WorldWideScience

Sample records for targets rna interference

  1. Inhibition of osteoclastogenesis by RNA interference targeting RANK

    Directory of Open Access Journals (Sweden)

    Ma Ruofan

    2012-08-01

    Full Text Available Abstract Background Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK ligand (RANKL as well as the macrophage colony-stimulating factor (M-CSF. The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. Methods Three pairs of short hairpin RNAs (shRNA targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs using the optimal shRNA by targeting RANK. Results Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p Conclusions These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

  2. Targeting genes in living mammals by RNA interference

    Czech Academy of Sciences Publication Activity Database

    Podolská, Kateřina; Svoboda, Petr

    2011-01-01

    Roč. 10, č. 4 (2011), s. 238-247 ISSN 2041-2649 R&D Projects: GA ČR GA204/09/0085; GA ČR GAP305/10/2215; GA MŠk ME09039 Grant - others:EMBO SDIG(XE) 1483 Institutional research plan: CEZ:AV0Z50520514 Keywords : RNAi * shRNA * siRNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.126, year: 2011

  3. A systematic analysis of the effect of target RNA structure on RNA interference

    NARCIS (Netherlands)

    Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest

  4. Effects of RNA interference targeting Smad7 on nerve cells ischemic ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-01-10

    Jan 10, 2012 ... Effects of RNA interference targeting Smad7 on nerve cells ischemic injury induced in PC12 cells. Chunli Mei1,2, Jing Mang1, Jinting He1, Guihua Xu1, Zhongshu Li1, Wenzhao Liang1 and. Zhongxin Xu1*. 1Department of Neurology, China-Japan Union Hospital, Jilin University, Changchun 130012, ...

  5. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

    DEFF Research Database (Denmark)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

  6. Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

    Science.gov (United States)

    Fernando, Deepani D; Marr, Edward J; Zakrzewski, Martha; Reynolds, Simone L; Burgess, Stewart T G; Fischer, Katja

    2017-06-10

    Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools. We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi). We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls. A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

  7. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Wang Jianing

    2011-05-01

    Full Text Available Abstract Background The human coagulation trigger tissue factor (TF is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. Methods The specific small interfering RNA (siRNA designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. Results TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. Conclusions Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

  8. Tumor-specific RNA interference targeting Pokemon suppresses tumor growth and induces apoptosis in prostate cancer.

    Science.gov (United States)

    Li, Yining; Xu, Shuxiong; Wang, Xiangwei; Shi, Hua; Sun, Zhaolin; Yang, Zhao

    2013-02-01

    To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

    Directory of Open Access Journals (Sweden)

    Feixiang Wu

    2012-01-01

    Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  10. The RNA interference revolution

    Directory of Open Access Journals (Sweden)

    G. Lenz

    2005-12-01

    Full Text Available 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.

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

    CSIR Research Space (South Africa)

    Meliopoulos, VA

    2012-01-01

    Full Text Available 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...

  12. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  13. piggyBac transposon-derived targeting shRNA interference against the Bombyx mori nucleopolyhedrovirus (BmNPV).

    Science.gov (United States)

    Zhou, Fang; Chen, Rui-ting; Lu, Yan; Liang, Shuang; Wang, Mei-xian; Miao, Yun-gen

    2014-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.

  14. Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR–Cas system by binding to target RNA and crRNA

    Science.gov (United States)

    Li, Yingjun; Zhang, Yan; Lin, Jinzhong; Pan, Saifu; Han, Wenyuan; Peng, Nan; Liang, Yun Xiang

    2017-01-01

    Abstract CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR–Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔβΔ1α, Δβ1α-M1 and Δβ1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR–Cas systems in thermophiles. PMID:28977458

  15. Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR-Cas system by binding to target RNA and crRNA.

    Science.gov (United States)

    Li, Yingjun; Zhang, Yan; Lin, Jinzhong; Pan, Saifu; Han, Wenyuan; Peng, Nan; Liang, Yun Xiang; She, Qunxin

    2017-11-02

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR-Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔβΔ1α, Δβ1α-M1 and Δβ1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR-Cas systems in thermophiles. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. RNA interference in Lepidoptera

    DEFF Research Database (Denmark)

    Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

    2011-01-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...

  17. Lentivirus-mediated shRNA interference targeting SLUG inhibits lung cancer growth and metastasis.

    Science.gov (United States)

    Wang, Yao-Peng; Wang, Ming-Zhao; Luo, Yi-Ren; Shen, Yi; Wei, Zhao-Xia

    2012-01-01

    Lung cancer is a deadly cancer, whose kills more people worldwide than any other malignancy. SLUG (SNAI2, Snail2) is involved in the epithelial mesenchymal transition in physiological and in pathological contexts and is implicated in the development and progression of lung cancer. We constructed a lentivirus vector with SLUG shRNA (LV-shSLUG). LV-shSLUG and a control lentivirus were infected into the non-small cell lung cancer cell A549 and real-time PCR, Western blot and IHC were applied to assess expression of the SLUG gene. Cell proliferation and migration were detected using MTT and clony formation methods. Real-time PCR, Western Blot and IHC results confirmed down-regulation of SLUG expression by its shRNA by about 80%~90% at both the mRNA and protein levels. Knockdown of SLUG significantly suppressed lung cancer cell proliferation. Furthermore, knockdown of SLUG significantly inhibited lung cancer cell invasion and metastasis. Finally, knockdown of SLUG induced the down-regulation of Bcl-2 and up-regulation of E-cadherin. These results indicate that SLUG is a newly identified gene associated with lung cancer growth and metastasis. SLUG may serve as a new therapeutic target for the treatment of lung cancer in the future.

  18. Gene expression: RNA interference in adult mice

    Science.gov (United States)

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

    2002-07-01

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

  19. RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans.

    Science.gov (United States)

    Grishok, Alla; Hoersch, Sebastian; Sharp, Phillip A

    2008-12-23

    In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.

  20. Systematic Identification and Assessment of Therapeutic Targets for Breast Cancer Based on Genome-Wide RNA Interference Transcriptomes

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-02-01

    Full Text Available With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA. Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.

  1. Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

    International Nuclear Information System (INIS)

    Xue Yan; Bi Feng; Zhang Xueyong; Pan Yanglin; Liu Na; Zheng Yi; Fan Daiming

    2004-01-01

    Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Rac1, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To further define Rac1 function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Rac1 in gastric cancer cell line AGS that expresses a high level of Rac1. Both the mRNA and protein levels of Rac1 in the AGS cells were decreased dramatically after transfection with a Rac1-specific siRNA vector. When the conditioned medium derived from the Rac1 downregulated AGS cells was applied to the human endothelial cells, it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-1α, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors, were upregulated in the Rac1 siRNA transfected cells. Our results suggest that Rac1 may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Rac1 GTPase

  2. RNA interference as a method for target-site screening in the Western corn rootworm, Diabrotica virgifera virgifera.

    Science.gov (United States)

    Alves, Analiza P; Lorenzen, Marcé D; Beeman, Richard W; Foster, John E; Siegfried, Blair D

    2010-01-01

    To test the efficacy of RNA interference (RNAi) as a method for target-site screening in Diabrotica virgifera virgifera LeConte (Coleptera: Chrysomelidae) larvae, genes were identified and tested for which clear RNAi phenotypes had been identified in the Coleopteran model, Tribolium castaneum. Here the cloning of the D. v. vergifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2) is reported. Injection of DvvLac2-specific double-stranded RNA resulted in prevention of post-molt cuticular tanning, while injection of DvvCHS2-specific dsRNA reduced chitin levels in midguts. Silencing of both DvvLac2 and DvvCHS2 was confirmed by RT-PCR and quantitative RT-PCR. As in T. castaneum, RNAi-mediated gene silencing is systemic in Diabrotica. The results indicate that RNAi-induced silencing of D. v. vergifera genes provides a powerful tool for identifying potential insecticide targets.

  3. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Generation of siRNA Nanosheets for Efficient RNA Interference

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Chen Jie

    2012-10-01

    Full Text Available Abstract 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.

  7. Hepatic-targeted RNA interference provides persistent knockdown of alpha-1 antitrypsin levels in ZZ patients.

    Science.gov (United States)

    Turner, Alice M; Stolk, Jan; Bals, Robert; Lickliter, Jason; Hamilton, James; Christianson, Dawn R; Given, Bruce D; Burdon, Jonathan G; Loomba, Rohit; Stoller, James K; Teckman, Jeffery H

    2018-03-20

    Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder causing pulmonary and liver disease. The PiZ mutation results in mis-folded alpha-1 antitrypsin protein (Z-AAT) leading to hepatocyte accumulation, fibrosis and cirrhosis. RNAi-based therapeutics silencing production of hepatic Z-AAT might benefit patients with AATD-associated liver disease. This study evaluated an RNAi therapeutic to silence production of alpha-1 antitrypsin. Part A of this double-blind first-in-human study randomized 54 healthy volunteers (HVs) into single dose cohorts (2 placebo: 4 active), receiving escalating doses of the investigational agent ARC-AAT from 0.38 to 8.0 mg/kg or placebo. Part B randomized 11 PiZZ genotype AATD patients who received up to 4.0 mg/kg or placebo. Patients with baseline FibroScan® >11 kPa or FEV 1 (forced expiratory volume in one second) < 60% were excluded. Assessments included safety, pharmacokinetics, and change in serum alpha-1 antitrypsin (AAT) concentrations. 36 healthy volunteers received ARC-AAT and 18 received placebo (Part A). Seven PiZZ individuals received ARC-AAT and 4 received placebo (Part B). A dose-response in serum AAT reduction was observed at doses ≥ 4 mg/kg with similar relative reductions in PiZZ patients and HVs at 4 mg/kg and a maximum reduction of 76.1% (HVs) vs. 78.8% (PiZZ) at this dose. Time for serum AAT return to baseline was similar for HV and PiZZ. There were no notable differences between HV and PiZZ safety parameters. The study was terminated early due to toxicity findings related to the delivery vehicle (ARC-EX1) seen in a non-human primate study. PiZZ and HV responded similarly to ARC-AAT. Deep and durable knockdown of hepatic AAT production based on observed reduction in serum AAT concentrations was demonstrated. Accumulation of abnormal proteins in the livers of patients with alpha-1 antitrypsin deficiency may lead to decreased liver function and potentially liver failure. Therapeutics targeting the production of

  8. RNA interference in plant parasitic nematodes

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... nematodes. RNAi should help identify gene and, hence, protein targets for nematode control strategies. Key words: RNA interference, RNAi, gene expression, plant parasitic nematodes. INTRODUCTION. Plant parasitic nematodes are found as pests of crops throughout the world with many having a severe ...

  9. Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi targets based on Gibbs free energy

    Directory of Open Access Journals (Sweden)

    Tiago Campos Pereira

    2007-01-01

    Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

  10. α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

    Directory of Open Access Journals (Sweden)

    Yuan-Fei Peng

    Full Text Available RNA interference (RNAi has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1 was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3 and non-HCC cell lines (L-02, Hela and SW1116 were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5 was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.The AFP-miRNA system could silence target gene (Beclin 1 but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1 in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA was successfully established. The system provides a usable tool for HCC-specific RNAi

  11. A Halloween gene shadow is a potential target for RNA-interference-based pest management in the small brown planthopper Laodelphax striatellus.

    Science.gov (United States)

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2015-02-01

    Laodelphax striatellus is an economically important rice pest in China. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. The cytochrome P450 monooxygenase Shadow (Sad) plays a critical role in ecdysteroidogenesis. Here, tests were conducted to establish whether Lssad was a potential target gene for RNA-interference-based management of L. striatellus. Lssad was cloned and characterised. LsSad had Helix-C, Helix-I, Helix-K, PERF and haem-binding motifs. Lssad is expressed at a higher level in the thorax, where prothoracic glands are located, compared with the level in the head or abdomen. It showed two expression peaks in day 2 and day 4-5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Oral delivery of double-stranded RNA (dsRNA) of Lssad at the nymph stage successfully knocked down the expression of the target gene, reduced the expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development in a dose-dependent manner. Ingestion of 20-hydroxyecdysone in Lssad-dsRNA-exposed nymphs did not increase Lssad expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. The ecdysteroidogenic pathway is conserved in L. striatellus. Lssad can serve as a possible target for dsRNA-based pesticides for planthopper control. © 2014 Society of Chemical Industry.

  12. RNA interference and Register Machines (extended abstract

    Directory of Open Access Journals (Sweden)

    Masahiro Hamano

    2012-11-01

    Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

  13. RNA interference in designing transgenic crops.

    Science.gov (United States)

    Ali, Nusrat; Datta, Swapan K; Datta, Karabi

    2010-01-01

    RNA interference (RNAi) is a sequence specific gene silencing mechanism, triggered by the introduction of dsRNA leading to mRNA degradation. It helps in switching on and off the targeted gene, which might have significant impact in developmental biology. Discovery of RNAi represents one of the most promising and rapidly advancing frontiers in plant functional genomics and in crop improvement by plant metabolic engineering and also plays an important role in reduction of allergenicity by silencing specific plant allergens. In plants the RNAi technology has been employed successfully in improvement of several plant species- by increasing their nutritional value, overall quality and by conferring resistance against pathogens and diseases. The review gives an insight to the perspective use of the technology in designing crops with innovation, to bring improvement to crop productivity and quality.

  14. Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

    Science.gov (United States)

    Xiao, Su; Chen, Yu Chi; Buehler, Eugen; Mandal, Swati; Mandal, Ajeet; Betenbaugh, Michael; Park, Myung Hee; Martin, Scott; Shiloach, Joseph

    2016-11-01

    For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. A 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport, and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1-was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Biotechnol. Bioeng. 2016;113: 2403-2415. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling

    Directory of Open Access Journals (Sweden)

    Craigon Marie

    2009-08-01

    Full Text Available Abstract Background Interferons (IFNs are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs. Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ. Results Transfection of murine bone-marrow derived macrophages (BMDMs with a non-targeting (control siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000 prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response. Conclusion Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated

  16. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  17. RNA interference against viruses: strike and counterstrike

    NARCIS (Netherlands)

    Haasnoot, Joost; Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral

  18. Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles.

    Science.gov (United States)

    Afonin, Kirill A; Viard, Mathias; Kagiampakis, Ioannis; Case, Christopher L; Dobrovolskaia, Marina A; Hofmann, Jen; Vrzak, Ashlee; Kireeva, Maria; Kasprzak, Wojciech K; KewalRamani, Vineet N; Shapiro, Bruce A

    2015-01-27

    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.

  19. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  20. RNA interference: its use as antiviral therapy

    NARCIS (Netherlands)

    Haasnoot, J.; Berkhout, B.

    2006-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more

  1. Characterization of RNA interference in rat PC12 cells

    DEFF Research Database (Denmark)

    Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia

    2004-01-01

    to achieve RNAi in a cultured rat neuronal cell line, PC12. Targeting of neuropeptide Y mRNA by synthetic siRNA results in knock down of the mRNA levels with an IC50 of approximately 0.1 nM. The mRNA knockdown lasts for at least 96 h and is not dependent on protein synthesis. Further, PC12 cells were ablated...... of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells.......Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous...

  2. Prokaryotic Argonautes – variations on the RNA interference theme

    Directory of Open Access Journals (Sweden)

    John van der Oost

    2014-04-01

    Full Text Available The discovery of RNA interference (RNAi has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago, an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes.

  3. Prokaryotic Argonautes - variations on the RNA interference theme

    Science.gov (United States)

    van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

    2014-01-01

    The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

  4. Small Molecule Modifiers of the microRNA and RNA Interference Pathway

    OpenAIRE

    Deiters, Alexander

    2009-01-01

    Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

  5. Symbiont-mediated RNA interference in insects

    Science.gov (United States)

    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

  6. In Vivo Imaging of RNA Interference

    OpenAIRE

    Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    RNA interference (RNAi), an effective technique for regulating/silencing specific genes, can be applied to treat various diseases. Multiple clinical trials using RNAi are ongoing and molecular imaging can serve as a powerful tool in RNAi-based therapies. This brief review will highlight the current progress on in vivo imaging of RNAi delivery and silencing effects. Incorporation of suitable molecular imaging techniques into future RNAi-based clinical trials will provide more pieces of the puz...

  7. RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema.

    Science.gov (United States)

    Kai, Yoshiro; Tomoda, Koichi; Yoneyama, Hiroyuki; Yoshikawa, Masanori; Kimura, Hiroshi

    2015-12-09

    Chondroitin sulfate proteoglycans are an important mediators in inflammation and leukocyte trafficking. However, their roles in pulmonary emphysema have not been explored. In a murine model of elastase-induced pulmonary emphysema, we found increased carbohydrate sulfotransferase 3 (CHST3), a specific enzyme that synthesizes chondroitin 6-sulfate proteoglycan (C6SPG). To elucidate the role of C6SPG, we investigated the effect of small interfering RNA (siRNA) targeting CHST3 that inhibits C6SPG-synthesis on the pathogenesis of pulmonary emphysema. Mice were intraperitoneally injected with CHST3 siRNA or negative control siRNA on day0 and 7 after intratracheal instillation of elastase. Histology, respiratory function, glycosaminoglycans (GAGs) content, bronchoalveolar lavage (BAL), elastin staining and gene expressions of tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-9 mRNA were evaluated on day7 and/or day21. CHST3 mRNA increased at day 7 and decreased thereafter in lung. CHST3 siRNA successfully inhibited the expression of CHST3 mRNA throughout the study and this was associated with significant reduction of GAGs and C6SPG. Airway destruction and respiratory function were improved by the treatment with CHST3 siRNA. CHST3 siRNA reduced the number of macrophages both in BAL and lung parenchyma and also suppressed the increased expressions of TNF-α and MMP-9 mRNA. Futhermore, CHST3 siRNA improved the reduction of the elastin in the alveolar walls. CHST3 siRNA diminishes accumulation of excessive macrophages and the mediators, leading to accelerate the functional recovery from airway damage by repair of the elastin network associated with pulmonary emphysema.

  8. Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

    Science.gov (United States)

    Shimizu, Takumi; Ogamino, Takumi; Hiraguri, Akihiro; Nakazono-Nagaoka, Eiko; Uehara-Ichiki, Tamaki; Nakajima, Masami; Akutsu, Katsumi; Omura, Toshihiro; Sasaya, Takahide

    2013-05-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

  9. A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

    Directory of Open Access Journals (Sweden)

    Kivork Christine

    2007-10-01

    Full Text Available Abstract Background Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.

  10. Domain motions of Argonaute, the catalytic engine of RNA interference

    Directory of Open Access Journals (Sweden)

    Wall Michael E

    2007-11-01

    Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

  11. RNA Interference Targeting Snail Inhibits the Transforming Growth Factor β2-Induced Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Ping Li

    2013-01-01

    Full Text Available Epithelial-msenchymal transition (EMT contributes to posterior capsule opacification (PCO type of cataract. Transcription factors Snail is a key trigger of EMT activated by transforming growth factor β (TGFβ. This study was done to investigate the effect of Snail targeting siRNA on TGFβ2-induced EMT in human lens epithelial cells. TGFβ2 treatment of cultured human epithelial cell line (HLEB3 upregulated the expression of Snail and the EMT relevant molecules such as vimentin and α-SMA but downregulated the expression of keratin and E-cadherin. After the stimulation of TGFβ2, the HLEB3 cells became fibroblast-like in morphology, and the junctions of cell-cell disappeared. TGFβ2 treatment also enhanced migration ability of HLEB3 cells. TGFβ2-induced Snail expression and EMT were significantly inhibited by Snail siRNA. By analyzing the response characteristics of HLEB3 in TGFβ2-induced EMT model with/without Snail-specific siRNA, we concluded that Snail is an element in the EMT of HLEB3 cells induced by TGFβ2. Snail siRNA targeting can block the induced EMT and therefore has the potential to suppress the development of PCO.

  12. RNA Interference and its therapeutic applications

    Directory of Open Access Journals (Sweden)

    Srinivasa Rao T

    2011-10-01

    Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

  13. Inhibition of Monkeypox virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

  14. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids...... was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones...

  15. Induction of RNA interference in dendritic cells.

    Science.gov (United States)

    Li, Mu; Qian, Hua; Ichim, Thomas E; Ge, Wei-Wen; Popov, Igor A; Rycerz, Katarzyna; Neu, John; White, David; Zhong, Robert; Min, Wei-Ping

    2004-01-01

    Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

  16. RNA-interference gene knockdown in the poultry red mite ...

    African Journals Online (AJOL)

    Lucy

    2013-03-20

    Mar 20, 2013 ... interference; dsRNA, double stranded RNA; FEC, faecal egg counts; siRNAs ... resistance, residues in meat and eggs and other environmental .... 1352 Afr. J. Biotechnol. Table 1. Total RNA extracted from varying number of mites. Number of mite. Concentration of RNA (ng/µl. Total RNA (ng). 1. 14.6. 146. 1.

  17. Role of RNA interference in plant improvement.

    Science.gov (United States)

    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.

  18. Role of RNA interference in plant improvement

    Science.gov (United States)

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

    2011-06-01

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

  19. Ran Involved in the Development and Reproduction Is a Potential Target for RNA-Interference-Based Pest Management in Nilaparvata lugens.

    Directory of Open Access Journals (Sweden)

    Kai-Long Li

    Full Text Available Ran (RanGTPase in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1 and vitellogenin, are involved. A putative Ran gene (NlRan was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1 Extended body form, and failed to molt; (2 The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control.

  20. RNA Interference - Towards RNA becoming a Medicine -42 ...

    Indian Academy of Sciences (India)

    RN Ai in Therapeutics and Research. RNAi possesses great potential as a therapeutic agent by its virtue to silence genes. Many diseases are being targeted, like. AIDS, tumors, Hepatitis C, Malaria, Polio to name a few. Inacti- vation of critical proteins involved in pathogenesis is the prin- ciple use of siRNA. In case of AIDS, ...

  1. RNA Interference in Fungi: Retention and Loss.

    Science.gov (United States)

    Nicolás, Francisco E; Garre, Victoriano

    2016-12-01

    RNA interference (RNAi) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding RNAs (sRNAs) of about 20 to 30 nucleotides. Its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. Initially described and characterized in Neurospora crassa, the RNAi is widespread in fungi, suggesting that it plays important functions in the fungal kingdom. Several RNAi-related mechanisms for maintenance of genome integrity, particularly protection against exogenous nucleic acids such as mobile elements, have been described in several fungi, suggesting that this is the main function of RNAi in the fungal kingdom. However, an increasing number of fungal sRNAs with regulatory functions generated by specific RNAi pathways have been identified. Several mechanistic aspects of the biogenesis of these sRNAs are known, but their function in fungal development and physiology is scarce, except for remarkable examples such as Mucor circinelloides, in which specific sRNAs clearly regulate responses to environmental and endogenous signals. Despite the retention of RNAi in most species, some fungal groups and species lack an active RNAi mechanism, suggesting that its loss may provide some selective advantage. This article summarizes the current understanding of RNAi functions in the fungal kingdom.

  2. RNA interference targeting CD147 inhibits the proliferation, invasiveness, and metastatic activity of thyroid carcinoma cells by down-regulating glycolysis.

    Science.gov (United States)

    Huang, Peng; Chang, Shi; Jiang, Xiaolin; Su, Juan; Dong, Chao; Liu, Xu; Yuan, Zhengtai; Zhang, Zhipeng; Liao, Huijun

    2015-01-01

    A high rate of glycolytic flux, even in the presence of oxygen, is a key metabolic hallmark of cancer cells. Lactate, the end product of glycolysis, decreases the extracellular pH and contributes to the proliferation, invasiveness and metastasis of tumor cells. CD147 play a crucial role in tumorigenicity, invasion and metastasis; and CD147 also interacts strongly and specifically with monocarboxylate transporter1 (MCT1) that mediates the transport of lactate. The objective of this study was to determine whether CD147 is involved, via its association with MCT1 to transport lactate, in glycolysis, contributing to the progression of thyroid carcinoma. The expression levels of CD147 in surgical specimens of normal thyroid, nodular goiter (NG), well-differentiated thyroid carcinoma (WDTC), and undifferentiated thyroid carcinoma (UDTC) were determined using immunohistochemical techniques. The effects of CD147 silencing on cell proliferation, invasiveness, metastasis, co-localization with MCT1, glycolysis rate and extracellular pH of thyroid cancer cells (WRO and FRO cell lines) were measured after CD147 was knocked-down using siRNA targeting CD147. Immunohistochemical analysis of thyroid carcinoma (TC) tissues revealed significant increases in signal for CD147 compared with normal tissue or NG, while UDTC expressed remarkably higher levels of CD147 compared with WDTC. Furthermore, silencing of CD147 in TC cells clearly abrogated the expression of MCT1 and its co-localization with CD147 and dramatically decreased both the glycolysis rate and extracellular pH. Thus, cell proliferation, invasiveness, and metastasis were all significantly decreased by siRNA. These results demonstrate in vitro that the expression of CD147 correlates with the degree of dedifferentiation of thyroid cancer, and show that CD147 interacts with MCT1 to regulate tumor cell glycolysis, resulting in the progression of thyroid carcinoma.

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

    Directory of Open Access Journals (Sweden)

    Jin Xu

    2016-10-01

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

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  6. Progress and challenges in RNA interference therapy for Huntington disease.

    Science.gov (United States)

    Harper, Scott Q

    2009-08-01

    Huntington disease is an incurable, dominant neurodegenerative disorder caused by polyglutamine repeat expansion in the huntingtin protein. Reducing mutant huntingtin expression may offer a treatment for Huntington disease. RNA interference has emerged as a powerful method to silence dominant disease genes. As such, it is being developed as a prospective Huntington disease therapy. Here I discuss the current progress and important remaining challenges of RNA interference therapy for Huntington disease.

  7. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2018-01-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

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

    Science.gov (United States)

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

    2015-05-01

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

  9. RNA Interference - Towards RNA becoming a Medicine -42 ...

    Indian Academy of Sciences (India)

    ph~nomenon in C.elegans. They were attempting'to use antisens'c'RNA as an approach to Inhibit gene expression. They found that sense and antisense RNA forming a double. stranded RNA was a better silencing trigger than antisense RNA. After the discovery ofRNAi in C.elegans, identification of the RNAi pathway was ...

  10. Role of RNA interference (RNAi) in the moss Physcomitrella patens

    KAUST Repository

    Arif, Muhammad Asif

    2013-01-14

    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. 2013 by the authors; licensee MDPI, Basel, Switzerland.

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

    Science.gov (United States)

    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…

  12. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2017-11-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

  13. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  14. The first discovery of RNA interference by RNA restriction enzymes to inhibit protein synthesis.

    Science.gov (United States)

    Inouye, Masayori

    2017-01-15

    In this article, I review how an RNA restriction enzyme, a highly sequence-specific endoribonuclease, was for the first time discovered in 2003 and how the concept of RNA interference using RNA restriction enzymes or mRNA interferases has been developed. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. mediated RNA interference in bovine fibroblast cells

    African Journals Online (AJOL)

    Jane

    2011-08-03

    Aug 3, 2011 ... Melanocortin receptor 4 (MC4R) is a key element in the mechanisms used to regulate both aspects of keeping the balance between energy uptake and energy expenditure. MC4R was knocked down by lentivirus-mediated shRNA expressing plasmids, which were controlled by the U6 promoter in bovine.

