis of Crohn’s disease. A comparison of hits from this screen with other “omics” data sets revealed interconnected networks of genes implicated in NF-κB signaling. Secondary assays, including the measurement of interleukin-8 secretion, served to validate many of the regulators. Knockdown of putative regulators in HEK293 cells followed by stimulation with tumor necrosis factor α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB–mediated inflammation. PMID:23322906

  15. Genome-Wide Associations between Genetic and Epigenetic Variation Influence mRNA Expression and Insulin Secretion in Human Pancreatic Islets

    PubMed Central

    Olsson, Anders H.; Volkov, Petr; Bacos, Karl; Dayeh, Tasnim; Hall, Elin; Nilsson, Emma A.; Ladenvall, Claes; Rönn, Tina; Ling, Charlotte

    2014-01-01

    Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic

  16. Lentiviral miR30-based RNA Interference against Heparanase Suppresses Melanoma Metastasis with Lower Liver and Lung Toxicity

    PubMed Central

    Liu, Xiao-yan; Tang, Qiu-su; Chen, Hong-chao; Jiang, Xiao-ling; Fang, Hong

    2013-01-01

    Aim: To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE. Methods: First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models. Results: Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-β1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated. Conclusion: These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications. PMID:23847439

  17. 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

  18. Genome-Wide Dissection of the MicroRNA Expression Profile in Rice Embryo during Early Stages of Seed Germination

    PubMed Central

    He, Dongli; Wang, Qiong; Wang, Kun; Yang, Pingfang

    2015-01-01

    The first 24 hours after imbibition (HAI) is pivotal for rice seed germination, during which embryo cells switch from a quiescent state to a metabolically active state rapidly. MicroRNAs (miRNAs) have increasingly been shown to play important roles in rice development. Nevertheless, limited knowledge about miRNA regulation has been obtained in the early stages of rice seed germination. In this study, the small RNAs (sRNAs) from embryos of 0, 12, and 24 HAI rice seeds were sequenced to investigate the composition and expression patterns of miRNAs. The bioinformatics analysis identified 289 miRNA loci, including 59 known and 230 novel miRNAs, and 35 selected miRNAs were confirmed by stem-loop real-time RT-PCR. Expression analysis revealed that the dry and imbibed seeds have unique miRNA expression patterns compared with other tissues, particularly for the dry seeds. Using three methods, Mireap, psRNATarget and degradome analyses, 1197 potential target genes of identified miRNAs involved in various molecular functions were predicted. Among these target genes, 39 had significantly negative correlations with their corresponding miRNAs as inferred from published transcriptome data, and 6 inversely expressed miRNA-target pairs were confirmed by 5ʹ-RACE assay. Our work provides an inventory of miRNA expression profiles and miRNA-target interactions in rice embryos, and lays a foundation for further studies of miRNA-mediated regulation in initial seed germination. PMID:26681181

  19. Genome-Wide Dissection of the MicroRNA Expression Profile in Rice Embryo during Early Stages of Seed Germination.

    PubMed

    He, Dongli; Wang, Qiong; Wang, Kun; Yang, Pingfang

    2015-01-01

    The first 24 hours after imbibition (HAI) is pivotal for rice seed germination, during which embryo cells switch from a quiescent state to a metabolically active state rapidly. MicroRNAs (miRNAs) have increasingly been shown to play important roles in rice development. Nevertheless, limited knowledge about miRNA regulation has been obtained in the early stages of rice seed germination. In this study, the small RNAs (sRNAs) from embryos of 0, 12, and 24 HAI rice seeds were sequenced to investigate the composition and expression patterns of miRNAs. The bioinformatics analysis identified 289 miRNA loci, including 59 known and 230 novel miRNAs, and 35 selected miRNAs were confirmed by stem-loop real-time RT-PCR. Expression analysis revealed that the dry and imbibed seeds have unique miRNA expression patterns compared with other tissues, particularly for the dry seeds. Using three methods, Mireap, psRNATarget and degradome analyses, 1197 potential target genes of identified miRNAs involved in various molecular functions were predicted. Among these target genes, 39 had significantly negative correlations with their corresponding miRNAs as inferred from published transcriptome data, and 6 inversely expressed miRNA-target pairs were confirmed by 5'-RACE assay. Our work provides an inventory of miRNA expression profiles and miRNA-target interactions in rice embryos, and lays a foundation for further studies of miRNA-mediated regulation in initial seed germination. PMID:26681181

  20. 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.

