WorldWideScience

Sample records for thaliana rna-dependent rna

  1. Identification of novel miRNAs and miRNA dependent developmental shifts of gene expression in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Shuhua Zhan

    Full Text Available microRNAs (miRNAs are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.

  2. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

    Science.gov (United States)

    Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P

    2016-03-16

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

  3. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

    Previously, an RNA-dependent RNA polymerase produced upon infection of

  4. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

    2015-01-01

    ) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

  5. miRNA-dependent translational repression in the Drosophila ovary.

    Directory of Open Access Journals (Sweden)

    John Reich

    Full Text Available The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary.Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed.Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

  6. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2007-06-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.

  7. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud

    2007-01-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination

  8. Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lydia J R Hunter

    Full Text Available RNA-dependent RNA polymerases (RDRs function in anti-viral silencing in Arabidopsis thaliana and other plants. Salicylic acid (SA, an important defensive signal, increases RDR1 gene expression, suggesting that RDR1 contributes to SA-induced virus resistance. In Nicotiana attenuata RDR1 also regulates plant-insect interactions and is induced by another important signal, jasmonic acid (JA. Despite its importance in defense RDR1 regulation has not been investigated in detail.In Arabidopsis, SA-induced RDR1 expression was dependent on 'NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1', indicating regulation involves the same mechanism controlling many other SA- defense-related genes, including pathogenesis-related 1 (PR1. Isochorismate synthase 1 (ICS1 is required for SA biosynthesis. In defensive signal transduction RDR1 lies downstream of ICS1. However, supplying exogenous SA to ics1-mutant plants did not induce RDR1 or PR1 expression to the same extent as seen in wild type plants. Analysing ICS1 gene expression using transgenic plants expressing ICS1 promoter:reporter gene (β-glucuronidase constructs and by measuring steady-state ICS1 transcript levels showed that SA positively regulates ICS1. In contrast, ICS2, which is expressed at lower levels than ICS1, is unaffected by SA. The wound-response hormone JA affects expression of Arabidopsis RDR1 but jasmonate-induced expression is independent of CORONATINE-INSENSITIVE 1, which conditions expression of many other JA-responsive genes. Transiently increased RDR1 expression following tobacco mosaic virus inoculation was due to wounding and was not a direct effect of infection. RDR1 gene expression was induced by ethylene and by abscisic acid (an important regulator of drought resistance. However, rdr1-mutant plants showed normal responses to drought.RDR1 is regulated by a much broader range of phytohormones than previously thought, indicating that it plays roles beyond those already suggested in virus

  9. In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

    NARCIS (Netherlands)

    Flore, P.H.

    1986-01-01

    The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with Sonchus yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the

  10. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Černá, B.; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Roč. 9, č. 5 (2014), e96070 E-ISSN 1932-6203 R&D Projects: GA ČR GAP502/11/2116; GA ČR GAP302/12/2490; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Q-BETA replicase * C virus RNA * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  11. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Dufresne, Philippe J.; Thivierge, Karine; Cotton, Sophie; Beauchemin, Chantal; Ide, Christine; Ubalijoro, Eliane; Laliberte, Jean-Francois; Fortin, Marc G.

    2008-01-01

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

  12. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

    Directory of Open Access Journals (Sweden)

    Rodrigo Jácome

    Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  13. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  14. Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: Evidence for phosphorylation

    International Nuclear Information System (INIS)

    Ransone, L.J.; Dasgupta, A.

    1989-01-01

    Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol

  15. A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

    Directory of Open Access Journals (Sweden)

    Jiqin Wu

    2015-06-01

    Full Text Available RNA-dependent RNA polymerases (RdRPs from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

  16. Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Ranjith-Kumar, C.T.; Santos, Jan Lee; Gutshall, Lester L.; Johnston, Victor K.; Juili, L.-G.; Kim, M.-J.; Porter, David J.; Maley, Derrick; Greenwood, Cathy; Earnshaw, David L.; Baker, Audrey; Gu Baohua; Silverman, Carol; Sarisky, Robert T.; Kao Cheng

    2003-01-01

    The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn 2+ concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp

  17. RNA-dependent RNA polymerase: Addressing Zika outbreak by a phylogeny-based drug target study.

    Science.gov (United States)

    Stephen, Preyesh; Lin, Sheng-Xiang

    2018-01-01

    Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. © 2017 John Wiley & Sons A/S.

  18. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Liu, Xinran; Musser, Derek M; Lee, Cheri A; Yang, Xiaorong; Arnold, Jamie J; Cameron, Craig E; Boehr, David D

    2015-10-26

    The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

  19. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

    Directory of Open Access Journals (Sweden)

    Xinran Liu

    2015-10-01

    Full Text Available The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I in the RNA-dependent RNA polymerase (RdRp. We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

  20. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

    Directory of Open Access Journals (Sweden)

    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  1. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

    Science.gov (United States)

    Heck, Julie A; Meng, Xiao; Frick, David N

    2009-04-01

    Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

  2. MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Sonia Molina-Pinelo

    Full Text Available Squamous cell lung cancer (SCC and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC, and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA and mRNA profiling (Whole Genome 44 K array G112A, Agilent was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708 and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1 were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  4. 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.

  5. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  6. A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias

    2016-01-01

    , critically depended on the interaction of cellular miR-17 and let-7 with the viral 3' UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de...... immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries...

  7. Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

    LENUS (Irish Health Repository)

    Paquet, Claire

    2009-02-01

    The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

  8. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  9. Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

    Science.gov (United States)

    Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

    2008-11-01

    The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

  10. Structural and functional characterisation of Aichi virus RNA dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 15, č. 1 (2017), s. 7-8 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : Aichi virus * RNA replication Subject RIV: CE - Biochemistry

  11. Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

    International Nuclear Information System (INIS)

    Rubach, Jon K.; Wasik, Brian R.; Rupp, Jonathan C.; Kuhn, Richard J.; Hardy, Richard W.; Smith, Janet L.

    2009-01-01

    The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA

  12. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

    Directory of Open Access Journals (Sweden)

    Eliane F. Meurs

    2012-10-01

    Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

  13. Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

    Science.gov (United States)

    Cajigas, Ivelisse; Leib, David E; Cochrane, Jesse; Luo, Hao; Swyter, Kelsey R; Chen, Sean; Clark, Brian S; Thompson, James; Yates, John R; Kingston, Robert E; Kohtz, Jhumku D

    2015-08-01

    Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains. © 2015. Published by The Company of Biologists Ltd.

  14. NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections.

    Science.gov (United States)

    Deore, R R; Chern, J-W

    2010-01-01

    Hepatitis C virus (HCV), a causative agent for non-A and non-B hepatitis, has infected approximately 3% of world's population. The current treatment option of ribavirin in combination with pegylated interferon possesses lower sustained virological response rates, and has serious disadvantages. Unfortunately, no prophylactic vaccine has been approved yet. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. HCV NS5B RNA dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells. More than 25 pharmaceutical companies and some research groups have developed ≈50 structurally diverse scaffolds to inhibit NS5B. Here we provide comprehensive account of the drug development process of these scaffolds. NS5B polymerase inhibitors have been broadly classified in nucleoside and non nucleoside inhibitors and are sub classified according to their mechanism of action and structural diversities. With some additional considerations about the inhibitor bound NS5B enzyme X-ray crystal structure information and pharmacological aspects of the inhibitors, this review summarizes the lead identification, structure activity relationship (SAR) studies leading to the most potent NS5B inhibitors with subgenomic replicon activity.

  15. Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

    Science.gov (United States)

    Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

    2017-12-01

    Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

  16. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    Science.gov (United States)

    Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

  17. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  18. Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

    2017-11-01

    Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used

  19. A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

    Science.gov (United States)

    Paeshuyse, Jan; Leyssen, Pieter; Mabery, Eric; Boddeker, Nina; Vrancken, Robert; Froeyen, Matheus; Ansari, Israrul H.; Dutartre, Hélène; Rozenski, Jef; Gil, Laura H. V. G.; Letellier, Carine; Lanford, Robert; Canard, Bruno; Koenen, Frank; Kerkhofs, Pierre; Donis, Ruben O.; Herdewijn, Piet; Watson, Julia; De Clercq, Erik; Puerstinger, Gerhard; Neyts, Johan

    2006-01-01

    We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 ± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed. PMID:16352539

  20. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp

    Directory of Open Access Journals (Sweden)

    Devendra K. Rai

    2012-07-01

    Full Text Available Bovine Rhinitis B Virus (BRBV is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp, which is also known as 3D polymerase (3Dpol. In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P, nucleotide tri-phosphate (NTP and pyrophosphate (PPi bound FMDV 3Dpol (PDB, 1U09 and 2E9Z. The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16 and rabbit hemorrhagic disease virus (RHDV 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the

  1. microRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus.

    Directory of Open Access Journals (Sweden)

    Sunantha Sethuraman

    2017-07-01

    Full Text Available Kaposi's sarcoma (KS is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL. In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs. The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

  2. RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

    Science.gov (United States)

    Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

    2010-05-01

    The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

  3. Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.

    Science.gov (United States)

    Helgadóttir, Sunna; Sinapah, Sylvie; Söll, Dieter; Ling, Jiqiang

    2012-01-02

    In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

    2010-10-22

    Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

  5. Expression analysis of argonaute, Dicer-like, and RNA-dependent ...

    Indian Academy of Sciences (India)

    DEFANG GAN

    the first report of expression analysis of all CsDCL, CsAGO and CsRDR family genes in cucumber under .... tissues and organs were detected using online data and semi- ...... Wheeler B. S. 2013 Small RNAs, big impact: small RNA pathways.

  6. RNA-Dependent Intergenerational Inheritance of Enhanced Synaptic Plasticity after Environmental Enrichment

    Directory of Open Access Journals (Sweden)

    Eva Benito

    2018-04-01

    Full Text Available Summary: Physical exercise in combination with cognitive training is known to enhance synaptic plasticity, learning, and memory and lower the risk for various complex diseases including Alzheimer’s disease. Here, we show that exposure of adult male mice to an environmental enrichment paradigm leads to enhancement of synaptic plasticity and cognition also in the next generation. We show that this effect is mediated through sperm RNA and especially miRs 212/132. In conclusion, our study reports intergenerational inheritance of an acquired cognitive benefit and points to specific miRs as candidates mechanistically involved in this type of transmission. : Environmental enrichment (EE, a combination of physical and mental exercise, has been shown to increase cognitive abilities in mice and in humans. Benito et al. find that offspring of male mice subjected to EE also show this increase. This effect is dependent on sperm RNA and involves microRNA212/132. Keywords: epigenetics, brain, microRNA, memory, intergenerational, transgenerational, exercise, environmental enrichment, cognition

  7. Environmental cues induce a long noncoding RNA-dependent remodeling of the nucleolus.

    Science.gov (United States)

    Jacob, Mathieu D; Audas, Timothy E; Uniacke, James; Trinkle-Mulcahy, Laura; Lee, Stephen

    2013-09-01

    The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate-positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.

  8. The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

    2017-12-01

    To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

  9. Evidence for Importance of tRNA-dependent Cytokinin Biosynthetic Pathway in the Moss Physcomitrella patens

    Czech Academy of Sciences Publication Activity Database

    Yevdakova, N.A.; Motyka, Václav; Malbeck, Jiří; Trávníčková, Alena; Novák, Ondřej; Strnad, Miroslav; von Schwartzenberg, K.

    2008-01-01

    Roč. 27, č. 3 (2008), s. 271-281 ISSN 0721-7595 R&D Projects: GA MŠk(CZ) LC06034; GA ČR GA206/05/0894; GA AV ČR IAA600380701 Institutional research plan: CEZ:AV0Z50380511 Keywords : Physcomitrella * ove mutant * Cytokinin biosynthesis * tRNA-isopentenyl transferase Subject RIV: ED - Physiology Impact factor: 2.109, year: 2008

  10. RNA-Dependent Intergenerational Inheritance of Enhanced Synaptic Plasticity after Environmental Enrichment.

    Science.gov (United States)

    Benito, Eva; Kerimoglu, Cemil; Ramachandran, Binu; Pena-Centeno, Tonatiuh; Jain, Gaurav; Stilling, Roman Manuel; Islam, Md Rezaul; Capece, Vincenzo; Zhou, Qihui; Edbauer, Dieter; Dean, Camin; Fischer, André

    2018-04-10

    Physical exercise in combination with cognitive training is known to enhance synaptic plasticity, learning, and memory and lower the risk for various complex diseases including Alzheimer's disease. Here, we show that exposure of adult male mice to an environmental enrichment paradigm leads to enhancement of synaptic plasticity and cognition also in the next generation. We show that this effect is mediated through sperm RNA and especially miRs 212/132. In conclusion, our study reports intergenerational inheritance of an acquired cognitive benefit and points to specific miRs as candidates mechanistically involved in this type of transmission. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Immunopurification of the suppressor tRNA dependent rabbit β-globin readthrough protein

    International Nuclear Information System (INIS)

    Hatfield, D.; Thorgeirsson, S.S.; Copeland, T.D.; Oroszlan, S.; Bustin, M.

    1988-01-01

    In mammalian cells, the rabbit β-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, the authors elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit β-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [ 35 S] methionine. Incorporation of [ 35 S] methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the β-globin readthrough protein is naturally occurring in rabbit reticulocytes

  12. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    International Nuclear Information System (INIS)

    Yap, Thai Leong; Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G.; Lescar, Julien

    2007-01-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration

  14. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Thai Leong [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G. [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); Lescar, Julien, E-mail: julien@ntu.edu.sg [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2007-02-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration.

  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. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  17. Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8.

    Directory of Open Access Journals (Sweden)

    Yen-Chin Liu

    2014-06-01

    Full Text Available The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D(pol also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D(pol enters the nucleus through the nuclear localization signal (NLS and targets the pre-mRNA processing factor 8 (Prp8 to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D(pol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D(pol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.

  18. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Science.gov (United States)

    Nicolas, Francisco Esteban; Moxon, Simon; de Haro, Juan P.; Calo, Silvia; Grigoriev, Igor V.; Torres-Martínez, Santiago; Moulton, Vincent; Ruiz-Vázquez, Rosa M.; Dalmay, Tamas

    2010-01-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi. PMID:20427422

  19. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

    2011-09-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

  20. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shan-Shan [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Jiang, Teng [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Wang, Yi; Gu, Li-Ze [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Wu, Hui-Wen [Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing (China); Tan, Lan [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Guo, Jun, E-mail: Guoj@njmu.edu.cn [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China)

    2014-01-17

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

  1. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    International Nuclear Information System (INIS)

    Chen, Shan-Shan; Jiang, Teng; Wang, Yi; Gu, Li-Ze; Wu, Hui-Wen; Tan, Lan; Guo, Jun

    2014-01-01

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM

  2. tRNA-dependent cysteine biosynthetic pathway represents a strategy to increase cysteine contents by preventing it from thermal degradation: thermal adaptation of methanogenic archaea ancestor.

    Science.gov (United States)

    Qu, Ge; Wang, Wei; Chen, Ling-Ling; Qian, Shao-Song; Zhang, Hong-Yu

    2009-10-01

    Although cysteine (Cys) is beneficial to stabilize protein structures, it is not prevalent in thermophiles. For instance, the Cys contents in most thermophilic archaea are only around 0.7%. However, methanogenic archaea, no matter thermophilic or not, contain relatively abundant Cys, which remains elusive for a long time. Recently, Klipcan et al. correlated this intriguing property of methanogenic archaea with their unique tRNA-dependent Cys biosynthetic pathway. But, the deep reasons underlying the correlation are ambiguous. Considering the facts that free Cys is thermally labile and the tRNA-dependent Cys biosynthesis avoids the use of free Cys, we speculate that the unique Cys biosynthetic pathway represents a strategy to increase Cys contents by preventing it from thermal degradation, which may be relevant to the thermal adaptation of methanogenic archaea ancestor.

  3. The Crystal Structure of the RNA-Dependent RNA Polymerase from Human Rhinovirus: A Dual Function Target for Common Cold Antiviral Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Love, Robert A.; Maegley, Karen A.; Yu, Xiu; Ferre, RoseAnn; Lingardo, Laura K.; Diehl, Wade; Parge, Hans E.; Dragovich, Peter S.; Fuhrman, Shella A. (Pfizer)

    2010-11-16

    Human rhinoviruses (HRV), the predominant members of the Picornaviridae family of positive-strand RNA viruses, are the major causative agents of the common cold. Given the lack of effective treatments for rhinoviral infections, virally encoded proteins have become attractive therapeutic targets. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) denoted 3D{sup pol}, which is responsible for replicating the viral genome and for synthesizing a protein primer used in the replication. Here the crystal structures for three viral serotypes (1B, 14, and 16) of HRV 3D{sup pol} have been determined. The three structures are very similar to one another, and to the closely related poliovirus (PV) 3D{sup pol} enzyme. Because the reported PV crystal structure shows significant disorder, HRV 3D{sup pol} provides the first complete view of a picornaviral RdRp. The folding topology of HRV 3D{sup pol} also resembles that of RdRps from hepatitis C virus (HCV) and rabbit hemorrhagic disease virus (RHDV) despite very low sequence homology.

  4. Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

    Directory of Open Access Journals (Sweden)

    Ester Sesmero

    2015-07-01

    Full Text Available Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV, human immunodeficiency virus (HIV and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

  5. Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus.

    Science.gov (United States)

    Silin, D; Lyubomska, O; Ludlow, M; Duprex, W P; Rima, B K

    2007-12-01

    We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

  6. Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

    2003-01-01

    A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

  7. The use of RNA-dependent RNA polymerase for the taxonomic assignment of Picorna-like viruses (order Picornavirales infecting Apis mellifera L. populations

    Directory of Open Access Journals (Sweden)

    Schroeder Declan C

    2008-01-01

    Full Text Available Abstract Background Single-stranded RNA viruses, infectious to the European honeybee, Apis mellifera L. are known to reside at low levels in colonies, with typically no apparent signs of infection observed in the honeybees. Reverse transcription-PCR (RT-PCR of regions of the RNA-dependent RNA polymerase (RdRp is often used to diagnose their presence in apiaries and also to classify the type of virus detected. Results Analysis of RdRp conserved domains was undertaken on members of the newly defined order, the Picornavirales; focusing in particular on the amino acid residues and motifs known to be conserved. Consensus sequences were compiled using partial and complete honeybee virus sequences published to date. Certain members within the iflaviruses, deformed wing virus (DWV, Kakugo virus (KV and Varroa destructor virus (VDV; and the dicistroviruses, acute bee paralysis virus (ABPV, Israeli paralysis virus (IAPV and Kashmir bee virus (KBV, shared greater than 98% and 92% homology across the RdRp conserved domains, respectively. Conclusion RdRp was validated as a suitable taxonomic marker for the assignment of members of the order Picornavirales, with the potential for use independent of other genetic or phenotypic markers. Despite the current use of the RdRp as a genetic marker for the detection of specific honeybee viruses, we provide overwhelming evidence that care should be taken with the primer set design. We demonstrated that DWV, VDV and KV, or ABPV, IAPV and KBV, respectively are all recent descendents or variants of each other, meaning caution should be applied when assigning presence or absence to any of these viruses when using current RdRp primer sets. Moreover, it is more likely that some primer sets (regardless of what gene is used are too specific and thus are underestimating the diversity of honeybee viruses.

  8. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice

    Directory of Open Access Journals (Sweden)

    Abu Musa Md Talimur Reza

    2018-02-01

    Full Text Available The Rag2 knockout (KO mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs and messenger RNAs (mRNAs in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  9. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

    Science.gov (United States)

    Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Kim, Jin-Hoi

    2018-02-27

    The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  10. Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

    Directory of Open Access Journals (Sweden)

    Xu Wanhong

    2008-12-01

    Full Text Available Abstract Background The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses. Results We determined the L2 genome segment nucleotide sequences, which encode the RdRp proteins, of two different avian reoviruses, strains ARV138 and ARV176 in order to define conserved and variable regions within reovirus RdRp proteins and to better delineate structure/function of this important enzyme. The ARV138 L2 genome segment was 3829 base pairs long, whereas the ARV176 L2 segment was 3830 nucleotides long. Both segments were predicted to encode λB RdRp proteins 1259 amino acids in length. Alignments of these newly-determined ARV genome segments, and their corresponding proteins, were performed with all currently available homologous mammalian reovirus (MRV and aquareovirus (AqRV genome segment and protein sequences. There was ~55% amino acid identity between ARV λB and MRV λ3 proteins, making the RdRp protein the most highly conserved of currently known orthoreovirus proteins, and there was ~28% identity between ARV λB and homologous MRV and AqRV RdRp proteins. Predictive structure/function mapping of identical and conserved residues within the known MRV λ3 atomic structure indicated most identical amino acids and conservative substitutions were located near and within predicted catalytic domains and lining RdRp channels, whereas non-identical amino acids were generally located on the molecule's surfaces. Conclusion The ARV λB and MRV λ3 proteins showed the highest ARV:MRV identity values (~55% amongst all currently known ARV and MRV

  11. StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Zemla, A; Lang, D; Kostova, T; Andino, R; Zhou, C

    2010-11-29

    Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory - still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could overcome these difficulties and facilitate the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV, a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus and demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique or that shared structural similarity with structures that are distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected

  12. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  13. In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

    Science.gov (United States)

    Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

    2006-11-01

    Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

  14. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region

    DEFF Research Database (Denmark)

    Pedersen, Margit; Nissen, Søren; Mitarai, Namiko

    2011-01-01

    Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5'-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described...

  15. MicroRNA-dependent regulation of metamorphosis and identification of microRNAs in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Wu, Wei; Xiong, Wenfeng; Li, Chengjun; Zhai, Mengfan; Li, Yao; Ma, Fei; Li, Bin

    2017-10-01

    To date, although some microRNAs (miRNAs) have been discovered in the holometabolism insect Tribolium castaneum, large numbers of miRNAs still require investigation. Knocking down Dicer-1 (Dcr-1) and Argonaute-1 (Ago-1) in late larvae impaired miRNA synthesis, affected the juvenile hormone pathway by up-regulating Methoprene-tolerant (Met) and Krüppel-homolog1 (Kr-h1) transcript levels, and resulted in a series of defects in T. castaneum development and metamorphosis. Thus, high-throughput Illumina/Solexa sequencing was performed with a mixed sample of eight key developmental stages of T. castaneum. In total, 1154 unique miRNAs were discovered containing 274 conserved miRNAs belong to 68 miRNA families, 108 known candidate miRNAs and 772 novel miRNAs. Genome locus analysis showed that miRNA clusters are more abundant in T. castaneum than other species. The results indicated that RNAi of Dcr-1 and Ago-1 in T. castaneum resulted in miRNA-induced metamorphosis defects. Furthermore, large numbers of novel miRNAs were discovered in T. castaneum and localized to T. castaneum genome loci. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

    Science.gov (United States)

    Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

    2010-10-15

    The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV

  17. Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

    Science.gov (United States)

    Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

    2003-10-01

    Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

  18. Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

    Science.gov (United States)

    Cacci, Emanuele; Negri, Rodolfo; Biagioni, Stefano; Lupo, Giuseppe

    2017-01-01

    Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.

  19. Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala

    Directory of Open Access Journals (Sweden)

    Tae-Yeon Eom

    2017-05-01

    Full Text Available Individuals with 22q11.2 deletion syndrome (22q11DS are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections. This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8–Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS.

  20. Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala.

    Science.gov (United States)

    Eom, Tae-Yeon; Bayazitov, Ildar T; Anderson, Kara; Yu, Jing; Zakharenko, Stanislav S

    2017-05-23

    Individuals with 22q11.2 deletion syndrome (22q11DS) are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections). This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8-Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples

    Directory of Open Access Journals (Sweden)

    Eleonora Ciarlo

    2013-03-01

    Recent studies indicated that sortilin-related receptor 1 (SORL1 is a risk gene for late-onset Alzheimer's disease (AD, although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc RNA (hereafter referred to as 51A that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP, leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

  2. Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Jixian Zhai

    2008-04-01

    Full Text Available Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC, a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42 were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

  3. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Habjan, Matthias; Pichlmair, Andreas; Elliott, Richard M; Overby, Anna K; Glatter, Timo; Gstaiger, Matthias; Superti-Furga, Giulio; Unger, Hermann; Weber, Friedemann

    2009-05-01

    Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

  4. The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U RNA.

    Directory of Open Access Journals (Sweden)

    Tiago Antonio de Souza

    Full Text Available Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC, a translin-associated factor X (CsTRAX, a VirE2-interacting protein (CsVIP2, a high mobility group (CsHMG and two poly(A-binding proteins (CsPABP1 and 2, interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

  5. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

    Directory of Open Access Journals (Sweden)

    Moes Lorin

    2007-11-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV and the more distantly related Hepatitis C virus (HCV pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical

  6. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon.

    Science.gov (United States)

    Moes, Lorin; Wirth, Manfred

    2007-11-22

    Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the

  7. Infection and RNA recombination of Brome mosaic virus in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Dzianott, Aleksandra; Bujarski, Jozef J.

    2004-01-01

    Ecotypes of Arabidopsis thaliana supported the replication and systemic spread of Brome mosaic virus (BMV) RNAs. Infection was induced either by manual inoculation with viral RNA or by BMV virions, demonstrating that virus disassembly did not prevent infection. When in vitro-transcribed BMV RNAs 1-3 were used, production of subgenomic RNA4 was observed, showing that BMV RNA replication and transcription had occurred. Furthermore, inoculations of the transgenic Arabidopsis line that expressed a suppressor of RNA interference (RNAi) pathway markedly increased the BMV RNA concentrations. Inoculations with designed BMV RNA3 recombination vectors generated both homologous and nonhomologous BMV RNA-RNA recombinants. Thus, all cellular factors essential for BMV RNA replication, transcription, and RNA recombination were shown to be present in Arabidopsis. The current scope of understanding of the model Arabidopsis plant system should facilitate the identification of these factors governing the BMV life cycle

  8. MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hafidh Said

    2009-12-01

    Full Text Available Abstract Background New generation sequencing technology has allowed investigation of the small RNA populations of flowering plants at great depth. However, little is known about small RNAs in their reproductive cells, especially in post-meiotic cells of the gametophyte generation. Pollen - the male gametophyte - is the specialised haploid structure that generates and delivers the sperm cells to the female gametes at fertilisation. Whether development and differentiation of the male gametophyte depends on the action of microRNAs and trans-acting siRNAs guiding changes in gene expression is largely unknown. Here we have used 454 sequencing to survey the various small RNA populations present in mature pollen of Arabidopsis thaliana. Results In this study we detected the presence of 33 different microRNA families in mature pollen and validated the expression levels of 17 selected miRNAs by Q-RT-PCR. The majority of the selected miRNAs showed pollen-enriched expression compared with leaves. Furthermore, we report for the first time the presence of trans-acting siRNAs in pollen. In addition to describing new patterns of expression for known small RNAs in each of these classes, we identified 7 putative novel microRNAs. One of these, ath-MIR2939, targets a pollen-specific F-box transcript and we demonstrate cleavage of its target mRNA in mature pollen. Conclusions Despite the apparent simplicity of the male gametophyte, comprising just two different cell types, pollen not only utilises many miRNAs and trans-acting siRNAs expressed in the somatic tissues but also expresses novel miRNAs.