  16. RNA Interference-Towards RNA becoming a Medicine

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 8; Issue 12. RNA Inteference – Towards RNA becoming a Medicine. Subhanjan Mondal. General Article Volume 8 Issue 12 December 2003 pp 42-49. Fulltext. Click here to view fulltext PDF. Permanent link:

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

    Science.gov (United States)

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

    2013-01-01

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

  18. Establishment and characterization of Prnp knockdown neuroblastoma cells using dual microRNA-mediated RNA interference

    Science.gov (United States)

    Lau, Agnes; Westaway, David; McKenzie, Debbie; Aiken, Judd; Kim, Yong-Sun

    2011-01-01

    Prion diseases are fatal transmissible neurodegenerative disorders. In the pathogenesis of the disease, the cellular prion protein (PrPC) is required for replication of abnormal prion (PrPSc), which results in accumulation of PrPSc. Although there have been extensive studies using Prnp knockout systems, the normal function of PrPC remains ambiguous. Compared with conventional germline knockout technologies and transient naked siRNA-dependent knockdown systems, newly constructed durable chained-miRNA could provide a cell culture model that is closer to the disease status and easier to achieve with no detrimental sequelae. The selective silencing of a target gene by RNA interference (RNAi) is a powerful approach to investigate the unknown function of genes in vitro and in vivo. To reduce PrPC expression, a novel dual targeting-microRNA (miRdual) was constructed. The miRdual, which targets N- and C-termini of Prnp simultaneously, more effectively suppressed PrPC expression compared with conventional single site targeting. Furthermore, to investigate the cellular change following PrPC depletion, gene expression analysis of PrPC interacting and/or associating genes and several assays including proliferation, viability and apoptosis were performed. The transcripts 670460F02Rik and Plk3, Ppp2r2b and Csnk2a1 increase in abundance and are reported to be involved in cell proliferation and mitochondrial-mediated apoptosis. Dual-targeting RNAi with miRdual against Prnp will be useful for analyzing the physiological function of PrPC in neuronal cell lines and may provide a potential therapeutic intervention for prion diseases in the future. PMID:21494092

  19. Toward a durable treatment of HIV-1 infection using RNA interference

    NARCIS (Netherlands)

    Eekels, Julia J. M.; Berkhout, Ben

    2011-01-01

    RNA interference (RNAi) is a cellular mechanism that mediates sequence-specific gene silencing at the posttranscriptional level. RNAi can be used as an antiviral approach against human pathogens. An attractive target for RNAi therapeutics is the human immunodeficiency virus type 1 (HIV-1), and the

  20. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    2014-07-31

    Jul 31, 2014 ... cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infec- tion. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach. [Gan D., Ding F., Zhuang D., Jiang H., ...

  1. From cross protection to RNA interference and DNA vaccination

    Indian Academy of Sciences (India)

    2012-10-18

    Oct 18, 2012 ... The award in 2006 of the Nobel Prize in Physiology or. Medicine to Andrew Fire and Craig Mello immediately aroused a controversy. They were rewarded for their discov- ery and characterization in C. elegans of the mechanisms of. RNA interference (Fire 2006; Mello 2006). A small group of.

  2. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 2. Application of RNA interference methodology to investigate and develop SCMV resistance in maize. Defang Gan Fei Ding Dan Zhuang Haiyang Jiang Tong Jiang Suwen Zhu Beijiu Cheng. Research Article Volume 93 Issue 2 August 2014 pp 305-311 ...

  3. RNA interference, arthropod-borne viruses, and mosquitoes.

    Science.gov (United States)

    Sanchez-Vargas, Irma; Travanty, Emily A; Keene, Kimberly M; Franz, Alexander W E; Beaty, Barry J; Blair, Carol D; Olson, Ken E

    2004-06-01

    RNA interference (RNAi) probably functions as an antiviral mechanism in most eukaryotic organisms. Variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. There are three lines of evidence that show the RNAi pathway exists in Aedes species that transmit arboviruses. The first is that recombinant Sindbis viruses expressing a RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) interfere with DENV-2 replication in Aedes aegypti mosquitoes by a mechanism similar to virus-induced gene silencing described in plants. The second is that transfection of C6/36 (Aedes albopictus) cells with either double-stranded RNA or synthetic small interfering RNAs derived from an arbovirus genome interferes with replication of the homologous virus. The third is that a hairpin DENV-2-specific RNA transcribed from a plasmid can generate virus-resistant C6/36 cells. We hypothesize that genetically modified mosquitoes can be generated that transcribe a flavivirus-specific dsRNA, triggering the RNAi response soon after ingestion of a blood meal. This could induce the RNAi pathway in the midgut prior to establishment of virus infection and profoundly change vector competence. Towards this goal, we are developing transgenic A. aegypti lines that are refractory to DENV by exploiting the RNAi pathway.

  4. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    Directory of Open Access Journals (Sweden)

    Gerlinde A. Metz

    2006-01-01

    Full Text Available In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt. Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They include targeting the symptoms of the disease, the progression of the disease and the cause of the disease. By using RNA interference (RNAi, the cause of the disease can be targeted. RNAi is a method that could potentially silence the formation of abnormal htt. This paper will discuss how RNAi could potentially cure Huntington's disease, by describing the genetic and proteinomic basis of Huntington's disease, the function of RNAi in Huntington's disease and the problems of benefits of RNAi. Preliminary work using RNAi in transgenic mice has shown a decrease in the behavioural expression of the mutant Huntington gene. There are several limitations associated with using RNAi as a gene therapy. For example, the effects of RNAi are short lived. A transposition system such as Sleeping Beauty can be used to increase the integration of the gene, however, for patients who currently have Huntington's disease, RNAi may potentially be used in combination with drugs or other treatments to target both symptoms and the underlying cause of Huntington's disease. This combination could eventually alleviate many painful symptoms associated with Huntington's disease and could even stop the progressive neurodegeneration of Huntington's disease. This review concludes that a substantial amount of new research is still necessary before RNAi is directly applicable to human patients with Huntington's disease.

  5. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision

    Directory of Open Access Journals (Sweden)

    Hubert Denise

    2014-01-01

    Full Text Available TT-034 (PF-05095808 is a recombinant adeno-associated virus serotype 8 (AAV8 agent expressing three short hairpin RNA (shRNA pro-drugs that target the hepatitis C virus (HCV RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA drugs. Using next-generation sequencing (NGS to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi enzymes Dicer and siRNA-induced silencing complex (siRISC.

  6. RNA Interference Prevents Autosomal-Dominant Hearing Loss.

    Science.gov (United States)

    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

    2016-06-02

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Sims, David; Mendes-Pereira, Ana M; Frankum, Jessica; Burgess, Darren; Cerone, Maria-Antonietta; Lombardelli, Cristina; Mitsopoulos, Costas; Hakas, Jarle; Murugaesu, Nirupa; Isacke, Clare M; Fenwick, Kerry; Assiotis, Ioannis; Kozarewa, Iwanka; Zvelebil, Marketa; Ashworth, Alan; Lord, Christopher J

    2011-10-21

    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.

  8. Host Recognition Responses of Western (Family: Chrysomelidae) Corn Rootworm Larvae to RNA Interference and Bt Corn

    OpenAIRE

    Zukoff, Sarah N.; Zukoff, Anthony L.

    2017-01-01

    Western corn rootworm Diabrotica virgifera virgifera LeConte is an important pest of corn whose larvae exhibit particular quantifiable patterns of locomotion after exposure to, and removal from, host roots and nonhost roots. Using EthoVision software, the behavior and locomotion of the western corn rootworm larvae was analyzed to determine the level of host recognition to germinated roots of differing corn hybrids containing either rootworm targeted Bt genes, RNA interference (RNAi) technolog...

  9. Intervention of radiation‐induced skin fibrosis by RNA interference

    DEFF Research Database (Denmark)

    Nawroth, Isabel

    Radiation therapy is a cornerstone of cancer treatment for many types of cancer, but is often limited by normal tissue toxicity. One of the most common and dose‐limiting long‐term adverse effects is radiation‐induced fibrosis (RIF), which is characterized by restricted tissue flexibility, reduced......‐induced skin fibrosis. Chitosan‐based nanoparticles (or polyplexes) formed by self‐assembly with siRNA were applied to overcome extracellular and intracellular barriers and deliver siRNA site‐specific. In this work we show that intraperitoneal administration of chitosan/DsiRNA nanoparticles targeting TNFα...

  10. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

    NARCIS (Netherlands)

    Westerhout, Ellen M.; Ooms, Marcel; Vink, Monique; Das, Atze T.; Berkhout, Ben

    2005-01-01

    HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed

  11. RNA interference by feeding in vitro synthesized double-stranded RNA to planarians: methodology and dynamics

    Science.gov (United States)

    Rouhana, Labib; Weiss, Jennifer A.; Forsthoefel, David J.; Lee, Hayoung; King, Ryan S.; Inoue, Takeshi; Shibata, Norito; Agata, Kiyokazu; Newmark, Phillip A.

    2013-01-01

    Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time, and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. PMID:23441014

  12. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

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

    Directory of Open Access Journals (Sweden)

    Valentina Vongrad

    Full Text Available 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.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.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.

  14. RNA-dependent RNA targeting by CRISPR-Cas9

    OpenAIRE

    Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A; Doudna, Jennifer A

    2018-01-01

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA...

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

    Science.gov (United States)

    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.

  16. RNA-dependent RNA targeting by CRISPR-Cas9.

    Science.gov (United States)

    Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A; Doudna, Jennifer A

    2018-01-05

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. © 2018, Strutt et al.

  17. Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

    International Nuclear Information System (INIS)

    Schulze-Bergkamen, Henning; Fleischer, Binje; Schuchmann, Marcus; Weber, Achim; Weinmann, Arndt; Krammer, Peter H; Galle, Peter R

    2006-01-01

    Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1

  18. RNA as a small molecule druggable target.

    Science.gov (United States)

    Rizvi, Noreen F; Smith, Graham F

    2017-12-01

    Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs.

    Directory of Open Access Journals (Sweden)

    Xiaoxue Li

    Full Text Available BACKGROUND: RNA interference (RNAi is a powerful method to inhibit gene expression in a sequence specific manner. Recently silencing the target gene through feeding has been successfully carried out in many insect species. METHODOLOGY/PRINCIPAL FINDINGS: Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting genes that encode ribosomal protein Rpl19, V type ATPase D subunit, the fatty acid elongase Noa and a small GTPase Rab11. qRT-PCR showed that mRNA level of four target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of each gene varied from 35% to 100%. Tissue specific examination indicated that RNAi could be observed not only in midgut but also in other tissues like the ovary, nervous system and fat body. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group in four tissues on day 7 after continuous feeding either dsRNA or engineered bacteria. The maximum over-expression is 21 times compared to ds-egfp control group. Up-regulation of rab11 mRNA level could be observed in testes on day 7 after continuous bacteria treatment and in midgut on day 2 after ds-rab11 treatment. This phenomenon could also be observed in rpl19 groups. CONCLUSIONS: Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in Bactrocera dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed.

  20. Co-expression of Argonaute2 enhances short hairpin RNA-induced RNA interference in Xenopus CNS neurons in vivo

    Directory of Open Access Journals (Sweden)

    Chih-ming Chen

    2009-07-01

    Full Text Available RNA interference (RNAi is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi and pReporter (pRe constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex (RISC, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30 enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

  1. RNA interference-based resistance against a legume mastrevirus

    Directory of Open Access Journals (Sweden)

    Mansoor Shahid

    2011-11-01

    Full Text Available Abstract Background RNA interference (RNAi is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan. Results Here a hairpin (hpRNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants. Conclusions The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.

  2. Retention and loss of RNA interference pathways in trypanosomatid protozoans.

    Directory of Open Access Journals (Sweden)

    Lon-Fye Lye

    2010-10-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    Directory of Open Access Journals (Sweden)

    Jinlong Guo

    2015-01-01

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

  5. Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification of Target Interference Motifs in Escherichia coli.

    Science.gov (United States)

    Almendros, Cristóbal; Mojica, Francisco J M

    2015-01-01

    Plasmid transformation into a bacterial host harboring a functional CRISPR-Cas system targeting a sequence in the transforming molecule can be specifically hindered by CRISPR-mediated interference. In this case, measurements of transformation efficacy will provide an estimation of CRISPR activity. However, in order to standardize data of conventional assays (using a single plasmid in the input DNA sample), transformation efficiencies have to be compared to those obtained for a reference molecule in independent experiments. Here we describe the use of a transforming mixture of plasmids that includes the non-targeted vector as an internal reference to obtain normalized data which are unbiased by empirical variations.

  6. Macros in microRNA target identification

    Science.gov (United States)

    Tarang, Shikha; Weston, Michael D

    2014-01-01

    MicroRNAs (miRNAs) are short RNA molecules that modulate post-transcriptional gene expression by partial or incomplete base-pairing to the complementary sequences on their target genes. Sequence-based miRNA target gene recognition enables the utilization of computational methods, which are highly informative in identifying a subset of putative miRNA targets from the genome. Subsequently, single miRNA–target gene binding is evaluated experimentally by in vitro assays to validate and quantify the transcriptional or post-transcriptional effects of miRNA–target gene interaction. Although ex vivo approaches are instructive in providing a basis for further analyses, in vivo genetic studies are critical to determine the occurrence and biological relevance of miRNA targets under physiological conditions. In the present review, we summarize the important features of each of the experimental approaches, their technical and biological limitations, and future challenges in light of the complexity of miRNA target gene recognition. PMID:24717361

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

    Science.gov (United States)

    Bouxsein, Nathan

    2005-03-01

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

  8. Long-term RNA interference from optimized siRNA expression constructs in adult mice.

    Science.gov (United States)

    Wooddell, Christine I; Van Hout, Cristopher V; Reppen, Thomas; Lewis, David L; Herweijer, Hans

    2005-08-19

    DNA constructs for small interfering RNA (siRNA) expression in mammalian cells have the potential for longer-term target gene knockdown than synthetic siRNAs. We compared in adult mice the efficacy and longevity of target gene knockdown from siRNA expression cassettes contained in plasmids, PCR-generated linear constructs or PCR constructs containing "dumbbell" ends using the hydrodynamic delivery method. Plasmid siRNA expression constructs were more effective than PCR constructs for target gene knockdown. The efficacy of the PCR constructs was improved by addition of short extensions beyond the transcription termination signal and greatly improved by addition of dumbbell ends. Constructs containing the H1 promoter were significantly less effective in mice than those containing the U6 promoter, whereas both promoters functioned equally well in cultured cells. Target gene knockdown perdured for at least 20 weeks in mice after delivery of either PCR or plasmid siRNA expression cassettes. These results will help guide RNAi vector design.

  9. Reversal of pathology in CHMP2B-mediated frontotemporal dementia patient cells using RNA interference

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup; Mizielinska, Sarah; Hasholt, Lis

    2012-01-01

    role in the pathogenesis of the disease. METHODS: In the present study, we used lentiviral vectors to efficiently knockdown CHMP2B by delivering microRNA embedded small hairpin RNAs. RESULTS: We show that CHMP2B can be efficiently knocked down in patient fibroblasts using an RNA interference approach......BACKGROUND: Frontotemporal dementia is the second most common form of young-onset dementia after Alzheimer's disease, and several genetic forms of frontotemporal dementia are known. A rare genetic variant is caused by a point mutation in the CHMP2B gene. CHMP2B is a component of the ESCRT......-III complex, which is involved in endosomal trafficking of proteins targeted for degradation in lysosomes. Mutations in CHMP2B result in abnormal endosomal structures in patient fibroblasts and patient brains, probably through a gain-of-function mechanism, suggesting that the endosomal pathway plays a central...

  10. The relationship between target quality and interference in sound zones

    DEFF Research Database (Denmark)

    Baykaner, Khan; Coleman, Phillip; Mason, Russell

    2015-01-01

    Sound zone systems aim to control sound fields in such a way that multiple listeners can enjoy different audio programs within the same room with minimal acoustic interference. Often, there is a trade-off between the acoustic contrast achieved between the zones and the fidelity of the reproduced...... audio program in the target zone. A listening test was conducted to obtain subjective measures of distraction, target quality, and overall quality of listening experience for ecologically valid programs within a sound zoning system. Sound zones were reproduced using acoustic contrast control, planarity...

  11. Targeting Promoter-Associated Noncoding RNA In Vivo.

    Science.gov (United States)

    Civenni, Gianluca

    2017-01-01

    There are many classes of noncoding RNAs (ncRNAs), with wide-ranging functionalities (e.g., RNA editing, mediation of mRNA splicing, ribosomal function). MicroRNAs (miRNAs) and long ncRNAs (lncRNAs) are implicated in a wide variety of cellular processes, including the regulation of gene expression. Incorrect expression or mutation of lncRNAs has been reported to be associated with several disease conditions, such a malignant transformation in humans. Importantly, pivotal players in tumorigenesis and cancer progression, such as c-Myc, may be regulated by lncRNA at promoter level. The function of lncRNA can be reduced with antisense oligonucleotides that sequester or degrade mature lncRNAs. In alternative, lncRNA transcription can be blocked by small interference RNA (RNAi), which had acquired, recently, broad interested in clinical applications. In vivo-jetPEI™ is a linear polyethylenimine mediating nucleic acid (DNA, shRNA, siRNA, oligonucelotides) delivery with high efficiency. Different in vivo delivery routes have been validated: intravenous (IV), intraperitoneal (IP), intratumoral, subcutaneous, topical, and intrathecal. High levels of nucleic acid delivery are achieved into a broad range of tissues, such as lung, salivary glands, heart, spleen, liver, and prostate upon systemic administration. In addition, in vivo-jetPEI™ is also an efficient carrier for local gene and siRNA delivery such as intratumoral or topical application on the skin. After systemic injection, siRNA can be detected and the levels can be validated in target tissues by qRT-PCR. Targeting promoter-associated lncRNAs with siRNAs (small interfering RNAs) in vivo is becoming an exciting breakthrough for the treatment of human disease.

  12. RNA interference against animal viruses: how morbilliviruses generate extended diversity to escape small interfering RNA control.

    Science.gov (United States)

    Holz, Carine L; Albina, Emmanuel; Minet, Cécile; Lancelot, Renaud; Kwiatek, Olivier; Libeau, Geneviève; Servan de Almeida, Renata

    2012-01-01

    Viruses are serious threats to human and animal health. Vaccines can prevent viral diseases, but few antiviral treatments are available to control evolving infections. Among new antiviral therapies, RNA interference (RNAi) has been the focus of intensive research. However, along with the development of efficient RNAi-based therapeutics comes the risk of emergence of resistant viruses. In this study, we challenged the in vitro propensity of a morbillivirus (peste des petits ruminants virus), a stable RNA virus, to escape the inhibition conferred by single or multiple small interfering RNAs (siRNAs) against conserved regions of the N gene. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications involved consisted of single or multiple point nucleotide mutations and a deletion of a stretch of six nucleotides, illustrating that this virus has an unusual genomic malleability.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Memory for found targets interferes with subsequent performance in multiple-target visual search.

    Science.gov (United States)

    Cain, Matthew S; Mitroff, Stephen R

    2013-10-01

    Multiple-target visual searches--when more than 1 target can appear in a given search display--are commonplace in radiology, airport security screening, and the military. Whereas 1 target is often found accurately, additional targets are more likely to be missed in multiple-target searches. To better understand this decrement in 2nd-target detection, here we examined 2 potential forms of interference that can arise from finding a 1st target: interference from the perceptual salience of the 1st target (a now highly relevant distractor in a known location) and interference from a newly created memory representation for the 1st target. Here, we found that removing found targets from the display or making them salient and easily segregated color singletons improved subsequent search accuracy. However, replacing found targets with random distractor items did not improve subsequent search accuracy. Removing and highlighting found targets likely reduced both a target's visual salience and its memory load, whereas replacing a target removed its visual salience but not its representation in memory. Collectively, the current experiments suggest that the working memory load of a found target has a larger effect on subsequent search accuracy than does its perceptual salience. PsycINFO Database Record (c) 2013 APA, all rights reserved.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jana L McCaskill

    Full Text Available 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.

  17. Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato.

    Science.gov (United States)

    Xiong, Ai-Sheng; Yao, Quan-Hong; Peng, Ri-He; Li, Xian; Han, Pei-Lai; Fan, Hui-Qin

    2005-02-01

    RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.

  18. RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes

    Directory of Open Access Journals (Sweden)

    Sagar Banerjee

    2017-05-01

    Full Text Available Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

  19. CRISPR/Cas13 as a Tool for RNA Interference

    KAUST Repository

    Ali, Zahir

    2018-03-28

    Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.

  20. Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi

    Directory of Open Access Journals (Sweden)

    Manev Hari

    2003-08-01

    Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should

  1. Engineering host-derived resistance against plant parasites through RNA interference: challenges and opportunities.

    Science.gov (United States)

    Runo, Steven

    2011-01-01

    RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

  2. Design, simplified cloning, and in-silico analysis of multisite small interfering RNA-targeting cassettes

    OpenAIRE

    Baghban-Kohnehrouz, Bahram; Nayeri, Shahnoush

    2016-01-01

    Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning...

  3. Frnakenstein: multiple target inverse RNA folding

    Directory of Open Access Journals (Sweden)

    Lyngsø Rune B

    2012-10-01

    Full Text Available Abstract Background RNA secondary structure prediction, or folding, is a classic problem in bioinformatics: given a sequence of nucleotides, the aim is to predict the base pairs formed in its three dimensional conformation. The inverse problem of designing a sequence folding into a particular target structure has only more recently received notable interest. With a growing appreciation and understanding of the functional and structural properties of RNA motifs, and a growing interest in utilising biomolecules in nano-scale designs, the interest in the inverse RNA folding problem is bound to increase. However, whereas the RNA folding problem from an algorithmic viewpoint has an elegant and efficient solution, the inverse RNA folding problem appears to be hard. Results In this paper we present a genetic algorithm approach to solve the inverse folding problem. The main aims of the development was to address the hitherto mostly ignored extension of solving the inverse folding problem, the multi-target inverse folding problem, while simultaneously designing a method with superior performance when measured on the quality of designed sequences. The genetic algorithm has been implemented as a Python program called Frnakenstein. It was benchmarked against four existing methods and several data sets totalling 769 real and predicted single structure targets, and on 292 two structure targets. It performed as well as or better at finding sequences which folded in silico into the target structure than all existing methods, without the heavy bias towards CG base pairs that was observed for all other top performing methods. On the two structure targets it also performed well, generating a perfect design for about 80% of the targets. Conclusions Our method illustrates that successful designs for the inverse RNA folding problem does not necessarily have to rely on heavy biases in base pair and unpaired base distributions. The design problem seems to become more

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    George Heath

    Full Text Available 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.

  6. Inhibition of full length Hepatitis C Virus particles of 1a genotype through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV, a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2. Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

  7. MicroRNA: an Emerging Therapeutic Target and Intervention Tool

    Directory of Open Access Journals (Sweden)

    Decheng Yang

    2008-06-01

    Full Text Available MicroRNAs (miRNAs are a class of short non-coding RNAs with posttranscriptional regulatory functions. To date, more than 600 human miRNAs have been experimentally identified, and estimated to regulate more than one third of cellular messenger RNAs. Accumulating evidence has linked the dysregulated expression patterns of miRNAs to a variety of diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases and viral infections. MiRNAs provide its particular layer of network for gene regulation, thus possessing the great potential both as a novel class of therapeutic targets and as a powerful intervention tool. In this regard, synthetic RNAs that contain the binding sites of miRNA have been shown to work as a “decoy” or “miRNA sponge” to inhibit the function of specific miRNAs. On the other hand, miRNA expression vectors have been used to restore or overexpress specific miRNAs to achieve a long-term effect. Further, double-stranded miRNA mimetics for transient replacement have been experimentally validated. Endogenous precursor miRNAs have also been used as scaffolds for the induction of RNA interference. This article reviews the recent progress on this emerging technology as a powerful tool for gene regulation studies and particularly as a rationale strategy for design of therapeutics.

  8. RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

    Science.gov (United States)

    Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

    2014-01-01

    The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

  9. RNA interference-based strategies for the inhibition of picornaviruses

    OpenAIRE

    Rothe, Diana

    2011-01-01

    Die Inhibition von Viren mittels RNA-Interferenz hat sich in den letzten Jahren als ein neuer und vielversprechender therapeutischer Ansatz erwiesen. Die vorliegende Arbeit beschreibt sowohl die Optimierung von siRNA-basierten antiviralen Ansätzen als auch neue Methoden, die eine therapeutische Anwendung vereinfachen können. 1. Für ihre Replikation synthetisieren Picornaviren einen RNA-Minus-Strang, der als Matrize für den genomischen Plus-Strang dient. In der vorliegenden Arbeit konnte mit H...

  10. Combinatorial microRNA target predictions

    DEFF Research Database (Denmark)

    Krek, Azra; Grün, Dominic; Poy, Matthew N.

    2005-01-01

    in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published micro...... suggest widespread coordinate control executed by microRNAs. In particular, we experimentally validate common regulation of Mtpn by miR-375, miR-124 and let-7b and thus provide evidence for coordinate microRNA control in mammals....

  11. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcription-polymerase 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 ...

  12. Application of RNA interference methodology to investigate and ...

    Indian Academy of Sciences (India)

    2014-07-31

    Jul 31, 2014 ... tion 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 infec- tion. Our findings provide a valuable tool for controlling plant ...

  13. Interplay between the RNA interference machinery and HIV-1

    NARCIS (Netherlands)

    Schopman, N.C.T.

    2012-01-01

    Resistente infecties zijn lastig te behandelen. Nick Schopman onderzocht een verbeterde RNA-interferentie (RNAi)-gebaseerde anti-hiv-1 gentherapie. Dit kan in de toekomst leiden tot een nieuwe aanpak van de behandeling van resistente infecties. Schopman beschrijft een nieuw ontwerp van een

  14. Application of RNA interference in treating human diseases

    Indian Academy of Sciences (India)

    specific mRNA degradation in the cytoplasm without dramatic changes in transcription of corresponding gene in nucleus. Both. TGS and PTGS are used to regulate endogenous genes. Interestingly, mechanisms for gene silencing also protect the genome from transposons and viruses. In this paper, we first review RNAi ...

  15. RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.

    Directory of Open Access Journals (Sweden)

    Christina Rauschhuber

    Full Text Available BACKGROUND: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. PRINCIPAL FINDINGS: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.

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

    Science.gov (United States)

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi

    2016-03-01

    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 (PJurkat cells, thus inhibiting cell proliferation.

  17. Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interference.

    Science.gov (United States)

    Carneiro, Joyce S; de la Bastide, Paul Y; Chabot, Meghan; Lerch, Lindsey; Hintz, William E

    2010-05-01

    The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity. Copyright 2010 Elsevier Inc. All rights reserved.

  18. RNA interference as a resistance mechanism against crop parasites in Africa: a 'Trojan horse' approach.

    Science.gov (United States)

    Runo, Steven; Alakonya, Amos; Machuka, Jesse; Sinha, Neelima

    2011-02-01

    Biological crop pests cause serious economic losses. In Africa, the most prevalent parasites are insect pests, plant pathogenic root-knot nematodes, viruses and parasitic plants. African smallholder farmers struggle to overcome these parasitic constraints to agricultural production. Crop losses and the host range of these parasites have continued to increase in spite of the use of widely advocated control methods. A sustainable method to overcome biological pests in Africa would be to develop crop germplasm resistant to parasites. This is achievable using either genetic modification (GM) or a non-GM approach. However, there is a paucity of resistant genes available for introduction. Additionally, the biological processes underpinning host parasite resistance are not sufficiently well understood. The authors review a technology platform for using RNA-mediated interference (RNAi) as bioengineered resistance to important crop parasites in Africa. To achieve acquired resistance, a host crop is stably transformed with a transgene that encodes a hairpin RNA targeting essential parasitic genes. The RNAi sequence is chosen in such a way that it shares no homology with the host's genes, so it remains 'inactive' until parasitism. Upon parasitism, the RNAi sequence enters the parasite and post-transcriptional gene silencing (PTGS) mechanisms are activated, leading to the death of the parasite. Copyright © 2010 Society of Chemical Industry.