  1. Molecular Genetic Maps in Wild Emmer Wheat, Triticum dicoccoides: Genome-Wide Coverage, Massive Negative Interference, and Putative Quasi-Linkage

    PubMed Central

    Peng, Junhua; Korol, Abraham B.; Fahima, Tzion; Röder, Marion S.; Ronin, Yefim I.; Li, Youchun C.; Nevo, Eviatar

    2000-01-01

    The main objectives of the study reported here were to construct a molecular map of wild emmer wheat, Triticum dicoccoides, to characterize the marker-related anatomy of the genome, and to evaluate segregation and recombination patterns upon crossing T. dicoccoides with its domesticated descendant Triticum durum (cultivar Langdon). The total map length exceeded 3000 cM and possibly covered the entire tetraploid genome (AABB). Clusters of molecular markers were observed on most of the 14 chromosomes. AFLP (amplified fragment length polymorphism) markers manifested a random distribution among homologous groups, but not among genomes and chromosomes. Genetic differentiation between T. dicoccoides and T. durum was attributed mainly to the B genome as revealed by AFLP markers. The segregation-distorted markers were mainly clustered on 4A, 5A, and 5B chromosomes. Homeoalleles, differentially conferring the vigor of gametes, might be responsible for the distortion on 5A and 5B chromosomes. Quasilinkage, deviation from free recombination between markers of nonhomologous chromosomes, was discovered. Massive negative interference was observed in most of the chromosomes (an excess of double crossovers in adjacent intervals relative to the expected rates on the assumption of no interference). The general pattern of distribution of islands of negative interference included near-centromeric location, spanning the centromere, and median/subterminal location. [An appendix describing the molecular marker loci is available as an online supplement at http://www.genome.org.] PMID:11042150

  2. 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

  3. Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq

    PubMed Central

    2013-01-01

    Background Butanol (n-butanol) has high values as a promising fuel source and chemical feedstock. Biobutanol is usually produced by the solventogenic clostridia through a typical biphasic (acidogenesis and solventogenesis phases) acetone-butanol-ethanol (ABE) fermentation process. It is well known that the acids produced in the acidogenic phase are significant and play important roles in the switch to solventogenesis. However, the mechanism that triggers the metabolic switch is still not clear. Results Sodium butyrate (40 mM) was supplemented into the medium for the ABE fermentation with Clostridium beijerinckii NCIMB 8052. With butyrate addition (reactor R1), solvent production was triggered early in the mid-exponential phase and completed quickly in < 50 h, while in the control (reactor R2), solventogenesis was initiated during the late exponential phase and took > 90 h to complete. Butyrate supplementation led to 31% improvement in final butanol titer, 58% improvement in sugar-based yield, and 133% improvement in butanol productivity, respectively. The butanol/acetone ratio was 2.4 versus 1.8 in the control, indicating a metabolic shift towards butanol production due to butyrate addition. Genome-wide transcriptional dynamics was investigated with RNA-Seq analysis. In reactor R1, gene expression related to solventogenesis was induced about 10 hours earlier when compared to that in reactor R2. Although the early sporulation genes were induced after the onset of solventogenesis in reactor R1 (mid-exponential phase), the sporulation events were delayed and uncoupled from the solventogenesis. In contrast, in reactor R2, sporulation genes were induced at the onset of solventogenesis, and highly expressed through the solventogenesis phase. The motility genes were generally down-regulated to lower levels prior to stationary phase in both reactors. However, in reactor R2 this took much longer and gene expression was maintained at comparatively higher levels