  9. Blue light alters miR167 expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants.

    Science.gov (United States)

    Pashkovskiy, Pavel P; Kartashov, Alexander V; Zlobin, Ilya E; Pogosyan, Sergei I; Kuznetsov, Vladimir V

    2016-07-01

    The effect of blue LED (450 nm) on the photomorphogenesis of Arabidopsis thaliana Col-0 plants and the transcript levels of several genes, including miRNAs, photoreceptors and auxin response factors (ARF) was investigated. It was observed that blue light accelerated the generative development, reduced the rosette leaf number, significantly reduced the leaf area, dry biomass and led to the disruption of conductive tissue formation. The blue LED differentially influenced the transcript levels of several phytochromes (PHY a, b, c, d, and e), cryptochromes (CRY 1 and 2) and phototropins (PHOT 1 and 2). At the same time, the blue LED significantly increased miR167 expression compared to a fluorescent lamp or white LEDs. This increase likely resulted in the enhanced transcription of the auxin response factor genes ARF4 and ARF8, which are regulated by this miRNA. These findings support the hypothesis that the effects of blue light on A. thaliana are mediated by auxin signalling pathway involving miRNA-dependent regulation of ARF gene expression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

  11. 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline inhibits pestivirus replication by targeting a hot spot drug binding pocket in the RNA-dependent RNA polymerase.

    Science.gov (United States)

    Musiu, Simone; Leyssen, Pieter; Froeyen, Mathy; Chezal, Jean-Michel; Neyts, Johan; Paeshuyse, Jan

    2016-05-01

    The compound 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline (CF02334) was identified as a selective inhibitor of the cytopathic effect (CPE) caused by bovine viral diarrhea virus (BVDV) in a virus-cell-based assay. The EC50-values for inhibition of CPE, viral RNA synthesis and the production of infectious virus progeny were 13.0 ± 0.6 μM, 2.6 ± 0.9 μM and 17.8 ± 0.6 μM, respectively. CF02334 was found to be inactive in the hepatitis C subgenomic replicon system. CF02334-resistant BVDV was obtained and was found to carry the N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). Molecular modeling revealed that N264D is located in a small cavity near the fingertip domain of the pestivirus polymerase. CF02334-resistant BVDV was proven to be cross-resistant to BPIP, AG110 and LZ37, inhibitors that have previously been described to target the same region of the BVDV RdRp. CF02334 did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of BVDV replication complexes. Taken together, these observations indicate that CF02334 likely interacts with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37, which marks this region of the viral polymerase as a "hot spot" for inhibition of pestivirus replication. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

    Science.gov (United States)

    Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

    2014-01-01

    Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

  13. Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

    Science.gov (United States)

    Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2016-06-01

    MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  14. Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase.

    Science.gov (United States)

    Musiu, Simone; Pürstinger, Gerhard; Stallinger, Sylvia; Vrancken, Robert; Haegeman, Andy; Koenen, Frank; Leyssen, Pieter; Froeyen, Mathy; Neyts, Johan; Paeshuyse, Jan

    2014-06-01

    2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA

    DEFF Research Database (Denmark)

    Porse, B T; Kirillov, S V; Awayez, M J

    1999-01-01

    The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within...... in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop...... the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA....

  16. Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

    Science.gov (United States)

    Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

    2018-04-06

    Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

  17. Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map.

    Science.gov (United States)

    Hardcastle, Thomas J; Müller, Sebastian Y; Baulcombe, David C

    2018-04-20

    Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.

  18. 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.

  19. Identification of an Arabidopsis thaliana protein that binds to tomato mosaic virus genomic RNA and inhibits its multiplication

    International Nuclear Information System (INIS)

    Fujisaki, Koki; Ishikawa, Masayuki

    2008-01-01

    The genomic RNAs of positive-strand RNA viruses carry RNA elements that play positive, or in some cases, negative roles in virus multiplication by interacting with viral and cellular proteins. In this study, we purified Arabidopsis thaliana proteins that specifically bind to 5' or 3' terminal regions of tomato mosaic virus (ToMV) genomic RNA, which contain important regulatory elements for translation and RNA replication, and identified these proteins by mass spectrometry analyses. One of these host proteins, named BTR1, harbored three heterogeneous nuclear ribonucleoprotein K-homology RNA-binding domains and preferentially bound to RNA fragments that contained a sequence around the initiation codon of the 130K and 180K replication protein genes. The knockout and overexpression of BTR1 specifically enhanced and inhibited, respectively, ToMV multiplication in inoculated A. thaliana leaves, while such effect was hardly detectable in protoplasts. These results suggest that BTR1 negatively regulates the local spread of ToMV

  20. Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2010-11-01

    Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

  1. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

    Directory of Open Access Journals (Sweden)

    Terro F

    2011-06-01

    Full Text Available Abstract Background Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD. There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ peptides, the nuclear factor-kappa B (NF-κB pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. Methods Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h, co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα, interleukin (IL-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test Results In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα and interleukin (IL-1β, and limited apoptosis. Conclusion In spite of the

  2. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Changho Eun

    Full Text Available RNA-directed DNA methylation (RdDM is a small interfering RNA (siRNA-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

  3. Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Daniel Lepe-Soltero

    2017-12-01

    Full Text Available The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana, ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

  4. Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana.

    Science.gov (United States)

    Lepe-Soltero, Daniel; Armenta-Medina, Alma; Xiang, Daoquan; Datla, Raju; Gillmor, C Stewart; Abreu-Goodger, Cei

    2017-12-01

    The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana , ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

  5. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    Science.gov (United States)

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  6. AthMethPre: a web server for the prediction and query of mRNA m6A sites in Arabidopsis thaliana.

    Science.gov (United States)

    Xiang, Shunian; Yan, Zhangming; Liu, Ke; Zhang, Yaou; Sun, Zhirong

    2016-10-18

    N 6 -Methyladenosine (m 6 A) is the most prevalent and abundant modification in mRNA that has been linked to many key biological processes. High-throughput experiments have generated m 6 A-peaks across the transcriptome of A. thaliana, but the specific methylated sites were not assigned, which impedes the understanding of m 6 A functions in plants. Therefore, computational prediction of mRNA m 6 A sites becomes emergently important. Here, we present a method to predict the m 6 A sites for A. thaliana mRNA sequence(s). To predict the m 6 A sites of an mRNA sequence, we employed the support vector machine to build a classifier using the features of the positional flanking nucleotide sequence and position-independent k-mer nucleotide spectrum. Our method achieved good performance and was applied to a web server to provide service for the prediction of A. thaliana m 6 A sites. The server also provides a comprehensive database of predicted transcriptome-wide m 6 A sites and curated m 6 A-seq peaks from the literature for query and visualization. The AthMethPre web server is the first web server that provides a user-friendly tool for the prediction and query of A. thaliana mRNA m 6 A sites, which is freely accessible for public use at .

  7. Genome-wide search for miRNA-target interactions in Arabidopsis thaliana with an integrated approach

    Directory of Open Access Journals (Sweden)

    Ding Jiandong

    2012-06-01

    Full Text Available Abstract Background MiRNA are about 22nt long small noncoding RNAs that post transcriptionally regulate gene expression in animals, plants and protozoa. Confident identification of MiRNA-Target Interactions (MTI is vital to understand their function. Currently, several integrated computational programs and databases are available for animal miRNAs, the mechanisms of which are significantly different from plant miRNAs. Methods Here we present an integrated MTI prediction and analysis toolkit (imiRTP for Arabidopsis thaliana. It features two important functions: (i combination of several effective plant miRNA target prediction methods provides a sufficiently large MTI candidate set, and (ii different filters allow for an efficient selection of potential targets. The modularity of imiRTP enables the prediction of high quality targets on genome-wide scale. Moreover, predicted MTIs can be presented in various ways, which allows for browsing through the putative target sites as well as conducting simple and advanced analyses. Results Results show that imiRTP could always find high quality candidates compared with single method by choosing appropriate filter and parameter. And we also reveal that a portion of plant miRNA could bind target genes out of coding region. Based on our results, imiRTP could facilitate the further study of Arabidopsis miRNAs in real use. All materials of imiRTP are freely available under a GNU license at (http://admis.fudan.edu.cn/projects/imiRTP.htm.

  8. Assessing the 5S ribosomal RNA heterogeneity in Arabidopsis thaliana using short RNA next generation sequencing data.

    Science.gov (United States)

    Szymanski, Maciej; Karlowski, Wojciech M

    2016-01-01

    In eukaryotes, ribosomal 5S rRNAs are products of multigene families organized within clusters of tandemly repeated units. Accumulation of genomic data obtained from a variety of organisms demonstrated that the potential 5S rRNA coding sequences show a large number of variants, often incompatible with folding into a correct secondary structure. Here, we present results of an analysis of a large set of short RNA sequences generated by the next generation sequencing techniques, to address the problem of heterogeneity of the 5S rRNA transcripts in Arabidopsis and identification of potentially functional rRNA-derived fragments.

  9. Selective mRNA translation coordinates energetic and metabolic adjustments to cellular oxygen deprivation and reoxygenation in Arabidopsis thaliana.

    Science.gov (United States)

    Branco-Price, Cristina; Kaiser, Kayla A; Jang, Charles J H; Larive, Cynthia K; Bailey-Serres, Julia

    2008-12-01

    Cellular oxygen deprivation (hypoxia/anoxia) requires an acclimation response that enables survival during an energy crisis. To gain new insights into the processes that facilitate the endurance of transient oxygen deprivation, the dynamics of the mRNA translation state and metabolites were quantitatively monitored in Arabidopsis thaliana seedlings exposed to a short (2 h) or prolonged (9 h) period of oxygen and carbon dioxide deprivation and following 1 h of re-aeration. Hypoxia stress and reoxygenation promoted adjustments in the levels of polyribosomes (polysomes) that were highly coordinated with cellular ATP content. A quantitative comparison of steady-state and polysomal mRNA populations revealed that over half of the cellular mRNAs were restricted from polysome complexes during the stress, with little or no change in abundance. This selective repression of translation was rapidly reversed upon reoxygenation. Comparison of the adjustment in gene transcripts and metabolites demonstrated that profiling of polysomal mRNAs strongly augments the prediction of cellular processes that are altered during cellular oxygen deprivation. The selective translation of a subset of mRNAs promotes the conservation of ATP and facilitates the transition to anaerobic metabolism during low-oxygen stress.

  10. A Novel Non-coding RNA Regulates Drought Stress Tolerance in Arabidopsis thaliana

    KAUST Repository

    Albesher, Nour H.

    2014-05-01

    Drought (soil water deficit) as a major adverse environmental condition can result in serious reduction in plant growth and crop production. Plants respond and adapt to drought stresses by triggering various signalling pathways leading to physiological, metabolic and developmental changes that may ultimately contribute to enhanced tolerance to the stress. Here, a novel non-coding RNA (ncRNA) involved in plant drought stress tolerance was identified. We showed that increasing the expression of this ncRNA led to enhanced sensitivity during seed germination and seedling growth to the phytohormone abscisic acid. The mutant seedlings are also more sensitive to osmotic stress inhibition of lateral root growth. Consistently, seedlings with enhanced expression of this ncRNA exhibited reduced transiprational water loss and were more drought-tolerant than the wild type. Future analyses of the mechanism for its role in drought tolerance may help us to understand how plant drought tolerance could be further regulated by this novel ncRNA.

  11. Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Il Lae Jung

    Full Text Available MicroRNAs (miRNAs are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism.

  12. Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana.

    Science.gov (United States)

    Plötner, Björn; Nurmi, Markus; Fischer, Axel; Watanabe, Mutsumi; Schneeberger, Korbinian; Holm, Svante; Vaid, Neha; Schöttler, Mark Aurel; Walther, Dirk; Hoefgen, Rainer; Weigel, Detlef; Laitinen, Roosa A E

    2017-07-01

    Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F 2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  13. Expression of Cucumber mosaic virus suppressor 2b alters FWA methylation and its siRNA accumulation in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Sadia Hamera

    2016-11-01

    Full Text Available The Cucumber mosaic virus (CMV suppressor 2b co-localizes with AGO4 in cytoplasmic and nuclear fractions of Arabidopsis thaliana. Biochemical fractionation of A. thaliana cellular extracts revealed that 2b and AGO4 coexist in multiple size exclusions. 2b transgenic A. thaliana exhibited an enhanced accumulation of 24nt siRNAs from flowering wageningen (FWA and other heterochromatic loci. These plants also exhibited hypo-methylation of an endogenous- as well as transgene-FWA promoter at non-CG sites. In corroboration, both transgenic 2b and CMV infection affected the regulation of transposons which mimics the ago4 phenotype. In conclusion, 2b perturbs plant defense by interfering with AGO4-regulated transcriptional gene silencing.

  14. RNA Processing Factor 5 is required for efficient 5' cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana.

    Science.gov (United States)

    Hauler, Aron; Jonietz, Christian; Stoll, Birgit; Stoll, Katrin; Braun, Hans-Peter; Binder, Stefan

    2013-05-01

    The 5' ends of many mitochondrial transcripts are generated post-transcriptionally. Recently, we identified three RNA PROCESSING FACTORs required for 5' end maturation of different mitochondrial mRNAs in Arabidopsis thaliana. All of these factors are pentatricopeptide repeat proteins (PPRPs), highly similar to RESTORERs OF FERTILTY (RF), that rescue male fertility in cytoplasmic male-sterile lines from different species. Therefore, we suggested a general role of these RF-like PPRPs in mitochondrial 5' processing. We now identified RNA PROCESSING FACTOR 5, a PPRP not classified as an RF-like protein, required for the efficient 5' maturation of the nad6 and atp9 mRNAs as well as 26S rRNA. The precursor molecules of these RNAs share conserved sequence elements, approximately ranging from positions -50 to +9 relative to mature 5' mRNA termini, suggesting these sequences to be at least part of the cis elements required for processing. The knockout of RPF5 has only a moderate influence on 5' processing of atp9 mRNA, whereas the generation of the mature nad6 mRNA and 26S rRNA is almost completely abolished in the mutant. The latter leads to a 50% decrease of total 26S rRNA species, resulting in an imbalance between the large rRNA and 18S rRNA. Despite these severe changes in RNA levels and in the proportion between the 26S and 18S rRNAs, mitochondrial protein levels appear to be unaltered in the mutant, whereas seed germination capacity is markedly reduced. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  15. Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information

    DEFF Research Database (Denmark)

    Hebsgaard, Stefan M.; Korning, Peter G.; Tolstrup, Niels

    1996-01-01

    Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local predicition of splice sites, is refin...

  16. Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that promotes infection in soybean and Arabidopsis thaliana.

    Science.gov (United States)

    Xiong, Qin; Ye, Wenwu; Choi, Duseok; Wong, James; Qiao, Yongli; Tao, Kai; Wang, Yuanchao; Ma, Wenbo

    2014-12-01

    The genus Phytophthora consists of notorious and emerging pathogens of economically important crops. Each Phytophthora genome encodes several hundreds of cytoplasmic effectors, which are believed to manipulate plant immune response inside the host cells. However, the majority of Phytophthora effectors remain functionally uncharacterized. We recently discovered two effectors from the soybean stem and root rot pathogen Phytophthora sojae with the activity to suppress RNA silencing in plants. These effectors are designated Phytophthora suppressor of RNA silencing (PSRs). Here, we report that the P. sojae PSR2 (PsPSR2) belongs to a conserved and widespread effector family in Phytophthora. A PsPSR2-like effector produced by P. infestans (PiPSR2) can also suppress RNA silencing in plants and promote Phytophthora infection, suggesting that the PSR2 family effectors have conserved functions in plant hosts. Using Agrobacterium rhizogenes-mediated hairy roots induction, we demonstrated that the expression of PsPSR2 rendered hypersusceptibility of soybean to P. sojae. Enhanced susceptibility was also observed in PsPSR2-expressing Arabidopsis thaliana plants during Phytophthora but not bacterial infection. These experiments provide strong evidence that PSR2 is a conserved Phytophthora effector family that performs important virulence functions specifically during Phytophthora infection of various plant hosts.

  17. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    Science.gov (United States)

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  18. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase

    OpenAIRE

    Heck, Julie A.; Meng, Xiao; Frick, David N.

    2009-01-01

    Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B (Mol. Cell 19:111). We examined the effects of pu...

  19. Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

    Science.gov (United States)

    Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

    2016-10-13

    In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various

  20. The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

    Directory of Open Access Journals (Sweden)

    Xuebin Zhang

    Full Text Available In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3 mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1 carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST, known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

  1. A Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in Arabidopsis thaliana NMD Mutants

    Science.gov (United States)

    Rayson, Samantha; Arciga-Reyes, Luis; Wootton, Lucie; De Torres Zabala, Marta; Truman, William; Graham, Neil; Grant, Murray; Davies, Brendan

    2012-01-01

    Nonsense-mediated mRNA decay (NMD) is a conserved mechanism that targets aberrant mRNAs for destruction. NMD has also been found to regulate the expression of large numbers of genes in diverse organisms, although the biological role for this is unclear and few evolutionarily conserved targets have been identified. Expression analyses of three Arabidopsis thaliana lines deficient in NMD reveal that the vast majority of NMD-targeted transcripts are associated with response to pathogens. Congruently, NMD mutants, in which these transcripts are elevated, confer partial resistance to Pseudomonas syringae. These findings suggest a biological rationale for the regulation of gene expression by NMD in plants and suggest that manipulation of NMD could offer a new approach for crop protection. Amongst the few non-pathogen responsive NMD-targeted genes, one potential NMD targeted signal, the evolutionarily conserved upstream open reading frame (CuORF), was found to be hugely over-represented, raising the possibility that this feature could be used to target specific physiological mRNAs for control by NMD. PMID:22384098

  2. NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

    Science.gov (United States)

    Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena

    2012-10-01

    Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.

  3. Impact of SO(2) on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing.

    Science.gov (United States)

    Hamisch, Domenica; Randewig, Dörte; Schliesky, Simon; Bräutigam, Andrea; Weber, Andreas P M; Geffers, Robert; Herschbach, Cornelia; Rennenberg, Heinz; Mendel, Ralf R; Hänsch, Robert

    2012-12-01

    High concentrations of sulfur dioxide (SO(2) ) as an air pollutant, and its derivative sulfite, cause abiotic stress that can lead to cell death. It is currently unknown to what extent plant fumigation triggers specific transcriptional responses. To address this question, and to test the hypothesis that sulfite oxidase (SO) is acting in SO(2) detoxification, we compared Arabidopsis wildtype (WT) and SO knockout lines (SO-KO) facing the impact of 600 nl l(-1) SO(2) , using RNAseq to quantify absolute transcript abundances. These transcriptome data were correlated to sulfur metabolism-related enzyme activities and metabolites obtained from identical samples in a previous study. SO-KO plants exhibited remarkable and broad regulative responses at the mRNA level, especially in transcripts related to sulfur metabolism enzymes, but also in those related to stress response and senescence. Focusing on SO regulation, no alterations were detectable in the WT, whereas in SO-KO plants we found up-regulation of two splice variants of the SO gene, although this gene is not functional in this line. Our data provide evidence for the highly specific coregulation between SO and sulfur-related enzymes like APS reductase, and suggest two novel candidates for involvement in SO(2) detoxification: an apoplastic peroxidase, and defensins as putative cysteine mass storages. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  4. Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress

    International Nuclear Information System (INIS)

    Revenkova, E.; Masson, J.; Koncz, C.; Afsar, K.; Jakovleva, L.; Paszkowski, J.

    1999-01-01

    A recessive Arabidopsis mutant with elevated sensitivity to DNA damaging treatments was identified in one out of 800 families generated by T-DNA insertion mutagenesis. The T-DNA generated a chromosomal deletion of 1287 bp in the promoter of one of three S27 ribosomal protein genes (ARS27A) preventing its expression. Seedlings of ars27A developed normally under standard growth conditions, suggesting wildtype proficiency of translation. However, growth was strongly inhibited in media supplemented with methyl methane sulfate (MMS) at a concentration not affecting the wild type. This inhibition was accompanied by the formation of tumor–like structures instead of auxiliary roots. Wild-type seedlings treated with increasing concentrations of MMS up to a lethal dose never displayed such a trait, neither was this phenotype observed in ars27A plants in the absence of MMS or under other stress conditions. Thus, the hypersensitivity and tumorous growth are mutant-specific responses to the genotoxic MMS treatment. Another important feature of the mutant is its inability to perform rapid degradation of transcripts after UV treatment, as seen in wild-type plants. Therefore, we propose that the ARS27A protein is dispensable for protein synthesis under standard conditions but is required for the elimination of possibly damaged mRNA after UV irradiation. (author)

  5. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Hercík, Kamil; Kozák, Jaroslav; Šála, Michal; Dejmek, Milan; Hřebabecký, Hubert; Zborníková, Eva; Smola, Miroslav; Růžek, Daniel; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 137, Jan (2017), s. 131-133 ISSN 0166-3542 R&D Projects: GA ČR GA15-09310S Institutional support: RVO:61388963 ; RVO:60077344 Keywords : hepatitis C virus * borne encephalitis virus * crystal structure Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.271, year: 2016

  6. The Dicer-like, Argonaute and RNA-dependent RNA polymerase ...

    Indian Academy of Sciences (India)

    15 days after cal ind. 15 days after callus induction. 4.796359765 5.345679052 2.367724275 2.373588326 8.296583735 8.470631911 10.40219234. 10.4819069. 3 days after shoot ind 3 days after the start of shoot induction 3.976837223 4.475576805 2.414958455 2.390819138 7.834091661 8.554726998 10.07637957.

  7. Translation initiation factor AteIF(iso4E is involved in selective mRNA translation in Arabidopsis thaliana seedlings.

    Directory of Open Access Journals (Sweden)

    Ana Valeria Martínez-Silva

    Full Text Available One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5'end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso4E knockout mutant [(iso4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1, Sucrose transporter 3 (SUC3, ABC transporter-like with ATPase activity (MRP11 and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso4E is relevant for Arabidopsis root development under normal growth conditions.

  8. Expression analysis of argonaute, Dicer-like, and RNA-dependent ...

    Indian Academy of Sciences (India)

    DEFANG GAN

    2Modern Agricultural Science and Technology Cooperative Extension Service Center of Mingguang City, Mingguang,. 239400, People's .... The quality and purity of the prepara- .... CsActin was used as an internal control to normalize the data.

  9. AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

    KAUST Repository

    Zhang, Runxuan

    2016-05-06

    Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

  10. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  11. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    Science.gov (United States)

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

  12. The Arabidopsis thaliana RNA editing factor SLO2, which affects the mitochondrial electron transport chain, participates in multiple stress and hormone responses.

    Science.gov (United States)

    Zhu, Qiang; Dugardeyn, Jasper; Zhang, Chunyi; Mühlenbock, Per; Eastmond, Peter J; Valcke, Roland; De Coninck, Barbara; Oden, Sevgi; Karampelias, Michael; Cammue, Bruno P A; Prinsen, Els; Van Der Straeten, Dominique

    2014-02-01

    Recently, we reported that the novel mitochondrial RNA editing factor SLO2 is essential for mitochondrial electron transport, and vital for plant growth through regulation of carbon and energy metabolism. Here, we show that mutation in SLO2 causes hypersensitivity to ABA and insensitivity to ethylene, suggesting a link with stress responses. Indeed, slo2 mutants are hypersensitive to salt and osmotic stress during the germination stage, while adult plants show increased drought and salt tolerance. Moreover, slo2 mutants are more susceptible to Botrytis cinerea infection. An increased expression of nuclear-encoded stress-responsive genes, as well as mitochondrial-encoded NAD genes of complex I and genes of the alternative respiratory pathway, was observed in slo2 mutants, further enhanced by ABA treatment. In addition, H2O2 accumulation and altered amino acid levels were recorded in slo2 mutants. We conclude that SLO2 is required for plant sensitivity to ABA, ethylene, biotic, and abiotic stress. Although two stress-related RNA editing factors were reported very recently, this study demonstrates a unique role of SLO2, and further supports a link between mitochondrial RNA editing events and stress response.

  13. Negative charge and membrane tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3Dpol

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 190 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : RdRp * ACBD3 Subject RIV: CE - Biochemistry

  14. Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

    Science.gov (United States)

    Li, Chen; Wang, Haiwei; Yuan, Tiangang; Woodman, Andrew; Yang, Decheng; Zhou, Guohui; Cameron, Craig E; Yu, Li

    2018-05-01

    Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3D pol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3D pol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...

  16. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  17. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  18. Purification and properties of cowpea mosaic virus RNA replicase

    NARCIS (Netherlands)

    Zabel, P.