  19. RNA interference: a promising technique for the improvement of traditional crops.

    Science.gov (United States)

    Katoch, Rajan; Thakur, Neelam

    2013-03-01

    RNA interference (RNAi) is a homology-dependent gene-silencing technology that involves double-stranded RNA directed against a target gene. This technique has emerged as powerful tool in understanding the functions of a number of genes in recent years. For the improvement in the nutritional status of the plants and reduction in the level of antinutrients, the conventional breeding methods were not completely successful in achieving the tissue-specific regulation of some genes. RNAi has shown successful results in a number of plant species for nutritional improvement, change in morphology and alteration in metabolite synthesis. This technology has been applied mostly in genetic engineering of important crop plants, and till date there are no reports of its application for the improvement of traditional/underutilized crops. In this study, we discuss current knowledge of RNAi function and concept and strategies for the improvement of traditional crops. Practical application. Although RNAi has been extensively used for the improvement of popular crops, no attention has been given for the use of this technology for the improvement of underutilized crops. This study describes the importance of use of this technology for the improvement of underutilized crops.

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

    Directory of Open Access Journals (Sweden)

    Yang Shi

    2013-01-01

    Full Text Available 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.

  1. Targeted Delivery of siRNA into Breast Cancer Cells via Phage Fusion Proteins

    OpenAIRE

    Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A.; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A.

    2013-01-01

    Nucleic acids including antisense oligonucleotides, small interfering RNA (siRNA), aptamers and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as target...

  2. RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.

    Science.gov (United States)

    Rouhana, Labib; Weiss, Jennifer A; Forsthoefel, David J; Lee, Hayoung; King, Ryan S; Inoue, Takeshi; Shibata, Norito; Agata, Kiyokazu; Newmark, Phillip A

    2013-06-01

    The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Copyright © 2013 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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

    2009-11-05

    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.

  4. Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference.

    Science.gov (United States)

    Zhang, Zhi-Yong; Yang, Lin; Zhou, Shu-Feng; Wang, Han-Guang; Li, Wan-Chen; Fu, Feng-Ling

    2011-05-20

    To overcome the low efficiency of agronomic protection from maize dwarf mosaic disease, susceptible maize inbred line was transformed by Agrobacterium tumefaciens harboring hpRNA expression vectors containing inverted-repeat sequences of different lengths targeting coat protein gene (CP) of maize dwarf mosaic virus (MDMV). After PCR screening and Southern blotting, the flanking sequences of the integration sites were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) and used for analysis of T-DNA integration patterns. The T₂ plant lines were evaluated for their MDMV resistance in field inoculation trials under two environments. Of the nineteen T₂ plant lines positive in Southern blotting, six were evaluated as resistant to MDMV, and four of them had resistance non-significantly different from the highly resistant control "H9-21", while the resistance of the other eleven was proved to be significantly improved when compared to their non-transformed parent line. These improvements in MDMV resistance were verified by the relative amount of virus CP gene expression measured by quantitative real time PCR. Comparing the results of Southern blotting and TAIL-PCR analysis, different integration patterns of one or two copies of the inverted-repeat sequences were identified from non-repetitive and repetitive sequences of the maize genome. The MDMV resistance mediated by RNA interference is relative to the length of the inverted-repeat sequence, the copy number of T-DNA integration and the repeatability of integration sites. A longer hpRNA expression construct shows more efficiency than a shorter one. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

    International Nuclear Information System (INIS)

    O'Grady, Michael; Raha, Debasish; Hanson, Bonnie J; Bunting, Michaeline; Hanson, George T

    2005-01-01

    The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC 50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. EGFR pathway components were qualified as

  6. Effects of downregulating TEAD4 transcripts by RNA interference on early development of bovine embryos

    OpenAIRE

    SAKURAI, Nobuyuki; TAKAHASHI, Kazuki; EMURA, Natsuko; HASHIZUME, Tsutomu; SAWAI, Ken

    2016-01-01

    Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was f...

  7. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Fatima Khaja

    2016-01-01

    Full Text Available Since its discovery, small interfering RNA (siRNA has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA not easily accessed by conventional drugs. Hence, RNA interference (RNAi therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs. This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF, an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP, showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  8. [Downregulation of HER2 by adenovirus-mediated RNA interference and its inhibitory effect on growth of SKBR3 breast cancer cell].

    Science.gov (United States)

    Cheng, Lian-sheng; Zha, Zhao; Xi, Jia-jia; Jiang, Bing; Liu, Jing; Yao, Xue-biao

    2007-08-01

    To explore the possibility of RNA interference (RNAi)-based gene therapy against HER2-overexpressing tumors using adenovirus-mediated vector. A plasmid named pHER2-GFP containing HER2 and green fluorescent protein (GFP) fusion was constructed and cotransfected into CHO-K1 cells respectively with nine small interference RNA (siRNA)-expressing plasmids targeting different regions of HER2. The siRNA-expressing plasmids with best interference effect were screened out and then used to identify the gene silence effect in HER2-overexpressing SKBR3 breast cancer cells. Subsequently, the siRNA-expressing cassettes were subcloned into adenoviral vectors. Downregulation of HER2 by adenovirus-mediated RNAi and its effect on SKBR3 cell proliferation were identified again. Two siRNA-expressing plasmids with best interference effect were screened out and HER2 was also efficiently downregulated in SKBR3 cells infected with the adenovirus containing these siRNA-expressing cassettes. Downregulation of HER2 resulted in the increase of cells in G1 phase and the induction of apoptosis. Furthermore, infection of adenovirus inhibited SKBR3 cell growth, which was confirmed by MTT and cell long-term proliferation assays. The adenovirus-mediated RNAi could downregulate the HER2 expression efficiently and exert an inhibitory effect on growth of HER2-overexpressing breast cancer cell.

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

    Science.gov (United States)

    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"…

  10. RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

    NARCIS (Netherlands)

    Limpens, E.H.M.; Ramos, J.; Franken, C.; Raz, V.; Compaan, B.; Franssen, H.; Bisseling, T.; Geurts, R.

    2004-01-01

    RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was

  11. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition

    NARCIS (Netherlands)

    Das, Atze T.; Brummelkamp, Thijn R.; Westerhout, Ellen M.; Vink, Monique; Madiredjo, Mandy; Bernards, René; Berkhout, Ben

    2004-01-01

    Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We

  12. Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

    NARCIS (Netherlands)

    Das, A.T.; Brummelkamp, T.R.; Westerhout, E.M.; Vink, M.; Madiredjo, M.; Bernards, R.A.; Berkhout, Ben

    2004-01-01

    Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We

  13. Production of high-amylose maize lines using RNA interference in ...

    African Journals Online (AJOL)

    To regulate the biosynthetic process of maize starch and produce high-amylose transgenic maize, RNA interference was used to inhibit the starch branching enzyme gene Sbe2. A construct with a 562 bp segment of Sbe2 (pRSBE2a) was cloned as inverted repeats. Highly efficient RNAi vector pRSBE2a was transferred to ...

  14. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

    DEFF Research Database (Denmark)

    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man

    2018-01-01

    a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining...

  15. RNA interference for the control of whiteflies (Bemisia tabaci) by oral ...

    Indian Academy of Sciences (India)

    [Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Borgio JF, Singh PK and Tuli R 2011 RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J. Biosci. 36 153–161] DOI 10.1007/s12038-011-9009-1. 1. Introduction. Cultivation of Bt-transgenic cotton has significantly reduced the usage of chemical ...

  16. Regulation of gene expression in neuronal tissue by RNA interference and editing

    DEFF Research Database (Denmark)

    Venø, Morten Trillingsgaard

    mediated by microRNAs are crucial for the correct function of the mammalian brain. We are addressing A-to-I editing and regulation by microRNAs with spatio-temporal resolution in the embryonic porcine brain by Solexa sequencing of microRNAs and 454 sequencing of edited neuronal messenger RNAs, resulting...... microRNA expression and targeting, specifically in neurons of knockout mice or transgenic mice, were also performed. One study revealed that disrupting the expression of Argonaute2, the main effector of miRNA function, leads to a reduction of the microRNA abundance for a specific subset of micro......RNAs is able to target the GLP 3’ untranslated region, causing reduced expression of the targeted transcript. In the third study a procedure for global detection of microRNA targeting specifically in neurons is demonstrated, using Solexa sequencing of microRNA and messenger RNA segments binding to Argonaute2...

  17. Engineering silkworms for resistance to baculovirus through multigene RNA interference.

    Science.gov (United States)

    Subbaiah, Edupalli V; Royer, Corinne; Kanginakudru, Sriramana; Satyavathi, Valluri V; Babu, Adari Sobhan; Sivaprasad, Vankadara; Chavancy, Gérard; Darocha, Martine; Jalabert, Audrey; Mauchamp, Bernard; Basha, Ibrahim; Couble, Pierre; Nagaraju, Javaregowda

    2013-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to sericulture.

  18. Improving model predictions for RNA interference activities that use support vector machine regression by combining and filtering features

    Directory of Open Access Journals (Sweden)

    Peek Andrew S

    2007-06-01

    Full Text Available Abstract Background RNA interference (RNAi is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM approach was used to quantitatively model RNA interference activities. Results Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (N-grams and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5' most base of the guide strand is the most informative. Conclusion The capacity of specific feature mapping methods and their ability to build predictive models of RNAi activity suggests a relative biological importance of these features. Some feature mapping methods are more informative in building predictive models and overall t-test filtering provides a method to remove some noisy features or make comparisons among datasets. Together, these features can yield predictive SVM regression models with increased predictive accuracy between predicted and observed activities both within datasets by cross validation, and between independently collected RNAi activity datasets. Feature filtering to remove features should be approached carefully in that it is possible to reduce feature set size without substantially reducing predictive models, but the features retained in the candidate models become increasingly distinct. Software to perform feature prediction and SVM training and testing on nucleic acid

  19. Host Recognition Responses of Western (Family: Chrysomelidae) Corn Rootworm Larvae to RNA Interference and Bt Corn.

    Science.gov (United States)

    Zukoff, Sarah N; Zukoff, Anthony L

    2017-01-01

    Western corn rootworm Diabrotica virgifera virgifera LeConte is an important pest of corn whose larvae exhibit particular quantifiable patterns of locomotion after exposure to, and removal from, host roots and nonhost roots. Using EthoVision software, the behavior and locomotion of the western corn rootworm larvae was analyzed to determine the level of host recognition to germinated roots of differing corn hybrids containing either rootworm targeted Bt genes, RNA interference (RNAi) technology, the stack of both Bt and RNAi, or the isoline of these. The behavior of the rootworm larvae indicated a significant host preference response to all corn hybrids (with or without insecticidal traits) compared to the filter paper and oat roots. A weaker host response to the RNAi corn roots was observed in the susceptible larvae when compared to the resistant larvae, but not for the Bt + RNAi vector stack. Additionally, the resistant larvae demonstrated a weaker host response to the isoline corn roots when compared to the susceptible larvae. Although weaker, these host responses were significantly different from those observed in the negative controls, indicating that all hybrids tested do contain the contact cues necessary to elicit a host preference response by both Cry3Bb1-resistant and Cry3Bb1-susceptible larvae that would work to hinder resistance development in refuge in a bag fields. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  20. RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

    KAUST Repository

    Cernilogar, Filippo M.

    2013-06-13

    Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

  1. [Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

    Science.gov (United States)

    Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

    2016-06-25

    To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Ptissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (Ptissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

  2. Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Heikkinen Liisa

    2008-06-01

    Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

  3. Design, simplified cloning, andin-silicoanalysis of multisite small interfering RNA-targeting cassettes.

    Science.gov (United States)

    Baghban-Kohnehrouz, Bahram; Nayeri, Shahnoush

    2016-03-01

    Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning steps. In this method, effective siRNA sites in the target messenger RNAs (mRNAs) were determined using in silico analysis and consecutively arranged to reduce length of inverted repeats. Here, we used one-step (polymerase chain reaction) PCR by designed long primer sets covering the selected siRNA sites. Rapid screening, cost-effective and shorten procedure are advantages of this method compare to PCR classic cloning. Validity of constructs was confirmed by optimal centroid secondary structures with high stability in plants.

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

    Science.gov (United States)

    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.

  5. Validating the use of short interfering RNA as a novel technique for cell-specific target gene knockdown in lung ischemia-reperfusion injury.

    Science.gov (United States)

    Merry, Heather E; Phelan, Patrick; Hwang, Billanna; Mulligan, Michael S

    2016-02-01

    Short interfering RNA is an effective method for target gene knockdown. However, concerns surround the design, administration, efficacy, specificity, and immunostimulatory potential. Although uptake by alveolar macrophages has been demonstrated, studies have not examined its use in lung ischemia-reperfusion injury. We describe the validation of short interference RNA as a novel technique for cell-specific target gene knockdown in our model of lung ischemia-reperfusion injury. Dose-response experiments were performed, and 3 distinct sequences of toll-like receptor-4, toll-like receptor-2, and myeloid differentiation factor-88 short interference RNA were tested for efficacy of knockdown. Saline, lipid vector, and noncoding short interference RNA controls were used. Similar experiments were performed in primary cultures of resident pulmonary cells. Target protein knockdown was assessed by Western blot. Rat serum and cell culture media were assessed for interferon and cytokine production. Biotin labeling was used to assess short interference RNA uptake. Target protein expression was significantly reduced using short interference RNA. However, toll-like receptor-4 knockdown was isolated to alveolar macrophages, and biotin labeling confirmed toll-like receptor-4 short interference RNA localization to alveolar macrophages. There was significant knockdown of toll-like receptor-4 expression in cultured cells treated with toll-like receptor-4 short interference RNA. There was no significant change in interferon production after short interference RNA treatment. There was effective target protein knockdown with each sequence used. Short interference RNA is a valid method for achieving target protein knockdown in alveolar macrophages and is an important tool in the evaluation of its role in the development of lung ischemia-reperfusion injury. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  6. EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

    Science.gov (United States)

    Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

    2016-11-01

    Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

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

    Science.gov (United States)

    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

  8. Is TNF-a-targeted short hairpin RNA (shRNA) a novel potential therapeutic tool in psoriasis treatment?

    DEFF Research Database (Denmark)

    Stenderup, Karin; Jakobsen, Maria; Rosada, Cecilia

    2008-01-01

      TNF-α is a well known target in psoriasis treatment and biological treatments targeting TNF-a are already clinically used against psoriasis and psoriasis arthritis. Attention is however given to a novel therapeutic tool: RNA interference that controls gene silencing. This study investigates...... the efficiency of targeting TNF-a with specific short hairpin RNA (shRNA) and explores its potential in treating psoriasis. ShRNAs targeting human TNF-α mRNA were generated. Their efficiency in down-regulating TNF-a protein expression was evaluated using a Renilla luciferase screening-assay and a transient co...... TNF-a shRNA was used to transduce HEK293 cells and verify vector-derived TNF-a knockdown in vitro. In vivo, psoriasis skin was exposed to lentiviral TNF-a shRNAs by a single intra-dermal injection. Psoriasis skin for the in vivo study was obtained from psoriatic plaque skin biopsies that were...

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

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2013-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Carol D. Blair

    2015-02-01

    Full Text Available 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 (dsRNA 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 (siRNA, micro (miRNA, and Piwi-interacting (piRNA 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.

  11. RNA Interference towards the Potato Psyllid, Bactericera cockerelli, Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV.

    Directory of Open Access Journals (Sweden)

    Hada Wuriyanghan

    Full Text Available The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli, is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum, which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum, tomatillo (Physalis philadelphica and tobacco (Nicotiana tabacum plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX and Tobacco rattle virus (TRV did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Zongli eHu

    2015-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  15. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    NARCIS (Netherlands)

    Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

    2005-01-01

    Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

  16. Identification of nonviable genes affecting touch sensitivity in Caenorhabditis elegans using neuronally enhanced feeding RNA interference.

    Science.gov (United States)

    Chen, Xiaoyin; Cuadros, Margarete Diaz; Chalfie, Martin

    2015-01-09

    Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions. Copyright © 2015 Chen et al.

  17. Common features of microRNA target prediction tools

    Directory of Open Access Journals (Sweden)

    Sarah M. Peterson

    2014-02-01

    Full Text Available The human genome encodes for over 1800 microRNAs, which are short noncoding RNA molecules that function to regulate gene expression post-transcriptionally. Due to the potential for one microRNA to target multiple gene transcripts, microRNAs are recognized as a major mechanism to regulate gene expression and mRNA translation. Computational prediction of microRNA targets is a critical initial step in identifying microRNA:mRNA target interactions for experimental validation. The available tools for microRNA target prediction encompass a range of different computational approaches, from the modeling of physical interactions to the incorporation of machine learning. This review provides an overview of the major computational approaches to microRNA target prediction. Our discussion highlights three tools for their ease of use, reliance on relatively updated versions of miRBase, and range of capabilities, and these are DIANA-microT-CDS, miRanda-mirSVR, and TargetScan. In comparison across all microRNA target prediction tools, four main aspects of the microRNA:mRNA target interaction emerge as common features on which most target prediction is based: seed match, conservation, free energy, and site accessibility. This review explains these features and identifies how they are incorporated into currently available target prediction tools. MicroRNA target prediction is a dynamic field with increasing attention on development of new analysis tools. This review attempts to provide a comprehensive assessment of these tools in a manner that is accessible across disciplines. Understanding the basis of these prediction methodologies will aid in user selection of the appropriate tools and interpretation of the tool output.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

    OpenAIRE

    Ahmad, Aftab; Zia-Ur-Rehman, Muhammad; Hameed, Usman; Qayyum Rao, Abdul; Ahad, Ammara; Yasmeen, Aneela; Akram, Faheem; Bajwa, Kamran Shahzad; Scheffler, Jodi; Nasir, Idrees Ahmad; Shahid, Ahmad Ali; Iqbal, Muhammad Javed; Husnain, Tayyab; Haider, Muhammad Saleem; Brown, Judith K.

    2017-01-01

    Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of ...

  20. psRNATarget: a plant small RNA target analysis server.

    Science.gov (United States)

    Dai, Xinbin; Zhao, Patrick Xuechun

    2011-07-01

    Plant endogenous non-coding short small RNAs (20-24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to 'open' secondary structure around small RNA's target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/.

  1. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.

    Directory of Open Access Journals (Sweden)

    Irma Sánchez-Vargas

    2009-02-01

    Full Text Available A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi, is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA, which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs. These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2 infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

  2. Small regulatory RNAs of the RNA interference (RNAi) pathway as a prophylactic treatment against fish pathogenic viruses

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Hajiabadi, Seyed Amir Hossein Jalali; Kristensen, Lasse Bøgelund Juel

    2011-01-01

    . The mechanism can be programmed with several types of small double stranded RNAs - the type of which defines the destiny of the target. One such class of regulatory RNAs called microRNAs are upregulated due to various physiological responses of the cell and they suppress many genes simultaneously believed...... septicemia virus (VHSV) and examples of some of our results on delivery and effect of siRNAs designed to target viral genes of VHSV. The VHS disease causes high mortalities in salmonid fish aquacultures why intervention strategies are highly in demand.......Small RNAs acting in the recently discovered gene regulatory mechanism called RNA interference has a potential as diagnostic signatures of disease and immunological state and when produced synthetically as prophylactic treatment of such diseases. In the RNAi mechanism the cell produces different...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Arif Muhammad Khan

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

  5. Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

    Science.gov (United States)

    Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

    2017-06-01

    Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

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

    Science.gov (United States)

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. miRNA-target prediction based on transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Fujiwara Toyofumi

    2013-02-01

    Full Text Available Abstract Background microRNAs (miRNAs are tiny endogenous RNAs that have been discovered in animals and plants, and direct the post-transcriptional regulation of target mRNAs for degradation or translational repression via binding to the 3'UTRs and the coding exons. To gain insight into the biological role of miRNAs, it is essential to identify the full repertoire of mRNA targets (target genes. A number of computer programs have been developed for miRNA-target prediction. These programs essentially focus on potential binding sites in 3'UTRs, which are recognized by miRNAs according to specific base-pairing rules. Results Here, we introduce a novel method for miRNA-target prediction that is entirely independent of existing approaches. The method is based on the hypothesis that transcription of a miRNA and its target genes tend to be co-regulated by common transcription factors. This hypothesis predicts the frequent occurrence of common cis-elements between promoters of a miRNA and its target genes. That is, our proposed method first identifies putative cis-elements in a promoter of a given miRNA, and then identifies genes that contain common putative cis-elements in their promoters. In this paper, we show that a significant number of common cis-elements occur in ~28% of experimentally supported human miRNA-target data. Moreover, we show that the prediction of human miRNA-targets based on our method is statistically significant. Further, we discuss the random incidence of common cis-elements, their consensus sequences, and the advantages and disadvantages of our method. Conclusions This is the first report indicating prevalence of transcriptional regulation of a miRNA and its target genes by common transcription factors and the predictive ability of miRNA-targets based on this property.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Amir Raza

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  11. Evolutionary Transitions of MicroRNA-Target Pairs

    KAUST Repository

    Nozawa, Masafumi

    2016-04-27

    How newly generated microRNA (miRNA) genes are integrated into gene regulatory networks during evolution is fundamental in understanding the molecular and evolutionary bases of robustness and plasticity in gene regulation. A recent model proposed that after the birth of a miRNA, the miRNA is generally integrated into the network by decreasing the number of target genes during evolution. However, this decreasing model remains to be carefully examined by considering in vivo conditions. In this study, we therefore compared the number of target genes among miRNAs with different ages, combining experiments with bioinformatics predictions. First, we focused on three Drosophila miRNAs with different ages. As a result, we found that an older miRNA has a greater number of target genes than a younger miRNA, suggesting the increasing number of targets for each miRNA during evolution (increasing model). To further confirm our results, we also predicted all target genes for all miRNAs in D. melanogaster, considering co-expression of miRNAs and mRNAs in vivo. The results obtained also do not support the decreasing model but are reasonably consistent with the increasing model of miRNA-target pairs. Furthermore, our large-scale analyses of currently available experimental data of miRNA-target pairs also showed a weak but the same trend in humans. These results indicate that the current decreasing model of miRNA-target pairs should be reconsidered and the increasing model may be more appropriate to explain the evolutionary transitions of miRNA-target pairs in many organisms.

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

    Science.gov (United States)

    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.

  13. Mechanism of microRNA-target interaction: molecular dynamics simulations and thermodynamics analysis.

    Science.gov (United States)

    Wang, Yonghua; Li, Yan; Ma, Zhi; Yang, Wei; Ai, Chunzhi

    2010-07-29

    MicroRNAs (miRNAs) are endogenously produced approximately 21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5'-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg(2+)) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.

  14. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

    Czech Academy of Sciences Publication Activity Database

    Sarnová, Lenka; Malík, Radek; Sedláček, Radislav; Svoboda, Petr

    2010-01-01

    Roč. 9, č. 8 (2010), s. 1-10 ISSN 1477-5751 R&D Projects: GA MŠk ME09039 Grant - others:EMBO SDIG(DE) project 1483 Institutional research plan: CEZ:AV0Z50520514 Keywords : transgenic RNAi * shRNA * oocyte Subject RIV: EB - Genetics ; Molecular Biology http://www.jnrbm.com/content/9/1/8

  15. Intervention modalities for targeting cognitive-motor interference in individuals with neurodegenerative disease: a systematic review.

    Science.gov (United States)

    Wajda, Douglas A; Mirelman, Anat; Hausdorff, Jeffrey M; Sosnoff, Jacob J

    2017-03-01

    Individuals with neurodegenerative disease (NDD) commonly have elevated cognitive-motor interference, change in either cognitive or motor performance (or both) when tasks are performed simultaneously, compared to healthy controls. Given that cognitive-motor interference is related to reduced community ambulation and elevated fall risk, it is a target of rehabilitation interventions. Areas covered: This review details the collective findings of previous dual task interventions in individuals with NDD. A total of 21 investigations focusing on 4 different neurodegenerative diseases and one NDD precursor (Parkinson's disease, multiple sclerosis, Alzheimer's disease (AD), dementia other than AD, and mild cognitive impairment) consisting of 721 participants were reviewed. Expert commentary: Preliminary evidence from interventions targeting cognitive-motor interference, both directly and indirectly, show promising results for improving CMI in individuals with neurodegenerative diseases. Methodological limitations, common to pilot investigations preclude firm conclusions. Well-designed randomized control trials targeting cognitive motor interference are warranted.

  16. MicroRNA target finding by comparative genomics.

    Science.gov (United States)

    Friedman, Robin C; Burge, Christopher B

    2014-01-01

    MicroRNAs (miRNAs) have been implicated in virtually every metazoan biological process, exerting a widespread impact on gene expression. MicroRNA repression is conferred by relatively short "seed match" sequences, although the degree of repression varies widely for individual target sites. The factors controlling whether, and to what extent, a target site is repressed are not fully understood. As an alternative to target prediction based on sequence alone, comparative genomics has emerged as an invaluable tool for identifying miRNA targets that are conserved by natural selection, and hence likely effective and important. Here we present a general method for quantifying conservation of miRNA seed match sites, separating it from background conservation, controlling for various biases, and predicting miRNA targets. This method is useful not only for generating predictions but also as a tool for empirically evaluating the importance of various target prediction criteria.

  17. MicroRNA as therapeutic targets for treatment of depression

    Directory of Open Access Journals (Sweden)

    Hansen KF

    2013-07-01

    Full Text Available Katelin F Hansen, Karl Obrietan Department of Neuroscience, Ohio State University, Columbus, OH, USA Abstract: Depression is a potentially life-threatening mental disorder affecting approximately 300 million people worldwide. Despite much effort, the molecular underpinnings of clinical depression remain poorly defined, and current treatments carry limited therapeutic efficacy and potentially burdensome side effects. Recently, small noncoding RNA molecules known as microRNA (miRNA have gained prominence as a target for therapeutic intervention, given their capacity to regulate neuronal physiology. Further, mounting evidence suggests a prominent role for miRNA in depressive molecular signaling. Recent studies have demonstrated that dysregulation of miRNA expression occurs in animal models of depression, and in the post-mortem tissue of clinically depressed patients. Investigations into depression-associated miRNA disruption reveals dramatic effects on downstream targets, many of which are thought to contribute to depressive symptoms. Furthermore, selective serotonin reuptake inhibitors, as well as other antidepressant drugs, have the capacity to reverse aberrant depressive miRNA expression and their downstream targets. Given the powerful effects that miRNA have on the central nervous system transcriptome, and the aforementioned studies, there is a compelling rationale to begin to assess the potential contribution of miRNA to depressive etiology. Here, we review the molecular biology of miRNA, our current understanding of miRNA in relation to clinical depression, and the utility of targeting miRNA for antidepressant treatment. Keywords: depression, microRNA, miRNA, BDNF, Dicer, serotonin

  18. Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

    Science.gov (United States)

    Bedi, Deepa; Gillespie, James W; Petrenko, Vasily A; Ebner, Andreas; Leitner, Michael; Hinterdorfer, Peter; Petrenko, Valery A

    2013-02-04

    Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

  19. Downregulation of the Adenosine A2b Receptor by RNA Interference Inhibits Hepatocellular Carcinoma Cell Growth

    OpenAIRE

    Xiang, Hong-Jun; Chai, Fu-Lu; Wang, De-Sheng; Dou, Ke-Feng

    2011-01-01

    To investigate the biological effect of adenosine A2b receptor (A2bR) on the human hepatocellular carcinoma cell line HepG2, three A2bR siRNA constructs were transiently transfected into HepG2 cells. The results showed that A2bR siRNA reduced the levels of A2bR mRNA and protein. In order to further detect the function of A2bR, we established a stable hepatocellular carcinoma cell line (HepG2) expressing siRNA targeting the adenosine A2b receptor. Targeted RNAi significantly inhibited tumor ce...