  4. Functional specificity of shuttling hnRNPs revealed by genome-wide analysis of their RNA binding profiles.

    PubMed

    Kim Guisbert, Karen; Duncan, Kent; Li, Hao; Guthrie, Christine

    2005-04-01

    Nab2, Npl3, and Nab4/Hrp1 are essential RNA binding proteins of the shuttling hnRNP class that are required for the efficient export of mRNA. To characterize the in vivo transcript specificity of these proteins, we identified their mRNA binding partners using a microarray-based assay. Each of the three proteins was coimmunoprecipitated with many different mRNA transcripts. Interestingly, each protein exhibits preferential associations with a distinct set of mRNAs. Notably, some of these appear to denote specific functional classes. For example, the ribosomal protein mRNAs and other highly expressed transcripts significantly favor association with Npl3 over Nab2, and Nab4/Hrp1 is strongly enriched with transcripts required for amino acid metabolism. Significantly, nab4 mutants showed a striking, desensitized growth phenotype when exposed to amino acid stress conditions suggesting a biological consequence to the associations we observed. Supporting the hypothesis that these proteins display transcript specificity, we identified a unique 7-nucleotide sequence overrepresented in the transcripts highly associated with Nab2 and Nab4/Hrp1 using the REDUCE algorithm. Validating our approach, our bioinformatics analysis correctly identified the known binding site for Nab4/Hrp1. These specialized associations of the hnRNP proteins of Saccharomyces cerevisiae suggest the opportunity to regulate the processing of particular transcripts between transcription and translation. PMID:15703440

  5. GENOME-WIDE RNA EXPRESSION ANALYSIS DURING CONIDIAL MATURATION AND GERMINATION IN THE FILAMENTOUS FUNGUS, FUSARIUM GRAMINEARUM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal plant pathogen, F. graminearum, causes Fusarium head blight disease of wheat and barley. To understand the early infection cycle of this organism, we monitored the RNA expression profiles in newly formed spores (macroconidia), in maturing spores and during the early stages of spore germin...

  6. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

    PubMed

    Nijhof, Ard M; Taoufik, Amar; de la Fuente, José; Kocan, Katherine M; de Vries, Erik; Jongejan, Frans

    2007-05-01

    The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis. PMID:17196597

  7. Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

    SciTech Connect

    Pang, Shi-Feng; Li, Xiao-Kun; Zhang, Qiang; Yang, Fang; Xu, Peilin

    2009-12-10

    Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

  8. 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

  9. dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42

    PubMed Central

    Fan, Ben; Förstner, Konrad; Vogel, Jörg; Borriss, Rainer; Wu, Xiao-Qin

    2015-01-01

    Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizosphere-mimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions. PMID:26540162

  10. 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

  11. Genome-Wide Investigation Using sRNA-Seq, Degradome-Seq and Transcriptome-Seq Reveals Regulatory Networks of microRNAs and Their Target Genes in Soybean during Soybean mosaic virus Infection

    PubMed Central

    Yu, Kangfu; Wang, Aiming

    2016-01-01

    MicroRNAs (miRNAs) play key roles in a variety of cellular processes through regulation of their target gene expression. Accumulated experimental evidence has demonstrated that infections by viruses are associated with the altered expression profile of miRNAs and their mRNA targets in the host. However, the regulatory network of miRNA-mRNA interactions during viral infection remains largely unknown. In this study, we performed small RNA (sRNA)-seq, degradome-seq and as well as a genome-wide transcriptome analysis to profile the global gene and miRNA expression in soybean following infections by three different Soybean mosaic virus (SMV) isolates, L (G2 strain), LRB (G2 strain) and G7 (G7 strain). sRNA-seq analyses revealed a total of 253 soybean miRNAs with a two-fold or greater change in abundance compared with the mock-inoculated control. 125 transcripts were identified as the potential cleavage targets of 105 miRNAs and validated by degradome-seq analyses. Genome-wide transcriptome analysis showed that total 2679 genes are differentially expressed in response to SMV infection including 71 genes predicted as involved in defense response. Finally, complex miRNA-mRNA regulatory networks were derived using the RNAseq, small RNAseq and degradome data. This work represents a comprehensive, global approach to examining virus-host interactions. Genes responsive to SMV infection are identified as are their potential miRNA regulators. Additionally, regulatory changes of the miRNAs themselves are described and the regulatory relationships were supported with degradome data. Taken together these data provide new insights into molecular SMV-soybean interactions and offer candidate miRNAs and their targets for further elucidation of the SMV infection process. PMID:26963095

  12. Genome-wide Long Non-coding RNA Analysis Identified Circulating LncRNAs as Novel Non-invasive Diagnostic Biomarkers for Gynecological Disease