    1978-01-01

    This thesis concerns the partial purification and properties of an RNA-dependent RNA polymerase (RNA replicase) produced upon infection of Vigna unguiculata plants with Cowpea Mosaic Virus (CPMV). The enzyme is believed to be coded, at least in part, by the virus genome and to

  19. MicroRNA related polymorphisms and breast cancer risk

    NARCIS (Netherlands)

    S. Khan (Sofia); D. Greco (Dario); K. Michailidou (Kyriaki); R.L. Milne (Roger); T.A. Muranen (Taru); T. Heikkinen (Tuomas); K. Aaltonen (Kirsimari); J. Dennis (Joe); M.K. Bolla (Manjeet); J. Liu (Jianjun); P. Hall (Per); A. Irwanto (Astrid); M.K. Humphreys (Manjeet); J. Li (Jingmei); K. Czene (Kamila); J. Chang-Claude (Jenny); R. Hein (Rebecca); A. Rudolph (Anja); P. Seibold (Petra); D. Flesch-Janys (Dieter); O. Fletcher (Olivia); J. Peto (Julian); I. dos Santos Silva (Isabel); N. Johnson (Nichola); L.J. Gibson (Lorna); A. Aitken; J.L. Hopper (John); H. Tsimiklis (Helen); M. Bui (Minh); E. Makalic (Enes); D.F. Schmidt (Daniel); M.C. Southey (Melissa); C. Apicella (Carmel); J. Stone (Jennifer); Q. Waisfisz (Quinten); E.J. Meijers-Heijboer (Hanne); M.A. Adank (Muriel); R.B. van der Luijt (Rob); A. Meindl (Alfons); R.K. Schmutzler (Rita); B. Müller-Myhsok (B.); P. Lichtner (Peter); C. Turnbull (Clare); N. Rahman (Nazneen); S.J. Chanock (Stephen); D. Hunter (David); A. Cox (Angela); S.S. Cross (Simon); M.W.R. Reed (Malcolm); M.K. Schmidt (Marjanka); A. Broeks (Annegien); L.J. van 't Veer (Laura); F.B.L. Hogervorst (Frans); P.A. Fasching (Peter); A. Schrauder (André); A.B. Ekici (Arif); M.W. Beckmann (Matthias); S.E. Bojesen (Stig); B.G. Nordestgaard (Børge); S.F. Nielsen (Sune); H. Flyger (Henrik); J. Benítez (Javier); P.M. Zamora (Pilar M.); J.I.A. Perez (Jose Ignacio Arias); C.A. Haiman (Christopher); B.E. Henderson (Brian); F.R. Schumacher (Fredrick); L.L. March (Loic Le); P.D.P. Pharoah (Paul); A.M. Dunning (Alison); M. Shah (Mitul); R.N. Luben (Robert); J. Brown (Judith); F.J. Couch (Fergus); X. Wang (X.); C. Vachon (Celine); J.E. Olson (Janet); D. Lambrechts (Diether); M. Moisse (Matthieu); R. Paridaens (Robert); M.R. Christiaens (Marie Rose); P. Guénel (Pascal); T. Truong (Thérèse); P. Laurent-Puig (Pierre); C. Mulot (Claire); F. Marme (Frederick); B. Burwinkel (Barbara); A. Schneeweiss (Andreas); C. Sohn (Christof); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); I.L. Andrulis (Irene); J.A. Knight (Julia); S. Tchatchou (Srine); A.-M. Mulligan (Anna-Marie); T. Dörk (Thilo); N.V. Bogdanova (Natalia); N.N. Antonenkova (Natalia); H. Anton-Culver (Hoda); H. Darabi (Hatef); M. Eriksson (Mats); M. García-Closas (Montserrat); J.D. Figueroa (Jonine); J. Lissowska (Jolanta); L.A. Brinton (Louise); P. Devilee (Peter); R.A.E.M. Tollenaar (Rob); C.M. Seynaeve (Caroline); C.J. van Asperen (Christi); V. Kristensen (Vessela); S. Slager (Susan); A.E. Tol (Ama E.); C.B. Ambrosone (Christine); D. Yannoukakos (Drakoulis); A. Lindblom (Annika); S. Margolin (Sara); P. Radice (Paolo); P. Peterlongo (Paolo); M. Barile (Monica); P. Mariani (Paolo); M.J. Hooning (Maartje); J.W.M. Martens (John); J.M. Collée (Margriet); A. Jager (Agnes); A. Jakubowska (Anna); J. Lubinski (Jan); K. Jaworska-Bieniek (Katarzyna); K. Durda (Katarzyna); G.G. Giles (Graham); C.A. McLean (Catriona Ann); H. Brauch (Hiltrud); T. Brüning (Thomas); Y.-D. Ko (Yon-Dschun); H. Brenner (Hermann); A.K. Dieffenbach (Aida Karina); V. Arndt (Volker); C. Stegmaier (Christa); A.J. Swerdlow (Anthony ); A. Ashworth (Alan); N. Orr (Nick); M. Jones (Michael); J. Simard (Jacques); M.S. Goldberg (Mark); F. Labrèche (France); M. Dumont (Martine); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); A. Mannermaa (Arto); U. Hamann (Ute); G. Chenevix-Trench (Georgia); C. Blomqvist (Carl); K. Aittomäki (Kristiina); D.F. Easton (Douglas); H. Nevanlinna (Heli)

    2014-01-01

    textabstractGenetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility

  20. Identification of Polyadenylation Sites within Arabidopsis Thaliana

    KAUST Repository

    Kalkatawi, Manal

    2011-09-01

    Machine Learning (ML) is a field of artificial intelligence focused on the design and implementation of algorithms that enable creation of models for clustering, classification, prediction, ranking and similar inference tasks based on information contained in data. Many ML algorithms have been successfully utilized in a variety of applications. The problem addressed in this thesis is from the field of bioinformatics and deals with the recognition of polyadenylation (poly(A)) sites in the genomic sequence of the plant Arabidopsis thaliana. During the RNA processing, a tail consisting of a number of consecutive adenine (A) nucleotides is added to the terminal nucleotide of the 3’- untranslated region (3’UTR) of the primary RNA. The process in which these A nucleotides are added is called polyadenylation. The location in the genomic DNA sequence that corresponds to the start of terminal A nucleotides (i.e. to the end of 3’UTR) is known as a poly(A) site. Recognition of the poly(A) sites in DNA sequence is important for better gene annotation and understanding of gene regulation. In this study, we built an artificial neural network (ANN) for the recognition of poly(A) sites in the Arabidopsis thaliana genome. Our study demonstrates that this model achieves improved accuracy compared to the existing predictive models for this purpose. The key factor contributing to the enhanced predictive performance of our ANN model is a distinguishing set of features used in creation of the model. These features include a number of physico-chemical characteristics of relevance, such as dinucleotide thermodynamic characteristics, electron-ion interaction potential, etc., but also many of the statistical properties of the DNA sequences from the region surrounding poly(A) site, such as nucleotide and polynucleotide properties, common motifs, etc. Our ANN model was compared in performance with several other ML models, as well as with the PAC tool that is specifically developed for

  1. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

    Science.gov (United States)

    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  2. The genome of Arabidopsis thaliana.

    OpenAIRE

    Goodman, H M; Ecker, J R; Dean, C

    1995-01-01

    Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae. It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project. The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented. Examples are given of genes isolated by using map-based cloning. The importance of the Arabidopsis project ...

  3. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV.

    Directory of Open Access Journals (Sweden)

    Julie A Law

    2011-07-01

    Full Text Available DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2, is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs through a pathway termed RNA-directed DNA methylation (RdDM. Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV. However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1, the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2, CLASSY1 (CLSY1, and RNA-directed DNA methylation 4 (RDM4, suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1, was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.

  4. Negative charge and membrane-tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3D(pol)

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 7, Dec 11 (2017), č. článku 17309. ISSN 2045-2322 R&D Projects: GA ČR GJ15-21030Y Institutional support: RVO:61388963 Keywords : 4-kinase III beta * in-vitro reconstitution * poliovirus protein 3AB Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.259, year: 2016 https://www.nature.com/articles/s41598-017-17621-6

  5. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences

    Czech Academy of Sciences Publication Activity Database

    Drexler, J. F.; Gloza-Rausch, F.; Glende, J.; Corman, V. M.; Muth, D.; Goettsche, M.; Seebens, A.; Niedrig, M.; Pfefferle, S.; Yordanov, S.; Zhelyazkov, L.; Hermanns, U.; Vallo, Peter; Lukashev, A.; Müller, M. A.; Deng, H.; Herrler, G.; Drosten, C.

    2010-01-01

    Roč. 84, č. 21 (2010), s. 11336-11349 ISSN 0022-538X Institutional research plan: CEZ:AV0Z60930519 Keywords : cross-species transmission * SARS-like coronavirus es * reservoir hosts * horseshoe bats Subject RIV: EE - Microbiology, Virology Impact factor: 5.189, year: 2010

  6. The fate of retrotransposed processed genes in Arabidopsis thaliana.

    Science.gov (United States)

    Abdelkarim, Basma T M; Maranda, Vincent; Drouin, Guy

    2017-04-20

    Processed genes are functional genes that have arisen as a result of the retrotransposition of mRNA molecules. We found 6 genes that generated processed genes in the common ancestor of five Brassicaceae species (Arabidopsis thaliana, Arabidopsis lyrata, Capsella rubella, Brassica rapa and Thellungiella parvula). These processed genes have therefore been kept for at least 30millionyears. Analyses of the Ka/Ks ratio of these genes, and of those having given rise to them, show that they evolve relatively slowly and suggest that the processed genes maintained the same function as that of their parental gene. There is a significant negative correlation between the number of ESTs and transcripts produced and the Ka/Ks ratios of the parental genes but not of the processed genes. This suggests that selection has not yet adapted the selective pressure the processed genes experience to their expression level. However, the A. thaliana processed genes tend to be expressed in the same tissues as that of their parental genes. Furthermore, most have a CAATT-box, a TATA-box and are located about 1kb from another protein-coding gene. Altogether, our results suggest that the processed genes found in the A. thaliana genome have been kept to produce more of the same product, and in the same tissues, as that encoded by their parental gene. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  7. Initiation, elongation, and realignment during influenza virus mRNA synthesis

    NARCIS (Netherlands)

    Velthuis, te Aartjan J.W.; Oymans, Judith

    2018-01-01

    The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to

  8. RNA binding and replication by the poliovirus RNA polymerase

    International Nuclear Information System (INIS)

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

  9. Bioavailability of nanoparticulate hematite to Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Marusenko, Yevgeniy; Shipp, Jessie; Hamilton, George A.; Morgan, Jennifer L.L.; Keebaugh, Michael; Hill, Hansina; Dutta, Arnab; Zhuo, Xiaoding; Upadhyay, Nabin; Hutchings, James; Herckes, Pierre; Anbar, Ariel D.; Shock, Everett; Hartnett, Hilairy E.

    2013-01-01

    The environmental effects and bioavailability of nanoparticulate iron (Fe) to plants are currently unknown. Here, plant bioavailability of synthesized hematite Fe nanoparticles was evaluated using Arabidopsis thaliana (A. thaliana) as a model. Over 56-days of growing wild-type A. thaliana, the nanoparticle-Fe and no-Fe treatments had lower plant biomass, lower chlorophyll concentrations, and lower internal Fe concentrations than the Fe-treatment. Results for the no-Fe and nanoparticle-Fe treatments were consistently similar throughout the experiment. These results suggest that nanoparticles (mean diameter 40.9 nm, range 22.3–67.0 nm) were not taken up and therefore not bioavailable to A. thaliana. Over 14-days growing wild-type and transgenic (Type I/II proton pump overexpression) A. thaliana, the Type I plant grew more than the wild-type in the nanoparticle-Fe treatment, suggesting Type I plants cope better with Fe limitation; however, the nanoparticle-Fe and no-Fe treatments had similar growth for all plant types. -- Highlights: ► Iron nanoparticles were synthesized and assessed for bioavailability to Arabidopsis. ► Arabidopsis grew better in the presence of EDTA-bound iron than nanoparticulate iron. ► Arabidopsis grew the same in the presence of nanoparticulate iron compared to no iron. -- Synthesized iron nanoparticles were not bioavailable to Arabidopsis thaliana in agar nutrient media

  10. Novel Ribonuclease Activity Differs between Fibrillarins from Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Ulises Rodriguez-Corona

    2017-10-01

    Full Text Available Fibrillarin is one of the most important nucleolar proteins that have been shown as essential for life. Fibrillarin localizes primarily at the periphery between fibrillar center and dense fibrillar component as well as in Cajal bodies. In most plants there are at least two different genes for fibrillarin. In Arabidopsis thaliana both genes show high level of expression in transcriptionally active cells. Here, we focus on two important differences between A. thaliana fibrillarins. First and most relevant is the enzymatic activity by AtFib2. The AtFib2 shows a novel ribonuclease activity that is not seen with AtFib1. Second is a difference in the ability to interact with phosphoinositides and phosphatidic acid between both proteins. We also show that the novel ribonuclease activity as well as the phospholipid binding region of fibrillarin is confine to the GAR domain. The ribonuclease activity of fibrillarin reveals in this study represents a new role for this protein in rRNA processing.

  11. Protists are an integral part of the Arabidopsis thaliana microbiome.

    Science.gov (United States)

    Sapp, Melanie; Ploch, Sebastian; Fiore-Donno, Anna M; Bonkowski, Michael; Rose, Laura E

    2018-01-01

    Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont. © 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

    OpenAIRE

    Fallath, Thorya; Kidd, Brendan N.; Stiller, Jiri; Davoine, Celine; Bj?rklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

    2017-01-01

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display do...

  13. Long noncoding RNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana.

    Science.gov (United States)

    Zhu, Qian-Hao; Stephen, Stuart; Taylor, Jennifer; Helliwell, Chris A; Wang, Ming-Bo

    2014-01-01

    Short noncoding RNAs have been demonstrated to play important roles in regulation of gene expression and stress responses, but the repertoire and functions of long noncoding RNAs (lncRNAs) remain largely unexplored, particularly in plants. To explore the role of lncRNAs in disease resistance, we used a strand-specific RNA-sequencing approach to identify lncRNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana. Antisense transcription was found in c. 20% of the annotated A. thaliana genes. Several noncoding natural antisense transcripts responsive to F. oxysporum infection were found in genes implicated in disease defense. While the majority of the novel transcriptionally active regions (TARs) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TARs, including 20 F. oxysporum-responsive lncTARs, were identified. Ten F. oxysporum-induced lncTARs were functionally characterized using T-DNA insertion or RNA-interference knockdown lines, and five were demonstrated to be related to disease development. Promoter analysis suggests that some of the F. oxysporum-induced lncTARs are direct targets of transcription factor(s) responsive to pathogen attack. Our results demonstrated that strand-specific RNA sequencing is a powerful tool for uncovering hidden levels of transcriptome and that IncRNAs are important components of the antifungal networks in A. thaliana. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  14. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance.

    Science.gov (United States)

    Niu, Qi-Wen; Lin, Shih-Shun; Reyes, Jose Luis; Chen, Kuan-Chun; Wu, Hui-Wen; Yeh, Shyi-Dong; Chua, Nam-Hai

    2006-11-01

    Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

  15. Arabidopsis thaliana mTERF proteins: evolution and functional classification

    Directory of Open Access Journals (Sweden)

    Tatjana eKleine

    2012-10-01

    Full Text Available Organellar gene expression (OGE is crucial for plant development, photosynthesis and respiration, but our understanding of the mechanisms that control it is still relatively poor. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming and editing of organellar RNAs, and regulate translation. In metazoans, proteins of the mitochondrial Transcription tERmination Factor (mTERF family interact with the mitochondrial chromosome and regulate transcriptional initiation and termination. Sequencing of the Arabidopsis thaliana genome led to the identification of a diversified MTERF gene family but, in contrast to mammalian mTERFs, knowledge about the function of these proteins in photosynthetic organisms is scarce. In this hypothesis article, I show that tandem duplications and one block duplication contributed to the large number of MTERF genes in A. thaliana, and propose that the expansion of the family is related to the evolution of land plants. The MTERF genes - especially the duplicated genes - display a number of distinct mRNA accumulation patterns, suggesting functional diversification of mTERF proteins to increase adaptability to environmental changes. Indeed, hypothetical functions for the different mTERF proteins can be predicted using co-expression analysis and gene ontology annotations. On this basis, mTERF proteins can be sorted into five groups. Members of the chloroplast and chloroplast-associated clusters are principally involved in chloroplast gene expression, embryogenesis and protein catabolism, while representatives of the mitochondrial cluster seem to participate in DNA and RNA metabolism in that organelle. Moreover, members of the mitochondrion-associated cluster and the low expression group may act in the nucleus and/or the cytosol. As proteins involved in OGE and presumably nuclear gene expression, mTERFs are ideal candidates for the coordination of the expression of organelle and nuclear

  16. ADN ribosomique 5S chez Arabidopsis thaliana : dynamique chromatinienne et ARN polymérase IV

    OpenAIRE

    Douet , Julien

    2008-01-01

    In Arabidopsis thaliana, 5S rRNA genes are found clustered at pericentromeric heterochromatin of chromosomes 3, 4 and 5. 5S rRNA genes transcription is epigenetically regulated through the formation of specific chromatin structure. In order to determine the events that lead to the establishment of such structures, a study during the first steps of post-germinative plant development was done. Unexpectedly, we observed a decondensation followed by a rapid "re"condensation of 5S rDNA chromatin. ...

  17. In vivo interactions between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA dependent polymerase VP1

    NARCIS (Netherlands)

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  18. Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent polymerase, VP1

    NARCIS (Netherlands)

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  19. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase

    NARCIS (Netherlands)

    Beisser, P.S.; Verzijl, D.; Gruijthuijsen, Y.K.; Beuken, E.V.; Smit, M.J.; Leurs, R.; Bruggeman, C.A.; Vink, C.

    2005-01-01

    Epstein-Barr vires (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesvirases. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has

  20. Accumulation of the coumarin scopolin under abiotic stress conditions is mediated by the Arabidopsis thaliana THO/TREX complex

    KAUST Repository

    Döll, Stefanie

    2017-12-09

    Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function.

  1. The mitochondrial phosphate transporters modulate plant responses to salt stress via affecting ATP and gibberellin metabolism in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Wei Zhu

    Full Text Available The mitochondrial phosphate transporter (MPT plays crucial roles in ATP production in plant cells. Three MPT genes have been identified in Arabidopsis thaliana. Here we report that the mRNA accumulations of AtMPTs were up-regulated by high salinity stress in A. thaliana seedlings. And the transgenic lines overexpressing AtMPTs displayed increased sensitivity to salt stress compared with the wild-type plants during seed germination and seedling establishment stages. ATP content and energy charge was higher in overexpressing plants than those in wild-type A. thaliana under salt stress. Accordingly, the salt-sensitive phenotype of overexpressing plants was recovered after the exogenous application of atractyloside due to the change of ATP content. Interestingly, Genevestigator survey and qRT-PCR analysis indicated a large number of genes, including those related to gibberellin synthesis could be regulated by the energy availability change under stress conditions in A. thaliana. Moreover, the exogenous application of uniconazole to overexpressing lines showed that gibberellin homeostasis was disturbed in the overexpressors. Our studies reveal a possible link between the ATP content mediated by AtMPTs and gibberellin metabolism in responses to high salinity stress in A. thaliana.

  2. Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana.

    Science.gov (United States)

    Havlová, Kateřina; Dvořáčková, Martina; Peiro, Ramon; Abia, David; Mozgová, Iva; Vansáčová, Lenka; Gutierrez, Crisanto; Fajkus, Jiří

    2016-11-01

    Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

  3. Isolation and characterization of CNGC17 gene from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Yamagami, Mutsumi; Kobayashi, Daisuke; Hisamatsu, Shun'ichi

    2007-01-01

    Phytoremediation is a possible countermeasure for cleaning up soil contaminated by 137 Cs, and development of plants which can effectively absorb 137 Cs is important for it. It is expected that capability of Cs extraction from soil can be strengthened by genetic alteration of the Cs + root-uptake mechanism of plants. This study aimed at elucidating the uptake mechanism of Cs + for future genetic engineering. Plant roots take up Cs + from the soil solution via transport proteins at the plasma membrane of root cells. Voltage-insensitive cation channels (VICCs) are a possible transfer route of Cs + , and they are encoded by cyclic-nucleotide gated channel (CNGC) and glutamate receptor (GLR) gene families. The genome of Arabidopsis thaliana contains 20 CNGC genes. We have cloned a putative AtCNGC17 gene from cDNAs which were generated with total-RNA obtained from leaves of Arabidopsis thaliana by RT-PCR. The cDNA contained 2163 bp with an ORF that encoded a protein consisting of 721 amino acids residues. The plasmid prepared by the insertion of the gene under a Taq promoter was used to transform an E. coli deficient in the three major K + uptake systems (Kdp, Trk, and Kup). Only the E. coli with AtCNGC17 gene grew in low K + concentration minimal medium. This result suggested that the AtCNGC17 protein has a function of K + uptake. Growth rates of the E. coli cells expressing the gene were strongly inhibited by CsCl in low K + concentration minimal medium, suggesting that the AtCNGC17 transporter also carries Cs + . (author)

  4. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    OpenAIRE

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand; Kumar, Aseem

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-strande...

  5. A Specific Hepatic Transfer RNA for Phosphoserine*

    Science.gov (United States)

    Mäenpää, Pekka H.; Bernfield, Merton R.

    1970-01-01

    Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

  6. The pattern of polymorphism in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.

  7. Genetic analysis of seed development in Arabidopsis thaliana = [Genetische analyse van de zaadontwikkeling in Arabidopsis thaliana

    NARCIS (Netherlands)

    Leon - Kloosterziel, K.

    1997-01-01


    This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection

  8. Expression analysis of the Arabidopsis thaliana AtSpen2 gene, and its relationship with other plant genes encoding Spen proteins

    OpenAIRE

    Solís-Guzmán, María Gloria; Argüello-Astorga, Gerardo; López-Bucio, José; Ruiz-Herrera, León Francisco; López-Meza, Joel; Sánchez-Calderón, Lenin; Carreón-Abud, Yazmín; Martínez-Trujillo, Miguel

    2017-01-01

    Abstract Proteins of the Split ends (Spen) family are characterized by an N-terminal domain, with one or more RNA recognition motifs and a SPOC domain. In Arabidopsis thaliana, the Spen protein FPA is involved in the control of flowering time as a component of an autonomous pathway independent of photoperiod. The A. thaliana genome encodes another gene for a putative Spen protein at the locus At4g12640, herein named AtSpen2. Bioinformatics analysis of the AtSPEN2 SPOC domain revealed low sequ...

  9. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    Science.gov (United States)

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  10. Ecology of Arabidopsis thaliana : local adaptation and interaction with herbivores

    NARCIS (Netherlands)

    Mosleh Arany, A.

    2006-01-01

    As first step the impact of herbivory and abiotic factors on population dynamics of Arabidopsis thaliana were studied. Ceutorhynchus atomus and C. contractus were identified as the major insect herbivores on A. thaliana population, reducing seed production by more than 40%. Mortality from February

  11. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

  12. Locus-specific ribosomal RNA gene silencing in nucleolar dominance.

    Directory of Open Access Journals (Sweden)

    Michelle S Lewis

    2007-08-01

    Full Text Available The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs, the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

  13. Stochastic gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Araújo, Ilka Schultheiß; Pietsch, Jessica Magdalena; Keizer, Emma Mathilde; Greese, Bettina; Balkunde, Rachappa; Fleck, Christian; Hülskamp, Martin

    2017-12-14

    Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner.

  14. Epithelial Distribution and Replication of Foot-and-Mouth Disease Virus RNA in Infected Pigs

    DEFF Research Database (Denmark)

    Durand, S.; Murphy, C.; Zhang, Z.

    2008-01-01

    experimentally with FMDV serotype O UKG 34/2001 and tissue samples were collected from I to 4 clays post-infection. Samples were stored at -70 degrees C and frozen sections were prepared for in-situ hybridization (ISH). A digoxigenin-labelled RNA probe complementary to a coding part of the RNA-dependent RNA...... negative strand RNA was observed in basal cells above the basement membrane and along the dermal papillae. The basal cells therefore demonstrate the highest signal for detection of the FMDV positive and negative strand RNAs in both tongue and foot epithelium. These novel results Suggest that the epithelial...

  15. The Hidden Geometries of the Arabidopsis thaliana Epidermis

    KAUST Repository

    Staff, Lee; Hurd, Patricia; Reale, Lara; Seoighe, Cathal; Rockwood, Alyn; Gehring, Christoph A

    2012-01-01

    The quest for the discovery of mathematical principles that underlie biological phenomena is ancient and ongoing. We present a geometric analysis of the complex interdigitated pavement cells in the Arabidopsis thaliana (Col.) adaxial epidermis

  16. Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

    NARCIS (Netherlands)

    Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

    2001-01-01

    Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

  17. The cis-acting replication signal at the 3' end of Flock House virus RNA2 is RNA3-dependent

    International Nuclear Information System (INIS)

    Albarino, Cesar G.; Eckerle, Lance D.; Ball, L. Andrew

    2003-01-01

    The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3' 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3' termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3

  18. Pseudogenes regulate parental gene expression via ceRNA network.

    Science.gov (United States)

    An, Yang; Furber, Kendra L; Ji, Shaoping

    2017-01-01

    The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  19. MicroRNA Related Polymorphisms and Breast Cancer Risk

    DEFF Research Database (Denmark)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer....... We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41......,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated...

  20. Investigating the Association between Flowering Time and Defense in the Arabidopsis thaliana-Fusarium oxysporum Interaction

    Science.gov (United States)

    Lyons, Rebecca; Rusu, Anca; Stiller, Jiri; Powell, Jonathan; Manners, John M.; Kazan, Kemal

    2015-01-01

    Plants respond to pathogens either by investing more resources into immunity which is costly to development, or by accelerating reproductive processes such as flowering time to ensure reproduction occurs before the plant succumbs to disease. In this study we explored the link between flowering time and pathogen defense using the interaction between Arabidopsis thaliana and the root infecting fungal pathogen Fusarium oxysporum. We report that F. oxysporum infection accelerates flowering time and regulates transcription of a number of floral integrator genes, including FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and GIGANTEA (GI). Furthermore, we observed a positive correlation between late flowering and resistance to F. oxysporum in A. thaliana natural ecotypes. Late-flowering gi and autonomous pathway mutants also exhibited enhanced resistance to F. oxysporum, supporting the association between flowering time and defense. However, epistasis analysis showed that accelerating flowering time by deletion of FLC in fve-3 or fpa-7 mutants did not alter disease resistance, suggesting that the effect of autonomous pathway on disease resistance occurs independently from flowering time. Indeed, RNA-seq analyses suggest that fve-3 mediated resistance to F. oxysporum is most likely a result of altered defense-associated gene transcription. Together, our results indicate that the association between flowering time and pathogen defense is complex and can involve both pleiotropic and direct effects. PMID:26034991

  1. ARA-PEPs: a repository of putative sORF-encoded peptides in Arabidopsis thaliana.

    Science.gov (United States)

    Hazarika, Rashmi R; De Coninck, Barbara; Yamamoto, Lidia R; Martin, Laura R; Cammue, Bruno P A; van Noort, Vera

    2017-01-17

    Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). However, there is increasing evidence for the translation of these sORFs into bioactive peptides with potent signaling, antimicrobial, developmental, antioxidant roles etc. Yet only a few peptides encoded by sORFs are annotated in the model organism Arabidopsis thaliana. To aid the functional annotation of these peptides, we have developed ARA-PEPs (available at http://www.biw.kuleuven.be/CSB/ARA-PEPs ), a repository of putative peptides encoded by sORFs in the A. thaliana genome starting from in-house Tiling arrays, RNA-seq data and other publicly available datasets. ARA-PEPs currently lists 13,748 sORF-encoded peptides with transcriptional evidence. In addition to existing data, we have identified 100 novel transcriptionally active regions (TARs) that might encode 341 novel stress-induced peptides (SIPs). To aid in identification of bioactivity, we add functional annotation and sequence conservation to predicted peptides. To our knowledge, this is the largest repository of plant peptides encoded by sORFs with transcript evidence, publicly available and this resource will help scientists to effortlessly navigate the list of experimentally studied peptides, the experimental and computational evidence supporting the activity of these peptides and gain new perspectives for peptide discovery.