  20. Mapping Long Noncoding RNA Chromatin Occupancy Using Capture Hybridization Analysis of RNA Targets (CHART).

    Science.gov (United States)

    Vance, Keith W

    2017-01-01

    Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22-28 nucleotide biotinylated antisense oligonucleotides, complementary to regions of the target RNA that are accessible for hybridization, to purify RNAs from a cross-linked chromatin extract. RNA-chromatin complexes are next immobilized on beads, washed, and specifically eluted using RNase H. Associated genomic DNA is then sequenced using high-throughput sequencing technologies and mapped to the genome to identify RNA-chromatin associations on a large scale. CHART-based strategies can be applied to determine the nature and extent of long noncoding RNA (long ncRNA) association with chromatin genome-wide and identify direct long ncRNA transcriptional targets.

  1. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  2. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Horáková, Eva; Van Den Burg, J.; Zíková, Alena; Ernst, N. L.; Stuart, K.; Benne, R.; Lukeš, Julius

    2005-01-01

    Roč. 280, č. 4 (2005), s. 2429-2438 ISSN 0021-9258 R&D Projects: GA AV ČR IAA6022903 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma brucei * RNA editing * interference RNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.854, year: 2005

  3. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells.

    Science.gov (United States)

    Maillard, Pierre V; Van der Veen, Annemarthe G; Deddouche-Grass, Safia; Rogers, Neil C; Merits, Andres; Reis E Sousa, Caetano

    2016-12-01

    RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  4. A fast, simple method for screening radiation susceptibility genes by RNA interference

    International Nuclear Information System (INIS)

    Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Otsuki, Marika; Miyagishi, Makoto; Taira, Kazunari; Imai, Takashi; Harada, Yoshi-nobu

    2005-01-01

    Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes

  5. miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

    DEFF Research Database (Denmark)

    Moore, Michael J; Scheel, Troels K H; Luna, Joseph M

    2015-01-01

    AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA-target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural...... analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences...

  6. Recent advances in developing small molecules targeting RNA.

    Science.gov (United States)

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

  7. Evaluation of metaphylactic RNA interference to prevent equine herpesvirus type 1 infection in experimental herpesvirus myeloencephalopathy in horses.

    Science.gov (United States)

    Perkins, Gillian A; Van de Walle, Gerlinde R; Pusterla, Nicola; Erb, Hollis N; Osterrieder, Nikolaus

    2013-02-01

    To evaluate metaphylactic RNA interference to prevent equine herpesvirus type 1 (EHV-1) infection in experimental herpesvirus myeloencephalopathy in horses and to determine whether horses infected with a neuropathogenic strain of the virus that develop equine herpesvirus myeloencephalopathy (EHM) have differences in viremia. 13 seronegative horses. EHV-1 strain Ab4 was administered intranasally on day 0, and small interfering RNAs (siRNAs [EHV-1 specific siRNAs {n = 7} or an irrelevant siRNA {6}]) were administered intranasally 24 hours before and 12, 24, 36, and 48 hours after infection. Physical and neurologic examinations, nasal swab specimens, and blood samples were collected for virus isolation and quantitative PCR assay. Data from the study were combined with data from a previous study of 14 horses. No significant difference was detected in clinical variables, viremia, or detection of EHV-1 in nasal swab specimens of horses treated with the EHV-1 targeted siRNAs (sigB3-siOri2) versus controls. No significant differences in viremia were detected between horses that developed EHM and those that did not. Administration of siRNAs targeted against EHV-1 around the time of EHV-1 infection was not protective with this experimental design. Horses infected with the neuropathogenic EHV-1 strain Ab4 that developed EHM did not have a more pronounced viremia.

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

    Directory of Open Access Journals (Sweden)

    Landmann Frédéric

    2012-01-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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. Copyright © 2016

  10. RNA targeting by small molecules: Binding of protoberberine ...

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Studies on RNA targeting by small molecules to specifically control certain cellular functions is an area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of ...

  11. Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Gabriel Rinaldi

    2008-07-01

    Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

  12. Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

    Science.gov (United States)

    Rinaldi, Gabriel; Morales, Maria E.; Cancela, Martín; Castillo, Estela; Brindley, Paul J.; Tort, José F.

    2008-01-01

    The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These

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

    Science.gov (United States)

    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

  14. Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

    Directory of Open Access Journals (Sweden)

    El Far Mohamed

    2009-05-01

    Full Text Available Abstract Background Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC. While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. Results We show that the TRBP binding site in Dicer is a 165 amino acid (aa region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsΔC4, co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsΔC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsΔC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsΔC4, rescued RNAi function. Conclusion The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsΔC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsΔC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.

  15. Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

    DEFF Research Database (Denmark)

    Hedman, Hanna K; Kirpekar, Finn; Elmroth, Sofi K C

    2011-01-01

    , cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized...... by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity...... of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays...

  16. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    International Nuclear Information System (INIS)

    Anesti, Anna-Maria; Simpson, Guy R; Price, Toby; Pandha, Hardev S; Coffin, Robert S

    2010-01-01

    Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF , we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

  17. Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

    Directory of Open Access Journals (Sweden)

    Alison L McCormack

    Full Text Available The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left vs. untreated (right hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i the number and phenotype of nigral dopaminergic neurons, and (ii the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

  18. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  19. Mutation of miRNA target sequences during human evolution

    DEFF Research Database (Denmark)

    Gardner, Paul P; Vinther, Jeppe

    2008-01-01

    It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits....

  20. Why Argonaute is needed to make microRNA target search fast and reliable

    NARCIS (Netherlands)

    Klein, M.; Chandradoss, S.D.; Depken, S.M.; Joo, C.

    2017-01-01

    MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the

  1. Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

    Science.gov (United States)

    Adelman, Zach N; Anderson, Michelle A E; Wiley, Michael R; Murreddu, Marta G; Samuel, Glady Hazitha; Morazzani, Elaine M; Myles, Kevin M

    2013-01-01

    The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery. We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses. This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

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

    Science.gov (United States)

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

    2008-01-17

    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. 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. 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.

  3. Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

    Directory of Open Access Journals (Sweden)

    Zach N Adelman

    Full Text Available The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

  4. Effects of RNA interference combined with ultrasonic irradiation and SonoVue microbubbles on expression of STAT3 gene in keratinocytes of psoriatic lesions.

    Science.gov (United States)

    Ran, Li-Wei; Wang, Hao; Lan, Dong; Jia, Hong-Xia; Yu, Si-Si

    2017-04-01

    The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out, and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose- and time-response were investigated. Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs, and the effects on STAT3 expression were detected, then the most effective siRNA was selected for the subsequent experiments. The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs, then the optimal parameters of ultrasonic irradiation were determined. The most effective siRNA carried by Li-pofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs, and the dose- and time-response of RNA interference was determined. The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Li-pofectamine 3000 (L group). The results showed that siRNA-3 achieved the highest silencing efficacy. 0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation. The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose- and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h, and on protein at 72 h after transfection. The LUS group achieved the highest silencing efficacy. It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3

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

    Energy Technology Data Exchange (ETDEWEB)

    Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

    2012-10-12

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

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

    Science.gov (United States)

    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.

  7. CRISPRTarget: bioinformatic prediction and analysis of crRNA targets

    NARCIS (Netherlands)

    Biswas, A.; Gagnon, J.N.; Brouns, S.J.J.; Fineran, P.C.; Brown, C.M.

    2013-01-01

    The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are

  8. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

    Science.gov (United States)

    Brettmann, Erin A; Shaik, Jahangheer S; Zangger, Haroun; Lye, Lon-Fye; Kuhlmann, F Matthew; Akopyants, Natalia S; Oschwald, Dayna M; Owens, Katherine L; Hickerson, Suzanne M; Ronet, Catherine; Fasel, Nicolas; Beverley, Stephen M

    2016-10-25

    Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

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

    Science.gov (United States)

    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

  10. Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

    DEFF Research Database (Denmark)

    Hansen, Klavs R.; Burns, G.; Mata, J.

    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...... 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...

  11. RNA interference for functional genomics and improvement of cotton (Gossypium spp.

    Directory of Open Access Journals (Sweden)

    Ibrokhim Y. Abdurakhmonov

    2016-02-01

    Full Text Available 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 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 commercialisation.

  12. RNA interference-based therapy for spinocerebellar ataxia type 7 retinal degeneration.

    Directory of Open Access Journals (Sweden)

    Pavitra S Ramachandran

    Full Text Available Spinocerebellar ataxia type 7 (SCA7 is an autosomal dominant neurodegenerative disease characterized by loss of motor coordination and retinal degeneration with no current therapies in the clinic. The causative mutation is an expanded CAG repeat in the ataxin-7 gene whose mutant protein product causes cerebellar and brainstem degeneration and retinal cone-rod dystrophy. Here, we reduced the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina by RNA interference and evaluated retinal function 23 weeks post injection. We observed a preservation of normal retinal function and no adverse toxicity with ≥50% reduction of mutant and wildtype ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 retinal therapy.

  13. A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs

    NARCIS (Netherlands)

    ter Brake, Olivier; Berkhout, Ben

    2005-01-01

    RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this

  14. RNA interference of gonadotropin-inhibitory hormone gene induces arousal in songbirds.

    Directory of Open Access Journals (Sweden)

    Takayoshi Ubuka

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

  15. Aedes aegypti uses RNA interference in defense against Sindbis virus infection

    Directory of Open Access Journals (Sweden)

    Wilusz Jeffrey

    2008-03-01

    Full Text Available Abstract Background RNA interference (RNAi is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus. Results SINV (TR339-eGFP (+ strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner. Conclusion We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

  16. Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers

    Directory of Open Access Journals (Sweden)

    Styliana Philippou

    2018-03-01

    Full Text Available Oligonucleotide gene therapy has shown great promise for the treatment of muscular dystrophies. Nevertheless, the selective delivery to affected muscles has shown to be challenging because of their high representation in the body and the high complexity of their cell membranes. Current trials show loss of therapeutic molecules to non-target tissues leading to lower target efficacy. Therefore, strategies that increase uptake efficiency would be particularly compelling. To address this need, we applied a cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment approach and identified a skeletal muscle-specific RNA aptamer. A01B RNA aptamer preferentially internalizes in skeletal muscle cells and exhibits decreased affinity for off-target cells. Moreover, this in vitro selected aptamer retained its functionality in vivo, suggesting a potential new approach for targeting skeletal muscles. Ultimately, this will aid in the development of targeted oligonucleotide therapies against muscular dystrophies.

  17. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    Directory of Open Access Journals (Sweden)

    Tina Y Liu

    Full Text Available CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus Type III-A Csm complex (TthCsm with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  18. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    Science.gov (United States)

    Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

    2017-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  19. Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference

    Directory of Open Access Journals (Sweden)

    Masaki Takahashi

    2015-01-01

    Full Text Available The α-synuclein (SNCA gene is a responsible gene for Parkinson's disease (PD; and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi; however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named “expression-control RNAi” (ExCont-RNAi. ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.

  20. RNA interference ofchitin synthasegenes inhibits chitin biosynthesis and affects larval performance inLeptinotarsa decemlineata(Say).

    Science.gov (United States)

    Shi, Ji-Feng; Mu, Li-Li; Chen, Xu; Guo, Wen-Chao; Li, Guo-Qing

    2016-01-01

    Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata . Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences ( LdChSAa , LdChSAb and LdChSB ) were cloned. LdChSAa and LdChSAb , two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed ds ChSA (derived from a common fragment of LdChSAa and LdChSAb ), ds ChSAa , ds ChSAb and ds ChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa + LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChS s are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata .

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

    Science.gov (United States)

    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

  2. RNA interference from multimeric shRNAs generated by rolling circle transcription.

    Science.gov (United States)

    Seyhan, Attila A; Vlassov, Alexander V; Johnston, Brian H

    2006-01-01

    Methods most commonly used for producing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are chemical synthesis and intracellular expression from engineered vectors. For shRNAs, chemical synthesis is very costly and construction of vectors is laborious. Synthesis by phage RNA polymerases from their natural promoters results in a 5 -terminal triphosphate that can trigger an interferon (IFN) response. Moreover, due to the requirement of phage promoters for 5 - GPuPuPu sequences for transcription initiation, shRNA transcripts may have extra 5 -nucleotides that can constrain the sequences that can be targeted. Also, the 3 ends may have an additional n + 1 nucleotide not encoded by the template. Here we present a novel approach for synthesizing functional shRNAs via rolling circle transcription (RCT) of small (approximately 70 nt) single-stranded DNA circles using T7 RNA polymerase, which avoids these issues. Due to internal pairing, these circles are dumbbell-shaped. RCT produces large transcripts (>10 kb in length) consisting of multimers (>150 copies) of shRNAs in the absence of promoter, terminator, or primer sequences. Dumbbells targeting red fluorescent protein (DsRed), human tumor necrosis factor-alpha (TNF-alpha) and hepatitis C virus (HCV) internal ribosome entry site (IRES) were prepared and transcribed. The resulting long transcripts are substrates for Dicer. When introduced into 293FT and Huh7 cells, the multimeric transcripts inhibited their target genes at levels similar to an equivalent mass of monomeric shRNAs, indicating that they can enter the RNAi pathway. Thus, rolling circle transcription of small DNA dumbbells provides a new source of biologically active interfering RNA.

  3. TARDIS, a targeted RNA directional sequencing method for rare RNA discovery.

    Science.gov (United States)

    Portal, Maximiliano M; Pavet, Valeria; Erb, Cathie; Gronemeyer, Hinrich

    2015-12-01

    High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap-based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    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 ({approx}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.

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  7. Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy.

    Science.gov (United States)

    Amorim, Rebeca Padrão; Araújo, Michelle Gasparetti Leão; Valero, Jorge; Lopes-Cendes, Iscia; Pascoal, Vinicius Davila Bitencourt; Malva, João Oliveira; da Silva Fernandes, Maria José

    2017-12-01

    Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested to be involved in epileptogenesis, via modulation of intracellular calcium levels, excitotoxicity, activation of inflammatory cascades, and cell death, among other mechanisms. These processes have been described to be involved in pilocarpine-induced status epilepticus (SE) and contribute to hyperexcitability, resulting in spontaneous and recurrent seizures. Here, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using RNA interference (RNAi) to inhibit the expression of this receptor. Small interfering RNA (siRNA) targeting P2X7R mRNA was injected into the lateral ventricles (icv) 6 h after SE. Four groups were studied: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle, and Pilo-siRNA. P2X7R was quantified by western blotting and neuronal death assessed by Fluoro-Jade B histochemistry. The hippocampal volume (edema) was determined 48 h following RNAi. Behavioral parameters as latency to the appearance of spontaneous seizures and the number of seizures were determined until 60 days after the SE onset. The Saline-siRNA and Pilo-siRNA groups showed a 43 and 37% reduction, respectively, in P2X7R protein levels compared to respective vehicle groups. Neuroprotection was observed in CA1 and CA3 of the Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the edema detected in the hilus, suprapyramidal dentate gyrus, CA1, and CA3; reduced mortality rate following SE; increased the time to onset of spontaneous seizure; and reduced the number of seizures, when compared to the Pilo-Vehicle group. Therefore, our data highlights the potential of P2X7R as a therapeutic target for the adjunct treatment of epilepsy.

  8. Effect of North Bicyclo[3.1.0]hexane 2'-Deoxypseudosugars on RNA Interference: A Novel Class of siRNA Modification | Center for Cancer Research

    Science.gov (United States)

    The inside cover picture shows how siRNAs modified with North bicyclo[3.1.0]hexane 2'-deoxy-pseudosugars are able to activate the RNA interference machinery. The paper confirms that the North conformation is critical for RNAi activity.

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

    Science.gov (United States)

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

  10. Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1

    Science.gov (United States)

    Zhou, Jiehua; Lazar, Daniel; Li, Haitang; Xia, Xin; Satheesan, Sangeetha; Charlins, Paige; O'Mealy, Denis; Akkina, Ramesh; Saayman, Sheena; Weinberg, Marc S.; Rossi, John J.; Morris, Kevin V.

    2018-01-01

    Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1. PMID:29556342

  11. RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Stéphane Mathis

    2018-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

  12. Targeting MYC in cancer therapy: RNA processing offers new opportunities

    Science.gov (United States)

    2016-01-01

    MYC is a transcription factor, which not only directly modulates multiple aspects of transcription and co‐transcriptional processing (e.g. RNA‐Polymerase II initiation, elongation, and mRNA capping), but also indirectly influences several steps of RNA metabolism, including both constitutive and alternative splicing, mRNA stability, and translation efficiency. As MYC is an oncoprotein whose expression is deregulated in multiple human cancers, identifying its critical downstream activities in tumors is of key importance for designing effective therapeutic strategies. With this knowledge and recent technological advances, we now have multiple angles to reach the goal of targeting MYC in tumors, ranging from the direct reduction of MYC levels, to the dampening of selected house‐keeping functions in MYC‐overexpressing cells, to more targeted approaches based on MYC‐induced secondary effects. PMID:26778668

  13. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  14. RNA targeting by small molecules: Binding of protoberberine ...

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... diseases particularly in viral infections like HIV, AIDS and hepatitis C has led to growing interest in RNA as a potential target for therapeutic intervention (Gallego and Varani 2001;. Foloppe et al. 2006; Liu et al. 2008; Fulle and Gohlke 2010). Furthermore, the recent discovery of a number of micro-.

  15. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-03-01

    Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

  16. shRNA target prediction informed by comprehensive enquiry (SPICE): a supporting system for high-throughput screening of shRNA library.

    Science.gov (United States)

    Kamatuka, Kenta; Hattori, Masahiro; Sugiyama, Tomoyasu

    2016-12-01

    RNA interference (RNAi) screening is extensively used in the field of reverse genetics. RNAi libraries constructed using random oligonucleotides have made this technology affordable. However, the new methodology requires exploration of the RNAi target gene information after screening because the RNAi library includes non-natural sequences that are not found in genes. Here, we developed a web-based tool to support RNAi screening. The system performs short hairpin RNA (shRNA) target prediction that is informed by comprehensive enquiry (SPICE). SPICE automates several tasks that are laborious but indispensable to evaluate the shRNAs obtained by RNAi screening. SPICE has four main functions: (i) sequence identification of shRNA in the input sequence (the sequence might be obtained by sequencing clones in the RNAi library), (ii) searching the target genes in the database, (iii) demonstrating biological information obtained from the database, and (iv) preparation of search result files that can be utilized in a local personal computer (PC). Using this system, we demonstrated that genes targeted by random oligonucleotide-derived shRNAs were not different from those targeted by organism-specific shRNA. The system facilitates RNAi screening, which requires sequence analysis after screening. The SPICE web application is available at http://www.spice.sugysun.org/.

  17. Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis.

    Science.gov (United States)

    Cho, Yoon Shin; Iguchi, Naoko; Yang, Juxiang; Handel, Mary Ann; Hecht, Norman B

    2005-10-01

    In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

  18. Mannan-Binding Lectin-Associated Serine Protease 1/3 Cleavage of Pro-Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference-Mediated Gene Silencing

    Science.gov (United States)

    Banda, Nirmal K.; Acharya, Sumitra; Scheinman, Robert I.; Mehta, Gaurav; Coulombe, Marilyne; Takahashi, Minoru; Sekine, Hideharu; Thiel, Steffen; Fujita, Teizo; Holers, V Michael

    2016-01-01

    The complement system is proposed to play an important role in the pathogenesis of rheumatoid arthritis (RA). The complement system mannan-binding lectin associated serine proteases 1 and 3 (MASP-1/3) cleave proDf (inactive) into Df (active), but it is unknown where this cleavage occurs and whether inhibition of MASP-1/3 is a relevant therapeutic strategy for RA. We show herein that the cleavage of proDf into Df by MASP-1/3 can occur in the circulation and that inhibition of MASP-1/3 by gene silencing is sufficient to ameliorate collagen antibody-induced arthritis (CAIA) in mice. Specifically, to examine the cleavage of proDf into Df, MASP-1/3 producing Df−/− liver tissue (donor) was transplanted under the kidney capsule of MASP-1/3−/− (recipient) mice. Five weeks after the liver transplantation, cleaved Df was present in the circulation of MASP-1/3−/− mice. To determine the individual effects of MASP-1/3 and Df gene silencing on CAIA, mice were injected with scrambled, MASP-1/3 targeted, or Df targeted siRNAs. The mRNA levels for MASP-1 and 3 decreased in the liver to 62% and 58%, respectively, in mice injected with MASP-1/3 siRNAs, and Df mRNA decreased to 53% in the adipose tissue of mice injected with Df siRNAs; additionally, circulating MASP-1/3 and Df protein levels were decreased. In mice injected with both siRNAs the clinical disease activity, histopathologic injury scores, C3 deposition, and synovial macrophage/ neutrophil infiltration were significantly decreased. Thus MASP-1/3 is a new therapeutic target for the treatment of RA, likely through both direct effects on the LP and indirect through the AP. PMID:27707997

  19. A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells

    DEFF Research Database (Denmark)

    Pedersen, Charlotte Demuth; Fang, J J; Pedersen, Anders Elm

    2009-01-01

    Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation...... with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show...... that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface...

  20. Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

    Science.gov (United States)

    Gentile, Carla; Rivas, Gustavo Bueno da S; Lima, José BP; Bruno, Rafaela Vieira; Peixoto, Alexandre Afranio

    2013-01-01

    Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti . PMID:24473806

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

    Science.gov (United States)

    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. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

  2. Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes

    KAUST Repository

    Zhu, Lizhe

    2016-10-05

    At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms.

  3. Discovering the first microRNA-targeted drug

    DEFF Research Database (Denmark)

    Lindow, Morten; Kauppinen, Sakari

    2012-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators of nearly every biological process in the cell and play key roles in the pathogenesis of human disease. As a result, there are many drug discovery programs that focus on developing miRNA-based therapeutics. The most advanced...... of these programs targets the liver-expressed miRNA-122 using the locked nucleic acid (LNA)–modified antisense oligonucleotide miravirsen. Here, we describe the discovery of miravirsen, which is currently in phase 2 clinical trials for treatment of hepatitis C virus (HCV) infection....

  4. Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Guosong Jiang

    Full Text Available Heparanase (HPA, an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA induces transcriptional gene silencing (TGS in human cells. In this study, transfection of siRNA against -9/+10 bp (siH3, but not -174/-155 bp (siH1 or -134/-115 bp (siH2 region relative to transcription start site (TSS locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2, histone H3 lysine 27 trimethylation (H3K27me3 or active chromatin marker acetylated histone H3 (AcH3. The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB, but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (-9/+10 bp into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells.

  5. Antisense-mediated RNA targeting: versatile and expedient genetic manipulation in the brain

    Directory of Open Access Journals (Sweden)

    Ioannis eZalachoras

    2011-07-01

    Full Text Available A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion, but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g. in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis.AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover 1. The principles of antisense-mediated techniques, chemistry and efficacy.2. The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial and temporal control over gene expression, toxicity and delivery issues.3. The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the CNS can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

  6. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

    Science.gov (United States)

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T; Cui, Jiajia; Cheng, Christopher J; Saltzman, W Mark

    2015-12-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.

  7. RFMirTarget: predicting human microRNA target genes with a random forest classifier.

    Directory of Open Access Journals (Sweden)

    Mariana R Mendoza

    Full Text Available MicroRNAs are key regulators of eukaryotic gene expression whose fundamental role has already been identified in many cell pathways. The correct identification of miRNAs targets is still a major challenge in bioinformatics and has motivated the development of several computational methods to overcome inherent limitations of experimental analysis. Indeed, the best results reported so far in terms of specificity and sensitivity are associated to machine learning-based methods for microRNA-target prediction. Following this trend, in the current paper we discuss and explore a microRNA-target prediction method based on a random forest classifier, namely RFMirTarget. Despite its well-known robustness regarding general classifying tasks, to the best of our knowledge, random forest have not been deeply explored for the specific context of predicting microRNAs targets. Our framework first analyzes alignments between candidate microRNA-target pairs and extracts a set of structural, thermodynamics, alignment, seed and position-based features, upon which classification is performed. Experiments have shown that RFMirTarget outperforms several well-known classifiers with statistical significance, and that its performance is not impaired by the class imbalance problem or features correlation. Moreover, comparing it against other algorithms for microRNA target prediction using independent test data sets from TarBase and starBase, we observe a very promising performance, with higher sensitivity in relation to other methods. Finally, tests performed with RFMirTarget show the benefits of feature selection even for a classifier with embedded feature importance analysis, and the consistency between relevant features identified and important biological properties for effective microRNA-target gene alignment.

  8. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Science.gov (United States)

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

  9. SeedVicious: Analysis of microRNA target and near-target sites.

    Science.gov (United States)

    Marco, Antonio

    2018-01-01

    Here I describe seedVicious, a versatile microRNA target site prediction software that can be easily fitted into annotation pipelines and run over custom datasets. SeedVicious finds microRNA canonical sites plus other, less efficient, target sites. Among other novel features, seedVicious can compute evolutionary gains/losses of target sites using maximum parsimony, and also detect near-target sites, which have one nucleotide different from a canonical site. Near-target sites are important to study population variation in microRNA regulation. Some analyses suggest that near-target sites may also be functional sites, although there is no conclusive evidence for that, and they may actually be target alleles segregating in a population. SeedVicious does not aim to outperform but to complement existing microRNA prediction tools. For instance, the precision of TargetScan is almost doubled (from 11% to ~20%) when we filter predictions by the distance between target sites using this program. Interestingly, two adjacent canonical target sites are more likely to be present in bona fide target transcripts than pairs of target sites at slightly longer distances. The software is written in Perl and runs on 64-bit Unix computers (Linux and MacOS X). Users with no computing experience can also run the program in a dedicated web-server by uploading custom data, or browse pre-computed predictions. SeedVicious and its associated web-server and database (SeedBank) are distributed under the GPL/GNU license.

  10. RNA editing in Drosophila melanogaster: new targets and functionalconsequences

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

    2006-09-05

    Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR including components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

  11. Interferência por RNA: uma nova alternativa para terapia nas doenças reumáticas RNA interference: a new alternative for rheumatic diseases therapy

    Directory of Open Access Journals (Sweden)

    Natália Regine de França

    2010-12-01

    Full Text Available A interferência por RNA (RNAi é um mecanismo de silenciamento gênico pós-transcricional conservado durante a evolução. Esse mecanismo, recentemente descrito, é mediado por pequenos RNAs de fita dupla (dsRNAs capazes de reconhecer especificamente uma sequência de mRNA-alvo e mediar sua clivagem ou repressão traducional. O emprego da RNAi como uma ferramenta de terapia gênica tem sido muito estudado, especialmente em infecções virais, câncer, desordens genéticas herdadas, doenças cardiovasculares e mesmo em doenças reumáticas. Aliados aos dados do genoma humano, os conhecimentos do silenciamento gênico mediado por RNAi podem permitir a determinação funcional de praticamente qualquer gene expresso em uma célula e sua implicação para o funcionamento e homeostase celular. Vários estudos terapêuticos in vitro e in vivo em modelos de doenças autoimunes vêm sendo realizados com resultados encorajadores. As vias de quebra de tolerância e inflamação são alvos potenciais para terapia com RNAi em doenças inflamatórias e autoimunes. Nesta revisão vamos recordar os princípios básicos da RNAi e discutir os aspectos que levaram ao desenvolvimento de propostas terapêuticas baseadas em RNAi, começando pelos estudos in vitro de desenvolvimento de ferramentas e identificação de alvos, chegando até os estudos pré-clínicos de disponibilização da droga in vivo, e testes em células humanas e modelos animais de doenças autoimunes. Por fim, vamos revisar os últimos avanços da experiência clínica da terapia com RNAiRNA interference (RNAi is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer

  12. Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

    Science.gov (United States)

    Chen, Y; Redinbaugh, M G; Michel, A P

    2015-06-01

    Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G. nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G. nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control. © 2015 The Royal Entomological Society.