    PubMed Central

    Wang, Wen-Tao; Sun, Yu-Meng; Huang, Wei; He, Bo; Zhao, Ya-Nan; Chen, Yue-Qin

    2016-01-01

    Increasing evidence indicates that long non-coding RNAs (lncRNAs) play important roles in human diseases. This study aimed to investigate the tissue and serum lncRNAs that are differentially expressed between patients with endometriosis, a gynecological disease, to evaluate the potential of these lncRNAs as non-invasive markers for the disease. The differentially expressed lncRNAs as competing endogenous RNAs (ceRNAs) were also analyzed to predict their functions in disease development. Genome-wide profiling of lncRNA expression patterns revealed that many lncRNAs were abnormally expressed between sera and tissuesof the patient samples. A set of aberrant differentially expressed lncRNAs were further validated in a validation cohort of 110 serum and 24 tissue samples. Functional analysis predicted that differentially expressed lncRNAs may participate in disease development through crosstalk between the ceRNAs of miRNAs and may be involved in a range of cellular pathways including steroid or hormone responses. We also found a unique set of lncRNAs that were associated with disease severity and progression, and their diagnostic values were also investigated. Our study demonstrated that lncRNAs could potentially serve as non-invasive biomarkers for the diagnosis of endometriosis and as important regulators in the progression of this disease. PMID:26987697

  13. Telomeric repeat-containing RNA/G-quadruplex-forming sequences cause genome-wide alteration of gene expression in human cancer cells in vivo

    PubMed Central

    Hirashima, Kyotaro; Seimiya, Hiroyuki

    2015-01-01

    Telomere erosion causes cell mortality, suggesting that longer telomeres enable more cell divisions. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than those of surrounding normal tissues. Recently, we showed that cancer cell telomere elongation represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated cancer cells. Using three different cell lines, we found that telomere elongation up-regulates TERRA signal and down-regulates innate immune genes such as STAT1, ISG15 and OAS3 in vivo. Ectopic TERRA oligonucleotides repressed these genes even in cells with short telomeres under three-dimensional culture conditions. This appeared to occur from the action of G-quadruplexes (G4) in TERRA, because control oligonucleotides had no effect and a nontelomeric G4-forming oligonucleotide phenocopied the TERRA oligonucleotide. Telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy by suppressing innate immune genes. PMID:25653161

  14. Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and tRNA Methylation Genes Involved in Life Span Regulation

    PubMed Central

    Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D.

    2010-01-01

    The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved. PMID:20657825

  15. A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways.

    PubMed

    Warner, Neil; Burberry, Aaron; Franchi, Luigi; Kim, Yun-Gi; McDonald, Christine; Sartor, Maureen A; Núñez, Gabriel

    2013-01-15

    The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor κB (NF-κB) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn's disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn's disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-κB signaling, thus supporting a role for NOD2 and NF-κB pathways in the pathogenesis of Crohn's disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-κB. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB-mediated inflammation. PMID:23322906

  16. Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of Their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy.

    PubMed

    Mohamed, Junaith S; Hajira, Ameena; Lopez, Michael A; Boriek, Aladin M

    2015-10-01

    Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle plays a major role in the progression of MD. To test our hypothesis, we performed a genome-wide expression profile of anisotropically regulated mechanomiRs and bioinformatically analyzed their target gene networks. We assessed their functional roles in the advancement of MD using diaphragm muscles from mdm (MD with myositis) mice, an animal model of human tibial MD (titinopathy), and their wild-type littermates. We were able to show that ex vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm muscles from WT and mdm mice; as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs, let-7e-5p and miR-98-5p, and their target genes associated with the extracellular matrix and TGF-β pathways, respectively, between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1, and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introduces additional biological players in the regulation of skeletal muscle structure and myogenesis that may contribute to unexplained disorders of MD. PMID:26272747

  17. Effects of RNA interference-mediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells.

    PubMed

    Guan, Hua-Peng; Sun, Jian-Zhong; Feng, Xiao-Lei; Chen, Jin-Shui; Chen, Fang-Jing; Cheng, Xiao-Fei; Liu, Xin-Wei; Ni, Bin

    2016-02-01

    MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol‑chitosan (mPEG‑CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG‑CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG‑CS‑livin shRNA, mPEG‑CS‑survivin shRNA and mPEG‑CS‑(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG‑63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription‑polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis‑inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG‑63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG‑63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG‑CS NPs significantly reduced livin and survivin expression in MG‑63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin. PMID:26708654

  18. 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.

  19. Genome-Wide Analyses in Bacteria Show Small-RNA Enrichment for Long and Conserved Intergenic Regions

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael

    2014-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5′ and 3′ untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms. PMID:25313390

  20. Uncovering the Genome-Wide Transcriptional Responses of the Filamentous Fungus Aspergillus niger to Lignocellulose Using RNA Sequencing

    PubMed Central

    Gaddipati, Sanyasi; Kokolski, Matthew; Malla, Sunir; Blythe, Martin J.; Ibbett, Roger; Campbell, Maria; Liddell, Susan; Aboobaker, Aziz; Tucker, Gregory A.; Archer, David B.