  2. A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

    Science.gov (United States)

    Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

    2015-01-29

    Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  4. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    Science.gov (United States)

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

  5. Structure of Arabidopsis thaliana Rubisco activase.

    Science.gov (United States)

    Hasse, Dirk; Larsson, Anna M; Andersson, Inger

    2015-04-01

    The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase from Arabidopsis thaliana is presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.

  6. Unearthing Bacillus endophytes from desert plants that enhance growth of Arabidopsis thaliana under abiotic stress conditions

    KAUST Repository

    Bokhari, Ameerah M

    2018-04-01

    Here, we embarked a bioprospecting project that focuses on the isolation and characterization of plant root endophytes, collected from the Thar Desert. A total of 381 endophytes were isolated and based on their 16S rRNA gene sequences, genus Bacillus (58 strains) was identified as the major taxon and only endophytes from this genus were isolated from all plant types. Of the 58 Bacillus strains, only 16 strains were selected for screening of plant growth promotion traits such as P and Zn solubilization, indole-3-acetic acid and siderophore production, and antimicrobial activity. Based on the presence of specific plant growth promotion traits 10 strains were shortlisted for further in vitro screening with A. thaliana; to confirm that these bacteria can confer resilience to plants under salt stress conditions. B. circulans (PK3-15 and PK3-109), B. cereus (PK6-15) B. subtilis (PK3-9) and B. licheniformis (PK5-26) displayed the ability to increased the fresh weight of A. thaliana under salt stress conditions by more than 50 % compared to the uninoculated control. An interesting observation was that B. circulans (PK3-109) (shown to produce IAA exopolysaccharide) and B. circulans (PK3-138) (shown to produce IAA) in vitro results were substantially different as B. circulans (PK3-138) decreased the total fresh weight of A. thaliana by 47 %, whilst B. circulans (PK3-109) was one of the best performing strains. Thus, the genomes of these two strains were sequences to unravel the molecular versatility of B. circulans strains, specifically with respect to their interaction with plants. Most of the genome of these strains is identical but the most interesting feature was the presence of 1/ the DegS–DegU two-component system that is known to mediate the salt stress response and DegU also represses toxin wapA similar to antitoxin wapI, and 2/ YxiG, a gene in the unique orthogroup of PK3-109 was found to be linked to WapI. Thus, PK3-138 substantially decreasing the total fresh

  7. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  8. The scale of population structure in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Alexander Platt

    2010-02-01

    Full Text Available The population structure of an organism reflects its evolutionary history and influences its evolutionary trajectory. It constrains the combination of genetic diversity and reveals patterns of past gene flow. Understanding it is a prerequisite for detecting genomic regions under selection, predicting the effect of population disturbances, or modeling gene flow. This paper examines the detailed global population structure of Arabidopsis thaliana. Using a set of 5,707 plants collected from around the globe and genotyped at 149 SNPs, we show that while A. thaliana as a species self-fertilizes 97% of the time, there is considerable variation among local groups. This level of outcrossing greatly limits observed heterozygosity but is sufficient to generate considerable local haplotypic diversity. We also find that in its native Eurasian range A. thaliana exhibits continuous isolation by distance at every geographic scale without natural breaks corresponding to classical notions of populations. By contrast, in North America, where it exists as an exotic species, A. thaliana exhibits little or no population structure at a continental scale but local isolation by distance that extends hundreds of km. This suggests a pattern for the development of isolation by distance that can establish itself shortly after an organism fills a new habitat range. It also raises questions about the general applicability of many standard population genetics models. Any model based on discrete clusters of interchangeable individuals will be an uneasy fit to organisms like A. thaliana which exhibit continuous isolation by distance on many scales.

  9. MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

    Science.gov (United States)

    Stiller, Jiri; Davoine, Celine; Björklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

    2017-01-01

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways. PMID:28441405

  10. Demographic history of european populations of Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Olivier François

    2008-05-01

    Full Text Available The model plant species Arabidopsis thaliana is successful at colonizing land that has recently undergone human-mediated disturbance. To investigate the prehistoric spread of A. thaliana, we applied approximate Bayesian computation and explicit spatial modeling to 76 European accessions sequenced at 876 nuclear loci. We find evidence that a major migration wave occurred from east to west, affecting most of the sampled individuals. The longitudinal gradient appears to result from the plant having spread in Europe from the east approximately 10,000 years ago, with a rate of westward spread of approximately 0.9 km/year. This wave-of-advance model is consistent with a natural colonization from an eastern glacial refugium that overwhelmed ancient western lineages. However, the speed and time frame of the model also suggest that the migration of A. thaliana into Europe may have accompanied the spread of agriculture during the Neolithic transition.

  11. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

    International Nuclear Information System (INIS)

    Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

    1986-01-01

    Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . Cs 2 SO 4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs

  12. The origin and effect of small RNA signaling in plants

    Directory of Open Access Journals (Sweden)

    Jean-Sébastien eParent

    2012-08-01

    Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

  13. New insights into siRNA amplification and RNAi.

    Science.gov (United States)

    Zhang, Chi; Ruvkun, Gary

    2012-08-01

    In the nematode Caenorhabditis elegans (C. elegans), gene inactivation by RNA interference can achieve remarkable potency due to the amplification of initial silencing triggers by RNA-dependent RNA polymerases (RdRPs). RdRPs catalyze the biogenesis of an abundant species of secondary small interfering RNAs (siRNAs) using the target mRNA as template. The interaction between primary siRNAs derived from the exogenous double-stranded RNA (dsRNA) trigger and the target mRNA is required for the recruitment of RdRPs. Other genetic requirements for RdRP activities have not been characterized. Recent studies have identified the RDE-10/RDE-11 complex which interacts with the primary siRNA bound target mRNA and acts upstream of the RdRPs. rde-10 and rde-11 mutants show an RNAi defective phenotype because the biogenesis of secondary siRNAs is completely abolished. In addition, the RDE-10/RDE-11 complex plays a similar role in the endogenous RNAi pathway for the biogenesis of a subset of siRNAs targeting recently acquired, duplicated genes.

  14. Highlights of meiotic genes in Arabidopsis thaliana | Consiglio ...

    African Journals Online (AJOL)

    Meiosis is a fascinating and complex phenomenon and, despite its central role in sexual plant reproduction, little is known on the molecular mechanisms involved in this process. We review the progress made in recent years using Arabidopsis thaliana mutants for isolating meiotic genes. In particular, emphasis is given on ...

  15. Adaptation of Arabidopsis thaliana to the Yangtze River basin.

    Science.gov (United States)

    Zou, Yu-Pan; Hou, Xing-Hui; Wu, Qiong; Chen, Jia-Fu; Li, Zi-Wen; Han, Ting-Shen; Niu, Xiao-Min; Yang, Li; Xu, Yong-Chao; Zhang, Jie; Zhang, Fu-Min; Tan, Dunyan; Tian, Zhixi; Gu, Hongya; Guo, Ya-Long

    2017-12-28

    Organisms need to adapt to keep pace with a changing environment. Examining recent range expansion aids our understanding of how organisms evolve to overcome environmental constraints. However, how organisms adapt to climate changes is a crucial biological question that is still largely unanswered. The plant Arabidopsis thaliana is an excellent system to study this fundamental question. Its origin is in the Iberian Peninsula and North Africa, but it has spread to the Far East, including the most south-eastern edge of its native habitats, the Yangtze River basin, where the climate is very different. We sequenced 118 A. thaliana strains from the region surrounding the Yangtze River basin. We found that the Yangtze River basin population is a unique population and diverged about 61,409 years ago, with gene flows occurring at two different time points, followed by a population dispersion into the Yangtze River basin in the last few thousands of years. Positive selection analyses revealed that biological regulation processes, such as flowering time, immune and defense response processes could be correlated with the adaptation event. In particular, we found that the flowering time gene SVP has contributed to A. thaliana adaptation to the Yangtze River basin based on genetic mapping. A. thaliana adapted to the Yangtze River basin habitat by promoting the onset of flowering, a finding that sheds light on how a species can adapt to locales with very different climates.

  16. Changes in leaf proteome profile of Arabidopsis thaliana in ...

    Indian Academy of Sciences (India)

    2013-04-25

    Apr 25, 2013 ... Changes in leaf proteome profile of Arabidopsis thaliana in response to salicylic ... ethylene (ET) plays a key role in plant defence response by controlling ..... Oryza sativa. ( japonica cultivar-group). 24. 33.855/5.85. RT8702.

  17. Metabolic changes in Arabidopsis thaliana plants overexpressing chalcone synthase

    NARCIS (Netherlands)

    Dao, Thi Thanh Hien

    2010-01-01

    The study has shown that it is possible to introduce the heterologous CHS gene in Arabidopsis thaliana and common multicopies of transgenes containing plants were obtained. Analysis of the change in metabolome of CHS transgenic plants, high expression transgenic lines can be identified by markers

  18. Uptake and metabolism of sulphur dioxide by Arabidopsis thaliana

    NARCIS (Netherlands)

    van der Kooij, T.A.W.; De Kok, L.J.; Haneklaus, S.; Schnug, E.

    Arabidopsis thaliana (L.) Heynh. was exposed to various concentrations of SO2 during almost the entire life cycle. No negative effects of SO2 on shoot biomass production were observed. There was a linear relation between the deposition of SO2 and the atmospheric SO2 concentration. Sulphur

  19. Cleaning the GenBank Arabidopsis thaliana data set

    DEFF Research Database (Denmark)

    Korning, Peter G.; Hebsgaard, Stefan M.; Rouze, Pierre

    1996-01-01

    Data driven computational biology relies on the large quantities of genomic data stored in international sequence data banks. However, the possibilities are drastically impaired if the stored data is unreliable. During a project aiming to predict splice sites in the dicot Arabidopsis thaliana, we...... through anonymous FTP....

  20. The influences of Hygromycin B on growth of Arabidopsis thaliana ...

    African Journals Online (AJOL)

    In this article, growth and development of Arabidopsis thaliana seedling cotyledon and leaf were evidently affected by Hygromycin B. As compared to the control, cotyledon of seedling on Murashige and Skoog (MS) with Hygromycin B was very small and its leaf was not formed. Along with increase in culture time, cells in the ...

  1. Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Kolář, Jan; Seňková, J.

    2008-01-01

    Roč. 165, č. 15 (2008), s. 1601-1609 ISSN 0176-1617 R&D Projects: GA AV ČR KJB600380510 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arabidopsis thaliana * Flowering * Landsberg erecta Subject RIV: EF - Botanics Impact factor: 2.437, year: 2008

  2. The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana

    Science.gov (United States)

    Espineda, Cromwell E.; Linford, Alicia S.; Devine, Domenica; Brusslan, Judy A.

    1999-01-01

    Chlorophyll b is synthesized from chlorophyll a and is found in the light-harvesting complexes of prochlorophytes, green algae, and both nonvascular and vascular plants. We have used conserved motifs from the chlorophyll a oxygenase (CAO) gene from Chlamydomonas reinhardtii to isolate a homologue from Arabidopsis thaliana. This gene, AtCAO, is mutated in both leaky and null chlorina1 alleles, and DNA sequence changes cosegregate with the mutant phenotype. AtCAO mRNA levels are higher in three different mutants that have reduced levels of chlorophyll b, suggesting that plants that do not have sufficient chlorophyll b up-regulate AtCAO gene expression. Additionally, AtCAO mRNA levels decrease in plants that are grown under dim-light conditions. We have also found that the six major Lhcb proteins do not accumulate in the null ch1-3 allele. PMID:10468639

  3. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

    Directory of Open Access Journals (Sweden)

    Peter Sarkies

    2015-02-01

    Full Text Available Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs. Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

  4. The Battle of RNA Synthesis: Virus versus Host.

    Science.gov (United States)

    Harwig, Alex; Landick, Robert; Berkhout, Ben

    2017-10-21

    Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

  5. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Böhm Siegfried

    2004-07-01

    Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

  6. The dynamics and efficacy of antiviral RNA silencing: A model study

    Directory of Open Access Journals (Sweden)

    Hogeweg Paulien

    2008-03-01

    Full Text Available Abstract Background Mathematical modeling is important to provide insight in the complicated pathway of RNA silencing. RNA silencing is an RNA based mechanism that is widely used by eukaryotes to fight viruses, and to control gene expression. Results We here present the first mathematical model that combines viral growth with RNA silencing. The model involves a plus-strand RNA virus that replicates through a double-strand RNA intermediate. The model of the RNA silencing pathway consists of cleavage of viral RNA into siRNA by Dicer, target cleavage of viral RNA via the RISC complex, and a secondary response. We found that, depending on the strength of the silencing response, different viral growth patterns can occur. Silencing can decrease viral growth, cause oscillations, or clear the virus completely. Our model can explain various observed phenomena, even when they seem contradictory at first: the diverse responses to the removal of RNA dependent RNA polymerase; different viral growth curves; and the great diversity in observed siRNA ratios. Conclusion The model presented here is an important step in the understanding of the natural functioning of RNA silencing in viral infections.

  7. Transmission of epi-alleles with MET1-dependent dense methylation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Michael Watson

    Full Text Available DNA methylation in plants targets cytosines in three sequence contexts, CG, CHG and CHH (H representing A, C or T. Each of these patterns has traditionally been associated with distinct DNA methylation pathways with CHH methylation being controlled by the RNA dependent DNA methylation (RdDM pathway employing small RNAs as a guide for the de novo DOMAINS REARRANGED METHYLTRANSFERASE (DRM2, and maintenance DNA METHYLTRANSFERASE1 (MET1 being responsible for faithful propagation of CG methylation. Here we report an unusual 'dense methylation' pattern under the control of MET1, with methylation in all three sequence contexts. We identified epi-alleles of dense methylation at a non coding RNA locus (At4g15242 in Arabidopsis ecotypes, with distinct dense methylation and expression characteristics, which are stably maintained and transmitted in genetic crosses and which can be heritably altered by depletion of MET1. This suggests that, in addition to its classical CG maintenance function, at certain loci MET1 plays a role in creating transcriptional diversity based on the generation of independent epi-alleles. Database inspection identified several other loci with MET1-dependent dense methylation patterns. Arabidopsis ecotypes contain distinct epi-alleles of these loci with expression patterns that inversely correlate with methylation density, predominantly within the transcribed region. In Arabidopsis, dense methylation appears to be an exception as it is only found at a small number of loci. Its presence does, however, highlight the potential for MET1 as a contributor to epigenetic diversity, and it will be interesting to investigate the representation of dense methylation in other plant species.

  8. Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana

    Science.gov (United States)

    Itoh, Takeshi; Tanaka, Tsuyoshi; Barrero, Roberto A.; Yamasaki, Chisato; Fujii, Yasuyuki; Hilton, Phillip B.; Antonio, Baltazar A.; Aono, Hideo; Apweiler, Rolf; Bruskiewich, Richard; Bureau, Thomas; Burr, Frances; Costa de Oliveira, Antonio; Fuks, Galina; Habara, Takuya; Haberer, Georg; Han, Bin; Harada, Erimi; Hiraki, Aiko T.; Hirochika, Hirohiko; Hoen, Douglas; Hokari, Hiroki; Hosokawa, Satomi; Hsing, Yue; Ikawa, Hiroshi; Ikeo, Kazuho; Imanishi, Tadashi; Ito, Yukiyo; Jaiswal, Pankaj; Kanno, Masako; Kawahara, Yoshihiro; Kawamura, Toshiyuki; Kawashima, Hiroaki; Khurana, Jitendra P.; Kikuchi, Shoshi; Komatsu, Setsuko; Koyanagi, Kanako O.; Kubooka, Hiromi; Lieberherr, Damien; Lin, Yao-Cheng; Lonsdale, David; Matsumoto, Takashi; Matsuya, Akihiro; McCombie, W. Richard; Messing, Joachim; Miyao, Akio; Mulder, Nicola; Nagamura, Yoshiaki; Nam, Jongmin; Namiki, Nobukazu; Numa, Hisataka; Nurimoto, Shin; O’Donovan, Claire; Ohyanagi, Hajime; Okido, Toshihisa; OOta, Satoshi; Osato, Naoki; Palmer, Lance E.; Quetier, Francis; Raghuvanshi, Saurabh; Saichi, Naomi; Sakai, Hiroaki; Sakai, Yasumichi; Sakata, Katsumi; Sakurai, Tetsuya; Sato, Fumihiko; Sato, Yoshiharu; Schoof, Heiko; Seki, Motoaki; Shibata, Michie; Shimizu, Yuji; Shinozaki, Kazuo; Shinso, Yuji; Singh, Nagendra K.; Smith-White, Brian; Takeda, Jun-ichi; Tanino, Motohiko; Tatusova, Tatiana; Thongjuea, Supat; Todokoro, Fusano; Tsugane, Mika; Tyagi, Akhilesh K.; Vanavichit, Apichart; Wang, Aihui; Wing, Rod A.; Yamaguchi, Kaori; Yamamoto, Mayu; Yamamoto, Naoyuki; Yu, Yeisoo; Zhang, Hao; Zhao, Qiang; Higo, Kenichi; Burr, Benjamin; Gojobori, Takashi; Sasaki, Takuji

    2007-01-01

    We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is ∼32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene. PMID:17210932

  9. Sequence analysis of L RNA of Lassa virus

    International Nuclear Information System (INIS)

    Vieth, Simon; Torda, Andrew E.; Asper, Marcel; Schmitz, Herbert; Guenther, Stephan

    2004-01-01

    The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses

  10. Matrin 3 binds and stabilizes mRNA.

    Directory of Open Access Journals (Sweden)

    Maayan Salton

    Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  11. Poliovirus RNA polymerase: in vitro enzymatic activities, fidelity of replication, and characterization of a temperature-sensitive RNA-negative mutant

    International Nuclear Information System (INIS)

    Stokes, M.A.M.

    1985-01-01

    The in vitro activities of the purified poliovirus RNA polymerase were investigated in this study. The polymerase was shown to be a strict RNA dependent RNA polymerase. It only copied RNA templates but used either a DNA or RNA primer to initiate RNA synthesis. Partially purified polymerase has some DNA polymerase activities. Additional purification of the enzyme and studies with a mutant poliovirus RNA polymerase indicated that the DNA polymerase activities were due to a cellular polymerase. The fidelity of RNA replication in vitro by the purified poliovirus RNA polymerase was studied by measuring the rate of misincorporation of noncomplementary ribonucleotide monophosphates on synthetic homopolymeric RNA templates. The results showed that the ratio of noncomplementary to complementary ribonucleotides incorporated was 1-5 x 10 -3 . The viral polymerase of a poliovirus temperature sensitive RNA-negative mutant, Ts 10, was isolated. This study confirmed that the mutant was viable 33 0 , but was RNA negative at 39 0 . Characterization of the Ts 10 polymerase showed it was significantly more sensitive to heat inactivation than was the old-type polymerase. Highly purified poliovirions were found to contain several noncapsid proteins. At least two of these proteins were labeled by [ 35 S]methionine infected cells and appeared to be virally encoded proteins. One of these proteins was immunoprecipitated by anti-3B/sup vpg/ antiserum. This protein had the approximate Mr = 50,000 and appeared to be one of the previously identified 3B/sup vpg/ precursor proteins

  12. Light responses in Photoperiodism in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Anthony R. Cashmore

    2006-08-01

    B. Nature 410, 487-490. Jarillo, J. A., Gabrys, H., Capel, J., Alonso, J. M., Ecker, J. R., and Cashmore, A. R. (2001b). Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410, 952-954. Kinoshita, T., Doi, M., Suetsugu, N., Kagawa, T., Wada, M., and Shimazaki Ki, K. (2001). phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414, 656-660. Mas, P., Kim, W. Y., Somers, D. E., and Kay, S. A. (2003). Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426, 567-570.

  13. Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans.

    Science.gov (United States)

    Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Lu, Rui

    2013-10-01

    Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

  14. 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.

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

    Science.gov (United States)

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-05-11

    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.

  16. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  17. Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana

    Science.gov (United States)

    2011-01-01

    Background All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing. Results This study provides a comprehensive analysis of the origins of lineage-specific genes (LSGs) in Arabidopsis thaliana that are restricted to the Brassicaceae family. In this study, lineage-specific genes within the nuclear (1761 genes) and mitochondrial (28 genes) genomes are identified. The evolutionary origins of two thirds of the lineage-specific genes within the Arabidopsis thaliana genome are also identified. Almost a quarter of lineage-specific genes originate from non-lineage-specific paralogs, while the origins of ~10% of lineage-specific genes are partly derived from DNA exapted from transposable elements (twice the proportion observed for non-lineage-specific genes). Lineage-specific genes are also enriched in genes that have overlapping CDS, which is consistent with such novel genes arising from overprinting. Over half of the subset of the 958 lineage-specific genes found only in Arabidopsis thaliana have alignments to intergenic regions in Arabidopsis lyrata, consistent with either de novo origination or differential gene loss and retention, with both evolutionary scenarios explaining the lineage-specific status of these genes. A smaller number of lineage-specific genes with an incomplete open reading frame across different Arabidopsis thaliana accessions are further identified as accession-specific genes, most likely of recent origin in Arabidopsis thaliana. Putative de novo origination for two of the Arabidopsis thaliana-only genes is identified via additional sequencing across accessions of Arabidopsis thaliana and closely related sister species

  18. Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

    Science.gov (United States)

    Mork-Jansson, Astrid Elisabeth; Gargano, Daniela; Kmiec, Karol; Furnes, Clemens; Shevela, Dmitriy; Eichacker, Lutz Andreas

    2015-10-07

    The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Fertilization-independent seed development in Arabidopsis thaliana

    OpenAIRE

    Chaudhury, Abdul M.; Ming, Luo; Miller, Celia; Craig, Stuart; Dennis, Elizabeth S.; Peacock, W. James

    1997-01-01

    We report mutants in Arabidopsis thaliana (fertilization-independent seed: fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers ...

  20. Functional genetics of intraspecific ecological interactions in Arabidopsis thaliana

    OpenAIRE

    Wolf, Jason B.; Mutic, Joshua J.; Kover, Paula X.

    2011-01-01

    Studying the genetic basis of traits involved in ecological interactions is a fundamental part of elucidating the connections between evolutionary and ecological processes. Such knowledge allows one to link genetic models of trait evolution with ecological models describing interactions within and between species. Previous work has shown that connections between genetic and ecological processes in Arabidopsis thaliana may be mediated by the fact that quantitative trait loci (QTL) with ‘direct...

  1. Collection of apoplastic fluids from Arabidopsis thaliana leaves

    DEFF Research Database (Denmark)

    Madsen, Svend Roesen; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann

    2016-01-01

    The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite conte...... in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described....

  2. Characterization of a Cytokinin Response Factor in Arabidopsis thaliana

    OpenAIRE

    Ketelsen, Bernd

    2012-01-01

    The papers of this thesis are not available in Munin: 1. Bernd Ketelsen, Rainer Schwacke, Kirsten Krause and Karsten Fischer: 'Transcriptional activation by Cytokinin Response Factor 5 is governed by an acidic Cterminus containing two conserved domains' (manuscript) 2. Bernd Ketelsen, Stian Olsen, Kirsten Krause and Karsten Fischer: 'Cytokinin responsive factor 5 (CRF5) is involved in root development, hormonal crosstalk and sugar metabolism in Arabidopsis thaliana' (manuscript) 3. Bernd K...

  3. Multiple reference genomes and transcriptomes for Arabidopsis thaliana

    KAUST Repository

    Gan, Xiangchao

    2011-08-28

    Genetic differences between Arabidopsis thaliana accessions underlie the plants extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions. ©2011 Macmillan Publishers Limited. All rights reserved.

  4. Multiple reference genomes and transcriptomes for Arabidopsis thaliana

    KAUST Repository

    Gan, Xiangchao; Stegle, Oliver; Behr, Jonas; Steffen, Joshua G.; Drewe, Philipp; Hildebrand, Katie L.; Lyngsoe, Rune; Schultheiss, Sebastian J.; Osborne, Edward J.; Sreedharan, Vipin T.; Kahles, André ; Bohnert, Regina; Jean, Gé raldine; Derwent, Paul; Kersey, Paul; Belfield, Eric J.; Harberd, Nicholas P.; Kemen, Eric; Toomajian, Christopher; Kover, Paula X.; Clark, Richard M.; Rä tsch, Gunnar; Mott, Richard

    2011-01-01

    Genetic differences between Arabidopsis thaliana accessions underlie the plants extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions. ©2011 Macmillan Publishers Limited. All rights reserved.

  5. Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks

    Science.gov (United States)

    2012-09-21

    Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks Paulo Shakarian1*, J. Kenneth Wickiser2 1 Paulo Shakarian...significantly attacked. Citation: Shakarian P, Wickiser JK (2012) Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks...to 00-00-2012 4. TITLE AND SUBTITLE Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks 5a. CONTRACT NUMBER 5b

  6. Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

    Science.gov (United States)

    Götze, Michael; Dufourt, Jérémy; Ihling, Christian; Rammelt, Christiane; Pierson, Stephanie; Sambrani, Nagraj; Temme, Claudia; Sinz, Andrea; Simonelig, Martine; Wahle, Elmar

    2017-10-01

    Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression. © 2017 Götze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. A meta-analysis reveals the commonalities and differences in Arabidopsis thaliana response to different viral pathogens.