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

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

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

  14. RNA interference-mediated silencing of eukaryotic translation initiation factor 3, subunit B (EIF3B) gene expression inhibits proliferation of colon cancer cells.

    Science.gov (United States)

    Wang, Zheng; Chen, Jinxian; Sun, Jianhua; Cui, Zhe; Wu, Hui

    2012-06-26

    A key factor underlying the control of the cellular growth, size and proliferation involves the regulation of the total protein synthesis. Most often, the initial stages of mRNA translation are rate limiting, which involves a group of eukaryotic translation initiation factors (EIFs). Research advances focused on the inhibition of their expression and activity hold the key to the initiation and progression of tumor and tumor prognosis. We performed RNA interference (RNAi) with the lentivirus vector system to silence the EIF3B gene using the colon cancer cell strain SW1116. The negative control included the normal target cells infected with the negative control virus whereas the knockdown cells included the normal target cells transfected with the RNAi target virus. We tested the inhibition resulting from the decreased expression of EIF3B gene on the proliferation rate of SW1116 cells, including the cell cycle, apoptosis and clonability. Compared with the negative control, the impact of EIF3B gene expression in SW1116 cells on the levels of mRNA and protein in the knockdown group, was significantly inhibited (P cell proliferation rate and clonability were also significantly inhibited (P cells in the G1 phase (P cells.

  15. RNA interference and the vaccine effect of a subolesin homolog from the tick Rhipicephalus haemaphysaloides.

    Science.gov (United States)

    Lu, Pengyun; Zhou, Yongzhi; Yu, Yingfang; Cao, Jie; Zhang, Houshuang; Gong, Haiyan; Li, Guoqing; Zhou, Jinlin

    2016-01-01

    Subolesin is a well-characterized protective antigen in many ticks and, thus, it is potentially useful in the development of a broad-spectrum vaccine or an autocidal gene silencing strategy to control tick infestations. A subolesin homolog was cloned from the tick Rhipicephalus haemaphysaloides, which is widespread in China, by rapid amplification of complementary DNA (cDNA) ends. Its full-length cDNA was 1386 base pairs (bp), containing a 483 bp open reading frame with a predicted molecular mass of 18.7 kilodaltons and an isoelectric point of 9.26. The subolesin protein had a typical nuclear localization signal in its amino-terminus. The full-length cDNA of R. haemaphysaloides showed 52 and 80% identities to those from Ixodes scapularis and R. microplus, respectively, whereas amino acid sequence alignments showed 80 and 97% identities, respectively. Native subolesin was recognized in the unfed tick midgut by an antibody against recombinant subolesin. Transcriptional analysis showed that subolesin was expressed in the tick's four developmental stages and in all of the tissues examined, except for the synganglion. The pathogen Babesia microti induced the subolesin transcript by fourfold. Subolesin gene silencing by RNA interference significantly decreased the larval engorgement rate, the attachment rate and body weight of engorged nymphs, and the body weight and attachment and engorgement rates of adults, as well as the egg weight per female tick. Vaccinating mice and rabbits with recombinant subolesin induced a significant protective effect, resulting in a reduction of blood feeding and oviposition. These results encourage further studies of using subolesin to control tick infestations in China.

  16. RNA interference can rebalance the nitrogen sink of maize seeds without losing hard endosperm.

    Directory of Open Access Journals (Sweden)

    Yongrui Wu

    Full Text Available BACKGROUND: One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2 could be restored in quality protein maize (QPM with quantitative trait loci called o2 modifiers (Mo2s, but those did not increase total protein levels. METHODS: The most predominant zeins are the 22- and 19-kDa α-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi against both α-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed. CONCLUSIONS: The α-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors

    Science.gov (United States)

    Ganesh, Shanthi; Iyer, Arun K.; Morrissey, David V.; Amiji, Mansoor M.

    2013-01-01

    Anticancer therapeutics employing RNA interference mechanism holds promising potentials for sequence-specific silencing of target genes. However targeted delivery of siRNAs to tumor tissues and cells and more importantly, their intracellular release at sites of interest still remains a major challenge that needs to be addressed before this technique could become a clinically viable option. In the current study, we have engineered and screened a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted siRNA delivery. The HA polymer was functionalized with lipids of varying carbon chain lengths/nitrogen content, as well as polyamines for assessing siRNA encapsulation. From the screens, several HA-derivatives were identified that could stably encapsulate/complex siRNAs and form self-assembled nanosystems, as determined by gel retardation assays and dynamic light scattering. Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3-labeled-siRNA loaded HA nano-assemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA-PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors. More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating (stem-) cells and metastatic lesions. PMID:23410679

  19. RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Abdelfatah Alhoot

    Full Text Available BACKGROUND: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. METHODOLOGY/PRINCIPAL FINDINGS: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78 and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4% and extracellular viral RNA load (71.4% compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7% in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells. CONCLUSIONS/SIGNIFICANCE: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS

  20. RNA interference of Trypanosoma brucei cathepsin B and L affects disease progression in a mouse model.

    Directory of Open Access Journals (Sweden)

    Maha-Hamadien Abdulla

    2008-09-01

    Full Text Available We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood-brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood-brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain.

  1. Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

    Directory of Open Access Journals (Sweden)

    Wang Haibo

    2010-09-01

    Full Text Available Abstract Background RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC in tumor-bearing nude mice in vivo. Methods To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 107 HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group: (1 normal saline(NS group, (2replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK group and (3replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse, Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse or PBS (30 ul/mouse was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was used to detect the death of cells. Results The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P RhoA and Rho

  2. Global mapping of miRNA-target interactions in cattle (Bos taurus)

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Moore, Michael J; Luna, Joseph M

    2017-01-01

    With roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential therapeutic target. Here we used cross-linking immunoprecipitation (CLIP) and ligation of miRNA-target chimeras on the Argonaute (AGO) protein to globally map miRNA interact...

  3. Free and total biotherapeutic evaluation in chromatographic assays: interference from targets and immunogenicity.

    Science.gov (United States)

    White, Joleen T; Bonilla, Leo E

    2012-10-01

    Measurement of drug concentrations is critical during drug development, supporting evaluation of safety and efficacy in the context of pharmacokinetics. Protein-based therapeutics have been historically measured by immunoassay methods. Technological advances provide new opportunities to measure these biotherapeutics using previously incompatible chromatographic techniques, such as MS. These advances are breaking down the barriers between 'large-molecule' and 'small-molecule' bioanalysis, and pushing scientists outside their comfort zones. One challenge in measuring biotherapeutic concentration is potential impact from other matrix components, such as therapeutic target or antidrug antibodies. Depending on the specific assay development objective, target interference could be either desired (favoring free measurement) or undesired (favoring total measurement). Orthogonal techniques provide additional tools to meet this challenge. The goal of this review is to introduce both small- and large-molecule bioanalytical scientists to the opportunities and challenges to consider while evaluating orthogonal methods for biotherapeutic bioanalysis.

  4. Anion-sensitive fluorophore identifies the Drosophila swell-activated chloride channel in a genome-wide RNA interference screen.

    Directory of Open Access Journals (Sweden)

    Stephanie C Stotz

    Full Text Available When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl(swell activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl(swell channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1 as the Drosophila Cl(swell channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl(swell current (I(Clswell. We further demonstrate the essential contribution of dBest1 to Drosophila I(Clswell with the introduction of a human Bestrophin disease-associated mutation (W94C. Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I(Clswell. We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293 cells creates a clearly identifiable Drosophila-like I(Clswell. In contrast, activation of mouse Bestrophin 2 (mBest2, the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl(swell channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.

  5. Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling.

    Science.gov (United States)

    Scheidt, Viktor; Jüdes, André; Bär, Christian; Klassen, Roland; Schaffrath, Raffael

    2014-11-29

    Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes ( tip41 ∆, sap190 ∆, ppm1 ∆, rrd1 ∆) and U34 modification defects ( elp3 ∆, kti 12∆, urm1 ∆, ncs2 ∆) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3 ∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3 ∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications ( elp3 ∆, urm1 ∆) enhances nuclear import of and NCR gene activation ( MEP2 , GAP1 ) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.

  7. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Yamanaka

    Full Text Available 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.

  8. Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

    Science.gov (United States)

    Kim, Juhyun; Won, Ranhui; Ban, Guyee; Ju, Mi Ha; Cho, Kyung Sook; Young Han, Sang; Jeong, Jin-Sook; Lee, Seong-Wook

    2015-07-20

    Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.

  9. The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2010-04-01

    Full Text Available Abstract Background The RNA interference (RNAi pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity. Results As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced ~50% between 1-7 days post-bloodmeal (pbm when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes. Conclusions We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both

  10. Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm.

    Directory of Open Access Journals (Sweden)

    Christiane Schaefer

    Full Text Available In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

  11. RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.

    Directory of Open Access Journals (Sweden)

    Kelly E Coller

    2009-12-01

    Full Text Available Hepatitis C virus (HCV enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.

  12. Identification and mapping of a region on the mRNA of Morbillivirus nucleoprotein susceptible to RNA interference.

    Science.gov (United States)

    Keita, Djénéba; Servan de Almeida, Renata; Libeau, Geneviève; Albina, Emmanuel

    2008-11-01

    The morbillivirus genus includes important pathogens such as measles virus (MV), peste des petits ruminants virus (PPRV), and rinderpest virus (RPV) and forms a group of antigenically related viruses. The viral nucleoprotein (N) is a well-conserved protein among the genus and plays a central role in the replication of the virus. Using a comprehensive approach for siRNA screening of the conserved sequences of the N gene, including sequence analysis and functional in vitro tests, we have identified a region for the design of siRNA effective for the control of PPRV, RPV, and MV replication. Silencing of the N mRNA efficiently shuts down the production of N transcripts, the expression of N protein, and the indirect inhibition of matrix protein, resulting in the inhibition of PPRV progeny by 10,000-fold. These results suggest that siRNA against this region should be further explored as a therapeutic strategy for morbillivirus infections.

  13. Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp.

    Science.gov (United States)

    Thammasorn, Thitiporn; Sangsuriya, Pakkakul; Meemetta, Watcharachai; Senapin, Saengchan; Jitrakorn, Sarocha; Rattanarojpong, Triwit; Saksmerprome, Vanvimon

    2015-12-01

    RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called "multi-WSSV dsRNA." A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners. For a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge. The present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

  14. Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

    Science.gov (United States)

    Alshehri, Abdullah; Grabowska, Anna; Stolnik, Snow

    2018-02-28

    Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

  15. Hypoxia-Induced MicroRNA-210 Targets Neurodegenerative Pathways

    Directory of Open Access Journals (Sweden)

    Michelle E. Watts

    2018-03-01

    Full Text Available Hypoxia-regulated microRNA-210 (miR-210 is a highly conserved microRNA, known to regulate various processes under hypoxic conditions. Previously we found that miR-210 is also involved in honeybee learning and memory, raising the questions of how neural activity may induce hypoxia-regulated genes and how miR-210 may regulate plasticity in more complex mammalian systems. Using a pull-down approach, we identified 620 unique target genes of miR-210 in humans, among which there was a significant enrichment of age-related neurodegenerative pathways, including Huntington’s, Alzheimer’s, and Parkinson’s diseases. We have also validated that miR-210 directly regulates various identified target genes of interest involved with neuronal plasticity, neurodegenerative diseases, and miR-210-associated cancers. This data suggests a potentially novel mechanism for how metabolic changes may couple plasticity to neuronal activity through hypoxia-regulated genes such as miR-210.

  16. RNA decay: a novel therapeutic target in bacteria.

    Science.gov (United States)

    Eidem, Tess M; Roux, Christelle M; Dunman, Paul M

    2012-01-01

    The need for novel antibiotics is greater now than perhaps any time since the pre-antibiotic era. Indeed, the recent collapse of most pharmaceutical antibacterial groups, combined with the emergence of hypervirulent and pan-antibiotic-resistant bacteria have, in effect, created a 'perfect storm' that has severely compromised infection treatment options and led to dramatic increases in the incidence and severity of bacterial infections. To put simply, it is imperative that we develop new classes of antibiotics for the therapeutic intervention of bacterial infections. In that regard, RNA degradation is an essential biological process that has not been exploited for antibiotic development. Herein we discuss the factors that govern bacterial RNA degradation, highlight members of this machinery that represent attractive antimicrobial drug development targets and describe the use of high-throughput screening as a means of developing antimicrobials that target these enzymes. Such agents would represent first-in-class antibiotics that would be less apt to inactivation by currently encountered enzymatic antibiotic-resistance determinants. Copyright © 2012 John Wiley & Sons, Ltd.

  17. RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of Machado-Joseph disease.

    Science.gov (United States)

    Nóbrega, Clévio; Nascimento-Ferreira, Isabel; Onofre, Isabel; Albuquerque, David; Déglon, Nicole; de Almeida, Luís Pereira

    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.

  18. RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach, Blattella germanica L.

    Science.gov (United States)

    Myers, A J; Gondhalekar, A D; Fardisi, M; Pluchar, K D; Saltzmann, K D; Bennett, G W; Scharf, M E

    2018-04-01

    German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha-amylase Blattella germanica Tergal Gland protein-1 (BGTG-1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG-1 protein. BGTG-1 gene expression profiles were determined by using quantitative real-time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG-1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG-1 gene expression by injecting BGTG-1 homologous double-stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG-1 dsRNA showed ∼90% lower BGTG-1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success. © 2017 The Royal Entomological Society.

  19. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence

    NARCIS (Netherlands)

    Semenova, E.V.; Jore, M.M.; Westra, E.R.; Oost, van der J.; Brouns, S.J.J.

    2011-01-01

    Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas

  20. Kinetic analysis of the effects of target structure on siRNA efficiency

    Science.gov (United States)

    Chen, Jiawen; Zhang, Wenbing

    2012-12-01

    RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

  1. Functional characterization of a juvenile hormone esterase related gene in the moth Sesamia nonagrioides through RNA interference.

    Directory of Open Access Journals (Sweden)

    Dimitrios Kontogiannatos

    Full Text Available Juvenile hormone esterase (JHE is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi. Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.

  2. Alleviation of Trigeminal Nociception Using p75 Neurotrophin Receptor Targeted Lentiviral Interference Therapy.

    Science.gov (United States)

    O'Leary, Valerie B; O'Connell, Marie; Antyborzec, Inga; Ntziachristos, Vasilis; Oliver Dolly, J; Ovsepian, Saak V

    2018-02-09

    Acute and chronic trigeminal (TG) neuropathies are the cause of considerable distress, with limited treatments available at present. Nociceptive neurons enriched with the vanilloid type 1 receptor (VR1) partake in pain sensation and sensitization in the TG system. While VR1 blockers with anti-nociceptive potential are of substantial medical interest, their use remains limited due to poor selectivity and lack of cell-targeting capabilities. This study describes a methodology for the alleviation of nociception via targeted depletion of VR1 in TG sensory neurons in rats. In cultured TG ganglion neurons, VR1 expression was virtually abolished by lentiviral short hairpin RNA (LV-VR1). By decorating GFP encoding LV (LV-GFP) and LV-VR1 with IgG192 for targeting TG sensory neurons enriched with the p75 neurotrophin receptor (p75NTR), transduction of a reporter GFP and VR1 depletion was achieved after injection of targeted vectors into the whisker pad. In IgG192/LV-VR1-injected rats, the behavioral response to capsaicin exposure as well as Erk 1/2 phosphorylation and VR1 current activation by capsaicin were significantly reduced. This pioneering investigation, thus, provides a proof of principle for a means of attenuating TG nociception, revealing therapeutic potential.

  3. Interference control theory : A new perspective on dual-task interference in memorizing and responding to visual targets

    NARCIS (Netherlands)

    Nieuwenstein, Mark; Scholz, Sabine; Broers, Nico

    2015-01-01

    In a recent study, Nieuwenstein and Wyble (2014) showed that the consolidation of a masked visual target can be disrupted for up to one second by a trailing 2-alternative forced choice task. Aside from demonstrating that working memory consolidation involves a time-consuming process that continues

  4. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh; Deng, Ling; She, Qunxin

    2016-01-01

    The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...

  5. Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

    Directory of Open Access Journals (Sweden)

    Mallory E. Harden

    2017-01-01

    Full Text Available The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs. While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Andrew S French

    2015-07-01

    Full Text Available 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 seven 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 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.

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

    Science.gov (United States)

    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.

  9. Macros in microRNA target identification: a comparative analysis of in silico, in vitro, and in vivo approaches to microRNA target identification.

    Science.gov (United States)

    Tarang, Shikha; Weston, Michael D

    2014-01-01

    MicroRNAs (miRNAs) are short RNA molecules that modulate post-transcriptional gene expression by partial or incomplete base-pairing to the complementary sequences on their target genes. Sequence-based miRNA target gene recognition enables the utilization of computational methods, which are highly informative in identifying a subset of putative miRNA targets from the genome. Subsequently, single miRNA-target gene binding is evaluated experimentally by in vitro assays to validate and quantify the transcriptional or post-transcriptional effects of miRNA-target gene interaction. Although ex vivo approaches are instructive in providing a basis for further analyses, in vivo genetic studies are critical to determine the occurrence and biological relevance of miRNA targets under physiological conditions. In the present review, we summarize the important features of each of the experimental approaches, their technical and biological limitations, and future challenges in light of the complexity of miRNA target gene recognition.

  10. Computational Challenges in miRNA Target Predictions: To Be or Not to Be a True Target?

    Directory of Open Access Journals (Sweden)

    Christian Barbato

    2009-01-01

    Full Text Available All microRNA (miRNA target—finder algorithms return lists of candidate target genes. How valid is that output in a biological setting? Transcriptome analysis has proven to be a useful approach to determine mRNA targets. Time course mRNA microarray experiments may reliably identify downregulated genes in response to overexpression of specific miRNA. The approach may miss some miRNA targets that are principally downregulated at the protein level. However, the high-throughput capacity of the assay makes it an effective tool to rapidly identify a large number of promising miRNA targets. Finally, loss and gain of function miRNA genetics have the clear potential of being critical in evaluating the biological relevance of thousands of target genes predicted by bioinformatic studies and to test the degree to which miRNA-mediated regulation of any “validated” target functionally matters to the animal or plant.

  11. Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.

    Directory of Open Access Journals (Sweden)

    John S Cho

    2009-09-01

    Full Text Available The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha. How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

  12. Micro RNA, A Review: Pharmacogenomic drug targets for complex diseases

    Directory of Open Access Journals (Sweden)

    Sandhya Bawa

    2010-01-01

    Full Text Available

    Micro RNAs (miRNAs are non-coding RNAs that can regulate gene expression to target several mRNAs in a gene regulatory network. MiRNA related Single Nucleotide Polymorphisms (S.N.P.s represent a newly identified type of genetic variability that can be of influence to the risk of certain human diseases and also affect how drugs can be activated and metabolized by patients. This will help in personalized medicines which are used for administrating the correct dosage of drug and drug efficacy. miRNA deregulated expression has been extensively described in a variety of diseases such as Cancer, Obesity , Diabetes, Schizophrenia and control and self renewal of stem cells. MiRNA can function as oncogenes and/or tumor suppressors. MiRNAs may act as key regulators of processes as diverse as early development, cell proliferation and cell death, apoptosis and fat metabolism and cell differentiation .miRNA expression have shown their role in brain development chronic lymphocytic leukemia, colonic adeno carcinoma, Burkiff’s lymphoma and viral infection. These show their links with viral disease, neurodevelopment and cancer. It has been shown that they play a key role in melanoma metastasis. These may be

  13. Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

    Directory of Open Access Journals (Sweden)

    Jaclyn C Scott

    2010-10-01

    Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

  14. Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Liou Louis S

    2010-04-01

    Full Text Available Abstract Background MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation. Results We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX, VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1

  15. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    Science.gov (United States)

    Wan, Pin-Jun; Jia, Shuang; Li, Na; Fan, Jin-Mei; Li, Guo-Qing

    2014-01-01

    Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase), encoded by the Halloween gene disembodied (dib), plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR) gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

  16. RNA interference depletion of the Halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus.

    Directory of Open Access Journals (Sweden)

    Pin-Jun Wan

    Full Text Available Sogatella furcifera and Laodelphax striatellus are economically important rice pests in China by acting as vectors of several rice viruses, sucking the phloem sap and blocking the phloem vessels. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. A cytochrome P450 monooxygenase CYP302A1 (22-hydroxylase, encoded by the Halloween gene disembodied (dib, plays a critical role in ecdysteroidogenesis. The objective of this study is to test whether dib genes are potential targets for RNA interference-based management of S. furcifera and L. striatellus. We cloned and characterized Sfdib and Lsdib. The open reading frame regions of dib genes were generated and used for designing and constructing dsRNA fragments. Experiments were conducted using oral delivery of dsdib to investigate the effectiveness of RNAi in S. furcifera and L. striatellus nymphs. Real-time quantitative reverse transcriptase-PCR analysis demonstrated that continuous ingestion of dsdib at the concentration of 0.01, 0.05 and 0.50 mg/ml diminished Sfdib expression levels by 35.9%, 45.1% and 66.2%, and ecdysone receptor (SfEcR gene mRNA levels by 34.0%, 36.2% and 58.5% respectively in S. furcifera, and decreased Lsdib expression level by 18.8%, 35.8% and 56.7%, and LsEcR mRNA levels by 25.2%, 46.8% and 68.8% respectively in L. striatellus. The reduction in dib and EcR transcript abundance resulted in observable phenotypes. The development of nymphs was impaired and the survival was negatively affected. Our data will enable the development of new insect control strategies and functional analysis of vital genes in S. furcifera and L. striatellus nymphs.

  17. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

    Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the

  18. Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Yao Hailan

    2012-08-01

    Full Text Available Abstract Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B expressed by a recombinant vector (pGCL-2B or a recombinant lentivirus (Lenti-2B were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

  19. Novel genes participating in the formation of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution and RNA interference knockdown.

    Directory of Open Access Journals (Sweden)

    Daisuke Funabara

    Full Text Available In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein, 000118, 000133 and 000411 (MSI60, which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

  20. Targeting the MicroRNA Passenger Strand for Regulating Therapeutic Transgenes.

    Science.gov (United States)

    Kim, Sung Jin; Lee, Chang Ho; Lee, Seong-Wook

    2015-08-01

    Gene therapy strategies have been developed, which can tissue or disease specifically regulate expression of exogenous transgenes by means of endogenous microRNA (miRNA) activity. However, the use of an endogenous guide strand to regulate an exogenous transgene could affect expression of endogenous miRNA target genes. In this study, we developed a new regulatory system of exogenous transgene expression by targeting the passenger strand. We constructed reporter constructs harboring miRNA-122 guide or passenger target sites with perfect or imperfect complementarity. We observed downregulation of an exogenous transgene harboring the miRNA-122 target sites against either the guide or passenger strand in cells expressing the cognate miRNA or cells stably expressing the miRNA target site. Moreover, the transgene activity as well as the gene expression level increased specifically by intracellular introduction of the antisense RNA against the corresponding strand. Endogenous target gene expression was induced by the transgene construct harboring the miRNA guide strand target sites, but not the passenger strand target sites. Importantly, the therapeutic transgene activity was efficiently regulated by targeting the passenger strand. These results suggested that an approach to passenger strand-regulated expression of therapeutic transgenes could be applied more safely as a therapeutic tool.

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

      Science.gov (United States)

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

      2013-08-01

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

    2. Generation of Constructs for DNA-Directed RNA Interference of Venezuelan Equine Encephalitis Virus Genes

      Science.gov (United States)

      2006-12-01

      the viral genome of the attenuated vaccine strain of VEE [14], TC-83, based on their suitability for siRNA design. Using the Silencer Express system...of an effective vaccine or therapeutic for VEE, a highly infectious virus, underscores the need for research in this area. In addition, the potential... poliovirus [5], influenza virus [6], Ebola virus [7], Coxsackievirus B3 [8], among others. RNAi involves sequence-specific gene silencing, or gene knockdown

    3. A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC.

      Directory of Open Access Journals (Sweden)

      Pieter J A Eichhorn

      2007-12-01

      Full Text Available Protein Phosphatase type 2A (PP2A represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2-terminal kinase (JNK activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.

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

      Science.gov (United States)

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

      2014-01-01

      Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) 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) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol 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. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

    5. Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

      Science.gov (United States)

      Denzler, Rémy; McGeary, Sean E; Title, Alexandra C; Agarwal, Vikram; Bartel, David P; Stoffel, Markus

      2016-11-03

      Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

    6. Proteomic analysis of differential proteins in pancreatic carcinomas: Effects of MBD1 knock-down by stable RNA interference

      Directory of Open Access Journals (Sweden)

      Ni Quanxing

      2008-04-01

      Full Text Available Abstract Background Methyl-CpG binding domain protein 1 (MBD1, a suppressor of gene transcription, may be involved in inactivation of tumor suppressor genes during tumorigenesis. Over-expression of MBD1 has been reported in human pancreatic carcinomas. Methods In this study, we established a MBD1-knock-down pancreatic cancer cell line (BxPC-3 using stable RNA interference, to compare the proteomic changes between control and MBD1-knock-down cells using two-dimensional gel electrophoresis and mass spectrometry. Results We identified five proteins that were up-regulated and nine proteins that were down-regulated. Most of the identified proteins are involved in tumorigenesis, some are prognostic biomarkers for human malignant tumors. Conclusion Our data suggest that these differential proteins may be associated with the function of MBD1, and provide some insight into the functional mechanism of MBD1 in the development of pancreatic cancer.

    7. Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNAseq, RNA interference and irradiation approach.

      Science.gov (United States)

      Solana, Jordi; Kao, Damian; Mihaylova, Yuliana; Jaber-Hijazi, Farah; Malla, Sunir; Wilson, Ray; Aboobaker, Aziz

      2012-01-01

      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. 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. 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.

    8. The RNA-dependent-RNA polymerase, an emerging antiviral drug target for the Hendra virus.

      Science.gov (United States)

      Velkov, Tony; Carbone, Vincenzo; Akter, Jesmin; Sivanesan, Sivashangarie; Li, Jian; Beddoe, Travis; Marsh, Glenn A

      2014-01-01

      Australia is facing a major national medical challenge with the emergence of the Hendra virus (HeV) as a medically and economically important pathogen of humans and animals. Clinical symptoms of human HeV infection can include fever, hypotension, dizziness, encephalitis, respiratory haemorrhage and edema. The window of opportunity for successful patient treatment remains unknown, but is likely to be very narrow. Currently, very few effective therapeutic options are available for the case management of severe HeV infections or the rapid silencing of local outbreaks. This underscores the need for more activity in the drug discovery arena to develop much needed therapeutics that specifically targets this deadly disease. The structural analysis of HeV is very much in its infancy, which leaves many gaps in our understanding of the biology of HeV and makes structure-guided drug design difficult. Structural studies of the viral RNAdependent- RNA polymerase (RdRp), which is the heart of the viral replication machinery, will set the stage for rational drug design and fill a major gap in our understanding of the HeV replication machinery. This review examines the current knowledge based on the multi-domain architecture of the Hendra RdRp and highlights which essential domain functions represent tangible targets for drug development against this deadly disease.

    9. Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

      Science.gov (United States)

      Lipscomb, Elizabeth A; Dugan, Aisling S; Rabinovitz, Isaac; Mercurio, Arthur M

      2003-01-01

      The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.