    2012-01-01

    A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall–degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions. PMID:22912594

  1. Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies

    PubMed Central

    Niu, Tianhua; Liu, Ning; Zhao, Ming; Xie, Guie; Zhang, Lei; Li, Jian; Pei, Yu-Fang; Shen, Hui; Fu, Xiaoying; He, Hao; Lu, Shan; Chen, Xiang-Ding; Tan, Li-Jun; Yang, Tie-Lin; Guo, Yan; Leo, Paul J.; Duncan, Emma L.; Shen, Jie; Guo, Yan-Fang; Nicholson, Geoffrey C.; Prince, Richard L.; Eisman, John A.; Jones, Graeme; Sambrook, Philip N.; Hu, Xiang; Das, Partha M.; Tian, Qing; Zhu, Xue-Zhen; Papasian, Christopher J.; Brown, Matthew A.; Uitterlinden, André G.; Wang, Yu-Ping; Xiang, Shuanglin; Deng, Hong-Wen

    2015-01-01

    MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNA target sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)- and femoral neck (FN)-bone mineral density (BMD). In stage I, 41 102 poly-miRTSs were meta-analyzed in seven cohorts with a genome-wide significance (GWS) α = 0.05/41 102 = 1.22 × 10−6. By applying α = 5 × 10−5 (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P = 7.67 × 10−6 and 1.58 × 10−5) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P = 5.08 × 10−3) at α = 0.10/11 = 9.09 × 10-3. PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P = 7.55 × 10−6) at α = 0.05/2 = 0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P = 8.87 × 10−12). Dual-luciferase reporter assays demonstrated that FGFRL1 3′ untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation. PMID:25941324

  2. Genome-wide analysis of KAP1, the 7SK snRNP complex, and RNA polymerase II

    PubMed Central

    McNamara, Ryan P.; Guzman, Carlos; Reeder, Jonathan E.; D'Orso, Iván

    2016-01-01

    The transition of RNA polymerase II (Pol II) from transcription initiation into productive elongation in eukaryotic cells is regulated by the P-TEFb kinase, which phosphorylates the C-terminal domain of paused Pol II at promoter-proximal regions. Our recent study found that P-TEFb (in an inhibited state bound to the 7SK snRNP complex) interacts with the KAP1/TRIM28 transcriptional regulator, and that KAP1 and the 7SK snRNP co-occupy most gene promoters containing paused Pol II. Here we provide a detailed experimental description and analysis of the ChIP-seq datasets that have been deposited into Gene Expression Omnibus (GEO): GS72622, so that independent groups can replicate and expand upon these findings. We propose these datasets would provide valuable information for researchers studying mechanisms of transcriptional regulation including Pol II pausing and pause release. PMID:26981421

  3. A Rationally Optimized Nanoparticle System for the Delivery of RNA Interference Therapeutics into Pancreatic Tumors in Vivo.

    PubMed

    Teo, Joann; McCarroll, Joshua A; Boyer, Cyrille; Youkhana, Janet; Sagnella, Sharon M; Duong, Hien T T; Liu, Jie; Sharbeen, George; Goldstein, David; Davis, Thomas P; Kavallaris, Maria; Phillips, Phoebe A

    2016-07-11

    Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition-fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (TUBB3/βIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced βIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors. PMID:27305597

  4. 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

  5. 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.

  6. Post-Natal Knockdown of Fukutin-Related Protein Expression in Muscle by Long-Term RNA Interference Induces Dystrophic Pathology

    PubMed Central

    Wang, Chi-Hsien; Chan, Yiumo Michael; Tang, Ru-Hang; Xiao, Bin; Lu, Peijuan; Keramaris-Vrantsis, Elizabeth; Zheng, Hui; Qiao, Chunping; Jiang, Jiangang; Li, Juan; Ma, Hsin-I.; Lu, Qilong; Xiao, Xiao

    2011-01-01

    Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology. PMID:21224063

  7. 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

  8. A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

    PubMed

    Su, Jianguo; Oanh, Dang T H; Lyons, Russell E; Leeton, Lisa; van Hulten, Marielle C W; Tan, Siok-Hwee; Song, Linsheng; Rajendran, K V; Walker, Peter J