    Directory of Open Access Journals (Sweden)

    Guillermo Rodrigo

    Full Text Available Understanding the mechanisms by which plants trigger host defenses in response to viruses has been a challenging problem owing to the multiplicity of factors and complexity of interactions involved. The advent of genomic techniques, however, has opened the possibility to grasp a global picture of the interaction. Here, we used Arabidopsis thaliana to identify and compare genes that are differentially regulated upon infection with seven distinct (+ssRNA and one ssDNA plant viruses. In the first approach, we established lists of genes differentially affected by each virus and compared their involvement in biological functions and metabolic processes. We found that phylogenetically related viruses significantly alter the expression of similar genes and that viruses naturally infecting Brassicaceae display a greater overlap in the plant response. In the second approach, virus-regulated genes were contextualized using models of transcriptional and protein-protein interaction networks of A. thaliana. Our results confirm that host cells undergo significant reprogramming of their transcriptome during infection, which is possibly a central requirement for the mounting of host defenses. We uncovered a general mode of action in which perturbations preferentially affect genes that are highly connected, central and organized in modules.

  8. Comparison of the spaceflight transcriptome of four commonly used Arabidopsis thaliana ecotypes

    Data.gov (United States)

    National Aeronautics and Space Administration — This experiment compared the spaceflight transcriptomes of four commonly used natural variants (ecotypes) of Arabidopsis thaliana using RNAseq. In nature Arabidopsis...

  9. Atomistic mechanism of microRNA translation upregulation via molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Wei Ye

    Full Text Available MicroRNAs are endogenous 23-25 nt RNAs that play important gene-regulatory roles in animals and plants. Recently, miR369-3 was found to upregulate translation of TNFα mRNA in quiescent (G0 mammalian cell lines. Knock down and immunofluorescence experiments suggest that microRNA-protein complexes (with FXR1 and AGO2 are necessary for the translation upregulation. However the molecular mechanism of microRNA translation activation is poorly understood. In this study we constructed the microRNA-mRNA-AGO2-FXR1 quadruple complex by bioinformatics and molecular modeling, followed with all atom molecular dynamics simulations in explicit solvent to investigate the interaction mechanisms for the complex. A combined analysis of experimental and computational data suggests that AGO2-FXR1 complex relocalize microRNA:mRNA duplex to polysomes in G0. The two strands of dsRNA are then separated upon binding of AGO2 and FXR1. Finally, polysomes may improve the translation efficiency of mRNA. The mutation research confirms the stability of microRNA-mRNA-FXR1 and illustrates importance of key residue of Ile304. This possible mechanism can shed more light on the microRNA-dependent upregulation of translation.

  10. Expression Profile of Stress-responsive Arabidopsis thaliana miRNAs and their Target Genes in Response to Inoculation with Pectobacterium carotovorum subsp. carotovorum.

    Science.gov (United States)

    Djami-Tchatchou, A T; Ntushelo, K

    2017-01-01

    Pectobacterium carotovorum subsp. carotovorum (Pcc) is a soft rot bacterium which upon entry into the plant macerates plant tissues by producing plant cell wall degrading enzymes. It has a wide host range which includes carrot, potato, tomato, leafy greens, squash and other cucurbits, onion, green peppers and cassava. During plant-microbe interactions, one of the ways of plant response to pathogen infection is through the small RNA silencing mechanism. Under pathogen attack the plant utilizes microRNAs to regulate gene expression by means of mediating gene silencing at transcriptional and post-transcriptional level. This study aims to assess for the first time, the expression profile of some stress-responsive miRNA and differential expression pattern of their target genes in Arabidopsis thaliana inoculated with Pcc. Leaves of five weeks old Arabidopsis thaliana plants were infected with Pcc and the quantitative real time-PCR, was used to investigate after 0, 24, 48 and 72 h post infection, the expression profiling of the stress-responsive miRNAs which include: miR156, miR159, miR169, miR393, miR396 miR398, miR399 and miR408 along with their target genes which include: Squamosa promoter-binding-like protein, myb domain protein 101, nuclear factor Y subunit A8, concanavalin A-like lectin protein kinase, growth regulating factor 4, copper superoxide dismutase, ubiquitin-protein ligase and plantacyanin respectively. The findings showed that the overexpression of 6 miRNAs at 24, 48 and 72 h after infection resulted in the repression of their target genes and the expression of 2 miRNAs didn't affect their target genes. These results provide the first indication of the miRNAs role in response to the infection of Pcc in A. thaliana and open new vistas for a better understanding of miRNA regulation of plant response to Pcc.

  11. Single feature polymorphism (SFP-based selective sweep identification and association mapping of growth-related metabolic traits in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Stitt Mark

    2010-03-01

    Full Text Available Abstract Background Natural accessions of Arabidopsis thaliana are characterized by a high level of phenotypic variation that can be used to investigate the extent and mode of selection on the primary metabolic traits. A collection of 54 A. thaliana natural accession-derived lines were subjected to deep genotyping through Single Feature Polymorphism (SFP detection via genomic DNA hybridization to Arabidopsis Tiling 1.0 Arrays for the detection of selective sweeps, and identification of associations between sweep regions and growth-related metabolic traits. Results A total of 1,072,557 high-quality SFPs were detected and indications for 3,943 deletions and 1,007 duplications were obtained. A significantly lower than expected SFP frequency was observed in protein-, rRNA-, and tRNA-coding regions and in non-repetitive intergenic regions, while pseudogenes, transposons, and non-coding RNA genes are enriched with SFPs. Gene families involved in plant defence or in signalling were identified as highly polymorphic, while several other families including transcription factors are depleted of SFPs. 198 significant associations between metabolic genes and 9 metabolic and growth-related phenotypic traits were detected with annotation hinting at the nature of the relationship. Five significant selective sweep regions were also detected of which one associated significantly with a metabolic trait. Conclusions We generated a high density polymorphism map for 54 A. thaliana accessions that highlights the variability of resistance genes across geographic ranges and used it to identify selective sweeps and associations between metabolic genes and metabolic phenotypes. Several associations show a clear biological relationship, while many remain requiring further investigation.

  12. AthaMap web tools for the analysis of transcriptional and posttranscriptional regulation of gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Hehl, Reinhard; Bülow, Lorenz

    2014-01-01

    The AthaMap database provides a map of verified and predicted transcription factor (TF) and small RNA-binding sites for the A. thaliana genome. The database can be used for bioinformatic predictions of putative regulatory sites. Several online web tools are available that address specific questions. Starting with the identification of transcription factor-binding sites (TFBS) in any gene of interest, colocalizing TFBS can be identified as well as common TFBS in a set of user-provided genes. Furthermore, genes can be identified that are potentially targeted by specific transcription factors or small inhibitory RNAs. This chapter provides detailed information on how each AthaMap web tool can be used online. Examples on how this database is used to address questions in circadian and diurnal regulation are given. Furthermore, complementary databases and databases that go beyond questions addressed with AthaMap are discussed.

  13. Zelltyp-spezifische Mikroanalyse von Arabidopsis thaliana-Blättern

    Science.gov (United States)

    Brandt, Stephan Peter

    2002-04-01

    -Zell-Kommunikation, Signalweiterleitung oder Pflanzen-Pathogen-Interaktionen führen. The subject of this thesis was the analysis of single plant cells in respect to their contents of i) transcripts, ii) inorganic cations and anions, iii) metabolites like amino acids and carbohydrates as well as iv) proteins. One task was the transfer of existing methods to single cell analysis on leaf tissues of the model plant Arabisopsis thaliana L., the second one was the refinement and the development, respectively, of new protocols for the analysis of such picoliter samples. For cell type specific sampling two different complimentary methods were applied: Using micro glass capillaries specific single cell contents could be harvested from intact plants, whereas typical sample volumes were in the picoliter range. Even the sampling of inner cell types such as companion cells could be demonstrated. Using mechanical micro dissection of embedded tissue a larger amount of homogenous tissue could be collected. Because single cell samples contain only femtogram amounts of mRNA, direct detection of transcripts is impossible. Therefore, two amplification protocols were applied to the cell samples: The first procedure makes use of specifically primed RT-PCR for amplification. Several genes derived from different plants and tissues could be detected after successful RT-PCR, including high as well as low expressed genes. The second method was developed to monitor the activity of many genes in parallel using array hybridisation with filters containing the cDNA of as many as 16.000 ESTs. For this purpose, unspecific RT-PCR as it is applied in the differential display was used to amplify different transcripts in just one reaction. However, in these tissue specific array hybridisations the expression patterns of several hundreds genes could be monitored. These included known tissue specific expression patterns (of mainly photosynthesis related genes) as well as a couple of unknown expression patterns. To verify the tissue

  14. Discovery of centrosomal RNA and centrosomal hypothesis of cellular ageing and differentiation.

    Science.gov (United States)

    Chichinadze, Konstantin; Tkemaladze, Jaba; Lazarashvili, Ann

    2012-01-01

    In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034-9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure-a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a "RNA-dependent mechanism." This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

  15. Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    Hélène Pelczar

    2010-12-01

    Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

  16. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  17. Genome-Wide Search for Translated Upstream Open Reading Frames in Arabidopsis Thaliana.

    Science.gov (United States)

    Hu, Qiwen; Merchante, Catharina; Stepanova, Anna N; Alonso, Jose M; Heber, Steffen

    2016-03-01

    Upstream open reading frames (uORFs) are open reading frames that occur within the 5' UTR of an mRNA. uORFs have been found in many organisms. They play an important role in gene regulation, cell development, and in various metabolic processes. It is believed that translated uORFs reduce the translational efficiency of the main coding region. However, only few uORFs are experimentally characterized. In this paper, we use ribosome footprinting together with a semi-supervised approach based on stacking classification models to identify translated uORFs in Arabidopsis thaliana. Our approach identified 5360 potentially translated uORFs in 2051 genes. GO terms enriched in genes with translated uORFs include catalytic activity, binding, transferase activity, phosphotransferase activity, kinase activity, and transcription regulator activity. The reported uORFs occur with a higher frequency in multi-isoform genes, and some uORFs are affected by alternative transcript start sites or alternative splicing events. Association rule mining revealed sequence features associated with the translation status of the uORFs. We hypothesize that uORF translation is a complex process that might be regulated by multiple factors. The identified uORFs are available online at:https://www.dropbox.com/sh/zdutupedxafhly8/AABFsdNR5zDfiozB7B4igFcja?dl=0. This paper is the extended version of our research presented at ISBRA 2015.

  18. Gene expression and hormone autonomy in radiation-induced tumors of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Persinger, S.M.; Town, C.D.

    1989-01-01

    In order to study the molecular genetics of factor controlling plant cell growth, we have isolated a group of radiation-induced tumors from Arabidopsis thaliana. Tumors appeared on plants derived from 60 Co gamma-irradiated seed or seedlings, and are capable of hormone-autonomous growth in culture. We have used vertebrate oncogene probes to explore the hypothesis that the tumors arose by the radiation-induced activation of growth-regulating plant oncogenes. One probe, int-2, was used to isolate cDNA clones representing an mRNA differentially expressed between tumors and hormone-dependent callus tissue. The genomic organization and function of this and other differentially expressed Arabidopsis sequences are being further characterized. A second area of study concerns the hormonal status of individual tumors. Tumor tissue varies in color, texture, and degree of differentiation: while some tumors appear undifferentiated, one consistently produces roots, and others occasionally develop shoots or leaflets. The tumors have characteristic growth rates on hormone-free medium, and growth in response to exogenous hormones differs among the tumors themselves and from wild-type. Characterization of the relationships between hormonal status, morphogenesis, and gene expression should yield valuable insights into the mechanisms regulating plant growth and development

  19. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

  20. Fusarium oxysporum Triggers Tissue-Specific Transcriptional Reprogramming in Arabidopsis thaliana

    Science.gov (United States)

    Lyons, Rebecca; Stiller, Jiri; Powell, Jonathan; Rusu, Anca; Manners, John M.; Kazan, Kemal

    2015-01-01

    Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant. PMID:25849296

  1. Fusarium oxysporum triggers tissue-specific transcriptional reprogramming in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Lyons

    Full Text Available Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant.

  2. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean; Rayapuram, Naganand; Bonhomme, Ludovic; Hirt, Heribert; Pflieger, Delphine

    2014-01-01

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

  3. Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana.

    Science.gov (United States)

    Rylott, E L; Hooks, M A; Graham, I A

    2001-05-01

    Molecular genetic approaches in the model plant Arabidopsis thaliana (Col0) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: beta-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolase-mediated steps of beta-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of beta-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

  4. Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

    OpenAIRE

    Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

    2011-01-01

    Abstract The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated p...

  5. Oxidative stress responses induced by uranium exposure at low pH in leaves of Arabidopsis thaliana plants

    International Nuclear Information System (INIS)

    Saenen, Eline; Horemans, Nele; Vanhoudt, Nathalie; Vandenhove, Hildegarde; Biermans, Geert; Hees, May van; Wannijn, Jean; Vangronsveld, Jaco; Cuypers, Ann

    2015-01-01

    Anthropogenic activities have led to a widespread uranium (U) contamination in many countries. The toxic effects of U at the cellular level have mainly been investigated at a pH around 5.5, the optimal pH for hydroponically grown plants. However, since the speciation of U, and hence its toxicity, is strongly dependent on environmental factors such as the pH, it is important to investigate the effects of U at different environmentally relevant pH levels. Although U is poorly translocated from the roots to the shoots, resulting in a low U concentration in the leaves, it has been demonstrated that toxic effects in the leaves were already visible after 1 day exposure at pH 5.5, although only when exposed to relatively high U concentrations (100 μM). Therefore, the present study aimed to analyse the effects of different U concentrations (ranging from 0 to 100 μM) at pH 4.5 in leaves of Arabidopsis thaliana plants. Results indicate that U induces early senescence in A. thaliana leaves as was suggested by a decreased expression of CAT2 accompanied by an induction of CAT3 expression, a decreased CAT capacity and an increased lipid peroxidation. In addition, miRNA398b/c is involved in the regulation of the SOD response in the leaves. As such, an increased MIR398b/c expression was observed leading to a decreased transcript level of CSD1/2. Finally, the biosynthesis of ascorbate was induced after U exposure. This can point towards an important role for this metabolite in the scavenging of reactive oxygen species under U stress. - Highlights: • Uranium induces early senescence in A. thaliana after uranium exposure. • Ascorbate is involved in scavenging reactive oxygen species under uranium stress. • miRNA398b/c is involved in the regulation of the SOD response under uranium stress.

  6. Characterization Of Laccase T-DNA Mutants In Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Andersen, Jeppe Reitan; Asp, Torben; Mansfield, Shawn

    2009-01-01

    Laccases (P-diphenol:O2 oxidoreductase; EC 1.10.3.2), also termed laccase-like multicopper oxidases, are blue copper-containing oxidases which comprise multigene families in plants. In the Arabidopsis thaliana genome, 17 laccase genes (LAC1 to LAC17) have been annotated. To identify laccases...... for LAC15 T-DNA mutant seeds and an approximate 24 hour delay in germination was observed for these seeds. An approximate 20% reduction in glucose, galactose, and xylose was observed in primary stem cell walls of the LAC2 T-DNA mutants while similar relative increases in xylose were observed for LAC8...

  7. Phomopsis longicolla RNA virus 1 - Novel virus at the edge of myco- and plant viruses.

    Science.gov (United States)

    Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

    2017-06-01

    The complete nucleotide sequence of a new RNA mycovirus in the KY isolate of Phomopsis longicolla Hobbs 1985 and its protoplasts subcultures p5, p9, and ME711 was discovered. The virus, provisionally named Phomopsis longicolla RNA virus 1 (PlRV1), was localized in mitochondria and was determined to have a genome 2822 nucleotides long. A single open reading frame could be translated in silico by both standard and mitochondrial genetic codes into a product featuring conservative domains for an RNA-dependent RNA polymerase (RdRp). The RdRp of PlRV1 has no counterpart among mycoviruses, but it is about 30% identical with the RdRp of plant ourmiaviruses. Recently, new mycoviruses related to plant ourmiaviruses and forming one clade with PlRV1 have been discovered. This separate clade could represent the crucial link between plant and fungal viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana

    KAUST Repository

    Gu, Jinbao

    2017-12-01

    Soil salinity is a significant threat to sustainable agricultural production worldwide. Plants must adjust their developmental and physiological processes to cope with salt stress. Although the capacity for adaptation ultimately depends on the genome, the exceptional versatility in gene regulation provided by the spliceosome-mediated alternative splicing (AS) is essential in these adaptive processes. However, the functions of the spliceosome in plant stress responses are poorly understood. Here, we report the in-depth characterization of a U1 spliceosomal protein, AtU1A, in controlling AS of pre-mRNAs under salt stress and salt stress tolerance in Arabidopsis thaliana. The atu1a mutant was hypersensitive to salt stress and accumulated more reactive oxygen species (ROS) than the wild-type under salt stress. RNA-seq analysis revealed that AtU1A regulates AS of many genes, presumably through modulating recognition of 5′ splice sites. We showed that AtU1A is associated with the pre-mRNA of the ROS detoxification-related gene ACO1 and is necessary for the regulation of ACO1 AS. ACO1 is important for salt tolerance because ectopic expression of ACO1 in the atu1a mutant can partially rescue its salt hypersensitive phenotype. Our findings highlight the critical role of AtU1A as a regulator of pre-mRNA processing and salt tolerance in plants.

  9. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  10. Current knowledge of microRNA-mediated regulation of drug metabolism in humans.

    Science.gov (United States)

    Nakano, Masataka; Nakajima, Miki

    2018-05-01

    Understanding the factors causing inter- and intra-individual differences in drug metabolism potencies is required for the practice of personalized or precision medicine, as well as for the promotion of efficient drug development. The expression of drug-metabolizing enzymes is controlled by transcriptional regulation by nuclear receptors and transcriptional factors, epigenetic regulation, such as DNA methylation and histone acetylation, and post-translational modification. In addition to such regulation mechanisms, recent studies revealed that microRNAs (miRNAs), endogenous ~22-nucleotide non-coding RNAs that regulate gene expression through the translational repression and degradation of mRNAs, significantly contribute to post-transcriptional regulation of drug-metabolizing enzymes. Areas covered: This review summarizes the current knowledge regarding miRNAs-dependent regulation of drug-metabolizing enzymes and transcriptional factors and its physiological and clinical significance. We also describe recent advances in miRNA-dependent regulation research, showing that the presence of pseudogenes, single-nucleotide polymorphisms, and RNA editing affects miRNA targeting. Expert opinion: It is unwavering fact that miRNAs are critical factors causing inter- and intra-individual differences in the expression of drug-metabolizing enzymes. Consideration of miRNA-dependent regulation would be a helpful tool for optimizing personalized and precision medicine.

  11. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    You Wanhui

    2012-04-01

    Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

  12. Impacts of high ATP supply from chloroplasts and mitochondria on the leaf metabolism of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Chao eLiang

    2015-10-01

    Full Text Available Chloroplasts and mitochondria are the major ATP producing organelles in plant leaves. Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2 is a phosphatase dually targeted to the outer membranes of both organelles and it plays a role in the import of selected nuclear-encoded proteins into these two organelles. Overexpression (OE of AtPAP2 in Arabidopsis thaliana accelerates plant growth and promotes flowering, seed yield and biomass at maturity. Measurement of ADP/ATP/NADP+/NADPH contents in the leaves of 20-day-old OE and wild-type lines at the end of night and at 1 and 8 h following illumination in a 16/8 h photoperiod revealed that the ATP levels and ATP/NADPH ratios were significantly increased in the OE line at all three time points. The AtPAP2 OE line is therefore a good model to investigate the impact of high energy on the global molecular status of Arabidopsis. In this study, transcriptome, proteome and metabolome profiles of the high ATP transgenic line were examined and compared with those of wild-type plants. A comparison of OE and WT at the end of the night provide valuable information on the impact of higher ATP output from mitochondria on plant physiology, as mitochondrial respiration is the major source of ATP in the dark in leaves. Similarly, comparison of OE and WT following illumination will provide information on the impact of higher energy output from chloroplasts on plant physiology. Overexpression of AtPAP2 was found to significantly affect the transcript and protein abundances of genes encoded by the two organellar genomes. For example, the protein abundances of many ribosomal proteins encoded by the chloroplast genome were higher in the AtPAP2 OE line under both light and dark conditions, while the protein abundances of multiple components of the photosynthetic complexes were lower. RNA-seq data also showed that the transcription of the mitochondrial genome is greatly affected by the availability of energy. These data

  13. Dehydration stress memory genes of Zea mays; comparison with Arabidopsis thaliana

    Science.gov (United States)

    2014-01-01

    Background Pre-exposing plants to diverse abiotic stresses may alter their physiological and transcriptional responses to a subsequent stress, suggesting a form of “stress memory”. Arabidopsis thaliana plants that have experienced multiple exposures to dehydration stress display transcriptional behavior suggesting “memory” from an earlier stress. Genes that respond to a first stress by up-regulating or down-regulating their transcription but in a subsequent stress provide a significantly different response define the ‘memory genes’ category. Genes responding similarly to each stress form the ‘non-memory’ category. It is unknown whether such memory responses exists in other Angiosperm lineages and whether memory is an evolutionarily conserved response to repeated dehydration stresses. Results Here, we determine the transcriptional responses of maize (Zea mays L.) plants that have experienced repeated exposures to dehydration stress in comparison with plants encountering the stress for the first time. Four distinct transcription memory response patterns similar to those displayed by A. thaliana were revealed. The most important contribution is the evidence that monocot and eudicot plants, two lineages that have diverged 140 to 200 M years ago, display similar abilities to ‘remember’ a dehydration stress and to modify their transcriptional responses, accordingly. The highly sensitive RNA-Seq analyses allowed to identify genes that function similarly in the two lineages, as well as genes that function in species-specific ways. Memory transcription patterns indicate that the transcriptional behavior of responding genes under repeated stresses is different from the behavior during an initial dehydration stress, suggesting that stress memory is a complex phenotype resulting from coordinated responses of multiple signaling pathways. Conclusions Structurally related genes displaying the same memory responses in the two species would suggest conservation

  14. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus

    International Nuclear Information System (INIS)

    Panavas, Tadas; Nagy, Peter D.

    2003-01-01

    Defective interfering (DI) RNA associated with Tomato bushy stunt virus (TBSV), which is a plus-strand RNA virus, requires p33 and p92 proteins of TBSV or the related Cucumber necrosis virus (CNV), for replication in plants. To test if DI RNA can replicate in a model host, we coexpressed TBSV DI RNA and p33/p92 of CNV in yeast. We show evidence for replication of DI RNA in yeast, including (i) dependence on p33 and p92 for DI replication; (ii) presence of active CNV RNA-dependent RNA polymerase in isolated membrane-containing preparations; (iii) increasing amount of DI RNA(+) over time; (iv) accumulation of (-)stranded DI RNA; (v) presence of correct 5' and 3' ends in DI RNA; (vi) inhibition of replication by mutations in the replication enhancer; and (vii) evolution of DI RNA over time, as shown by sequence heterogeneity. We also produced evidence supporting the occurrence of DI RNA recombinants in yeast. In summary, development of yeast as a host for replication of TBSV DI RNA will facilitate studies on the roles of viral and host proteins in replication/recombination

  15. Functional characterization of Arabidopsis thaliana transthyretin-like protein.

    Science.gov (United States)

    Pessoa, João; Sárkány, Zsuzsa; Ferreira-da-Silva, Frederico; Martins, Sónia; Almeida, Maria R; Li, Jianming; Damas, Ana M

    2010-02-18

    Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. The Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

  16. Functional characterization of Arabidopsis thaliana transthyretin-like protein

    Directory of Open Access Journals (Sweden)

    Almeida Maria R

    2010-02-01

    Full Text Available Abstract Background Arabidopsis thaliana transthyretin-like (TTL protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU decarboxylase (N-terminal domain and 5-hydroxyisourate (5-HIU hydrolase (C-terminal domain. TTL is a member of the transthyretin-related protein family (TRP, which comprises a number of proteins with sequence homology to transthyretin (TTR and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. Results The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. Conclusions The Arabidopsis thaliana transthyretin-like (TTL protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

  17. Allyl isothiocyanate affects the cell cycle of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Signe Elisabeth Åsberg

    2015-05-01

    Full Text Available Isothiocyanates (ITCs are degradation products of glucosinolates present in members of the Brassicaceae family acting as herbivore repellents and antimicrobial compounds. Recent results indicate that allyl ITC (AITC has a role in defense responses such as glutathione depletion, ROS generation and stomatal closure. In this study we show that exposure to non-lethal concentrations of AITC causes a shift in the cell cycle distribution of Arabidopsis thaliana leading to accumulation of cells in S-phases and a reduced number of cells in non-replicating phases. Furthermore, transcriptional analysis revealed an AITC-induced up-regulation of the gene encoding cyclin-dependent kinase A while several genes encoding mitotic proteins were down-regulated, suggesting an inhibition of mitotic processes. Interestingly, visualization of DNA synthesis indicated that exposure to AITC reduced the rate of DNA replication. Taken together, these results indicate that non-lethal concentrations of AITC induce cells of A. thaliana to enter the cell cycle and accumulate in S-phases, presumably as a part of a defensive response. Thus, this study suggests that AITC has several roles in plant defense and add evidence to the growing data supporting a multifunctional role of glucosinolates and their degradation products in plants.

  18. Nuclear micro-probe analysis of Arabidopsis thaliana leaves

    International Nuclear Information System (INIS)

    Ager, F.J.; Ynsa, M.D.; Dominguez-Solis, J.R.; Lopez-Martin, M.C.; Gotor, C.; Romero, L.C.

    2003-01-01

    Phytoremediation is a cost-effective plant-based approach for remediation of soils and waters which takes advantage of the remarkable ability of some plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues, such as toxic heavy metals and organic pollutants. Nowadays, phytoremediation technology is becoming of paramount importance when environmental decontamination is concerned, due to the emerging knowledge of its physiological and molecular mechanisms and the new biological and engineering strategies designed to optimize and improve it. In addition, the feasibility of using plants for environmental cleanup has been confirmed by many different trials around the world. Arabidopsis thaliana plants can be used for basic studies to improve the technology on phytoremediation. Making use of nuclear microscopy techniques, in this paper we study leaves of wild type and transgenic A. thaliana plants grown in a cadmium-rich environment under different conditions. Micro-PIXE, RBS and SEM analyses, performed on the scanning proton micro-probe at the CNA in Seville (Spain), prove that cadmium is preferentially sequestered in the central region of epidermal trichome and allow comparing the effects of genetic modifications

  19. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

    DEFF Research Database (Denmark)

    Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto

    2009-01-01

    of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both...... suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized...