    10. DIANA-microT web server: elucidating microRNA functions through target prediction.

      Science.gov (United States)

      Maragkakis, M; Reczko, M; Simossis, V A; Alexiou, P; Papadopoulos, G L; Dalamagas, T; Giannopoulos, G; Goumas, G; Koukis, E; Kourtis, K; Vergoulis, T; Koziris, N; Sellis, T; Tsanakas, P; Hatzigeorgiou, A G

      2009-07-01

      Computational microRNA (miRNA) target prediction is one of the key means for deciphering the role of miRNAs in development and disease. Here, we present the DIANA-microT web server as the user interface to the DIANA-microT 3.0 miRNA target prediction algorithm. The web server provides extensive information for predicted miRNA:target gene interactions with a user-friendly interface, providing extensive connectivity to online biological resources. Target gene and miRNA functions may be elucidated through automated bibliographic searches and functional information is accessible through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The web server offers links to nomenclature, sequence and protein databases, and users are facilitated by being able to search for targeted genes using different nomenclatures or functional features, such as the genes possible involvement in biological pathways. The target prediction algorithm supports parameters calculated individually for each miRNA:target gene interaction and provides a signal-to-noise ratio and a precision score that helps in the evaluation of the significance of the predicted results. Using a set of miRNA targets recently identified through the pSILAC method, the performance of several computational target prediction programs was assessed. DIANA-microT 3.0 achieved there with 66% the highest ratio of correctly predicted targets over all predicted targets. The DIANA-microT web server is freely available at www.microrna.gr/microT.

    11. Ribosomal RNA: nature's own polymerase-amplified target for diagnosis.

      Science.gov (United States)

      Waters, A P; McCuthan, T F

      1990-02-01

      The technological criteria for diagnostic tests for tropical diseases are strict and limited. One method of considerable potential for field use is the exploitation of regions of species-specific sequences of ribosomal RNA (rRNA). Oligodeoxynucleotide probes can be used to detect the specific RNa regions in question. Andy Waters and Tom McCutchan describe how simple rRNA methodologies for Plasmodium can detect and identify parasites as well as follow different stages in their development. The potential of this methodology extends to monitoring both epidemiological parameters and the efficacy of novel drug regimes.

    12. The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

      NARCIS (Netherlands)

      van der Linden, Lonneke; Vives-Adrián, Laia; Selisko, Barbara; Ferrer-Orta, Cristina; Liu, Xinran; Lanke, Kjerstin; Ulferts, Rachel; De Palma, Armando M; Tanchis, Federica; Goris, Nesya; Lefebvre, David; De Clercq, Kris; Leyssen, Pieter; Lacroix, Céline; Pürstinger, Gerhard; Coutard, Bruno; Canard, Bruno; Boehr, David D; Arnold, Jamie J; Cameron, Craig E; Verdaguer, Nuria; Neyts, Johan; van Kuppeveld, Frank J M

      2015-01-01

      The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy.

    13. Methods to enable the design of bioactive small molecules targeting RNA.

      Science.gov (United States)

      Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

      2014-02-21

      RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

    14. RNA targeting by small molecules: Binding of protoberberine ...

      Indian Academy of Sciences (India)

      For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are ...

    15. RNA interference mediated knockdown of the KDEL receptor and COPB2 inhibits digestion and reproduction in the parasitic copepod Lepeophtheirus salmonis.

      Science.gov (United States)

      Tröße, Christiane; Nilsen, Frank; Dalvin, Sussie

      2014-04-01

      Retrograde transport of proteins from the endoplasmic reticulum to the Golgi is an essential part of the secretory pathway that all newly synthesised secreted and membrane proteins in eukaryotic cells undergo. The aim of this study was to characterise two components of the retrograde transport pathway in the parasitic copepod Lepeophtheirus salmonis (salmon louse) on a molecular and functional level. LsKDELR and LsCOPB2 were confirmed to be the salmon louse homologues of the chosen target proteins by sequence similarity. Ontogenetic analysis by qRT-PCR revealed the highest expression levels of both genes in adult females and the earliest larval stage. LsKDELR and LsCOPB2 localisation in adult females was detected by immunofluorescence and in situ hybridisation, respectively. Both LsKDELR and LsCOPB2 were found in the ovaries, the oocytes and the gut. LsKDELR and LsCOPB2 were knocked down by RNA interference in preadult females, which was confirmed by qRT-PCR. LsCOPB2 knockdown lice had a significantly higher mortality and failed to develop normally, while both LsCOPB2 and LsKDELR knockdown caused disturbed digestion and the absence of egg strings. This shows the potential of LsKDELR and LsCOPB2 as suitable target candidates for new pest control methods. Copyright © 2014 Elsevier Inc. All rights reserved.

    16. RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

      Science.gov (United States)

      Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

      2014-06-19

      The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

    17. RNA Interference of 1-Aminocyclopropane-1-carboxylic Acid Oxidase (ACO1 and ACO2 Genes Expression Prolongs the Shelf Life of Eksotika (Carica papaya L. Papaya Fruit

      Directory of Open Access Journals (Sweden)

      Rogayah Sekeli

      2014-06-01

      Full Text Available The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6. Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

    18. Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression

      Directory of Open Access Journals (Sweden)

      Aftab Ahmad

      2017-09-01

      Full Text Available Cotton leaf curl virus disease (CLCuD is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB. The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

    19. RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

      Directory of Open Access Journals (Sweden)

      Li eJiang

      2014-04-01

      Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

    20. SHARP-2 gene silencing by lentiviral-based short hairpin RNA interference prolonged rat kidney transplant recipients' survival time.

      Science.gov (United States)

      Shou, Z; Xiao, H; Xu, Y; Wang, Y; Yang, Y; Jiang, H; Chen, J; Yamada, K; Miyamoto, K

      2009-01-01

      Split- and hairy-related protein-2 (SHARP-2) controls the expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), which both play a key role in transplant rejection. This study was designed to investigate whether SHARP-2 short hairpin RNA interference (shRNAi) could prolong the survival of rat kidney transplant recipients. A lentiviral-based shRNAi construct, LV-SHARP-2iC, showed a SHARP-2 gene silencing efficiency of 84% in normal rat kidney cells. In activated T-cells, SHARP-2 gene silencing with the LV-SHARP-2iC construct resulted in 61% and 69% down-regulation of IL-2 and IFN-gamma, respectively, compared with a scramble control construct. When donor kidney was perfused with 5 x 10(7) transforming units of the LV-SHARP-2iC construct, the median survival time of the transplant recipients was prolonged by 4 - 5 days compared with control groups. In conclusion, recombinant lentiviral LV-SHARP-2iC construct effectively silenced SHARP-2 gene expression, which reduced IL-2 and IFN-gamma mRNA expression and prolonged rat kidney transplant recipients' survival.

    1. [RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].

      Science.gov (United States)

      Wei, Min; Zhang, Yan-Ling; Chen, Lan; Cai, Cui-Xia; Wang, Han-Duo

      2016-02-20

      To explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms. Three HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting. Transfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (PHela cells, increased the apoptosis rate (PHela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

    2. In silico cloning and annotation of genes involved in the digestion, detoxification and RNA interference mechanism in the midgut of Bactrocera dorsalis [Hendel (Diptera: Tephritidae)].

      Science.gov (United States)

      Shen, G-M; Dou, W; Huang, Y; Jiang, X-Z; Smagghe, G; Wang, J-J

      2013-08-01

      As the second largest organ in insects, the insect midgut is the major tissue involved in the digestion of food and detoxification of xenobiotics, such as insecticides, and the first barrier and target for oral RNA interference (RNAi). In this study, we performed a midgut-specific transcriptome analysis in the oriental fruit fly, Bactrocera dorsalis, an economically important worldwide pest, with many populations showing high levels of insecticide resistance. Using high-throughput sequencing, 52 838 060 short reads were generated and assembled to 25 236 unigenes with a mean length of 758 bp. Interestingly, 34 unique sequences encoding digestion enzymes were newly described and these included aminopeptidase and trypsin, genes associated with Bacillus thuringiensis resistance and fitness cost. Second, 41 transcripts were annotated to particular detoxification genes such as glutathione S-transferases, carboxylesterases and cytochrome P450s, and the subsequent phylogenetic analysis indicated homology with tissue-specific and insecticide resistance-related genes of Drosophila melanogaster. Third, we identified the genes involved in the mechanism of RNAi and the uptake of double-stranded RNA. The sequences encoding Dicer-2, R2D2, AGO2, and Eater were confirmed, but SID and SR-CI were absent in the midgut transcriptome. In conclusion, the results provide basic molecular information to better understand the mechanisms of food digestion, insecticide resistance and oral RNAi in this important pest insect in agriculture. Specific genes in these systems can be used in the future as potential targets for pest control, for instance, with RNAi technology. © 2013 Royal Entomological Society.

    3. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

      Directory of Open Access Journals (Sweden)

      Jae-Su Moon

      Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

    4. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

      Directory of Open Access Journals (Sweden)

      Hongmei Wang

      Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

    5. Accurate microRNA target prediction correlates with protein repression levels

      Directory of Open Access Journals (Sweden)

      Simossis Victor A

      2009-09-01

      Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

    6. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

      Directory of Open Access Journals (Sweden)

      Fangquan Wang

      2016-05-01

      Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

    7. Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

      Science.gov (United States)

      Farazi, Thalia A; Leonhardt, Carl S; Mukherjee, Neelanjan; Mihailovic, Aleksandra; Li, Song; Max, Klaas E A; Meyer, Cindy; Yamaji, Masashi; Cekan, Pavol; Jacobs, Nicholas C; Gerstberger, Stefanie; Bognanni, Claudia; Larsson, Erik; Ohler, Uwe; Tuschl, Thomas

      2014-07-01

      Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed. © 2014 Farazi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

    8. Knockdown of Eag1 Expression by RNA Interference Increases Chemosensitivity to Cisplatin in Ovarian Cancer Cells.

      Science.gov (United States)

      Hui, Chen; Lan, Zhang; Yue-li, Lin; Li-lin, Hong; Li-lin, Huang

      2015-12-01

      Ether á go-go 1 (Eag1) is frequently highly expressed in various malignant cancers and its excessive expression is correlated with poor prognosis in various cancers. However, the relationship of Eag1 expression with the clinical outcome of patients having ovarian cancer treated with cisplatin-based adjuvant chemotherapy is still unknown. In this study, we measured the expression of Eag1 in ovarian cancer and investigated the association between cisplatin chemosensitivity of ovarian cancer cells and Eag1 expression level. We demonstrate that decreased expression of Eag1 correlates with favorable prognosis in patients treated with cisplatin-based adjuvant chemotherapy and predicts higher cisplatin sensitivity in ovarian cancer cells. In vitro, knockdown of Eag1 by small interfering RNA facilitated the sensitivity of ovarian cancer cells (SKOV3 and TYK) to cisplatin-induced apoptosis via nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) pathway. Furthermore, knockdown of Eag1 expression was associated with decreased expression of the P-glycoprotein without affecting multidrug resistance-associated protein 1 expression. Taken together, Eag1 may serve as a potential indicator to predict Eag1 chemosensitivity, and silencing Eag1 may represent a potential therapeutic strategy for ovarian cancer. © The Author(s) 2015.

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

      Czech Academy of Sciences Publication Activity Database

      Štefanič, S.; Dvořák, Jan; Horn, Martin; Braschi, S.; Sojka, Daniel; Ruelas, D.; Suzuki, B.; Lim, K.-C.; Hopkins, S. D.; McKerrow, J. H.; Caffrey, C. R.

      2010-01-01

      Roč. 4, č. 10 (2010), e850 ISSN 1935-2735 R&D Projects: GA ČR GA203/09/1585; GA AV ČR KJB600960911; GA MŠk(CZ) LC06009; GA MŠk(CZ) ME10011 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z60220518 Keywords : Tropical disease * Schistosoma * schistosomula * RNAi * protease * tegument * drug target * drug discovery Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.752, year: 2010

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

      Energy Technology Data Exchange (ETDEWEB)

      Green, Pamela J. [Univ. of Delaware, Newark, DE (United States)

      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.

    11. Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality

      NARCIS (Netherlands)

      van Haaften, Gijs; Vastenhouw, Nadine L; Nollen, Ellen A A; Plasterk, Ronald H A; Tijsterman, Marcel

      2004-01-01

      Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect

    12. Analysis of small-sample clinical genomics studies using multi-parameter shrinkage: application to high-throughput RNA interference screening

      NARCIS (Netherlands)

      van de Wiel, M.; Menezes, R.; van Olst, E.; van Beusechem, V.W.

      2013-01-01

      High-throughput (HT) RNA interference (RNAi) screens are increasingly used for reverse genetics and drug discovery. These experiments are laborious and costly, hence sample sizes are often very small. Powerful statistical techniques to detect siRNAs that potentially enhance treatment are currently

    13. RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR

      Science.gov (United States)

      Asian longhorned beetle (ALB), Anoplophora glabripennis, is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi)technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes in...

    14. RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

      Science.gov (United States)

      Subramanya, Sandesh; Kim, Sang-Soo; Manjunath, N; Shankar, Premlata

      2010-02-01

      Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV. We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 - 3 years. HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use. Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

    15. Reduction of interferences in the analysis of Children's Dimetapp using ultraviolet spectroscopy data and target factor analysis

      Science.gov (United States)

      Msimanga, Huggins Z.; Lam, Truong Thach Ho; Latinwo, Nathaniel; Song, Mihyang Kristy; Tavakoli, Newsha

      2018-03-01

      A calibration matrix has been developed and successfully applied to quantify actives in Children's Dimetapp®, a cough mixture whose active components suffer from heavy spectral interference. High-performance liquid chromatography/photodiode array instrument was used to identify the actives and any other UV-detectable excipients that might contribute to interferences. The instrument was also used to obtain reference data on the actives, instead of relying on the manufacturer's claims. Principal component analysis was used during the developmental stages of the calibration matrix to highlight any mismatch between the calibration and sample spectra, making certain that "apples" were not compared with "oranges". The prediction model was finally calculated using target factor analysis and partial least squares regression. In addition to the actives in Children's Dimetapp® (brompheniramine maleate, phenylephrine hydrogen chloride, and dextromethorphan hydrogen bromide), sodium benzoate was identified as the major and FD&C Blue #1, FD&C Red #40, and methyl anthranilate as minor spectral interferences. Model predictions were compared before and after the interferences were included into the calibration matrix. Before including interferences, the following results were obtained: brompheniramine maleate = 481.3 mg L- 1 ± 134% RE; phenylephrine hydrogen chloride = 1041 mg L- 1 ± 107% RE; dextromethorphan hydrogen bromide = 1571 mg L- 1 ± 107% RE, where % RE = percent relative error based on the reference HPLC data. After including interferences, the results were as follows: brompheniramine maleate = 196.3 mg L- 1 ± 4.4% RE; phenylephrine hydrogen chloride = 501.3 mg L- 1 ± 0.10% RE; dextromethorphan hydrogen bromide = 998.7 mg L- 1 ± 1.6% RE as detailed in Table 6.

    16. In vitro and in vivo inhibition of rabies virus replication by RNA interference

      Directory of Open Access Journals (Sweden)

      Ekaterina A. Durymanova Ono

      2013-09-01

      Full Text Available Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV (Mononegavirales: Rhabdoviridae: Lyssavirus. Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

    17. In vitro and in vivo inhibition of rabies virus replication by RNA interference.

      Science.gov (United States)

      Durymanova Ono, Ekaterina A; Iamamoto, Keila; Castilho, Juliana G; Carnieli, Pedro; de Novaes Oliveira, Rafael; Achkar, Samira M; Carrieri, Maria L; Kotait, Ivanete; Brandão, Paulo E

      2013-01-01

      Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

    18. Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

      Directory of Open Access Journals (Sweden)

      Maria Patrizia Somma

      2008-07-01

      Full Text Available RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

    19. Selective RNA targeting and regulated signaling by RIG-I is controlled by coordination of RNA and ATP binding.

      Science.gov (United States)

      Fitzgerald, Megan E; Rawling, David C; Potapova, Olga; Ren, Xiaoming; Kohlway, Andrew; Pyle, Anna Marie

      2017-02-17

      RIG-I is an innate immune receptor that detects and responds to infection by deadly RNA viruses such as influenza, and Hepatitis C. In the cytoplasm, RIG-I is faced with a difficult challenge: it must sensitively detect viral RNA while ignoring the abundance of host RNA. It has been suggested that RIG-I has a ‘proof-reading’ mechanism for rejecting host RNA targets, and that disruptions of this selectivity filter give rise to autoimmune diseases. Here, we directly monitor RNA proof-reading by RIG-I and we show that it is controlled by a set of conserved amino acids that couple RNA and ATP binding to the protein (Motif III). Mutations of this motif directly modulate proof-reading by eliminating or enhancing selectivity for viral RNA, with major implications for autoimmune disease and cancer. More broadly, the results provide a physical explanation for the ATP-gated behavior of SF2 RNA helicases and receptor proteins.

    20. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

      Energy Technology Data Exchange (ETDEWEB)

      Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko, E-mail: amasaki@mail.ecc.u-tokyo.ac.jp

      2014-08-15

      Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

    1. Optimizing prognosis-related key miRNA-target interactions responsible for cancer metastasis.

      Science.gov (United States)

      Zhao, Hongying; Yuan, Huating; Hu, Jing; Xu, Chaohan; Liao, Gaoming; Yin, Wenkang; Xu, Liwen; Wang, Li; Zhang, Xinxin; Shi, Aiai; Li, Jing; Xiao, Yun

      2017-12-12

      Increasing evidence suggests that the abnormality of microRNAs (miRNAs) and their downstream targets is frequently implicated in the pathogenesis of human cancers, however, the clinical benefit of causal miRNA-target interactions has been seldom studied. Here, we proposed a computational method to optimize prognosis-related key miRNA-target interactions by combining transcriptome and clinical data from thousands of TCGA tumors across 16 cancer types. We obtained a total of 1,956 prognosis-related key miRNA-target interactions between 112 miRNAs and 1,443 their targets. Interestingly, these key target genes are specifically involved in tumor progression-related functions, such as 'cell adhesion' and 'cell migration'. Furthermore, they are most significantly correlated with 'tissue invasion and metastasis', a hallmark of metastasis, in ten distinct types of cancer through the hallmark analysis. These results implicated that the prognosis-related key miRNA-target interactions were highly associated with cancer metastasis. Finally, we observed that the combination of these key miRNA-target interactions allowed to distinguish patients with good prognosis from those with poor prognosis both in most TCGA cancer types and independent validation sets, highlighting their roles in cancer metastasis. We provided a user-friendly database named miRNATarget (freely available at http://biocc.hrbmu.edu.cn/miRNATar/), which provides an overview of the prognosis-related key miRNA-target interactions across 16 cancer types.

    2. A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

      Energy Technology Data Exchange (ETDEWEB)

      Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.; Scuderi, M. José; Bashkin, James K.; Luo, Ming; Dutch, Rebecca Ellis

      2018-02-07

      Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.

      IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit

    3. RNA Interference (RNAi) as a Potential Tool for Control of Mycotoxin Contamination in Crop Plants: Concepts and Considerations.

      Science.gov (United States)

      Majumdar, Rajtilak; Rajasekaran, Kanniah; Cary, Jeffrey W

      2017-01-01

      Mycotoxin contamination in food and feed crops is a major concern worldwide. Fungal pathogens of the genera Aspergillus. Fusarium , and Penicillium are a major threat to food and feed crops due to production of mycotoxins such as aflatoxins, 4-deoxynivalenol, patulin, and numerous other toxic secondary metabolites that substantially reduce the value of the crop. While host resistance genes are frequently used to introgress disease resistance into elite germplasm, either through traditional breeding or transgenic approaches, such resistance is often compromised by the evolving pathogen over time. RNAi-based host-induced gene silencing of key genes required by the pathogen for optimal growth, virulence and/or toxin production, can serve as an alternative, pre-harvest approach for disease control. RNAi represents a robust and efficient tool that can be used in a highly targeted, tissue specific manner to combat mycotoxigenic fungi infecting crop plants. Successful transgenic RNAi implementation depends on several factors including (1) designing vectors to produce double-stranded RNAs (dsRNAs) that will generate small interfering RNA (siRNA) species for optimal gene silencing and reduced potential for off-target effects; (2) availability of ample target siRNAs at the infection site; (3) efficient uptake of siRNAs by the fungus; (4) siRNA half-life and (5) amplification of the silencing effect. This review provides a critical and comprehensive evaluation of the published literature on the use of RNAi-based approaches to control mycotoxin contamination in crop plants. It also examines experimental strategies used to better understand the mode of action of RNAi with the aim of eliminating mycotoxin contamination, thereby improving food and feed safety.

    4. Tracing fingerprints of young type interferences in angular distributions of ejected electrons from molecular targets

      Energy Technology Data Exchange (ETDEWEB)

      Stia, C R; Fojon, O A; Rivarola, R D [Instituto de Fisica Rosario, CONICET and Universidad Nacional de Rosario, Av. Pellegrini 250, 2000 Rosario (Argentina); Chatterjee, S; Misra, D; Tribedi, L C, E-mail: rivarola@fceia.unr.edu.a [Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005 (India)

      2010-02-01

      Interference patterns in angular distributions of ionized electrons due to coherent emission from the proximities of the nuclei of molecular hydrogen are investigated in (e,2e) collisions. It is shown that double and single differential cross sections show signatures of this effect.

    5. Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

      Science.gov (United States)

      Childs-Disney, Jessica L; Disney, Matthew D

      2016-01-01

      RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets.

    6. Review of the RNA Interference Pathway in Molluscs Including Some Possibilities for Use in Bivalves in Aquaculture

      Directory of Open Access Journals (Sweden)

      Leigh Owens

      2015-03-01

      Full Text Available Generalised reviews of RNA interference (RNAi in invertebrates, and for use in aquaculture, have taken for granted that RNAi pathways operate in molluscs, but inspection of such reviews show little specific evidence of such activity in molluscs. This review was to understand what specific research had been conducted on RNAi in molluscs, particularly with regard to aquaculture. There were questions of whether RNAi in molluscs functions similarly to the paradigm established for most eukaryotes or, alternatively, was it more similar to the ecdozoa and how RNAi may relate to disease control in aquaculture? RNAi in molluscs appears to have been only investigated in about 14 species, mostly as a gene silencing phenomenon. We can infer that microRNAs including let-7 are functional in molluscs. The genes/proteins involved in the actual RNAi pathways have only been rudimentarily investigated, so how homologous the genes and proteins are to other metazoa is unknown. Furthermore, how many different genes for each activity in the RNAi pathway are also unknown? The cephalopods have been greatly overlooked with only a single RNAi gene-silencing study found. The long dsRNA-linked interferon pathways seem to be present in molluscs, unlike some other invertebrates and could be used to reduce disease states in aquaculture. In particular, interferon regulatory factor genes have been found in molluscs of aquacultural importance such as Crassostrea, Mytilus, Pinctada and Haliotis. Two possible aquaculture scenarios are discussed, zoonotic norovirus and ostreid herpesvirus 1 to illustrate the possibilities. The entire field of RNAi in molluscs looks ripe for scientific exploitation and practical application.

    7. The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

      Directory of Open Access Journals (Sweden)

      Michal Lubas

      2015-01-01

      Full Text Available The RNA exosome complex constitutes the major nuclear eukaryotic 3′-5′ exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs, enhancer RNAs (eRNAs, and 3′-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3′ ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3′-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3′ ends.

    8. Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

      Science.gov (United States)

      Tants, Jan-Niklas; Fesser, Stephanie; Kern, Thomas; Stehle, Ralf; Geerlof, Arie; Wunderlich, Christoph; Juen, Michael; Hartlmüller, Christoph; Böttcher, Romy; Kunzelmann, Stefan; Lange, Oliver; Kreutz, Christoph; Förstemann, Klaus; Sattler, Michael

      2017-12-01

      RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

    9. Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual MicroRNAs.

      Science.gov (United States)

      Matkovich, Scot J; Dorn, Gerald W; Grossenheider, Tiffani C; Hecker, Peter A

      2015-12-01

      MicroRNAs are key players in cardiac stress responses, but the mRNAs, whose abundance and translational potential are primarily affected by changes in cardiac microRNAs, are not well defined. Stimulus-induced, large-scale alterations in the cardiac transcriptome, together with consideration of the law of mass action, further suggest that the mRNAs most substantively targeted by individual microRNAs will vary between unstressed and stressed conditions. To test the hypothesis that microRNA target profiles differ in health and disease, we traced the fate of empirically determined miR-133a and miR-378 targets in mouse hearts undergoing pressure overload hypertrophy. Ago2 immunoprecipitation with RNA sequencing (RNA-induced silencing complex sequencing) was used for unbiased definition of microRNA-dependent and microRNA-independent alterations occurring among ≈13 000 mRNAs in response to transverse aortic constriction (TAC). Of 37 direct targets of miR-133a defined in unstressed hearts (fold change ≥25%, false discovery rate the effect of TAC on microRNA direct target selection resulted in widespread alterations of signaling function. Numerous microRNA-mediated regulatory events occurring exclusively during pressure overload revealed signaling networks that may be responsive to the endogenous decreases in miR-133a during TAC. Pressure overload-mediated changes in overall cardiac RNA content alter microRNA targeting profiles, reinforcing the need to define microRNA targets in tissue-, cell-, and status-specific contexts. © 2015 American Heart Association, Inc.

    10. Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

      Science.gov (United States)

      Chung, H-C; Nguyen, V-G; Oh, W-T; Huynh, T-M-L; Moon, H-J; Lee, J-H; Kim, H-K; Park, S-J; Park, B-K

      2017-11-01

      There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes: long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line. Copyright © 2017. Published by Elsevier Inc.

    11. PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

      Science.gov (United States)

      Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

      2017-08-01

      Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host

    12. Construction of lentiviral shRNA expression vector targeting ...

      African Journals Online (AJOL)

      DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and ...

    13. Treatment of HCV Infection by Targeting MicroRNA

      NARCIS (Netherlands)

      Janssen, Harry L. A.; Reesink, Hendrik W.; Lawitz, Eric J.; Zeuzem, Stefan; Rodriguez-Torres, Maribel; Patel, Keyur; van der Meer, Adriaan J.; Patick, Amy K.; Chen, Alice; Zhou, Yi; Persson, Robert; King, Barney D.; Kauppinen, Sakari; Levin, Arthur A.; Hodges, Michael R.

      2013-01-01

      BACKGROUND The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122

    14. OmniSearch: a semantic search system based on the Ontology for MIcroRNA Target (OMIT) for microRNA-target gene interaction data.

      Science.gov (United States)

      Huang, Jingshan; Gutierrez, Fernando; Strachan, Harrison J; Dou, Dejing; Huang, Weili; Smith, Barry; Blake, Judith A; Eilbeck, Karen; Natale, Darren A; Lin, Yu; Wu, Bin; Silva, Nisansa de; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming; Ruttenberg, Alan

      2016-01-01

      As a special class of non-coding RNAs (ncRNAs), microRNAs (miRNAs) perform important roles in numerous biological and pathological processes. The realization of miRNA functions depends largely on how miRNAs regulate specific target genes. It is therefore critical to identify, analyze, and cross-reference miRNA-target interactions to better explore and delineate miRNA functions. Semantic technologies can help in this regard. We previously developed a miRNA domain-specific application ontology, Ontology for MIcroRNA Target (OMIT), whose goal was to serve as a foundation for semantic annotation, data integration, and semantic search in the miRNA field. In this paper we describe our continuing effort to develop the OMIT, and demonstrate its use within a semantic search system, OmniSearch, designed to facilitate knowledge capture of miRNA-target interaction data. Important changes in the current version OMIT are summarized as: (1) following a modularized ontology design (with 2559 terms imported from the NCRO ontology); (2) encoding all 1884 human miRNAs (vs. 300 in previous versions); and (3) setting up a GitHub project site along with an issue tracker for more effective community collaboration on the ontology development. The OMIT ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/omit.owl. The OmniSearch system is also free and open to all users, accessible at: http://omnisearch.soc.southalabama.edu/index.php/Software.