    2008-02-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. PMID:18166489

  9. Genome-Wide Identification and Characterization of Long Non-Coding RNAs from Mulberry (Morus notabilis) RNA-seq Data.

    PubMed

    Song, Xiaobo; Sun, Liang; Luo, Haitao; Ma, Qingguo; Zhao, Yi; Pei, Dong

    2016-01-01

    Numerous sources of evidence suggest that most of the eukaryotic genome is transcribed into protein-coding mRNAs and also into a large number of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs), a group consisting of ncRNAs longer than 200 nucleotides, have been found to play critical roles in transcriptional, post-transcriptional, and epigenetic gene regulation across all kingdoms of life. However, lncRNAs and their regulatory roles remain poorly characterized in plants, especially in woody plants. In this paper, we used a computational approach to identify novel lncRNAs from a published RNA-seq data set and analyzed their sequences and expression patterns. In total, 1133 novel lncRNAs were identified in mulberry, and 106 of these lncRNAs displayed a predominant tissue-specific expression in the five major tissues investigated. Additionally, functional predictions revealed that tissue-specific lncRNAs adjacent to protein-coding genes might play important regulatory roles in the development of floral organ and root in mulberry. The pipeline used in this study would be useful for the identification of lncRNAs obtained from other deep sequencing data. Furthermore, the predicted lncRNAs would be beneficial towards an understanding of the variations in gene expression in plants. PMID:26938562

  10. Genome-Wide Identification and Characterization of Long Non-Coding RNAs from Mulberry (Morus notabilis) RNA-seq Data

    PubMed Central

    Song, Xiaobo; Sun, Liang; Luo, Haitao; Ma, Qingguo; Zhao, Yi; Pei, Dong

    2016-01-01

    Numerous sources of evidence suggest that most of the eukaryotic genome is transcribed into protein-coding mRNAs and also into a large number of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs), a group consisting of ncRNAs longer than 200 nucleotides, have been found to play critical roles in transcriptional, post-transcriptional, and epigenetic gene regulation across all kingdoms of life. However, lncRNAs and their regulatory roles remain poorly characterized in plants, especially in woody plants. In this paper, we used a computational approach to identify novel lncRNAs from a published RNA-seq data set and analyzed their sequences and expression patterns. In total, 1133 novel lncRNAs were identified in mulberry, and 106 of these lncRNAs displayed a predominant tissue-specific expression in the five major tissues investigated. Additionally, functional predictions revealed that tissue-specific lncRNAs adjacent to protein-coding genes might play important regulatory roles in the development of floral organ and root in mulberry. The pipeline used in this study would be useful for the identification of lncRNAs obtained from other deep sequencing data. Furthermore, the predicted lncRNAs would be beneficial towards an understanding of the variations in gene expression in plants. PMID:26938562

  11. Genome-wide identification of novel genetic markers from RNA sequencing assembly of diverse Aegilops tauschii accessions.

    PubMed

    Nishijima, Ryo; Yoshida, Kentaro; Motoi, Yuka; Sato, Kazuhiro; Takumi, Shigeo

    2016-08-01

    The wild species in the Triticeae tribe are tremendous resources for crop breeding due to their abundant natural variation. However, their huge and highly repetitive genomes have hindered the establishment of physical maps and the completeness of their genome sequences. To develop molecular markers for the efficient utilization of their valuable traits while avoiding their genome complexity, we assembled RNA sequences of ten representative accessions of Aegilops tauschii, the progenitor of the wheat D genome, and estimated single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). The deduced unigenes were anchored to the chromosomes of Ae. tauschii and barley. The SNPs and indels in the anchored unigenes, covering entire chromosomes, were sufficient for linkage map construction, even in combinations between the genetically closest accessions. Interestingly, the resolution of SNP and indel distribution on barley chromosomes was slightly higher than on Ae. tauschii chromosomes. Since barley chromosomes are regarded as virtual chromosomes of Triticeae species, our strategy allows capture of genetic markers arranged on the chromosomes in order based on the conserved synteny. The resolution of these genetic markers will be comparable to that of the Ae. tauschii whose draft genome sequence is available. Our procedure should be applicable to marker development for Triticeae species, which have no draft sequences available. PMID:27142109

  12. RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109

    PubMed Central

    ZHANG, HEPING; LI, JIANCHENG; CHENG, WENFANG; LIU, DI; CHEN, CHENG; WANG, XIAOYING; LU, XUJING; ZHOU, XIFA

    2015-01-01

    The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity

  13. 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.