  20. Nitrite reductase expression is regulated at the post-transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana.

    Science.gov (United States)

    Crété, P; Caboche, M; Meyer, C

    1997-04-01

    Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post-transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild-type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium-containing medium. These results suggest that, together with transcriptional control, post-transcriptional regulation by the nitrogen source is operating on NiR expression. This post-transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.

  1. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Science.gov (United States)

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Multifaceted regulation of translational readthrough by RNA replication elements in a tombusvirus.

    Directory of Open Access Journals (Sweden)

    Peter A Cimino

    2011-12-01

    Full Text Available Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii mediating cis-preferential replication of viral genomes, and (iii coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.

  3. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    2016-05-01

    Full Text Available Plants have varying abilities to tolerate chilling (low but not freezing temperatures, and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

  4. Enhancement of RNA synthesis by promoter duplication in tombusviruses

    International Nuclear Information System (INIS)

    Panavas, T.; Panaviene, Z.; Pogany, J.; Nagy, P.D.

    2003-01-01

    Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants , Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses

  5. MicroRNA Related Polymorphisms and Breast Cancer Risk

    Science.gov (United States)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L.; Muranen, Taru A.; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K.; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L.; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F.; Southey, Melissa C.; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A.; van der Luijt, Rob B.; Meindl, Alfons; Schmutzler, Rita K.; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J.; Hunter, David J.; Cox, Angela; Cross, Simon S.; Reed, Malcolm W. R.; Schmidt, Marjanka K.; Broeks, Annegien; Veer, Laura J. V. a. n't.; Hogervorst, Frans B.; Fasching, Peter A.; Schrauder, Michael G.; Ekici, Arif B.; Beckmann, Matthias W.; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M.; Perez, Jose I. A.; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D. P.; Dunning, Alison M.; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J.; Wang, Xianshu; Vachon, Celine; Olson, Janet E.; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Andrulis, Irene L.; Knight, Julia A.; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A. E. M.; Seynaeve, Caroline; van Asperen, Christi J.; Kristensen, Vessela N.; Slager, Susan; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J.; Martens, John W. M.; Collée, J. Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G.; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F.; Nevanlinna, Heli

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88–0.96), rs1052532 (OR 0.97; 95% CI: 0.95–0.99), rs10719 (OR 0.97; 95% CI: 0.94–0.99), rs4687554 (OR 0.97; 95% CI: 0.95–0.99, and rs3134615 (OR 1.03; 95% CI: 1.01–1.05) located in the 3′ UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects. PMID:25390939

  6. MicroRNA related polymorphisms and breast cancer risk.

    Science.gov (United States)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L; Muranen, Taru A; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F; Southey, Melissa C; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A; van der Luijt, Rob B; Meindl, Alfons; Schmutzler, Rita K; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J; Hunter, David J; Cox, Angela; Cross, Simon S; Reed, Malcolm W R; Schmidt, Marjanka K; Broeks, Annegien; Van't Veer, Laura J; Hogervorst, Frans B; Fasching, Peter A; Schrauder, Michael G; Ekici, Arif B; Beckmann, Matthias W; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M; Perez, Jose I A; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D P; Dunning, Alison M; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J; Wang, Xianshu; Vachon, Celine; Olson, Janet E; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Andrulis, Irene L; Knight, Julia A; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V; Antonenkova, Natalia N; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline; van Asperen, Christi J; Kristensen, Vessela N; Slager, Susan; Toland, Amanda E; Ambrosone, Christine B; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J; Martens, John W M; Collée, J Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F; Nevanlinna, Heli

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

  7. MicroRNA related polymorphisms and breast cancer risk.

    Directory of Open Access Journals (Sweden)

    Sofia Khan

    Full Text Available Genetic variations, such as single nucleotide polymorphisms (SNPs in microRNAs (miRNA or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS. Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC. Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR 0.92; 95% confidence interval (CI: 0.88-0.96, rs1052532 (OR 0.97; 95% CI: 0.95-0.99, rs10719 (OR 0.97; 95% CI: 0.94-0.99, rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05 located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

  8. A Regulatory Network Analysis of Orphan Genes in Arabidopsis Thaliana

    Science.gov (United States)

    Singh, Pramesh; Chen, Tianlong; Arendsee, Zebulun; Wurtele, Eve S.; Bassler, Kevin E.

    Orphan genes, which are genes unique to each particular species, have recently drawn significant attention for their potential usefulness for organismal robustness. Their origin and regulatory interaction patterns remain largely undiscovered. Recently, methods that use the context likelihood of relatedness to infer a network followed by modularity maximizing community detection algorithms on the inferred network to find the functional structure of regulatory networks were shown to be effective. We apply improved versions of these methods to gene expression data from Arabidopsis thaliana, identify groups (clusters) of interacting genes with related patterns of expression and analyze the structure within those groups. Focusing on clusters that contain orphan genes, we compare the identified clusters to gene ontology (GO) terms, regulons, and pathway designations and analyze their hierarchical structure. We predict new regulatory interactions and unravel the structure of the regulatory interaction patterns of orphan genes. Work supported by the NSF through Grants DMR-1507371 and IOS-1546858.

  9. Human intrinsic factor expressed in the plant Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Laursen, Niels B; Nexø, Ebba

    2003-01-01

    and contamination by other B12 binders. We tested the use of recombinant plants for large-scale production of pathogen-free human recombinant IF. Human IF was successfully expressed in the recombinant plant Arabidopsis thaliana. Extract from fresh plants possessed high B12-binding capacity corresponding to 70 mg...... to recombinant IF and gastric IF were alike, as was the interaction of recombinant and native IF with the specific receptor cubilin. The data presented show that recombinant plants have a great potential as a large-scale source of human IF for analytical and therapeutic purposes.......Intrinsic factor (IF) is the gastric protein that promotes the intestinal uptake of vitamin B12. Gastric IF from animal sources is used in diagnostic tests and in vitamin pills. However, administration of animal IF to humans becomes disadvantageous because of possible pathogenic transmission...

  10. Human intrinsic factor expressed in the plant Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Laursen, Niels B; Nexø, Ebba

    2003-01-01

    and contamination by other B12 binders. We tested the use of recombinant plants for large-scale production of pathogen-free human recombinant IF. Human IF was successfully expressed in the recombinant plant Arabidopsis thaliana. Extract from fresh plants possessed high B12-binding capacity corresponding to 70 mg......Intrinsic factor (IF) is the gastric protein that promotes the intestinal uptake of vitamin B12. Gastric IF from animal sources is used in diagnostic tests and in vitamin pills. However, administration of animal IF to humans becomes disadvantageous because of possible pathogenic transmission...... IF per 1 kg wet weight. The dried plants still retained 60% of the IF activity. The purified IF preparation consisted of a 50-kDa glycosylated protein with the N-terminal sequence of mature IF. Approximately one-third of the protein was cleaved at the internal site em leader PSNP downward arrow GPGP...

  11. Multimodal nonlinear imaging of arabidopsis thaliana root cell

    Science.gov (United States)

    Jang, Bumjoon; Lee, Sung-Ho; Woo, Sooah; Park, Jong-Hyun; Lee, Myeong Min; Park, Seung-Han

    2017-07-01

    Nonlinear optical microscopy has enabled the possibility to explore inside the living organisms. It utilizes ultrashort laser pulse with long wavelength (greater than 800nm). Ultrashort pulse produces high peak power to induce nonlinear optical phenomenon such as two-photon excitation fluorescence (TPEF) and harmonic generations in the medium while maintaining relatively low average energy pre area. In plant developmental biology, confocal microscopy is widely used in plant cell imaging after the development of biological fluorescence labels in mid-1990s. However, fluorescence labeling itself affects the sample and the sample deviates from intact condition especially when labelling the entire cell. In this work, we report the dynamic images of Arabidopsis thaliana root cells. This demonstrates the multimodal nonlinear optical microscopy is an effective tool for long-term plant cell imaging.

  12. Interstitial telomere-like repeats in the Arabidopsis thaliana genome.

    Science.gov (United States)

    Uchida, Wakana; Matsunaga, Sachihiro; Sugiyama, Ryuji; Kawano, Shigeyuki

    2002-02-01

    Eukaryotic chromosomal ends are protected by telomeres, which are thought to play an important role in ensuring the complete replication of chromosomes. On the other hand, non-functional telomere-like repeats in the interchromosomal regions (interstitial telomeric repeats; ITRs) have been reported in several eukaryotes. In this study, we identified eight ITRs in the Arabidopsis thaliana genome, each consisting of complete and degenerate 300- to 1200-bp sequences. The ITRs were grouped into three classes (class IA-B, class II, and class IIIA-E) based on the degeneracy of the telomeric repeats in ITRs. The telomeric repeats of the two ITRs in class I were conserved for the most part, whereas the single ITR in class II, and the five ITRs in class III were relatively degenerated. In addition, degenerate ITRs were surrounded by common sequences that shared 70-100% homology to each other; these are named ITR-adjacent sequences (IAS). Although the genomic regions around ITRs in class I lacked IAS, those around ITRs in class II contained IAS (IASa), and those around five ITRs in class III had nine types of IAS (IASb, c, d, e, f, g, h, i, and j). Ten IAS types in classes II and III showed no significant homology to each other. The chromosomal locations of ITRs and IAS were not category-related, but most of them were adjacent to, or part of, a centromere. These results show that the A. thaliana genome has undergone chromosomal rearrangements, such as end-fusions and segmental duplications.

  13. Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana.

    Science.gov (United States)

    Christian, Jan-Ole; Braginets, Rostyslav; Schulze, Waltraud X; Walther, Dirk

    2012-01-01

    The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation "hotspots" has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.

  14. Population genomics of the Arabidopsis thaliana flowering time gene network.

    Science.gov (United States)

    Flowers, Jonathan M; Hanzawa, Yoshie; Hall, Megan C; Moore, Richard C; Purugganan, Michael D

    2009-11-01

    The time to flowering is a key component of the life-history strategy of the model plant Arabidopsis thaliana that varies quantitatively among genotypes. A significant problem for evolutionary and ecological genetics is to understand how natural selection may operate on this ecologically significant trait. Here, we conduct a population genomic study of resequencing data from 52 genes in the flowering time network. McDonald-Kreitman tests of neutrality suggested a strong excess of amino acid polymorphism when pooling across loci. This excess of replacement polymorphism across the flowering time network and a skewed derived frequency spectrum toward rare alleles for both replacement and noncoding polymorphisms relative to synonymous changes is consistent with a large class of deleterious polymorphisms segregating in these genes. Assuming selective neutrality of synonymous changes, we estimate that approximately 30% of amino acid polymorphisms are deleterious. Evidence of adaptive substitution is less prominent in our analysis. The photoperiod regulatory gene, CO, and a gibberellic acid transcription factor, AtMYB33, show evidence of adaptive fixation of amino acid mutations. A test for extended haplotypes revealed no examples of flowering time alleles with haplotypes comparable in length to those associated with the null fri(Col) allele reported previously. This suggests that the FRI gene likely has a uniquely intense or recent history of selection among the flowering time genes considered here. Although there is some evidence for adaptive evolution in these life-history genes, it appears that slightly deleterious polymorphisms are a major component of natural molecular variation in the flowering time network of A. thaliana.

  15. Distinct modes of adventitious rooting in Arabidopsis thaliana.

    Science.gov (United States)

    Correa, L da Rocha; Troleis, J; Mastroberti, A A; Mariath, J E A; Fett-Neto, A G

    2012-01-01

    The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.

  16. Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

    Science.gov (United States)

    Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

    1989-01-01

    Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

  17. A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

    Science.gov (United States)

    Kenesi, Erzsébet; Carbonell, Alberto; Lózsa, Rita; Vértessy, Beáta; Lakatos, Lóránt

    2017-07-27

    In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Tsung-Fu Yu

    Full Text Available The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1 encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

  19. Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

    Science.gov (United States)

    Kaye, C; Crawford, N M; Malmberg, R L

    1997-04-01

    We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

  20. Efficiency and fidelity of cell-free protein synthesis by transfer RNA from aged mice

    Energy Technology Data Exchange (ETDEWEB)

    Foote, R.S.; Stulberg, M.P.

    1980-01-01

    Transfer RNAs (tRNAs) from heart, kidney, liver, and spleen of mature (10 to 12 months old) and aged (29 months old) C57BL/6 mice were tested for their ability to translate encephalomyocarditis viral RNA in a tRNA-dependent cell-free system derived from mouse ascites tumor cells. The rates of in vitro protein synthesis were compared as a function of tRNA concentration, and the fidelity of translation was examined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing of the viral polypeptides synthesized in vitro. No significant age-related differences in either the efficiency or fidelity of synthesis were discovered, indicating that alternations in tRNAs are probably not involved in the cellular aging of these tissues.

  1. Adsorption and inhibition of CuO nanoparticles on Arabidopsis thaliana root

    Science.gov (United States)

    Xu, Lina

    2018-02-01

    CuO NPs, the size ranging from 20 to 80 nm were used to detect the adsorption and inhibition on the Arabidopsis thaliana roots. In this study, CuO NPs were adsorbed and agglomerated on the surface of root top after exposed for 7 days. With the increasing of CuO NPs concentrations, CuO NPs also adsorbed on the meristernatic zone. The growth of Arabidopsis thaliana lateral roots were also inhibited by CuO NPs exposure. The Inhibition were concentration dependent. The number of root top were 246, 188 and 123 per Arabidopsis thaliana, respectively. The number of root tops after CuO NPs exposure were significantly decreased compared with control groups. This results suggested the phytotoxicity of CuO NPs on Arabidopsis thaliana roots.

  2. Partial Purification and Characterization of RNase P from Arabidopsis Thaliana Tissue

    National Research Council Canada - National Science Library

    2000-01-01

    ...) molecules to give mature 5, ends has been isolated from Arabidopsis thaliana tissue. The RNase P activity was isolated by ammonium sulfate precipitation of a tissue homogenate and further purified by anion exchange chromatography...

  3. Inference of the Genetic Network Regulating Lateral Root Initiation in Arabidopsis thaliana

    KAUST Repository

    Muraro, D.; Voss, U.; Wilson, M.; Bennett, M.; Byrne, H.; De Smet, I.; Hodgman, C.; King, J.

    2013-01-01

    thaliana is stimulated by a cascade of regulators of which only the interactions of its initial elements have been identified. Using simulated gene expression data with known network topology, we compare the performance of inference algorithms, based

  4. Genome-scale cold stress response regulatory networks in ten Arabidopsis thaliana ecotypes

    DEFF Research Database (Denmark)

    Barah, Pankaj; Jayavelu, Naresh Doni; Rasmussen, Simon

    2013-01-01

    available from Arabidopsis thaliana 1001 genome project, we further investigated sequence polymorphisms in the core cold stress regulon genes. Significant numbers of non-synonymous amino acid changes were observed in the coding region of the CBF regulon genes. Considering the limited knowledge about......BACKGROUND: Low temperature leads to major crop losses every year. Although several studies have been conducted focusing on diversity of cold tolerance level in multiple phenotypically divergent Arabidopsis thaliana (A. thaliana) ecotypes, genome-scale molecular understanding is still lacking....... RESULTS: In this study, we report genome-scale transcript response diversity of 10 A. thaliana ecotypes originating from different geographical locations to non-freezing cold stress (10°C). To analyze the transcriptional response diversity, we initially compared transcriptome changes in all 10 ecotypes...

  5. Signals of speciation within Arabidopsis thaliana in comparison with its relatives

    NARCIS (Netherlands)

    Alcazar, R.; Pecinka, A.; Aarts, M.G.M.; Fransz, P.F.; Koornneef, M.

    2012-01-01

    The species within the now well-defined Arabidopsis genus provide biological materials suitable to investigate speciation and the development of reproductive isolation barriers between related species. Even within the model species A. thaliana, genetic differentiation between populations due to

  6. Transcriptional repression of BODENLOS by HD-ZIP transcription factor HB5 in Arabidopsis thaliana.

    NARCIS (Netherlands)

    Smet, De I.; Lau, S.; Ehrismann, J.S.; Axiotis, I.; Kolb, M.; Kientz, M.; Weijers, D.; Jürgens, G.

    2013-01-01

    In Arabidopsis thaliana, the phytohormone auxin is an important patterning agent during embryogenesis and post-embryonic development, exerting effects through transcriptional regulation. The main determinants of the transcriptional auxin response machinery are AUXIN RESPONSE FACTOR (ARF)

  7. Ethylene-induced hyponastic growth in Arabidopsis thaliana is controlled by ERECTA

    NARCIS (Netherlands)

    Zanten, van M.; Snoek, L.B.; Eck-Stouten, van E.; Proveniers, M.C.G.; Torii, K.U.; Voesenek, L.A.C.J.; Peeters, A.J.M.; Millenaar, F.F.

    2010-01-01

    Plants can respond quickly and profoundly to detrimental changes in their environment. For example, Arabidopsis thaliana can induce an upward leaf movement response through differential petiole growth (hyponastic growth) to outgrow complete submergence. This response is induced by accumulation of

  8. Re-establishment of desiccation tolerance by PEG in germinated Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Maia de Oliveira, Julio; Dias Costa, Maria; Ligterink, Wilco; Hilhorst, Henk

    2015-01-01

    Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose DT while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with polyethylene glycol (PEG).

  9. Universal primers that amplify RNA from all three flavivirus subgroups

    Directory of Open Access Journals (Sweden)

    Barnard Ross T

    2008-01-01

    Full Text Available Abstract Background Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. Results Bioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5 coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA. Conclusion Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.

  10. Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum.

    Science.gov (United States)

    Khoudi, Habib; Maatar, Yafa; Brini, Faïçal; Fourati, Amine; Ammar, Najoua; Masmoudi, Khaled

    2013-01-01

    Phosphogypsum (PG) is a by-product of the phosphorus-fertiliser industry and represents an environmental concern since it contains pollutants such as cadmium (Cd). We have recently shown that the overexpression of a proton pump gene (TaVP1) in transgenic tobacco (Nicotiana tabacum) led to an enhanced Cd tolerance and accumulation. The aim of this study was to evaluate the potential of transgenic Arabidopsis thaliana plants harbouring the TaVP1 gene to phytoremediate phosphogypsum. A pot experiment was carried out under greenhouse conditions. Transgenic A. thaliana plants harbouring the TaVP1 gene were grown on various substrates containing phosphogypsum (0, 25, 50 and 100 %) for 40 days. At the end of the growth period, we examined the growth (germination, root length, fresh weight) and physiological parameters (chlorophyll and protein contents, catalase activity and proteolysis) as well as the cadmium, Mg, Ca, and P contents of the A. thaliana plants. In order to evaluate Cd tolerance of the A. thaliana lines harbouring the TaVP1 gene, an in vitro experiment was also carried out. One week-old seedlings were transferred to Murashige and Skoog agar plates containing various concentrations of cadmium; the germination, total leaf area and root length were determined. The growth and physiological parameters of all A. thaliana plants were significantly altered by PG. The germination capacity, root growth and biomass production of wild-type (WT) plants were more severely inhibited by PG compared with the TaVP1 transgenic A. thaliana lines. In addition, TaVP1 transgenic A. thaliana plants maintained a higher antioxidant capacity than the WT. Interestingly, elemental analysis of leaf material derived from plants grown on PG revealed that the transgenic A. thaliana line accumulated up to ten times more Cd than WT. Despite its higher Cd content, the transgenic A. thaliana line performed better than the WT counterpart. In vitro evaluation of Cd tolerance showed that TaVP1

  11. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  12. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Effects of pH on uranium uptake and oxidative stress responses induced in Arabidopsis thaliana

    OpenAIRE

    Saenen, Eline; Horemans, Nele; Vanhoudt, Nathalie; Vandenhove, Hildegarde; Biermans, Geert; Van Hees, May; Wannijn, Jean; Vangronsveld, Jaco; Cuypers, Ann

    2013-01-01

    Uranium (U) causes oxidative stress in Arabidopsis thaliana plants grown at pH 5.5. However, U speciation and its toxicity strongly depend on environmental parameters, for example pH. It is unknown how different U species determine U uptake and translocation within plants and how they might affect the oxidative defense mechanisms of these plants. The present study analyzed U uptake and oxidative stress-related responses in A. thaliana (Columbia ecotype) under contrasted U chemical speciation ...

  14. Rclick: a web server for comparison of RNA 3D structures.

    Science.gov (United States)

    Nguyen, Minh N; Verma, Chandra

    2015-03-15

    RNA molecules play important roles in key biological processes in the cell and are becoming attractive for developing therapeutic applications. Since the function of RNA depends on its structure and dynamics, comparing and classifying the RNA 3D structures is of crucial importance to molecular biology. In this study, we have developed Rclick, a web server that is capable of superimposing RNA 3D structures by using clique matching and 3D least-squares fitting. Our server Rclick has been benchmarked and compared with other popular servers and methods for RNA structural alignments. In most cases, Rclick alignments were better in terms of structure overlap. Our server also recognizes conformational changes between structures. For this purpose, the server produces complementary alignments to maximize the extent of detectable similarity. Various examples showcase the utility of our web server for comparison of RNA, RNA-protein complexes and RNA-ligand structures. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Studies on the role of NonA in mRNA biogenesis

    International Nuclear Information System (INIS)

    Kozlova, Natalia; Braga, Jose; Lundgren, Josefin; Rino, Jose; Young, Patrick; Carmo-Fonseca, Maria; Visa, Neus

    2006-01-01

    The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A) + RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles

  16. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  17. Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

    2009-04-08

    Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

  18. Establishment of an Indirect Genetic Transformation Method for Arabidopsis thaliana ecotype Bangladesh

    Directory of Open Access Journals (Sweden)

    Bulbul AHMED

    2011-11-01

    Full Text Available Arabidopsis thaliana is a small flowering plant belonging to the Brassicaceae family, which is adopted as a model plant for genetic research. Agrobacterium tumifaciensmediated transformation method for A. thaliana ecotype Bangladesh was established. Leaf discs of A. thaliana were incubated with A. tumefaciens strain LBA4404 containing chimeric nos. nptII. nos and intron-GUS genes. Following inoculation and co-cultivation, leaf discs were cultured on selection medium containing 50 mg/l kanamycin + 50 mg/l cefotaxime + 1.5 mg/l NAA and kanamycin resistant shoots were induced from the leaf discs after two weeks. Shoot regeneration was achieved after transferring the tissues onto fresh medium of the same combination. Finally, the shoots were rooted on MS medium containing 50 mg/l kanamycin. Incorporation and expression of the transgenes were confirmed by PCR analysis. Using this protocol, transgenic A. thaliana plants can be obtained and indicates that genomic transformation in higher plants is possible through insertion of desired gene. Although Agrobacterium mediated genetic transformation is established for A. thaliana, this study was the conducted to transform A. thaliana ecotype Bangladesh.

  19. ABCE1 is a highly conserved RNA silencing suppressor.

    Directory of Open Access Journals (Sweden)

    Kairi Kärblane

    Full Text Available ATP-binding cassette sub-family E member 1 (ABCE1 is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

  20. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  1. Composition and function of P bodies in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Luis David Maldonado-Bonilla

    2014-05-01

    Full Text Available mRNA accumulation is tightly regulated by diverse molecular pathways. The identification and characterization of enzymes and regulatory proteins involved in controlling the fate of mRNA offers the possibility to broaden our understanding of posttranscriptional gene regulation. Processing bodies (P bodies, PB are cytoplasmic protein complexes involved in degradation and translational arrest of mRNA. Composition and dynamics of these subcellular structures have been studied in animal systems, yeasts and in the model plant Arabidopsis. Their assembly implies the aggregation of specific factors related to decapping, deadenylation and exoribonucleases that operate synchronously to regulate certain mRNA targets during development and adaptation to stress. Although the general function of PB along with the flow of genetic information is understood, several questions still remain open. This review summarizes data on the composition, potential molecular roles, and biological significance of PB and potentially related proteins in Arabidopsis.

  2. 50/50 Expressional Odds of Retention Signifies the Distinction between Retained Introns and Constitutively Spliced Introns in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Rui Mao

    2017-10-01

    Full Text Available Intron retention, one of the most prevalent alternative splicing events in plants, can lead to introns retained in mature mRNAs. However, in comparison with constitutively spliced introns (CSIs, the relevantly distinguishable features for retained introns (RIs are still poorly understood. This work proposes a computational pipeline to discover novel RIs from multiple next-generation RNA sequencing (RNA-Seq datasets of Arabidopsis thaliana. Using this pipeline, we detected 3,472 novel RIs from 18 RNA-Seq datasets and re-confirmed 1,384 RIs which are currently annotated in the TAIR10 database. We also use the expression of intron-containing isoforms as a new feature in addition to the conventional features. Based on these features, RIs are highly distinguishable from CSIs by machine learning methods, especially when the expressional odds of retention (i.e., the expression ratio of the RI-containing isoforms relative to the isoforms without RIs for the same gene reaches to or larger than 50/50. In this case, the RIs and CSIs can be clearly separated by the Random Forest with an outstanding performance of 0.95 on AUC (the area under a receiver operating characteristics curve. The closely related characteristics to the RIs include the low strength of splice sites, high similarity with the flanking exon sequences, low occurrence percentage of YTRAY near the acceptor site, existence of putative intronic splicing silencers (ISSs, i.e., AG/GA-rich motifs and intronic splicing enhancers (ISEs, i.e., TTTT-containing motifs, and enrichment of Serine/Arginine-Rich (SR proteins and heterogeneous nuclear ribonucleoparticle proteins (hnRNPs.