    15. Silencing of Entamoeba histolytica Glucosamine 6-Phosphate Isomerase by RNA Interference Inhibits the Formation of Cyst-Like Structures

      Directory of Open Access Journals (Sweden)

      Hugo Aguilar-Díaz

      2013-01-01

      Full Text Available Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

    16. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

      Science.gov (United States)

      Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

      2002-08-15

      Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.

    17. Overexpression and RNA interference of TwDXR regulate the accumulation of terpenoid active ingredients in Tripterygium wilfordii.

      Science.gov (United States)

      Zhang, Yifeng; Zhao, Yujun; Wang, Jiadian; Hu, Tianyuan; Tong, Yuru; Zhou, Jiawei; Song, Yadi; Gao, Wei; Huang, Luqi

      2018-02-01

      To examine the putative regulatory role of TwDXR in terpenoid biosynthesis and terpenoid biosynthetic pathway-related gene expression, through overexpression and RNA interference with TwDXR. We obtained 1410 and 454 bp TwDXR-specific fragments to construct overexpression and RNAi vectors. qRT-PCR was used to detect the expression of TwDXR and terpenoid biosynthesis pathway-related genes. The overexpression of TwDXR led to a 285% upregulation and the TwDXR RNAi led to a reduction to 26% of the control (empty vector-transformed cells) levels. However, pathway-related genes displayed different trends. When TwDXR was overexpressed, TwDXS expression decreased by 31% but increased to 198% when TwDXR expression was inhibited. The accumulation of terpenoids was also assayed. In the overexpression group, differences were not significant whereas the contents of triptolide and celastrol in the TwDXR RNAi samples were diminished by 27.3 and 24.0%, respectively. The feedback regulation of gene transcription and the accumulation of terpenoids in terpenoid biosynthesis in Tripterygium wilfordii were verified by TwDXR overexpression and RNAi experiments.

    18. microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus

      DEFF Research Database (Denmark)

      Leucci, Eleonora; Patella, Francesca; Waage, Johannes

      2013-01-01

      microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non......-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our...... findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions....

    19. Significant impact of miRNA-target gene networks on genetics of human complex traits.

      Science.gov (United States)

      Okada, Yukinori; Muramatsu, Tomoki; Suita, Naomasa; Kanai, Masahiro; Kawakami, Eiryo; Iotchkova, Valentina; Soranzo, Nicole; Inazawa, Johji; Tanaka, Toshihiro

      2016-03-01

      The impact of microRNA (miRNA) on the genetics of human complex traits, especially in the context of miRNA-target gene networks, has not been fully assessed. Here, we developed a novel analytical method, MIGWAS, to comprehensively evaluate enrichment of genome-wide association study (GWAS) signals in miRNA-target gene networks. We applied the method to the GWAS results of the 18 human complex traits from >1.75 million subjects, and identified significant enrichment in rheumatoid arthritis (RA), kidney function, and adult height (P impact of miRNA-target gene networks on the genetics of human complex traits, and provided resources which should contribute to drug discovery and nucleic acid medicine.

    20. An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences

      Directory of Open Access Journals (Sweden)

      Richard L. Moyle

      2017-09-01

      Full Text Available Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.

    1. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

      Science.gov (United States)

      Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

      2017-05-23

      The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

    2. Systems biology approach to study the role of miRNA in promoter targeting during megakaryopoiesis.

      Science.gov (United States)

      Sahu, Itishri; Hebalkar, Rucha; Kar, Sonika; Ts, SreeVidya; Gutti, Usha; Gutti, Ravi Kumar

      2018-05-15

      The distinct process of megakaryopoiesis requires occurrence of endomitosis for polyploidization of the megakaryocytes. Although, Cyclins, CDKs and have been described to regulate endomitosis, the exact mechanism still remains an enigma. miRNA which were otherwise known as post transcriptional gene silencers are now emerging with various non-canonical functions including gene regulation at pre-transcriptional level by miRNA binding at promoter region. Out of the many processes they regulate, miRNA have been manifested to play a role in megakaryocyte differentiation. In this study an attempt has been made to identify miRNA that could regulate cell cycle genes (Cyclins and CDKs) by targeting their promoters, during megakaryopoiesis. A new computational algorithm was implemented using Perl programming to identify putative targets of miRNA in CDK and Cyclin promoters. Perl script was also used to check nuclear localizing miRNA based on the presence of a consensus sequence. Real-time PCR was performed to analyze the expression of miRNA and their predicted targets in Dami vs. PMA treated Dami cells. Putative targets of miRNAs with longest, high complementarity matches in CDK/Cyclin promoters were obtained. We identified two significant miRNA, miR-1273g-3p and miR-619-5p with longest seed sequence matches. We further identified three main targets (CDK10, CDK11, Cyclin F) through which these two miRNA could regulate cell cycle during megakaryopoiesis. Our results reinforce the role of promoting targeting miRNA in regulation of cell cycle through certain CDK/Cyclins to support the process of endomitosis during megakaryopoiesis. Copyright © 2018 Elsevier Inc. All rights reserved.

    3. RNA interference in adult Ascaris suum--an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite.

      Science.gov (United States)

      McCoy, Ciaran J; Warnock, Neil D; Atkinson, Louise E; Atcheson, Erwan; Martin, Richard J; Robertson, Alan P; Maule, Aaron G; Marks, Nikki J; Mousley, Angela

      2015-09-01

      The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

    4. Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown

      Science.gov (United States)

      Bilgi, Vineeta; Fosu-Nyarko, John; Jones, Michael G. K.

      2017-01-01

      RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs. PMID:28054949

    5. Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown

      Directory of Open Access Journals (Sweden)

      Vineeta Bilgi

      2017-01-01

      Full Text Available RNA interference (RNAi is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (dsRNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae. Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02% were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8. There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs.

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

      Science.gov (United States)

      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

    7. Novel modeling of combinatorial miRNA targeting identifies SNP with potential role in bone density.

      Directory of Open Access Journals (Sweden)

      Claudia Coronnello

      Full Text Available MicroRNAs (miRNAs are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting, a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential

    8. Functionalized silicon quantum dots tailored for targeted siRNA delivery

      Energy Technology Data Exchange (ETDEWEB)

      Klein, S. [Department Chemistry and Pharmacy, Physical Chemistry I and ICMM, Friedrich-Alexander University of Erlangen-Nuremberg, Egerlandstr. 3, D-91058 Erlangen (Germany); Zolk, O.; Fromm, M.F. [Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander University of Erlangen-Nuremberg, Fahrstr. 17, D-91054 Erlangen (Germany); Schroedl, F. [Institute of Anatomy I, Friedrich-Alexander University of Erlangen-Nuremberg, Krankenhausstr. 9, D-91054 Erlangen (Germany); Departments of Anatomy/Ophthalmology, Paracelsus Medical University, Strubergasse 21, A-5020 Salzburg (Austria); Neuhuber, W. [Institute of Anatomy I, Friedrich-Alexander University of Erlangen-Nuremberg, Krankenhausstr. 9, D-91054 Erlangen (Germany); Kryschi, C., E-mail: kryschi@chemie.uni-erlangen.de [Department Chemistry and Pharmacy, Physical Chemistry I and ICMM, Friedrich-Alexander University of Erlangen-Nuremberg, Egerlandstr. 3, D-91058 Erlangen (Germany)

      2009-09-11

      For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.

    9. RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus.

      Science.gov (United States)

      Jia, Shuang; Wan, Pin-Jun; Zhou, Li-Tao; Mu, Li-Li; Li, Guo-Qing

      2015-04-01

      Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus. © 2013 Institute of Zoology, Chinese Academy of Sciences.

    10. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

      Directory of Open Access Journals (Sweden)

      Jie Chen

      Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

    11. Construction and Biological Evaluation of a Novel Integrin ανβ3-Specific Carrier for Targeted siRNA Delivery In Vitro

      Directory of Open Access Journals (Sweden)

      Xueqi Chen

      2017-02-01

      Full Text Available (1 Background: The great potential of RNA interference (RNAi-based gene therapy is premised on the effective delivery of small interfering RNAs (siRNAs to target tissues and cells. Hence, we aimed at developing and examining a novel integrin αvβ3-specific delivery carrier for targeted transfection of siRNA to malignant tumor cells; (2 Methods: Arginine-glycine-aspartate motif (RGD was adopted as a tissue target for specific recognition of integrin αvβ3. To enable siRNA binding, a chimeric peptide was synthesized by adding nonamer arginine residues (9R at the carboxy terminus of cyclic-RGD dimer, designated as c(RGD2-9R. The efficiency of 9R peptide transferring siRNA was biologically evaluated in vitro by flow cytometry, confocal microscopy, and Western blot; (3 Results: An optimal 10:1 molar ratio of c(RGD2-9R to siRNA was confirmed by the electrophoresis on agarose gels. Both the flow cytometry and confocal microscopy results testified that transfection of c(RGD2-9R as an siRNA delivery carrier was obviously higher than the naked-siRNA group. The results of Western blot demonstrated that these 9R peptides were able to transduce siRNA to HepG2 cells in vitro, resulting in efficient gene silencing; and (4 Conclusion: The chimeric peptide of c(RGD2-9R can be developed as an effective siRNA delivery carrier and shows potential as a new strategy for RNAi-based gene therapy.

    12. Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

      Directory of Open Access Journals (Sweden)

      Siyun Xu

      2014-10-01

      Full Text Available RNA interference (RNAi is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4, which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3 were designed to target the coding sequence (CDS of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55% in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

    13. RNA interference of cytochrome P450 CYP6F subfamily genes affects susceptibility to different insecticides in Locusta migratoria.

      Science.gov (United States)

      Guo, Yanqiong; Wu, Haihua; Zhang, Xueyao; Ma, Enbo; Guo, Yaping; Zhu, Kun Yan; Zhang, Jianzhen

      2016-11-01

      Many insect cytochrome P450s (CYPs) play critical roles in detoxification of insecticides. The CYP6 family is unique to the class Insecta, and its biochemical function has essentially been associated with the metabolism of xenobiotics. In this study, we sequenced and characterised the full-length cDNAs of five CYP genes from Locusta migratoria, a highly destructive agricultural pest worldwide. The five genes were predominantly expressed in brain, guts, fat bodies or Malpighian tubules. CYP6FE1, CYP6FF1 and CYP6FG1 were expressed at higher levels in fourth-instar nymphs than in other developmental stages. CYPFD2 is specifically expressed in adults, whereas CYP6FD1, CYP6FD2 and CYP6FE1 showed significantly lower expression in eggs than in other developmental stages. Deltamethrin suppressed CYP6FD1 expression in third-instar nymphs and upregulated the expression level of CYP6FD2, CYP6FF1 and CYP6FG1 at the dose of LD 10 . Efficient RNA interference-mediated gene silencing was established for four of the five CYP genes. Silencing of CYP6FF1 increased the nymphal mortality from 23 to 50% in response to deltamethrin. Silencing of CYP6FD2 and CYP6FE1 increased the nymphal mortality from 32 to 72 and 66%, respectively, to carbaryl. Three of the four CYP6F subfamily genes in L. migratoria were associated with the detoxification of deltamethrin or carbaryl. The role of CYPs in insecticide detoxification appears to be both gene and insecticide specific. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

    14. Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

      International Nuclear Information System (INIS)

      Carbonell, Alberto; Martinez de Alba, Angel-Emilio; Flores, Ricardo; Gago, Selma

      2008-01-01

      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

    15. A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

      Directory of Open Access Journals (Sweden)

      Marco Todesco

      2010-07-01

      Full Text Available Many targets of plant microRNAs (miRNAs are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

    16. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

      DEFF Research Database (Denmark)

      Sittka, A; Lucchini, S; Papenfort, K

      2008-01-01

      Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial...... transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co...... would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high...

    17. Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor.

      Science.gov (United States)

      Du, Junzheng; Guo, Xinbing; Gao, Shandian; Luo, Jihuai; Gong, Xiuli; Hao, Chunxia; Yang, Bo; Lin, Tong; Shao, Junjun; Cong, Guozheng; Chang, Huiyun

      2014-10-10

      Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice. Copyright © 2014 Elsevier B.V. All rights reserved.

    18. Human health and ecological risk assessments for SmartStax PRO (MON 89034 x TC1507 x MON 87411 x DAS-59122-7), a plant-incorporated protectant intended to control corn rootworm through ribonucleic acid (RNA) interference

      Science.gov (United States)

      The use of RNA interference (RNAi) gene silencing technology, particularly RNAi for pesticidal purposes to control macroorganism pests, is a relatively recent innovation. Post-transcriptional silencing of gene function is a very rapid process where double-stranded RNA (dsRNA) dir...

    19. Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets.

      Science.gov (United States)

      Bischler, Thorsten; Hsieh, Ping-Kun; Resch, Marcus; Liu, Quansheng; Tan, Hock Siew; Foley, Patricia L; Hartleib, Anika; Sharma, Cynthia M; Belasco, Joel G

      2017-02-03

      RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

    20. MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

      International Nuclear Information System (INIS)

      Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

      2011-01-01

      Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

    1. Mycoplasma non-coding RNA: identification of small RNAs and targets

      Directory of Open Access Journals (Sweden)

      Franciele Maboni Siqueira

      2016-10-01

      Full Text Available Abstract Background Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. Results A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. Conclusion This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.

    2. saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

      Science.gov (United States)

      Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

      2016-03-01

      Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

    3. microPIR: an integrated database of microRNA target sites within human promoter sequences.

      Directory of Open Access Journals (Sweden)

      Jittima Piriyapongsa

      Full Text Available BACKGROUND: microRNAs are generally understood to regulate gene expression through binding to target sequences within 3'-UTRs of mRNAs. Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism. METHODOLOGY: We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource. microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program. >15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomic features. Furthermore, functional information of target genes including gene ontologies, KEGG pathways, and OMIM associations are provided. The built-in genome browser of microPIR provides a comprehensive view of multidimensional genomic data. Finally, microPIR incorporates a PCR primer design module to facilitate experimental validation. CONCLUSIONS: The proposed micro

    4. Whole-Genome Thermodynamic Analysis Reduces siRNA Off-Target Effects

      Science.gov (United States)

      Chen, Xi; Liu, Peng; Chou, Hui-Hsien

      2013-01-01

      Small interfering RNAs (siRNAs) are important tools for knocking down targeted genes, and have been widely applied to biological and biomedical research. To design siRNAs, two important aspects must be considered: the potency in knocking down target genes and the off-target effect on any nontarget genes. Although many studies have produced useful tools to design potent siRNAs, off-target prevention has mostly been delegated to sequence-level alignment tools such as BLAST. We hypothesize that whole-genome thermodynamic analysis can identify potential off-targets with higher precision and help us avoid siRNAs that may have strong off-target effects. To validate this hypothesis, two siRNA sets were designed to target three human genes IDH1, ITPR2 and TRIM28. They were selected from the output of two popular siRNA design tools, siDirect and siDesign. Both siRNA design tools have incorporated sequence-level screening to avoid off-targets, thus their output is believed to be optimal. However, one of the sets we tested has off-target genes predicted by Picky, a whole-genome thermodynamic analysis tool. Picky can identify off-target genes that may hybridize to a siRNA within a user-specified melting temperature range. Our experiments validated that some off-target genes predicted by Picky can indeed be inhibited by siRNAs. Similar experiments were performed using commercially available siRNAs and a few off-target genes were also found to be inhibited as predicted by Picky. In summary, we demonstrate that whole-genome thermodynamic analysis can identify off-target genes that are missed in sequence-level screening. Because Picky prediction is deterministic according to thermodynamics, if a siRNA candidate has no Picky predicted off-targets, it is unlikely to cause off-target effects. Therefore, we recommend including Picky as an additional screening step in siRNA design. PMID:23484018

    5. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

      Directory of Open Access Journals (Sweden)

      Stephanie R. Coffman

      2017-03-01

      Full Text Available Dicer enzymes process virus-specific double-stranded RNA (dsRNA into small interfering RNAs (siRNAs to initiate specific antiviral defense by related RNA interference (RNAi pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1, not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I-like receptors (RLRs, intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.

    6. RNA sequencing reveals a depletion of collagen targeting microRNAs in Dupuytren's disease.

      Science.gov (United States)

      Riester, Scott M; Arsoy, Diren; Camilleri, Emily T; Dudakovic, Amel; Paradise, Christopher R; Evans, Jared M; Torres-Mora, Jorge; Rizzo, Marco; Kloen, Peter; Julio, Marianna Kruithof-de; van Wijnen, Andre J; Kakar, Sanjeev

      2015-10-07

      Dupuytren's disease is an inherited disorder in which patients develop fibrotic contractures of the hand. Current treatment strategies include surgical excision or enzymatic digestion of fibrotic tissue. MicroRNAs, which are key posttranscriptional regulators of genes expression, have been shown to play an important regulatory role in disorders of fibrosis. Therefore in this investigation, we apply high throughput next generation RNA sequencing strategies to characterize microRNA expression in diseased and healthy palmar fascia to elucidate molecular mechanisms responsible for pathogenic fibrosis. We applied high throughput RNA sequencing techniques to quantify the expression of all known human microRNAs in Dupuytren's and control palmar fascia. MicroRNAs that were differentially expressed between diseased and healthy tissue samples were used for computational target prediction using the bioinformatics tool ComiR. Molecular pathways that were predicted to be differentially expressed based on computational analysis were validated by performing RT-qPCR on RNA extracted from diseased and non-diseased palmar fascia biopsies. A comparison of microRNAs expressed in Dupuytren's fascia and control fascia identified 74 microRNAs with a 2-fold enrichment in Dupuytren's tissue, and 32 microRNAs with enrichment in control fascia. Computational target prediction for differentially expressed microRNAs indicated preferential targeting of collagens and extracellular matrix related proteins in control palmar fascia. RT-qPCR confirmed the decreased expression of microRNA targeted collagens in control palmar fascia tissues. Control palmar fascia show decreased expression of mRNAs encoding collagens that are preferentially targeted by microRNAs enriched in non-diseased fascia. Thus alterations in microRNA regulatory networks may play an important role in driving the pathogenic fibrosis seen in Dupuytren's disease via direct regulatory effects on extracellular matrix protein synthesis

    7. Molecular characterization, RNA interference and recombinant protein approach to study the function of the putative Molt Inhibiting Hormone (FmMIH1) gene from the shrimp Fenneropenaeus merguiensis.

      Science.gov (United States)

      Liang, Huafang; Liu, Yan; Zhou, Ting Ting; Li, Xiaoyuan; Li, Bin; Chan, Siuming F

      2017-12-14

      The Molt Inhibiting Hormone gene and cDNA of the banana shrimp Fenneropenaeus merguiensis (FmMIH1) has been cloned and characterized. FmMIH1 possesses most of the characteristics of the eyestalk CHH/MIH/GIH family subtype-II neuropeptides. FmMIH1 open reading frame consists of 315 bp encoding for 105 amino acid residues. The mature peptide of FmMIH1 consists of 76 amino acid residues, a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position. In addition to eyestalk, high levels of FmMIH1 transcript could also be detected in the intestine. FmMIH1 transcript level is low throughout the post-molt, early to mid-intermolt and premolt. However, a sharp increase could be observed in late intermolt (C3 stage). Both alignment and phylogenetic analysis reveal that FmMIH1 is most similar to the MIH1 of other shrimps. For functional assay, RNA interference results show that a significant 2.3 days (P < 0.05) reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection. Surprisingly, injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle (average 1.9 days, P < 0.05). We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH for carrier protein binding and consequently reduces the amount of biological MIH that could reach the targets. In conclusion, the result of this study will provide us new insight in molting/growth control in crustacean. Copyright © 2017. Published by Elsevier Inc.

    8. Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7.

      Science.gov (United States)

      Ravon, Morgane; Berrera, Marco; Ebeling, Martin; Certa, Ulrich

      2012-01-01

      We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3'-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.

    9. Targeted delivery of mutant tolerant anti-coxsackievirus artificial microRNAs using folate conjugated bacteriophage Phi29 pRNA.

      Directory of Open Access Journals (Sweden)

      Xin Ye

      Full Text Available Myocarditis is the major heart disease in infants and young adults. It is very commonly caused by coxsackievirus B3 (CVB3 infection; however, no specific treatment or vaccine is available at present. RNA interference (RNAi-based anti-viral therapy has shown potential to inhibit viral replication, but this strategy faces two major challenges; viral mutational escape from drug suppression and targeted delivery of the reagents to specific cell populations.In this study, we designed artificial microRNAs (AmiRs targeting the 3'untranslated region (3'UTR of CVB3 genome with mismatches to the central region of their targeting sites. Antiviral evaluation showed that AmiR-1 and AmiR-2 reduced CVB3 (Kandolf and CG strains replication approximately 100-fold in both HeLa cells and HL-1 cardiomyocytes. To achieve specific delivery, we linked AmiRs to the folate-conjugated bacterial phage packaging RNA (pRNA and delivered the complexes into HeLa cells, a folate receptor positive cancer cells widely used as an in vitro model for CVB3 infection, via folate-mediated specific internalization. We found that our designed pRNA-AmiRs conjugates were tolerable to target mutations and have great potential to suppress viral mutational escape with little effect on triggering interferon induction.This study provides important clues for designing AmiRs targeting the 3'UTR of viral genome. It also proves the feasibility of specific deliver of AmiRs using conjugated pRNA vehicles. These small AmiRs combined with pRNA-folate conjugates could form a promising system for antiviral drug development.

    10. An RGD-modified MRI-visible polymeric vector for targeted siRNA delivery to hepatocellular carcinoma in nude mice.

      Directory of Open Access Journals (Sweden)

      Chun Wu

      Full Text Available RNA interference (RNAi has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC. Targeted vectors are able to deliver small interfering RNA (siRNA into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION, was constructed as a magnetic resonance imaging (MRI-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.

    11. An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

      Science.gov (United States)

      Shen, Min; Zhu, Kangshun; Cheng, Du; Liu, Zhihao; Shan, Hong

      2013-01-01

      RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe. PMID:23922634

    12. The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia

      Directory of Open Access Journals (Sweden)

      Zhang Xiaolu

      2012-08-01

      Full Text Available Abstract Background Recent evidence has accumulated that MicroRNA (miRNA dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML and may contribute to onco-/leukemo-genesis. Methods The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1st complete remission (CR and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1. Results We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells. Conclusions Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.

    13. An integrated miRNA functional screening and target validation method for organ morphogenesis

      OpenAIRE

      Rebustini, Ivan T.; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A.; Maas, Richard L.

      2016-01-01

      The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our ...

    14. Ranking of microRNA target prediction scores by Pareto front analysis.

      Science.gov (United States)

      Sahoo, Sudhakar; Albrecht, Andreas A

      2010-12-01

      Over the past ten years, a variety of microRNA target prediction methods has been developed, and many of the methods are constantly improved and adapted to recent insights into miRNA-mRNA interactions. In a typical scenario, different methods return different rankings of putative targets, even if the ranking is reduced to selected mRNAs that are related to a specific disease or cell type. For the experimental validation it is then difficult to decide in which order to process the predicted miRNA-mRNA bindings, since each validation is a laborious task and therefore only a limited number of mRNAs can be analysed. We propose a new ranking scheme that combines ranked predictions from several methods and - unlike standard thresholding methods - utilises the concept of Pareto fronts as defined in multi-objective optimisation. In the present study, we attempt a proof of concept by applying the new ranking scheme to hsa-miR-21, hsa-miR-125b, and hsa-miR-373 and prediction scores supplied by PITA and RNAhybrid. The scores are interpreted as a two-objective optimisation problem, and the elements of the Pareto front are ranked by the STarMir score with a subsequent re-calculation of the Pareto front after removal of the top-ranked mRNA from the basic set of prediction scores. The method is evaluated on validated targets of the three miRNA, and the ranking is compared to scores from DIANA-microT and TargetScan. We observed that the new ranking method performs well and consistent, and the first validated targets are elements of Pareto fronts at a relatively early stage of the recurrent procedure, which encourages further research towards a higher-dimensional analysis of Pareto fronts. Copyright © 2010 Elsevier Ltd. All rights reserved.

    15. Comparison and Integration of Target Prediction Algorithms for microRNA Studies

      Directory of Open Access Journals (Sweden)

      Zhang Yanju

      2010-12-01

      Full Text Available microRNAs are short RNA fragments that have the capacity of regulating hundreds of target gene expression. Currently, due to lack of high-throughput experimental methods for miRNA target identification, a collection of computational target prediction approaches have been developed. However, these approaches deal with different features or factors are weighted differently resulting in diverse range of predictions. The prediction accuracy remains uncertain. In this paper, three commonly used target prediction algorithms are evaluated and further integrated using algorithm combination, ranking aggregation and Bayesian Network classification. Our results revealed that each individual prediction algorithm displays its advantages as was shown on different test data sets. Among different integration strategies, the application of Bayesian Network classifier on the features calculated from multiple prediction methods significantly improved target prediction accuracy.

    16. MicroRNA as Therapeutic Targets for Chronic Wound Healing.

      Science.gov (United States)

      Mulholland, Eoghan J; Dunne, Nicholas; McCarthy, Helen O

      2017-09-15

      Wound healing is a highly complex biological process composed of three overlapping phases: inflammation, proliferation, and remodeling. Impairments at any one or more of these stages can lead to compromised healing. MicroRNAs (miRs) are non-coding RNAs that act as post-transcriptional regulators of multiple proteins and associated pathways. Thus, identification of the appropriate miR involved in the different phases of wound healing could reveal an effective third-generation genetic therapy in chronic wound care. Several miRs have been shown to be upregulated or downregulated during the wound healing process. This article examines the biological processes involved in wound healing, the miR involved at each stage, and how expression levels are modulated in the chronic wound environment. Key miRs are highlighted as possible therapeutic targets, either through underexpression or overexpression, and the healing benefits are interrogated. These are prime miR candidates that could be considered as a gene therapy option for patients suffering from chronic wounds. The success of miR as a gene therapy, however, is reliant on the development of an appropriate delivery system that must be designed to overcome both extracellular and intracellular barriers. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

    17. MicroRNA as Therapeutic Targets for Chronic Wound Healing

      Directory of Open Access Journals (Sweden)

      Eoghan J. Mulholland

      2017-09-01

      Full Text Available Wound healing is a highly complex biological process composed of three overlapping phases: inflammation, proliferation, and remodeling. Impairments at any one or more of these stages can lead to compromised healing. MicroRNAs (miRs are non-coding RNAs that act as post-transcriptional regulators of multiple proteins and associated pathways. Thus, identification of the appropriate miR involved in the different phases of wound healing could reveal an effective third-generation genetic therapy in chronic wound care. Several miRs have been shown to be upregulated or downregulated during the wound healing process. This article examines the biological processes involved in wound healing, the miR involved at each stage, and how expression levels are modulated in the chronic wound environment. Key miRs are highlighted as possible therapeutic targets, either through underexpression or overexpression, and the healing benefits are interrogated. These are prime miR candidates that could be considered as a gene therapy option for patients suffering from chronic wounds. The success of miR as a gene therapy, however, is reliant on the development of an appropriate delivery system that must be designed to overcome both extracellular and intracellular barriers.