  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.

    PubMed

    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

  16. A Genome-Wide mRNA Expression Profile in Caenorhabditis elegans under Prolonged Exposure to 1750MHz Radiofrequency Fields

    PubMed Central

    Gao, Dawen; Yu, Zhoulong; Wu, Tongning; Zhang, Chenggang

    2016-01-01

    Objective C. elegans has been used as a biomonitor for microwave-induced stress. However, the RF (radiofrequency) fields that have been used in previous studies were weak (≤1.8W/kg), and the bio-effects on C. elegans were mostly negative or ambiguous. Therefore, this study used more intense RF fields (SAR = 3W/kg) and longer time course of exposure (60h at 25°C, L1 stage through adult stage) to investigate the biological consequences of 1750 MHz RF fields in wild-type worms. Methods The growth rates and lifespans of RF-exposure group and the control group were carefully recorded. RNA samples were collected at L4 (35h) and gravid adult (50h) stages for further high-throughput sequencing, focusing on differences between the RF-exposure and the sham control groups. Results The RF-exposed and sham control groups developed at almost the same rate and had similar longevity curves. In L4 stage worms, 94 up-regulated and 17 down-regulated genes were identified, while 186 up-regulated and 3 down-regulated genes were identified in adult stage worms. GO analysis showed that the differentially expressed genes at 35h were associated with growth, body morphogenesis and collagen and cuticle-based development. Genes that were linked to growth rate and reproductive development were differentially expressed at 50h. Some embryonic and larval development genes in the offspring were also differentially expressed at 50h. Ten genes were differentially expressed at both 35h and 50h, most of which were involved in both embryonic and larval developmental processes. Although prolonged RF fields did not induce significant temperature increase in RF exposure groups, the temperature inside worms during exposure was unknown. Conclusions No harmful effects were observed in prolonged exposure to 1750 MHz RF fields at SAR of 3W/kg on development and longevity of C. elegans. Although some differentially expressed genes were found after prolonged RF exposure, these differences were ascribed to

  17. 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

  18. Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

    PubMed Central

    CAO, Huibi; WANG, Anan; MARTIN, Bernard; KOEHLER, David R.; ZEITLIN, Pamela L.; TANAWELL, A. Keith; HU, Jim

    2015-01-01

    Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases. PMID:15740640

  19. The RNA interference proteins and vasa locus are involved in the silencing of retrotransposons in the female germline of Drosophila melanogaster.

    PubMed

    Vagin, Vasily V; Klenov, Mikhail S; Kalmykova, Alla I; Stolyarenko, Anastasia D; Kotelnikov, Roman N; Gvozdev, Vladimir A

    2004-05-01

    RNA interference (RNAi) is considered as a defense against expansion of transposable elements. The proteins related to RNA helicase and Argonaute families are involved in RNAi process in different organisms. It was shown that Argonaute AUBERGINE and putative RNA helicase SPINDLE-E proteins were essential for RNAi in Drosophila. Here, we describe the role of aubergine (aub) and spindle-E (spn-E) genes in the control of LTR retrotransposon copia and nonLTR telomeric Het-A and I retrotransposons in ovaries. spn-E mutation causes a drastically increased lacZ expression driven by copia LTR. For the first time we show the involvement of AUBERGINE protein and VASA RNA helicase, essential for oocyte patterning, in the retrotransposon silencing. spn-E, vasa and aub mutations cause similar accumulation of both I element and Het-A transcripts in the developing oocyte. VASA and AUBERGINE proteins are known as components of perinuclear ribonucleoprotein particles in germ cells, and spn-E mutation disturbs protein content of the particles. We suggest participation of these proteins in the same silencing pathway. PMID:17194939

  20. Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive Gα Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

    PubMed Central

    Margas, Wojciech; Sedeek, Khaled; Ruiz-Velasco, Victor

    2008-01-01

    The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Gαi/o subunits. However, the identification of the specific Gα subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Gα subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Gα subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Gα protein was examined by RT-PCR experiments. Fluorescence imaging showed that Gαi1, Gαi3, and Gαo were expressed in SG neurons. The transfection of Gαi1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Gαi3 or Gαo was without effect. Taken together, these results suggest that in SG neurons Gαi1 subunits selectively couple NOP receptors to N-type Ca2+ channels. PMID:18562551