  3. Acclimation of Arabidopsis thaliana to long-term CO{sub 2} enrichment and nitrogen supply is basically a matter of growth rate adjustment

    Energy Technology Data Exchange (ETDEWEB)

    Tocquin, P.; Ormenese, S.; Pieltain, A.; Detry, N.; Bernier, G.; Perilleux, C. [Univ. of Liege, Dept. of Life Sciences, Lab. of Plant Physiology, Liege (Belgium)

    2006-12-15

    The long-term response of Arabidopsis thaliana to increasing CO{sub 2} was evaluated in plants grown in 800 {mu}l l{sup -1} CO{sub 2} from sowing and maintained, in hydroponics, on three nitrogen supplies: 'low', 'medium' and 'high'. The global response to high CO{sub 2} and N-supply was evaluated by measuring growth parameters in parallel with photosynthetic activity, leaf carbohydrates, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) messenger RNA and protein, stomatal conductance (g-s) and density. CO{sub 2} enrichment was found to stimulate biomass production, whatever the N-supply. This stimulation was transient on low N-supply and persisted throughout the whole vegetative growth only in high N-supply. Acclimation on low N-high C0{sub 2} was not associated with carbohydrate accumulation or with a strong reduction in Rubisco amount or activity. At high N-supply, growth stimulation by high CO{sub 2} was mainly because of the acceleration of leaf production and expansion while other parameters such as specific leaf area, root/shoot ratio and g{sub s} appeared to be correlated with total leaf area. Our results thus suggest that, in strictly controlled and stable growing conditions, acclimation of A. thaliana to long-term CO{sub 2} enrichment is mostly controlled by growth rate adjustment. (au)

  4. Multiple paths to similar germination behavior in Arabidopsis thaliana.

    Science.gov (United States)

    Burghardt, Liana T; Edwards, Brianne R; Donohue, Kathleen

    2016-02-01

    Germination timing influences plant fitness, and its sensitivity to temperature may cause it to change as climate shifts. These changes are likely to be complex because temperatures that occur during seed maturation and temperatures that occur post-dispersal interact to define germination timing. We used the model organism Arabidopsis thaliana to determine how flowering time (which defines seed-maturation temperature) and post-dispersal temperature influence germination and the expression of genetic variation for germination. Germination responses to temperature (germination envelopes) changed as seeds aged, or after-ripened, and these germination trajectories depended on seed-maturation temperature and genotype. Different combinations of genotype, seed-maturation temperature, and after-ripening produced similar germination envelopes. Likewise, different genotypes and seed-maturation temperatures combined to produce similar germination trajectories. Differences between genotypes were most likely to be observed at high and low germination temperatures. The germination behavior of some genotypes responds weakly to maternal temperature but others are highly plastic. We hypothesize that weak dormancy induction could synchronize germination of seeds dispersed at different times. By contrast, we hypothesize that strongly responsive genotypes may spread offspring germination over several possible germination windows. Considering germination responses to temperature is important for predicting phenology expression and evolution in future climates. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  5. Adaptation response of Arabidopsis thaliana to random positioning

    Science.gov (United States)

    Kittang, A.-I.; Winge, P.; van Loon, J. J. W. A.; Bones, A. M.; Iversen, T.-H.

    2013-10-01

    Arabidopsis thaliana seedlings were exposed on a Random Positioning Machine (RPM) under light conditions for 16 h and the samples were analysed using microarray techniques as part of a preparation for a space experiment on the International Space Station (ISS). The results demonstrated a moderate to low regulation of 55 genes (genes). Genes encoding proteins associated with the chaperone system (e.g. heat shock proteins, HSPs) and enzymes in the flavonoid biosynthesis were induced. Most of the repressed genes were associated with light and sugar responses. Significant up-regulation of selected HSP genes was found by quantitative Real-Time PCR in 1 week old plants after the RPM exposure both in light and darkness. Higher quantity of DPBA (diphenylboric acid 2-amino-ethyl ester) staining was observed in the whole root and in the root elongation zone of the seedlings exposed on the RPM by use of fluorescent microscopy, indicating higher flavonoid content. The regulated genes and an increase of flavonoids are related to several stresses, but increased occurrence of HSPs and flavonoids are also representative for normal growth (e.g. gravitropism). The response could be a direct stress response or an integrated response of the two signal pathways of light and gravity resulting in an overall light response.

  6. Lagging adaptation to warming climate in Arabidopsis thaliana.

    Science.gov (United States)

    Wilczek, Amity M; Cooper, Martha D; Korves, Tonia M; Schmitt, Johanna

    2014-06-03

    If climate change outpaces the rate of adaptive evolution within a site, populations previously well adapted to local conditions may decline or disappear, and banked seeds from those populations will be unsuitable for restoring them. However, if such adaptational lag has occurred, immigrants from historically warmer climates will outperform natives and may provide genetic potential for evolutionary rescue. We tested for lagging adaptation to warming climate using banked seeds of the annual weed Arabidopsis thaliana in common garden experiments in four sites across the species' native European range: Valencia, Spain; Norwich, United Kingdom; Halle, Germany; and Oulu, Finland. Genotypes originating from geographic regions near the planting site had high relative fitness in each site, direct evidence for broad-scale geographic adaptation in this model species. However, genotypes originating in sites historically warmer than the planting site had higher average relative fitness than local genotypes in every site, especially at the northern range limit in Finland. This result suggests that local adaptive optima have shifted rapidly with recent warming across the species' native range. Climatic optima also differed among seasonal germination cohorts within the Norwich site, suggesting that populations occurring where summer germination is common may have greater evolutionary potential to persist under future warming. If adaptational lag has occurred over just a few decades in banked seeds of an annual species, it may be an important consideration for managing longer-lived species, as well as for attempts to conserve threatened populations through ex situ preservation.

  7. Fertilization-independent seed development in Arabidopsis thaliana

    Science.gov (United States)

    Chaudhury, Abdul M.; Ming, Luo; Miller, Celia; Craig, Stuart; Dennis, Elizabeth S.; Peacock, W. James

    1997-01-01

    We report mutants in Arabidopsis thaliana (fertilization-independent seed: fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, ≈50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization. PMID:9108133

  8. Fertilization-independent seed development in Arabidopsis thaliana.

    Science.gov (United States)

    Chaudhury, A M; Ming, L; Miller, C; Craig, S; Dennis, E S; Peacock, W J

    1997-04-15

    We report mutants in Arabidopsis thaliana (fertilization-independent seed:fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, approximately 50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.

  9. Transcriptional responses of Arabidopsis thaliana plants to As (V stress

    Directory of Open Access Journals (Sweden)

    Yuan Joshua S

    2008-08-01

    Full Text Available Abstract Background Arsenic is toxic to plants and a common environmental pollutant. There is a strong chemical similarity between arsenate [As (V] and phosphate (Pi. Whole genome oligonucleotide microarrays were employed to investigate the transcriptional responses of Arabidopsis thaliana plants to As (V stress. Results Antioxidant-related genes (i.e. coding for superoxide dismutases and peroxidases play prominent roles in response to arsenate. The microarray experiment revealed induction of chloroplast Cu/Zn superoxide dismutase (SOD (at2g28190, Cu/Zn SOD (at1g08830, as well as an SOD copper chaperone (at1g12520. On the other hand, Fe SODs were strongly repressed in response to As (V stress. Non-parametric rank product statistics were used to detect differentially expressed genes. Arsenate stress resulted in the repression of numerous genes known to be induced by phosphate starvation. These observations were confirmed with qRT-PCR and SOD activity assays. Conclusion Microarray data suggest that As (V induces genes involved in response to oxidative stress and represses transcription of genes induced by phosphate starvation. This study implicates As (V as a phosphate mimic in the cell by repressing genes normally induced when available phosphate is scarce. Most importantly, these data reveal that arsenate stress affects the expression of several genes with little or unknown biological functions, thereby providing new putative gene targets for future research.

  10. Desensitization and recovery of phototropic responsiveness in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Janoudi, A.K.; Poff, K.L.

    1993-01-01

    Phototropism is induced by blue light, which also induces desensitization, a partial or total loss of phototropic responsiveness. The fluence and fluence-rate dependence of densensitization and recovery from desensitization have been measured for etiolated and red light (669-nm) preirradiated Arabidopsis thaliana seedlings. The extent of desensitization increased as the fluence of the desensitizing 450-nm light was increased from 0.3 to 60 μmol m -2 s -1 . At equal fluences, blue light caused more desensitization when given at a fluence rate of 1.0 μmol m -2 s -1 than at 0.3 μmol m -2 s -1 . In addition, seedlings irradiated with blue light at the higher fluence rate required a longer recovery time than seedlings irradiated at the lower fluence rate. A red light preirradiation, probably mediated via phytochrome, decreased the time required for recovery from desensitization. The minimum time for detectable recovery was about 65 s, and the maximum time observed was about 10 min. It is proposed that the descending arm of the fluence-response relationship for first positive phototropism is a consequence of desensitization, and that the time threshold for second positive phototropism establishes a period during which recovery from desensitization occurs. 11 refs., 6 figs

  11. Desensitization and recovery of phototropic responsiveness in Arabidopsis thaliana

    Science.gov (United States)

    Janoudi, A. K.; Poff, K. L.

    1993-01-01

    Phototropism is induced by blue light, which also induces desensitization, a partial or total loss of phototropic responsiveness. The fluence and fluence-rate dependence of desensitization and recovery from desensitization have been measured for etiolated and red light (669-nm) preirradiated Arabidopsis thaliana seedlings. The extent of desensitization increased as the fluence of the desensitizing 450-nm light was increased from 0.3 to 60 micromoles m-2 s-1. At equal fluences, blue light caused more desensitization when given at a fluence rate of 1.0 micromole m-2 s-1 than at 0.3 micromole m-2 s-1. In addition, seedlings irradiated with blue light at the higher fluence rate required a longer recovery time than seedlings irradiated at the lower fluence rate. A red light preirradiation, probably mediated via phytochrome, decreased the time required for recovery from desensitization. The minimum time for detectable recovery was about 65 s, and the maximum time observed was about 10 min. It is proposed that the descending arm of the fluence-response relationship for first positive phototropism is a consequence of desensitization, and that the time threshold for second positive phototropism establishes a period during which recovery from desensitization occurs.

  12. Role of methyl salicylate on oviposition deterrence in Arabidopsis thaliana.

    Science.gov (United States)

    Groux, Raphaël; Hilfiker, Olivier; Gouhier-Darimont, Caroline; Peñaflor, Maria Fernanda Gomes Villalba; Erb, Matthias; Reymond, Philippe

    2014-07-01

    Plants attacked by herbivores have evolved different strategies that fend off their enemies. Insect eggs deposited on leaves have been shown to inhibit further oviposition through visual or chemical cues. In some plant species, the volatile methyl salicylate (MeSA) repels gravid insects but whether it plays the same role in the model species Arabidopsis thaliana is currently unknown. Here we showed that Pieris brassicae butterflies laid fewer eggs on Arabidopsis plants that were next to a MeSA dispenser or on plants with constitutively high MeSA emission than on control plants. Surprisingly, the MeSA biosynthesis mutant bsmt1-1 treated with egg extract was still repellent to butterflies when compared to untreated bsmt1-1. Moreover, the expression of BSMT1 was not enhanced by egg extract treatment but was induced by herbivory. Altogether, these results provide evidence that the deterring activity of eggs on gravid butterflies is independent of MeSA emission in Arabidopsis, and that MeSA might rather serve as a deterrent in plants challenged by feeding larvae.

  13. Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome.

    Science.gov (United States)

    Cerveau, Delphine; Kraut, Alexandra; Stotz, Henrik U; Mueller, Martin J; Couté, Yohann; Rey, Pascal

    2016-11-01

    Peroxiredoxins are ubiquitous thiol-dependent peroxidases for which chaperone and signaling roles have been reported in various types of organisms in recent years. In plants, the peroxidase function of the two typical plastidial 2-Cys peroxiredoxins (2-Cys PRX A and B) has been highlighted while the other functions, particularly in ROS-dependent signaling pathways, are still elusive notably due to the lack of knowledge of interacting partners. Using an ex vivo approach based on co-immunoprecipitation of leaf extracts from Arabidopsis thaliana wild-type and mutant plants lacking 2-Cys PRX expression followed by mass spectrometry-based proteomics, 158 proteins were found associated with 2-Cys PRXs. Already known partners like thioredoxin-related electron donors (Chloroplastic Drought-induced Stress Protein of 32kDa, Atypical Cysteine Histidine-rich Thioredoxin 2) and enzymes involved in chlorophyll synthesis (Protochlorophyllide OxidoReductase B) or carbon metabolism (Fructose-1,6-BisPhosphatase) were identified, validating the relevance of the approach. Bioinformatic and bibliographic analyses allowed the functional classification of the identified proteins and revealed that more than 40% are localized in plastids. The possible roles of plant 2-Cys PRXs in redox signaling pathways are discussed in relation with the functions of the potential partners notably those involved in redox homeostasis, carbon and amino acid metabolisms as well as chlorophyll biosynthesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

    DEFF Research Database (Denmark)

    Nayak, A.; Goodfellow, I. G.; Woolaway, K. E.

    2006-01-01

    The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3D(pol)), the precursor 3CD, and an RNA template containing the cre....../bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV X protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg...... precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues...

  15. Functional characterization of a heterologously expressed Brassica napus WRKY41-1 transcription factor in regulating anthocyanin biosynthesis in Arabidopsis thaliana.

    Science.gov (United States)

    Duan, Shaowei; Wang, Jianjun; Gao, Chenhao; Jin, Changyu; Li, Dong; Peng, Danshuai; Du, Guomei; Li, Yiqian; Chen, Mingxun

    2018-03-01

    Previous studies have shown that a plant WRKY transcription factor, WRKY41, has multiple functions, and regulates seed dormancy, hormone signaling pathways, and both biotic and abiotic stress responses. However, it is not known about the roles of AtWRKY41 from the model plant, Arabidopsis thaliana, and its ortholog, BnWRKY41, from the closely related and important oil-producing crop, Brassica napus, in the regulation of anthocyanin biosynthesis. Here, we found that the wrky41 mutation in A. thaliana resulted in a significant increase in anthocyanin levels in rosette leaves, indicating that AtWRKY41 acts as repressor of anthocyanin biosynthesis. RNA sequencing and quantitative real-time PCR analysis revealed increased expression of three regulatory genes AtMYB75, AtMYB111, and AtMYBD, and two structural genes, AT1G68440 and AtGSTF12, all of which contribute to anthocyanin biosynthesis, in the sixth rosette leaves of wrky41-2 plants at 20 days after germination. We cloned the full length complementary DNA of BnWRKY41-1 from the C2 subgenome of the B. napus genotype Westar and observed that, when overexpressed in tobacco leaves as a fusion protein with green fluorescent protein, BnWRKY41-1 is localized to the nucleus. We further showed that overexpression of BnWRKY41-1 in the A. thaliana wrky41-2 mutant rescued the higher anthocyanin content phenotype in rosette leaves of the mutant. Moreover, the elevated expression levels in wrky41-2 rosette leaves of several important regulatory and structural genes regulating anthocyanin biosynthesis were not observed in the BnWRKY41-1 overexpressing lines. These results reveal that BnWRKY41-1 has a similar role with AtWRKY41 in regulating anthocyanin biosynthesis when overexpressed in A. thaliana. This gene represents a promising target for genetically manipulating B. napus to increase the amounts of anthocyanins in rosette leaves. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Complete sequence of RNA1 of grapevine Anatolian ringspot virus.

    Science.gov (United States)

    Digiaro, Michele; Nahdi, Sabrine; Elbeaino, Toufic

    2012-10-01

    The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B(Hel)), the viral genome-linked protein (1C(VPg)), the proteinase (1D(Prot)), the RNA-dependent RNA polymerase (1E(Pol)), and of the protease cofactor (1A(Pro-cof)) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5'- and 3'-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.

  17. Production and processing of siRNA precursor transcripts from the highly repetitive maize genome.

    Directory of Open Access Journals (Sweden)

    Christopher J Hale

    2009-08-01

    Full Text Available Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA-directed DNA methylation (RdDM factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA-dependent RNA polymerase, RDR2 (MOP1. Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II-based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species.

  18. Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.

    Science.gov (United States)

    Kim, Hyein; Park, Dongbin; Hahn, Yoonsoo

    2018-01-05

    Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Novel Positive-Sense, Single-Stranded RNA (+ssRNA) Virus with Di-Cistronic Genome from Intestinal Content of Freshwater Carp (Cyprinus carpio)

    Science.gov (United States)

    Pankovics, Péter; Simmonds, Peter

    2011-01-01

    A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature. PMID:22195010

  20. Cytoplasmic Control of Sense-Antisense mRNA Pairs

    Directory of Open Access Journals (Sweden)

    Flore Sinturel

    2015-09-01

    Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

  1. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    Science.gov (United States)

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. 1001 Proteomes: a functional proteomics portal for the analysis of Arabidopsis thaliana accessions.

    Science.gov (United States)

    Joshi, Hiren J; Christiansen, Katy M; Fitz, Joffrey; Cao, Jun; Lipzen, Anna; Martin, Joel; Smith-Moritz, A Michelle; Pennacchio, Len A; Schackwitz, Wendy S; Weigel, Detlef; Heazlewood, Joshua L

    2012-05-15

    The sequencing of over a thousand natural strains of the model plant Arabidopsis thaliana is producing unparalleled information at the genetic level for plant researchers. To enable the rapid exploitation of these data for functional proteomics studies, we have created a resource for the visualization of protein information and proteomic datasets for sequenced natural strains of A. thaliana. The 1001 Proteomes portal can be used to visualize amino acid substitutions or non-synonymous single-nucleotide polymorphisms in individual proteins of A. thaliana based on the reference genome Col-0. We have used the available processed sequence information to analyze the conservation of known residues subject to protein phosphorylation among these natural strains. The substitution of amino acids in A. thaliana natural strains is heavily constrained and is likely a result of the conservation of functional attributes within proteins. At a practical level, we demonstrate that this information can be used to clarify ambiguously defined phosphorylation sites from phosphoproteomic studies. Protein sets of available natural variants are available for download to enable proteomic studies on these accessions. Together this information can be used to uncover the possible roles of specific amino acids in determining the structure and function of proteins in the model plant A. thaliana. An online portal to enable the community to exploit these data can be accessed at http://1001proteomes.masc-proteomics.org/

  3. Characterization of carbon ion-induced mutations in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Shikazono, N.; Suzuki, C.; Kitamura, S.; Watanabe, H.; Tano, S.; Tanaka, A.

    2003-01-01

    Full text: Irradiation of Arabidopsis thaliana by carbon ions was carried out to investigate the mutational effect of ion particles in higher plants. The averaged mutation rate of carbon ions was 2.0 X 10 -6 / Gy, which was 18-fold higher than that of electrons. PCR analysis of the carbon ion-induced mutants showed that, out of 28 mutant alleles, 14 had point-like mutations within the gene, while 14 contained large structural alterations. In the case of 12 electron-induced mutants, 9 had point-like mutations within the gene, while 3 contained large structural alterations. These results suggest that carbon ions are more likely to induce large structural alterations compared with electrons. Further sequence analysis revealed that most of the point-like mutations induced by carbon ions were short deletions. In the case of rearrangements, DNA strand breaks were found to be rejoined using, if present, short homologous sequences for both types of radiation. After carbon ion-irradiation, small deletions were frequently observed around the breakpoints, whereas duplications of terminal sequence were found after electron-irradiation. These results suggest that non-homologous end joining (NHEJ) pathway operates after plant cells are exposed to both ion particles and electrons but that different mode of rejoining deals with the broken ends produced by each radiation. From the present results, it seems reasonable to assume that carbon ions could predominantly induce null mutations in Arabidopsis. The fact that the molecular nature of carbon ion-induced mutation was different from that of electrons and that the molecular mechanisms of cells to induce mutations appeared to be also different implicates that ion particle is not only valuable as a new mutagen but also useful as a new tool to study repair mechanisms of certain types of DNA damage

  4. Control of seed development in Arabidopsis thaliana by atmospheric oxygen

    Science.gov (United States)

    Kuang, A.; Crispi, M.; Musgrave, M. E.

    1998-01-01

    Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5.1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2.5, 5.1, 10.1, 16.2 and 21.3 kPa O2, 0.035 kPa CO2 and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O2 treatments below 16.2 kPa, and seeds from plants grown in 2.5 kPa O2 did not germinate at all. Fewer than 25% of the seeds from plants grown in 5.1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5.1 kPa O2, at around the curled cotyledon stage in 10.1 kPa O2, and at the premature stage in 16.2 kPa O2. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2.5 kPa O2. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis.

  5. Multiwall carbon nanotubes modulate paraquat toxicity in Arabidopsis thaliana.

    Science.gov (United States)

    Fan, Xiaoji; Xu, Jiahui; Lavoie, Michel; Peijnenburg, W J G M; Zhu, Youchao; Lu, Tao; Fu, Zhengwei; Zhu, Tingheng; Qian, Haifeng

    2018-02-01

    Carbon nanotubes can be either toxic or beneficial to plant growth and can also modulate toxicity of organic contaminants through surface sorption. The complex interacting toxic effects of carbon nanotubes and organic contaminants in plants have received little attention in the literature to date. In this study, the toxicity of multiwall carbon nanotubes (MWCNT, 50 mg/L) and paraquat (MV, 0.82 mg/L), separately or in combination, were evaluated at the physiological and the proteomic level in Arabidopsis thaliana for 7-14 days. The results revealed that the exposure to MWCNT had no inhibitory effect on the growth of shoots and leaves. Rather, MWCNT stimulated the relative electron transport rate and the effective photochemical quantum yield of PSII value as compared to the control by around 12% and lateral root production up to nearly 4-fold as compared to the control. The protective effect of MWCNT on MV toxicity on the root surface area could be quantitatively explained by the extent of MV adsorption on MWCNT and was related to stimulation of photosynthesis, antioxidant protection and number and area of lateral roots which in turn helped nutrient assimilation. The influence of MWCNT and MV on photosynthesis and oxidative stress at the physiological level was consistent with the proteomics analysis, with various over-expressed photosynthesis-related proteins (by more than 2 folds) and various under-expressed oxidative stress related proteins (by about 2-3 folds). This study brings new insights into the interactive effects of two xenobiotics (MWCNT and MV) on the physiology of a model plant. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Gene transposition causing natural variation for growth in Arabidopsis thaliana.

    Science.gov (United States)

    Vlad, Daniela; Rappaport, Fabrice; Simon, Matthieu; Loudet, Olivier

    2010-05-13

    A major challenge in biology is to identify molecular polymorphisms responsible for variation in complex traits of evolutionary and agricultural interest. Using the advantages of Arabidopsis thaliana as a model species, we sought to identify new genes and genetic mechanisms underlying natural variation for shoot growth using quantitative genetic strategies. More quantitative trait loci (QTL) still need be resolved to draw a general picture as to how and where in the pathways adaptation is shaping natural variation and the type of molecular variation involved. Phenotypic variation for shoot growth in the Bur-0 x Col-0 recombinant inbred line set was decomposed into several QTLs. Nearly-isogenic lines generated from the residual heterozygosity segregating among lines revealed an even more complex picture, with major variation controlled by opposite linked loci and masked by the segregation bias due to the defective phenotype of SG3 (Shoot Growth-3), as well as epistasis with SG3i (SG3-interactor). Using principally a fine-mapping strategy, we have identified the underlying gene causing phenotypic variation at SG3: At4g30720 codes for a new chloroplast-located protein essential to ensure a correct electron flow through the photosynthetic chain and, hence, photosynthesis efficiency and normal growth. The SG3/SG3i interaction is the result of a structural polymorphism originating from the duplication of the gene followed by divergent paralogue's loss between parental accessions. Species-wide, our results illustrate the very dynamic rate of duplication/transposition, even over short periods of time, resulting in several divergent--but still functional-combinations of alleles fixed in different backgrounds. In predominantly selfing species like Arabidopsis, this variation remains hidden in wild populations but is potentially revealed when divergent individuals outcross. This work highlights the need for improved tools and algorithms to resolve structural variation

  7. The Hidden Geometries of the Arabidopsis thaliana Epidermis

    KAUST Repository

    Staff, Lee

    2012-09-11

    The quest for the discovery of mathematical principles that underlie biological phenomena is ancient and ongoing. We present a geometric analysis of the complex interdigitated pavement cells in the Arabidopsis thaliana (Col.) adaxial epidermis with a view to discovering some geometric characteristics that may govern the formation of this tissue. More than 2,400 pavement cells from 10, 17 and 24 day old leaves were analyzed. These interdigitated cells revealed a number of geometric properties that remained constant across the three age groups. In particular, the number of digits per cell rarely exceeded 15, irrespective of cell area. Digit numbers per 100 ?m2 cell area reduce with age and as cell area increases, suggesting early developmental programming of digits. Cell shape proportions as defined by length:width ratios were highly conserved over time independent of the size and, interestingly, both the mean and the medians were close to the golden ratio 1.618034. With maturity, the cell area:perimeter ratios increased from a mean of 2.0 to 2.4. Shape properties as defined by the medial axis transform (MAT) were calculated and revealed that branch points along the MAT typically comprise one large and two small angles. These showed consistency across the developmental stages considered here at 140° (± 5°) for the largest angles and 110° (± 5°) for the smaller angles. Voronoi diagram analyses of stomatal center coordinates revealed that giant pavement cells (?500 ?m2) tend to be arranged along Voronoi boundaries suggesting that they could function as a scaffold of the epidermis. In addition, we propose that pavement cells have a role in spacing and positioning of the stomata in the growing leaf and that they do so by growing within the limits of a set of \\'geometrical rules\\'. © 2012 Staff et al.

  8. Functional genetics of intraspecific ecological interactions in Arabidopsis thaliana.

    Science.gov (United States)

    Wolf, Jason B; Mutic, Joshua J; Kover, Paula X

    2011-05-12

    Studying the genetic basis of traits involved in ecological interactions is a fundamental part of elucidating the connections between evolutionary and ecological processes. Such knowledge allows one to link genetic models of trait evolution with ecological models describing interactions within and between species. Previous work has shown that connections between genetic and ecological processes in Arabidopsis thaliana may be mediated by the fact that quantitative trait loci (QTL) with 'direct' effects on traits of individuals also have pleiotropic 'indirect' effects on traits expressed in neighbouring plants. Here, we further explore these connections by examining functional relationships between traits affected directly and indirectly by the same QTL. We develop a novel approach using structural equation models (SEMs) to determine whether observed pleiotropic effects result from traits directly affected by the QTL in focal individuals causing the changes in the neighbours' phenotypes. This hypothesis was assessed using SEMs to test whether focal plant phenotypes appear to mediate the connection between the focal plants' genotypes and the phenotypes of their neighbours, or alternatively, whether the connection between the focal plants' genotypes and the neighbours' phenotypes is mediated by unmeasured traits. We implement this analysis using a QTL of major effect that maps to the well-characterized flowering locus, FRIGIDA. The SEMs support the hypothesis that the pleiotropic indirect effects of this locus arise from size and developmental timing-related traits in focal plants affecting the expression of developmental traits in their neighbours. Our findings provide empirical insights into the genetics and nature of intraspecific ecological interactions. Our technique holds promise in directing future work into the genetic basis and functional relationship of traits mediating and responding to ecological interactions.