    18. Quantification of non-coding RNA target localization diversity and its application in cancers.

      Science.gov (United States)

      Cheng, Lixin; Leung, Kwong-Sak

      2018-01-30

      Subcellular localization is pivotal for RNAs and proteins to implement biological functions. The localization diversity of protein interactions has been studied as a crucial feature of proteins, considering the protein-protein interactions take place in various subcellular locations. Nevertheless, the localization diversity of non-coding RNA (ncRNA) target proteins has not been systematically studied, especially its characteristics in cancers. In this study, we provide a new algorithm, non-coding RNA target localization coefficient (ncTALENT), to quantify the target localization diversity of ncRNAs based on the ncRNA-protein interaction and protein subcellular localization data. ncTALENT can be used to calculate the target localization coefficient of ncRNAs and measure how diversely their targets are distributed among the subcellular locations in various scenarios. We focus our study on long non-coding RNAs (lncRNAs), and our observations reveal that the target localization diversity is a primary characteristic of lncRNAs in different biotypes. Moreover, we found that lncRNAs in multiple cancers, differentially expressed cancer lncRNAs, and lncRNAs with multiple cancer target proteins are prone to have high target localization diversity. Furthermore, the analysis of gastric cancer helps us to obtain a better understanding that the target localization diversity of lncRNAs is an important feature closely related to clinical prognosis. Overall, we systematically studied the target localization diversity of the lncRNAs and uncovered its association with cancer. © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

    19. Interactome of Radiation-Induced microRNA-Predicted Target Genes

      Directory of Open Access Journals (Sweden)

      Tenzin W. Lhakhang

      2012-01-01

      Full Text Available The microRNAs (miRNAs function as global negative regulators of gene expression and have been associated with a multitude of biological processes. The dysfunction of the microRNAome has been linked to various diseases including cancer. Our laboratory recently reported modulation in the expression of miRNA in a variety of cell types exposed to ionizing radiation (IR. To further understand miRNA role in IR-induced stress pathways, we catalogued a set of common miRNAs modulated in various irradiated cell lines and generated a list of predicted target genes. Using advanced bioinformatics tools we identified cellular pathways where miRNA predicted target genes function. The miRNA-targeted genes were found to play key roles in previously identified IR stress pathways such as cell cycle, p53 pathway, TGF-beta pathway, ubiquitin-mediated proteolysis, focal adhesion pathway, MAPK signaling, thyroid cancer pathway, adherens junction, insulin signaling pathway, oocyte meiosis, regulation of actin cytoskeleton, and renal cell carcinoma pathway. Interestingly, we were able to identify novel targeted pathways that have not been identified in cellular radiation response, such as aldosterone-regulated sodium reabsorption, long-term potentiation, and neutrotrophin signaling pathways. Our analysis indicates that the miRNA interactome in irradiated cells provides a platform for comprehensive modeling of the cellular stress response to IR exposure.

    20. The concept of multiple-target anti-miRNA antisense oligonucleotide technology.

      Science.gov (United States)

      Wang, Zhiguo

      2011-01-01

      The multiple-target AMO technology or MT-AMO technology is an innovative strategy, which confers on a single AMO fragment the capability of targeting multiple miRNAs. This modified AMO is single-stranded 2'-O-methyl-modified oligoribonucleotides carrying multiple AMO units, which are engineered into a single unit and are able to simultaneously silence multiple-target miRNAs or multiple miRNA seed families. Studies suggest that the MT-AMO is an improved approach for miRNA target finding and miRNA function validation; it not only enhances the effectiveness of targeting miRNAs but also confers diversity of actions. It has been successfully used to identify target genes and cellular function of several oncogenic miRNAs and of the muscle-specific miRNAs (Lu et al., Nucleic Acids Res 37:e24-e33, 2009). This novel strategy may find its broad application as a useful tool in miRNA research for exploring biological processes involving multiple miRNAs and multiple genes, and the potential as an miRNA therapy for human disease such as cancer and cardiac disorders. This technology was developed by my research laboratory in collaboration with Yang's group (Lu et al., Nucleic Acids Res 37:e24-e33, 2009), and it is similar but distinct from the miRNA Sponge technology developed by Sharp's laboratory in 2007 (Ebert et al., Nat Methods 4:721-726, 2007) and modified by Gentner et al. (Nat Methods 6:63-66, 2009).

    1. Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment.

      Science.gov (United States)

      Gomes-da-Silva, Lígia C; Santos, Adriana O; Bimbo, Luís M; Moura, Vera; Ramalho, José S; Pedroso de Lima, Maria C; Simões, Sérgio; Moreira, João N

      2012-09-15

      The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety. The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes. Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level. Copyright © 2012 Elsevier B.V. All rights reserved.

    2. Combining Results from Distinct MicroRNA Target Prediction Tools Enhances the Performance of Analyses.

      Science.gov (United States)

      Oliveira, Arthur C; Bovolenta, Luiz A; Nachtigall, Pedro G; Herkenhoff, Marcos E; Lemke, Ney; Pinhal, Danillo

      2017-01-01

      Target prediction is generally the first step toward recognition of bona fide microRNA (miRNA)-target interactions in living cells. Several target prediction tools are now available, which use distinct criteria and stringency to provide the best set of candidate targets for a single miRNA or a subset of miRNAs. However, there are many false-negative predictions, and consensus about the optimum strategy to select and use the output information provided by the target prediction tools is lacking. We compared the performance of four tools cited in literature-TargetScan (TS), miRanda-mirSVR (MR), Pita, and RNA22 (R22), and we determined the most effective approach for analyzing target prediction data (individual, union, or intersection). For this purpose, we calculated the sensitivity, specificity, precision, and correlation of these approaches using 10 miRNAs (miR-1-3p, miR-17-5p, miR-21-5p, miR-24-3p, miR-29a-3p, miR-34a-5p, miR-124-3p, miR-125b-5p, miR-145-5p, and miR-155-5p) and 1,400 genes (700 validated and 700 non-validated) as targets of these miRNAs. The four tools provided a subset of high-quality predictions and returned few false-positive predictions; however, they could not identify several known true targets. We demonstrate that union of TS/MR and TS/MR/R22 enhanced the quality of in silico prediction analysis of miRNA targets. We conclude that the union rather than the intersection of the aforementioned tools is the best strategy for maximizing performance while minimizing the loss of time and resources in subsequent in vivo and in vitro experiments for functional validation of miRNA-target interactions.

    3. C-mii: a tool for plant miRNA and target identification

      Directory of Open Access Journals (Sweden)

      Numnark Somrak

      2012-12-01

      Full Text Available Abstract Background MicroRNAs (miRNAs have been known to play an important role in several biological processes in both animals and plants. Although several tools for miRNA and target identification are available, the number of tools tailored towards plants is limited, and those that are available have specific functionality, lack graphical user interfaces, and restrict the number of input sequences. Large-scale computational identifications of miRNAs and/or targets of several plants have been also reported. Their methods, however, are only described as flow diagrams, which require programming skills and the understanding of input and output of the connected programs to reproduce. Results To overcome these limitations and programming complexities, we proposed C-mii as a ready-made software package for both plant miRNA and target identification. C-mii was designed and implemented based on established computational steps and criteria derived from previous literature with the following distinguishing features. First, software is easy to install with all-in-one programs and packaged databases. Second, it comes with graphical user interfaces (GUIs for ease of use. Users can identify plant miRNAs and targets via step-by-step execution, explore the detailed results from each step, filter the results according to proposed constraints in plant miRNA and target biogenesis, and export sequences and structures of interest. Third, it supplies bird's eye views of the identification results with infographics and grouping information. Fourth, in terms of functionality, it extends the standard computational steps of miRNA target identification with miRNA-target folding and GO annotation. Fifth, it provides helper functions for the update of pre-installed databases and automatic recovery. Finally, it supports multi-project and multi-thread management. Conclusions C-mii constitutes the first complete software package with graphical user interfaces enabling

    4. Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

      Science.gov (United States)

      Hale, Caryn R.; Cocozaki, Alexis; Li, Hong

      2014-01-01

      The effector complex of the Cmr/type III-B CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) system cleaves RNAs recognized by the CRISPR RNA (crRNA) of the complex and includes six protein subunits of unknown functions. Using reconstituted Pyrococcus furiosus Cmr complexes, we found that each of the six Cmr proteins plays a critical role in either crRNA interaction or target RNA capture. Cmr2, Cmr3, Cmr4, and Cmr5 are all required for formation of a crRNA-containing complex detected by native gel electrophoresis, and the conserved 5′ repeat sequence tag and 5′-OH group of the crRNA are essential for the interaction. Interestingly, capture of the complementary target RNA additionally requires both Cmr1 and Cmr6. In detailed functional studies, we determined that P. furiosus Cmr complexes cleave target RNAs at 6-nucleotide (nt) intervals in the region of complementarity, beginning 5 nt downstream from the crRNA tag and continuing to within ∼14 nt of the 3′ end of the crRNA. Our findings indicate that Cmr3 recognizes the signature crRNA tag sequence (and depends on protein–protein interactions with Cmr2, Cmr4, and Cmr5), each Cmr4 subunit mediates a target RNA cleavage, and Cmr1 and Cmr6 mediate an essential interaction between the 3′ region of the crRNA and the target RNA. PMID:25367038

    5. The Limitations of Existing Approaches in Improving MicroRNA Target Prediction Accuracy.

      Science.gov (United States)

      Loganantharaj, Rasiah; Randall, Thomas A

      2017-01-01

      MicroRNAs (miRNAs) are small (18-24 nt) endogenous RNAs found across diverse phyla involved in posttranscriptional regulation, primarily downregulation of mRNAs. Experimentally determining miRNA-mRNA interactions can be expensive and time-consuming, making the accurate computational prediction of miRNA targets a high priority. Since miRNA-mRNA base pairing in mammals is not perfectly complementary and only a fraction of the identified motifs are real binding sites, accurately predicting miRNA targets remains challenging. The limitations and bottlenecks of existing algorithms and approaches are discussed in this chapter.A new miRNA-mRNA interaction algorithm was implemented in Python (TargetFind) to capture three different modes of association and to maximize detection sensitivity to around 95% for mouse (mm9) and human (hg19) reference data. For human (hg19) data, the prediction accuracy with any one feature among evolutionarily conserved score, multiple targets in a UTR or changes in free energy varied within a close range from 63.5% to 66%. When the results of these features are combined with majority voting, the expected prediction accuracy increases to 69.5%. When all three features are used together, the average best prediction accuracy with tenfold cross validation from the classifiers naïve Bayes, support vector machine, artificial neural network, and decision tree were, respectively, 66.5%, 67.1%, 69%, and 68.4%. The results reveal the advantages and limitations of these approaches.When comparing different sets of features on their strength in predicting true hg19 targets, evolutionarily conserved score slightly outperformed all other features based on thermostability, and target multiplicity. The sophisticated supervised learning algorithms did not improve the prediction accuracy significantly compared to a simple threshold based approach on conservation score or combining the results of each feature with majority agreements. The targets from randomly

    6. MicroRNA-145 targets YES and STAT1 in colon cancer cells

      DEFF Research Database (Denmark)

      Gregersen, Lea H; Jacobsen, Anders B; Frankel, Lisa

      2010-01-01

      BACKGROUND: MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. METHODOLOGY/PRINCIPAL FINDINGS: To investigate...... miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2......, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets. CONCLUSIONS/SIGNIFICANCE: The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor...

    7. One cell model establishment to inhibit CaMKIIγmRNA expression in the dorsal root ganglion neuron by RNA interfere.

      Science.gov (United States)

      Wen, Xianjie; Li, Xiaohong; Liang, Hua; Yang, Chenxiang; Zhong, Jiying; Wang, Hanbing; Liu, Hongzhen

      2017-09-01

      CaMKII γ in dorsal root ganglion neurons is closely related to the neuropathic pain, neuron injury induced by local anesthetics. To get great insight into the function of CaMKII γ in dorsal root ganglion neurons, we need one cell model to specially inhibit the CaMKII γ mRNA expression. The present study was aimed to establish one cell model to specially inhibit the CaMKII γ mRNA expression. We designed the CaMKII γ shRNA sequence and connected with pYr-1.1 plasmid. The ligation product of the CaMKII γ shRNA and pYr-1.1 plasmid was recombined with pAd/PL-DEST vector into pAD-CaMKIIγ-shRNA. adenovirus vector. pAD-CaMKIIγ-shRNA. adenovirus vector infected the dorsal root ganglion neuron to inhibit the CaMKII γ mRNA expression in vitro. The pAD-CaMKIIγ-shRNA adenovirus vector was verified to be correct by the digestion, sequence. And pAD-CaMKIIγ-shRNA. adenovirus vector can infect the DRG cells to inhibit the CaMKII γ mRNA or protein expression by the real-time polymerase chain reaction (PCR) or western blotting. Those results showed that we successfully constructed one adenovirus vector that can infect the dorsal root ganglion neuron to inhibit the CaMKII γ mRNA and protein expression. That will supply with one cell model for the CaMKII γ function study.

    8. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

      Science.gov (United States)

      Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

      2017-07-01

      Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C222 1 , with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

    9. Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment.

      Science.gov (United States)

      Qi, Ruixue; Qiao, Tiankui; Zhuang, Xibing

      2016-01-01

      This study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer. RNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability. Our results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA-directed S100A4 knockdown may

    10. miRNA signatures and transcriptional regulation of their target genes in vitiligo.

      Science.gov (United States)

      Mansuri, Mohmmad Shoab; Singh, Mala; Begum, Rasheedunnisa

      2016-10-01

      miRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression. We have earlier reported the skin miRNA expression profiling in patients with non-segmental vitiligo. In the present study, we show the expression of previously identified skin miRNAs signatures in blood and their target genes in whole blood and PBMCs as well as skin micro-environment of vitiligo patients and controls. miRNA expression profiling in whole blood was performed using customized TaqMan ® Low Density Array. We predicted the potential targets of differentially expressed miRNAs and investigated their expression levels in skin, whole blood and PBMCs from patients and controls using Real-time PCR. Our results showed miR-1, miR-184, miR-328, miR-383 and miR-577 hold similar pattern of expression as of skin, suggesting their potent eminence for being putative markers for vitiligo. In silico target prediction revealed miR-1 targets EDN1, G6PD, HSP60, HSP70, SERP1, SIRT1 & TYR; miR-184 targets EZR & LAMP1; miR-328 targets IL1B, POLH & TRPM1; miR-383 targets EDN1 & TYRP1; and miR-577 targets PTPN22 & TYRP1 which were corroborated by our validation study. In conclusion, the present study for the first time provides new insights into the crucial role of miRNA regulated gene network involved in oxidative stress, autoimmunity and ER stress mediated pathogenesis of vitiligo. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

    11. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

      Energy Technology Data Exchange (ETDEWEB)

      Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

      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.

    12. Synthesis and preliminary evaluation of pro-RNA 2'-O-masked with biolabile pivaloyloxymethyl groups in an RNA interference assay.

      Science.gov (United States)

      Lavergne, Thomas; Baraguey, Carine; Dupouy, Christelle; Parey, Nora; Wuensche, Winfried; Sczakiel, Georg; Vasseur, Jean-Jacques; Debart, Françoise

      2011-07-15

      The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery problem. With the aim to prepare RNA-based prodrugs bearing biolabile protections which facilitate cellular uptake and are prone to be removed enzymatically inside cells in order to release functional RNA, we synthesized pro-RNA totally or partially masked in 2'-OH position with pivaloyloxymethyl (PivOM) groups. A suitable strategy has been developed to synthesize and to purify base-sensitive mixed 2'-OH/2'-O-PivOM oligoribonucleotides, and to include them in siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A and C), the TBS group as 2'-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking some 2'-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi assay and preliminary promising data are discussed.

    13. A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

      Science.gov (United States)

      Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

      2010-10-01

      Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

    14. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

      Science.gov (United States)

      Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

      2010-01-01

      Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1–EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3′ addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3′ adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake. PMID:20719920

    15. Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi

      Directory of Open Access Journals (Sweden)

      Rohde Jörg

      2012-07-01

      Full Text Available Abstract Background The Orf virus (ORFV, a zoonotic Parapoxvirus, causes pustular skin lesions in small ruminants (goat and sheep. Intriguingly, ORFV can repeatedly infect its host, despite the induction of a specific immunity. These immune modulating and immune evading properties are still unexplained. Results Here, we describe that ORFV infection of permissive cells impairs the intracellular transport of MHC class I molecules (MHC I as a result of structural disruption and fragmentation of the Golgi apparatus. Depending on the duration of infection, we observed a pronounced co-localization of MHC I and COP-I vesicular structures as well as a reduction of MHC I surface expression of up to 50%. These subversion processes are associated with early ORFV gene expression and are accompanied by disturbed carbohydrate trimming of post-ER MHC I. The MHC I population remaining on the cell surface shows an extended half-life, an effect that might be partially controlled also by late ORFV genes. Conclusions The presented data demonstrate that ORFV down-regulates MHC I surface expression in infected cells by targeting the late vesicular export machinery and the structure and function of the Golgi apparatus, which might aid to escape cellular immune recognition.

    16. Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

      DEFF Research Database (Denmark)

      Peng, Wenfang; Li, Huan; Hallstrøm, Søren

      2013-01-01

      -adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

    17. Preferential microRNA targeting revealed by in vivo competitive binding and differential Argonaute immunoprecipitation.

      Science.gov (United States)

      Werfel, Stanislas; Leierseder, Simon; Ruprecht, Benjamin; Kuster, Bernhard; Engelhardt, Stefan

      2017-09-29

      MicroRNAs (miRNAs) have been described to simultaneously inhibit hundreds of targets, albeit to a modest extent. It was recently proposed that there could exist more specific, exceptionally strong binding to a subgroup of targets. However, it is unknown, whether this is the case and how such targets can be identified. Using Argonaute2-ribonucleoprotein immunoprecipitation and in vivo competitive binding assays, we demonstrate for miRNAs-21, -199-3p and let-7 exceptional regulation of a subset of targets, which are characterized by preferential miRNA binding. We confirm this finding by analysis of independent quantitative proteome and transcriptome datasets obtained after miRNA silencing. Our data suggest that mammalian miRNA activity is guided by preferential binding of a small set of 3'-untranslated regions, thereby shaping a steep gradient of regulation between potential targets. Our approach can be applied for transcriptome-wide identification of such targets independently of the presence of seed complementary sequences or other predictors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

    18. Targeting microRNA-122: walking on cutting edge of hepatitis C virus infection therapy.

      Science.gov (United States)

      Motavaf, M; Safari, S; Alavian, S M

      2014-01-01

      Hepatitis C virus (HCV) infection, with an estimated 170 million carriers worldwide, remains a major cause of chronic liver disease. Current anti-HCV treatments have significant side effects and have met with only partial success. Therefore, a more effective therapeutic modality for HCV infection is needed. The stability and propagation of HCV is dependent on the interaction between its genome and a highly abundant liver microRNA (miRNA), known as microRNA-122 (miR-122). As a conserved host factor that would not be expected to evolve resistance mutations, miR-122 makes an attractive antiviral target. In this review we will discuss how targeting miR-122, using antisense oligonucleotides (ASOs), can be a new anti-HCV treatment.

    19. Systematic Study of Drosophila MicroRNA Functions Using a Collection of Targeted Knockout Mutations

      DEFF Research Database (Denmark)

      Chen, Ya-Wen; Song, Shilin; Weng, Ruifen

      2014-01-01

      MicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection inclu...... analysis of the biological roles of Drosophila miRNAs....

    20. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

      DEFF Research Database (Denmark)

      Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

      2011-01-01

      intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

    1. Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

      NARCIS (Netherlands)

      Taylor, D.W.; Zhu, Y.; Staals, R.H.J.; Kornfeld, J.E.; Shinkai, A.; Oost, van der J.; Nogales, E.; Doudna, J.A.

      2015-01-01

      Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas

    2. DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

      Directory of Open Access Journals (Sweden)

      Bodo C. Melnik

      2017-09-01

      Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

    3. MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing

      Science.gov (United States)

      Chen, Dan; Fang, Zong Juan; Zhao, Yan; Dragas, Dragan; Dai, Yang; Marucha, Phillip T.; Zhou, Xiaofeng

      2013-01-01

      Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3′-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling. PMID:23724047

    4. Stathmin1 plays oncogenic role and is a target of microRNA-223 in gastric cancer.

      Directory of Open Access Journals (Sweden)

      Wei Kang

      Full Text Available Stathmin1 (STMN1 is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (p = 0.043, T stage (p = 0.004 and lymph node metastasis (p = 0.046. Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (p = 0.01. STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001, reduced monolayer colony formation (p<0.001, inhibited cell invasion and migration ability (p<0.001 and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01. We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer.

    5. Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication

      Science.gov (United States)

      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.

    6. Targeting microRNA-122 to Treat Hepatitis C Virus Infection.

      Science.gov (United States)

      Jopling, Catherine L

      2010-07-01

      An important host factor for hepatitis C virus (HCV) is microRNA-122 (miR-122). miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5' untranslated region (UTR) of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.

    7. Targeting microRNA-122 to Treat Hepatitis C Virus Infection

      Directory of Open Access Journals (Sweden)

      Catherine L. Jopling

      2010-07-01

      Full Text Available An important host factor for hepatitis C virus (HCV is microRNA-122 (miR-122. miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5′ untranslated region (UTR of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.

    8. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection.

      Science.gov (United States)

      Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

      2015-05-05

      A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

    9. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

      International Nuclear Information System (INIS)

      Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

      2011-01-01

      Highlights: → NDUFB6 is required for activity of mitochondrial complex I in human cell lines. → Lentivirus based RNA interference results in frequent off target insertions. → Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

    10. The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

      Energy Technology Data Exchange (ETDEWEB)

      Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

      2011-10-22

      Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

    11. Optimization Of A High-Throughput Transcriptomic (HTTr) Bioactivity Screen In MCF7 Cells Using Targeted RNA-Seq (SOT)

      Science.gov (United States)

      Recent advances in targeted RNA-Seq technology allow researchers to efficiently and cost-effectively obtain whole transcriptome profiles using picograms of mRNA from human cell lysates. Low mRNA input requirements and sample multiplexing capabilities has made time- and concentrat...

    12. New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

      Directory of Open Access Journals (Sweden)

      Christiane Margue

      Full Text Available The non-coding microRNAs (miRNA have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF. By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

    13. A new method for discovering disease-specific MiRNA-target regulatory networks.

      Directory of Open Access Journals (Sweden)

      Miriam Baglioni

      Full Text Available Genes and their expression regulation are among the key factors in the comprehension of the genesis and development of complex diseases. In this context, microRNAs (miRNAs are post-transcriptional regulators that play an important role in gene expression since they are frequently deregulated in pathologies like cardiovascular disease and cancer. In vitro validation of miRNA--targets regulation is often too expensive and time consuming to be carried out for every possible alternative. As a result, a tool able to provide some criteria to prioritize trials is becoming a pressing need. Moreover, before planning in vitro experiments, the scientist needs to evaluate the miRNA-target genes interaction network. In this paper we describe the miRable method whose purpose is to identify new potentially relevant genes and their interaction networks associate to a specific pathology. To achieve this goal miRable follows a system biology approach integrating together general-purpose medical knowledge (literature, Protein-Protein Interaction networks, prediction tools and pathology specific data (gene expression data. A case study on Prostate Cancer has shown that miRable is able to: 1 find new potential miRNA-targets pairs, 2 highlight novel genes potentially involved in a disease but never or little studied before, 3 reconstruct all possible regulatory subnetworks starting from the literature to expand the knowledge on the regulation of miRNA regulatory mechanisms.

    14. Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

      Directory of Open Access Journals (Sweden)

      Wengel Jesper

      2006-06-01

      Full Text Available Abstract Background DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. Results We investigated how incorporation of LNA (locked nucleic acid monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes. For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1. Conclusion The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

    15. RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells

      International Nuclear Information System (INIS)

      Beck, Markus; Strand, Michael R.

      2003-01-01

      The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes

    16. Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

      Directory of Open Access Journals (Sweden)

      Yu-Ping Zheng

      2016-01-01

      Full Text Available AIM: To investigate the effects of lentivirus (LV mediated integrin-linked kinase (ILK RNA interference (RNAi on biological behaviors of human lens epithelial cells (LECs. METHODS: Human cataract LECs and immortalized human LEC line, human lens epithelial (HLE B-3 cells were transfected by lentiviral vector expressing ILK-specific short hairpin RNA (shRNA and then stimulated by transforming growth factor- (TGF-, the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction (RT-PCR and Western blot methods; biological behaviors including cell cycle and apoptosis, cell morphology, -smooth muscle actin (SMA stress fiber formation and cell migration were examined. RESULTS: Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-shRNA vector; flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells. Less -SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs. CONCLUSION: The present study demonstrated that ILK was an important regulator for LECs proliferation and migration. LV mediated ILK RNAi is an effective way to decrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis, as well as, to prevent cell migration by inhibiting TGF- induced -SMA stress fiber formation. Thus, LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

    17. Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm.

      Science.gov (United States)

      Zhou, Hong; Zhou, Michael; Li, Daisy; Manthey, Joseph; Lioutikova, Ekaterina; Wang, Hong; Zeng, Xiao

      2017-11-17

      The beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA. Using human genome and Streptococcus pyogenes Cas9 (SpCas9) as an example, this article mathematically analyzed the probabilities of off-target homologies of sgRNAs and discovered that for large genome size such as human genome, potential off-target homologies are inevitable for sgRNA selection. A highly efficient computationl algorithm was developed for whole genome sgRNA design and off-target homology searches. By means of a dynamically constructed sequence-indexed database and a simplified sequence alignment method, this algorithm achieves very high efficiency while guaranteeing the identification of all existing potential off-target homologies. Via this algorithm, 1,876,775 sgRNAs were designed for the 19,153 human mRNA genes and only two sgRNAs were found to be free of off-target homology. By means of the novel and efficient sgRNA homology search algorithm introduced in this article, genome wide sgRNA design and off-target analysis were conducted and the results confirmed the mathematical analysis that for a sgRNA sequence, it is almost impossible to escape potential off-target homologies. Future innovations on the CRISPR Cas9 gene editing technology need to focus on how to eliminate the Cas9 off-target activity.

    18. Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats

      Science.gov (United States)

      Chen, Changxing; Li, Houxuan; Jiang, Jun; Zhang, Qian; Yan, Fuhua

      2017-01-01

      Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues. PMID:29069818

    19. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

      Science.gov (United States)

      Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

      2010-12-01

      Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

    20. Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement.

      Science.gov (United States)

      Kim, Sung Jin; Kim, Ju Hyun; Yang, Bitna; Jeong, Jin-Sook; Lee, Seong-Wook

      2017-02-01

      Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

    1. Discovery of microRNA-target modules of African rice (Oryza glaberrima) under salinity stress.

      Science.gov (United States)

      Mondal, Tapan Kumar; Panda, Alok Kumar; Rawal, Hukam C; Sharma, Tilak Raj

      2018-01-12

      Oryza glaberrima is the second edible rice in the genus Oryza. It is grown in the African countries. miRNAs are regulatory molecules that are involved in every domains of gene expression including salinity stress response. Although several miRNAs have been reported from various species of Oryza, yet none of them are from this species. Salt treated (200 mM NaCl for 48 h) and control smallRNA libraries of RAM-100, a salt tolerant genotype, each with 2 replications generated 150 conserve and 348 novel miRNAs. We also used smallRNAseq data of NCBI of O. glaberrima to discover additional 246 known miRNAs. Totally, 29 known and 32 novel miRNAs were differentially regulated under salinity stress. Gene ontology and KEGG analysis indicated several targets were involved in vital biological pathways of salinity stress tolerance. Expression of selected miRNAs as indicated by Illumina data were found to be coherent with real time-PCR analysis. However, target gene expression was inversely correlated with their corresponding miRNAs. Finally based upon present results as well as existing knowledge of literature, we proposed the miRNA-target modules that were induced by salinity stress. Therefore, the present findings provide valuable information about miRNA-target networks in salinity adaption of O. glaberrima.