  9. Alanine aminotransferase variants conferring diverse NUE phenotypes in Arabidopsis thaliana.

    Science.gov (United States)

    McAllister, Chandra H; Good, Allen G

    2015-01-01

    Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.

  10. Adaptive diversification of growth allometry in the plant Arabidopsis thaliana.

    Science.gov (United States)

    Vasseur, François; Exposito-Alonso, Moises; Ayala-Garay, Oscar J; Wang, George; Enquist, Brian J; Vile, Denis; Violle, Cyrille; Weigel, Detlef

    2018-03-27

    Seed plants vary tremendously in size and morphology; however, variation and covariation in plant traits may be governed, at least in part, by universal biophysical laws and biological constants. Metabolic scaling theory (MST) posits that whole-organismal metabolism and growth rate are under stabilizing selection that minimizes the scaling of hydrodynamic resistance and maximizes the scaling of resource uptake. This constrains variation in physiological traits and in the rate of biomass accumulation, so that they can be expressed as mathematical functions of plant size with near-constant allometric scaling exponents across species. However, the observed variation in scaling exponents calls into question the evolutionary drivers and the universality of allometric equations. We have measured growth scaling and fitness traits of 451 Arabidopsis thaliana accessions with sequenced genomes. Variation among accessions around the scaling exponent predicted by MST was correlated with relative growth rate, seed production, and stress resistance. Genomic analyses indicate that growth allometry is affected by many genes associated with local climate and abiotic stress response. The gene with the strongest effect, PUB4 , has molecular signatures of balancing selection, suggesting that intraspecific variation in growth scaling is maintained by opposing selection on the trade-off between seed production and abiotic stress resistance. Our findings suggest that variation in allometry contributes to local adaptation to contrasting environments. Our results help reconcile past debates on the origin of allometric scaling in biology and begin to link adaptive variation in allometric scaling to specific genes. Copyright © 2018 the Author(s). Published by PNAS.

  11. Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

    Science.gov (United States)

    Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

    2016-03-15

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

  12. Efficient cellular release of Rift Valley fever virus requires genomic RNA.

    Directory of Open Access Journals (Sweden)

    Mary E Piper

    2011-03-01

    Full Text Available The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies.

  13. Changes in Gene Expression of Arabidopsis Thaliana Cell Cultures Upon Exposure to Real and Simulated Partial- g Forces

    Science.gov (United States)

    Fengler, Svenja; Spirer, Ina; Neef, Maren; Ecke, Margret; Hauslage, Jens; Hampp, Rüdiger

    2016-06-01

    Cell cultures of the plant model organism Arabidopsis thaliana were exposed to partial- g forces during parabolic flight and clinostat experiments (0.16 g, 0.38 g and 0.5 g were tested). In order to investigate gravity-dependent alterations in gene expression, samples were metabolically quenched by the fixative RNA later Ⓡ to stabilize nucleic acids and used for whole-genome microarray analysis. An attempt to identify the potential threshold acceleration for the gravity-dependent response showed that the smaller the experienced g-force, the greater was the susceptibility of the cell cultures. Compared to short-term μ g during a parabolic flight, the number of differentially expressed genes under partial- g was lower. In addition, the effect on the alteration of amounts of transcripts decreased during partial- g parabolic flight due to the sequence of the different parabolas (0.38 g, 0.16 g and μ g). A time-dependent analysis under simulated 0.5 g indicates that adaptation occurs within minutes. Differentially expressed genes (at least 2-fold up- or down-regulated in expression) under real flight conditions were to some extent identical with those affected by clinorotation. The highest number of homologuous genes was detected within seconds of exposure to 0.38 g (both flight and clinorotation). To a considerable part, these genes deal with cell wall properties. Additionally, responses specific for clinorotation were observed.

  14. Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling.

    Science.gov (United States)

    Karpinska, Barbara; Alomrani, Sarah Owdah; Foyer, Christine H

    2017-09-26

    Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction-oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1 , WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Authors.

  15. MhYTP1 and MhYTP2 from Apple Confer Tolerance to Multiple Abiotic Stresses in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Na Wang

    2017-08-01

    Full Text Available The first YTH domain-containing RNA binding protein (YTP was found in rat, where it was related to oxidative stress. Unlike characterizations in yeast and animals, functions of plant YTPs are less clear. Malus hupehensis (Pamp. Rehd. YTP1 and YTP2 (MhYTP1 and MhYTP2 are known to be active in leaf senescence and fruit ripening. However, no research has been published about their roles in stress responses. Here, we investigate the stress-related functions of MhYTP1 and MhYTP2 in Arabidopsis thaliana. Both of the two genes participated in salicylic acid (SA, jasmonic acid (JA, and abscisic acid (ABA signaling and play roles in plant responses to oxidative stress, chilling, high temperature, high salinity, and mannitol induced physiological drought stress. Moreover, MhYTP1 plays leading roles in SA and ABA signaling, and MhYTP2 plays leading roles in JA signaling and oxidative stress responses. These results will fill a gap in our knowledge about plant YTPs and stress responses and provide a foundation for future attempts to improve stress tolerance in apple.

  16. Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

    Science.gov (United States)

    Rugh, C L; Wilde, H D; Stack, N M; Thompson, D M; Summers, A O; Meagher, R B

    1996-01-01

    With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution. Images Fig. 2 Fig. 3 Fig. 4 PMID:8622910

  17. Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

    Science.gov (United States)

    Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

    2017-01-01

    In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

  18. Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots.

    Science.gov (United States)

    Bressan, Mélanie; Roncato, Marie-Anne; Bellvert, Floriant; Comte, Gilles; Haichar, Feth Zahar; Achouak, Wafa; Berge, Odile

    2009-11-01

    A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched (13)CO(2) atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.

  19. Effect of nickel on the organization of actin filaments in Arabidopsis thaliana primary root cells

    International Nuclear Information System (INIS)

    Goryunova, I.I.; Krasilenko, Yu.A.; Emets, A.I.; Blyum, Ya.B.

    2016-01-01

    The influence of one of the most toxic heavy metals - nickel (Ni 2+ ) - on the organization of actin filaments (microfilaments) of different types of Arabidopsis thaliana (L.) root cells is studied in living cells by the laser scanning microscopy. To visualize microfilaments, the A. thaliana line expressing chimeric gene gfp-fabd2 was used. Ni 2+ leads to a significant inhibition of the growth of the main root and disturbs its morphology, causing the swelling of epidermal cells and inducing a large number of abnormally long root hairs. For the first time, it has been shown that Ni 2+ disturbs the organization of actin filaments in cells, leading to morphological changes of a root as the main organ, being the first exposed to the intoxication by soil pollutants. It is found that the most sensitive to its action are actin filaments of epidermal cells of all growth zones of A. thaliana root

  20. 3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3.

    Science.gov (United States)

    Yoshikawa, Manabu; Iki, Taichiro; Tsutsui, Yasuhiro; Miyashita, Kyoko; Poethig, R Scott; Habu, Yoshiki; Ishikawa, Masayuki

    2013-03-05

    trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)-mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1-RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3' cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3' cleavage fragment. When the 3' nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3' cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1-RISC via the double-stranded RNA formed by the 3'-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1-RISC molecular surface, (ii) SGS3 protects the 3' cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3' fragment of TAS2 RNA is key to tasiRNA production.

  1. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  2. Characterization of a novel single-stranded RNA mycovirus in pleurotus ostreatus

    International Nuclear Information System (INIS)

    Yu, Hyun Jae; Lim, Dongbin; Lee, Hyun-Sook

    2003-01-01

    A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group

  3. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

    Science.gov (United States)

    Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

    2015-02-13

    Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

  4. 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

  5. Mutations in ribosomal proteins, RPL4 and RACK1, suppress the phenotype of a thermospermine-deficient mutant of Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kakehi

    Full Text Available Thermospermine acts in negative regulation of xylem differentiation and its deficient mutant of Arabidopsis thaliana, acaulis5 (acl5, shows excessive xylem formation and severe dwarfism. Studies of two dominant suppressors of acl5, sac51-d and sac52-d, have revealed that SAC51 and SAC52 encode a transcription factor and a ribosomal protein L10 (RPL10, respectively, and these mutations enhance translation of the SAC51 mRNA, which contains conserved upstream open reading frames in the 5' leader. Here we report identification of SAC53 and SAC56 responsible for additional suppressors of acl5. sac53-d is a semi-dominant allele of the gene encoding a receptor for activated C kinase 1 (RACK1 homolog, a component of the 40S ribosomal subunit. sac56-d represents a semi-dominant allele of the gene for RPL4. We show that the GUS reporter activity driven by the CaMV 35S promoter plus the SAC51 5' leader is reduced in acl5 and restored by sac52-d, sac53-d, and sac56-d as well as thermospermine. Furthermore, the SAC51 mRNA, which may be a target of nonsense-mediated mRNA decay, was found to be stabilized in these ribosomal mutants and by thermospermine. These ribosomal proteins are suggested to act in the control of uORF-mediated translation repression of SAC51, which is derepressed by thermospermine.

  6. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  7. Recent advances in biological effect and molecular mechanism of arabidopsis thaliana irradiated by ion beams

    International Nuclear Information System (INIS)

    Wu Dali; Hou Suiwen; Li Wenjian

    2008-01-01

    Newly research progresses were summarized in effect of ion beams on seed surface, biological effect, growth, development, gravitropism and so on. Furthermore, mutation molecular mechanism of Arabidopsis thaliana was discussed, for example, alteration of DNA bases, DNA damage, chromosomal recombination, characteristics of mutant transmissibility, etc. Meanwhile, the achievements of transfer- ring extraneous gene to Arabidopsis thaliana by ion beams were reviewed in the paper. At last, the future prospective are also discussed here in mutation molecular mechanism and the potential application of biological effect of heavy ion beams. (authors)

  8. RAPD analysis of Arabidopsis thaliana transferred with total DNA of cabbage by ion beam

    International Nuclear Information System (INIS)

    Bian Po; Yu Zengliang; Qin Guangyong; Huo Yuping; Wang Yan

    2003-01-01

    Two mutants were found among the Arabidopsis thaliana transferred with total DNA of cabbage. Variation of genome of T6 and its offspring were analyzed by RAPD-PCR with 40 random primers. The result from S168 primer was different from the CK, indicating that variation of genome can be made by total DNA transferring by use of ion beam, and this variation is hereditary. It is found that S 168-1850 is included within the gene of ABC transporter by aligning with genome of Arabidopsis thaliana in TAIT

  9. Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

    Science.gov (United States)

    Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

    2015-09-01

    Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

  10. The UEA Small RNA Workbench: A Suite of Computational Tools for Small RNA Analysis.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Applegate, Christopher Steven; Folkes, Leighton; Moulton, Vincent

    2017-01-01

    transcripts and a summary of their corresponding patterns. Finally we describe individual analysis tools such as PAREsnip, for the analysis of PARE (degradome) data or CoLIde for the identification of sRNA loci based on their expression patterns and the visualization of the results using the software. We illustrate the features of the UEA sRNA Workbench on Arabidopsis thaliana and Homo sapiens datasets.

  11. Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination.

    Science.gov (United States)

    Lewkowicz, Przemysław; Cwiklińska, Hanna; Mycko, Marcin P; Cichalewska, Maria; Domowicz, Małgorzata; Lewkowicz, Natalia; Jurewicz, Anna; Selmaj, Krzysztof W

    2015-05-13

    MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS. Copyright © 2015 the authors 0270-6474/15/357521-17$15.00/0.

  12. Genetic Analysis of Gravity Signal Transduction in Arabidopsis thaliana Seedlings

    Science.gov (United States)

    Boonsirichai, K.; Harrison, B.; Stanga, J.; Young, L.-S.; Neal, C.; Sabat, G.; Murthy, N.; Harms, A.; Sedbrook, J.; Masson, P.

    The primary roots of Arabidopsis thaliana seedlings respond to gravity stimulation by developing a tip curvature that results from differential cellular elongation on opposite flanks of the elongation zone. This curvature appears modulated by a lateral gradient of auxin that originates in the gravity-perceiving cells (statocytes) of the root cap through an apparent lateral repositioning of a component the auxin efflux carrier complex within these cells (Friml et al, 2002, Nature 415: 806-809). Unfortunately, little is known about the molecular mechanisms that govern early phases of gravity perception and signal transduction within the root-cap statocytes. We have used a molecular genetic approach to uncover some of these mechanisms. Mutations in the Arabidopsis ARG1 and ARL2 genes, which encode J-domain proteins, resulted in specific alterations in root and hypocotyl gravitropism, without pleiotropic phenotypes. Interestingly, ARG1 and ARL2 appear to function in the same genetic pathway. A combination of molecular genetic, biochemical and cell-biological approaches were used to demonstrate that ARG1 functions in early phases of gravity signal transduction within the root and hypocotyl statocytes, and is needed for efficient lateral auxin transport within the cap. The ARG1 protein is associated with components of the secretory and/or endosomal pathways, suggesting its role in the recycling of components of the auxin efflux carrier complex between plasma membrane and endosome (Boonsirichai et al, 2003, Plant Cell 15:2612-2625). Genetic modifiers of arg1-2 were isolated and shown to enhance the gravitropic defect of arg1-2, while resulting in little or no gravitropic defects in a wild type ARG1 background. A slight tendency for arg1-2;mar1-1 and arg1-2;mar2-1 double-mutant organs to display an opposite gravitropic response compared to wild type suggests that all three genes contribute to the interpretation of the gravity-vector information by seedling organs. The

  13. A circadian clock-regulated toggle switch explains AtGRP7 and AtGRP8 oscillations in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Christoph Schmal

    Full Text Available The circadian clock controls many physiological processes in higher plants and causes a large fraction of the genome to be expressed with a 24h rhythm. The transcripts encoding the RNA-binding proteins AtGRP7 (Arabidopsis thaliana Glycine Rich Protein 7 and AtGRP8 oscillate with evening peaks. The circadian clock components CCA1 and LHY negatively affect AtGRP7 expression at the level of transcription. AtGRP7 and AtGRP8, in turn, negatively auto-regulate and reciprocally cross-regulate post-transcriptionally: high protein levels promote the generation of an alternative splice form that is rapidly degraded. This clock-regulated feedback loop has been proposed to act as a molecular slave oscillator in clock output. While mathematical models describing the circadian core oscillator in Arabidopsis thaliana were introduced recently, we propose here the first model of a circadian slave oscillator. We define the slave oscillator in terms of ordinary differential equations and identify the model's parameters by an optimization procedure based on experimental results. The model successfully reproduces the pertinent experimental findings such as waveforms, phases, and half-lives of the time-dependent concentrations. Furthermore, we obtain insights into possible mechanisms underlying the observed experimental dynamics: the negative auto-regulation and reciprocal cross-regulation via alternative splicing could be responsible for the sharply peaking waveforms of the AtGRP7 and AtGRP8 mRNA. Moreover, our results suggest that the AtGRP8 transcript oscillations are subordinated to those of AtGRP7 due to a higher impact of AtGRP7 protein on alternative splicing of its own and of the AtGRP8 pre-mRNA compared to the impact of AtGRP8 protein. Importantly, a bifurcation analysis provides theoretical evidence that the slave oscillator could be a toggle switch, arising from the reciprocal cross-regulation at the post-transcriptional level. In view of this

  14. Gravity-regulated gene expression in Arabidopsis thaliana

    Science.gov (United States)

    Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

    Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

  15. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  16. Phytotoxicity of chiral herbicide bromacil: Enantioselectivity of photosynthesis in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Chen, Zunwei; Zou, Yuqin; Wang, Jia; Li, Meichao; Wen, Yuezhong

    2016-01-01

    With the wide application of chiral herbicides and the frequent detection of photosystem II (PSII) herbicides, it is of great importance to assess the direct effects of PSII herbicides on photosynthesis in an enantiomeric level. In the present study, the enantioselective phytotoxicity of bromacil (BRO), typical photosynthesis inhibition herbicide, on Arabidopsis thaliana was investigated. The results showed that S-BRO exhibited a greater inhibition of electron transmission in photosystem I (PSI) of A. thaliana than R-BRO by inhibiting the transcription of fnr 1. S-BRO also changed the chlorophyll fluorescence parameters Y (II), Y (NO), and Y (NPQ) to a greater extent than R-Bro. Transcription of genes psbO2, Lhcb3 and Lhcb6 was down-regulated in an enantioselective rhythm and S-BRO caused more serious influence, indicating that S-BRO did worse damage to the photosystem II (PSII) of A. thaliana than R-BRO. This study suggested that S-BRO disturbed the photosynthesis of plants to a larger extent than R-BRO and provided a new sight to evaluate the phytotoxicity of chiral herbicides. - Highlights: • It is necessary to assess the direct effects of PSII herbicides on photosynthesis. • Phytotoxicity of bromacil is investigated in an enantiomeric level. • Bromacil disturbed enantioselectively the photosystem II of Arabidopsis thaliana. • S-bromacil caused severer damage to photosynthesis of Arabidopsis than R-bromacil. • Photosynthesis should be considered for phytotoxicity assessment of herbicides.

  17. Pleiotropic effects of flowering time genes in the annual crucifer Arabidopsis thaliana (Brassicaceae)

    NARCIS (Netherlands)

    Van Tienderen, P.H.; Hammad, I.; Zwaal, F.C.

    1996-01-01

    Variation in flowering time of Arabidopsis thaliana was studied in an experiment with mutant lines. The pleiotropic effects of flowering time genes on morphology and reproductive yield were assessed under three levels of nutrient supply. At all nutrient levels flowering time and number of rosette

  18. Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Scheres, B.J.G.; Zaal, B.J. van der; Hooykaas, P.J.J.

    1996-01-01

    Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a λ clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions

  19. YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana

    KAUST Repository

    Kim, Jeong Im; Murphy, Angus S.; Baek, Dongwon; Lee, Shin-Woo; Yun, Dae-Jin; Bressan, Ray A.; Narasimhan, Meena L.

    2011-01-01

    The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical

  20. Contribution of the cytochrome and alternative pathways to growth respiration and maintenance respiration in Arabidopsis thaliana

    NARCIS (Netherlands)

    Florez-Sarasa, I.D.; Bouma, T.J.; Medrano, H.; Azcon-Bieto, J.; Ribas-Carbo, M.

    2007-01-01

    The activities of the cytochrome and alternative respiratory pathways were measured during the growth cycle in Arabidopsis thaliana using a newly developed Isotope Ratio Mass Spectrometer (IRMS) dual-inlet system that allows very precise measurements of oxygen-isotope fractionation under low oxygen

  1. Unearthing Bacillus endophytes from desert plants that enhance growth of Arabidopsis thaliana under abiotic stress conditions

    KAUST Repository

    Bokhari, Ameerah

    2018-01-01

    that these bacteria can confer resilience to plants under salt stress conditions. B. circulans (PK3-15 and PK3-109), B. cereus (PK6-15) B. subtilis (PK3-9) and B. licheniformis (PK5-26) displayed the ability to increased the fresh weight of A. thaliana under salt

  2. A composite transcriptional signature differentiates responses towards closely related herbicides in Arabidopsis thaliana and brassica napus

    Science.gov (United States)

    In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide co...

  3. Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Toorop, P.E.; Barroco, R.M.; Engler, G.; Groot, S.P.C.; Hilhorst, H.W.M.

    2005-01-01

    Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was

  4. An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana

    NARCIS (Netherlands)

    Aarts, M.G.M.

    1996-01-01


    At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the

  5. Phytotoxicity of chiral herbicide bromacil: Enantioselectivity of photosynthesis in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zunwei; Zou, Yuqin; Wang, Jia [MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China); Li, Meichao [Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou 310032 (China); Wen, Yuezhong, E-mail: wenyuezhong@zju.edu.cn [MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China)

    2016-04-01

    With the wide application of chiral herbicides and the frequent detection of photosystem II (PSII) herbicides, it is of great importance to assess the direct effects of PSII herbicides on photosynthesis in an enantiomeric level. In the present study, the enantioselective phytotoxicity of bromacil (BRO), typical photosynthesis inhibition herbicide, on Arabidopsis thaliana was investigated. The results showed that S-BRO exhibited a greater inhibition of electron transmission in photosystem I (PSI) of A. thaliana than R-BRO by inhibiting the transcription of fnr 1. S-BRO also changed the chlorophyll fluorescence parameters Y (II), Y (NO), and Y (NPQ) to a greater extent than R-Bro. Transcription of genes psbO2, Lhcb3 and Lhcb6 was down-regulated in an enantioselective rhythm and S-BRO caused more serious influence, indicating that S-BRO did worse damage to the photosystem II (PSII) of A. thaliana than R-BRO. This study suggested that S-BRO disturbed the photosynthesis of plants to a larger extent than R-BRO and provided a new sight to evaluate the phytotoxicity of chiral herbicides. - Highlights: • It is necessary to assess the direct effects of PSII herbicides on photosynthesis. • Phytotoxicity of bromacil is investigated in an enantiomeric level. • Bromacil disturbed enantioselectively the photosystem II of Arabidopsis thaliana. • S-bromacil caused severer damage to photosynthesis of Arabidopsis than R-bromacil. • Photosynthesis should be considered for phytotoxicity assessment of herbicides.

  6. Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

    DEFF Research Database (Denmark)

    Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.

    2005-01-01

    A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...

  7. The genetics of some planthormones and photoreceptors in Arabidopsis thaliana (L.) Heynh.

    NARCIS (Netherlands)

    Koornneef, M.

    1982-01-01

    This thesis describes the isolation and characterization in Arabidopsis thaliana (L.) Heynh. of induced mutants, deficient for gibberellins (GA's), abscisic acid (ABA) and photoreceptors.

    These compounds are known to regulate various facets of plant growth and

  8. Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties

    Czech Academy of Sciences Publication Activity Database

    Kowalska, M.; Galuszka, Petr; Frébortová, Jitka; Šebela, M.; Béres, Tibor; Hluska, T.; Šmehilová, M.; Bilyeu, K. D.; Frébort, Ivo

    2010-01-01

    Roč. 71, č. 17 (2010), s. 1970-1978 ISSN 0031-9422 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arabidopsis thaliana * Pichia pastoris expression system * Electron acceptor Subject RIV: CE - Biochemistry Impact factor: 3.150, year: 2010

  9. Sekvencování a funkční genomika Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Ondřej, M.; Kocábek, Tomáš

    2003-01-01

    Roč. 68, - (2003), s. 109-133 ISSN 0366-0486 R&D Projects: GA ČR GA521/00/D036 Institutional research plan: CEZ:AV0Z5051902 Keywords : Arabidopsis thaliana, genome Subject RIV: EB - Genetics ; Molecular Biology

  10. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely ...

  11. A previously undescribed jasmonate compound in flowering Arabidopsis thaliana - The identification of cis-(+)-OPDA-Ile

    Czech Academy of Sciences Publication Activity Database

    Floková, K.; Feussner, K.; Herrfurth, C.; Miersch, O.; Mik, V.; Tarkowská, Danuše; Strnad, Miroslav; Feussner, I.; Wasternack, Claus; Novák, Ondřej

    2016-01-01

    Roč. 122, FEB (2016), s. 230-237 ISSN 0031-9422 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk LK21306 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana (Brassicaceae) * Jasmonates * Cis-(+)-12-oxo-phytodienoyl-L- iso leucine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.205, year: 2016

  12. Electron transfer reactivity of the Arabidopsis thaliana sulfhydryl oxidase AtErv1

    DEFF Research Database (Denmark)

    Farver, Ole; Vitu, Elvira; Wherland, Scot

    2009-01-01

    The redox reactivity of the three disulfide bridges and the flavin present in each protomer of the wild-type Arabidopsis thaliana mitochondrial sulfhydryl oxidase (AtErv1) homodimer has been investigated. Pulse radiolytically produced CO2- radical ions were found to reduce the disulfide bridges...

  13. Numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and Arabidopsis thaliana

    NARCIS (Netherlands)

    Ji, X.

    2014-01-01

    Numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and Arabidopsis thaliana.

    I studied numerical and structural chromosome aberrations in cauliflower (Brassica oleracea var. botrytis) and

  14. Natural variation and QTL analysis for cationic mineral content in seeds of Arabidopsis thaliana.

    NARCIS (Netherlands)

    Vreugdenhil, D.; Aarts, M.G.M.; Koornneef, M.; Nelissen, H.J.M.; Ernst, W.H.O.

    2004-01-01

    Naturally occurring genetic variation for contents of cationic minerals in seeds of Arabidopsis thaliana was studied by screening a series of accessions (ecotypes) for Ca, Fe, K, Mg, Mn, Na, Zn, and for total contents of P. Variation was observed for all minerals and correlations between contents of

  15. Characterization and identification of microRNA core promoters in four model species.

    Directory of Open Access Journals (Sweden)

    Xuefeng Zhou

    2007-03-01

    Full Text Available MicroRNAs are short, noncoding RNAs that play important roles in post-transcriptional gene regulation. Although many functions of microRNAs in plants and animals have been revealed in recent years, the transcriptional mechanism of microRNA genes is not well-understood. To elucidate the transcriptional regulation of microRNA genes, we study and characterize, in a genome scale, the promoters of intergenic microRNA genes in Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Oryza sativa. We show that most known microRNA genes in these four species have the same type of promoters as protein-coding genes have. To further characterize the promoters of microRNA genes, we developed a novel promoter prediction method, called common query voting (CoVote, which is more effective than available promoter prediction methods. Using this new method, we identify putative core promoters of most known microRNA genes in the four model species. Moreover, we characterize the promoters of microRNA genes in these four species. We discover many significant, characteristic sequence motifs in these core promoters, several of which match or resemble the known cis-acting elements for transcription initiation. Among these motifs, some are conserved across different species while some are specific to microRNA genes of individual species.

  16. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  17. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    Science.gov (United States)

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-01-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.

  18. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  19. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2001-12-01

    Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

  20. Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana*S⃞

    Science.gov (United States)

    Kissen, Ralph; Bones, Atle M.

    2009-01-01

    Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and

  1. In silico comparison of transcript abundances during Arabidopsis thaliana and Glycine max resistance to Fusarium virguliforme

    Directory of Open Access Journals (Sweden)

    Iqbal M Javed

    2008-09-01

    Full Text Available Abstract Background Sudden death syndrome (SDS of soybean (Glycine max L. Merr. is an economically important disease, caused by the semi-biotrophic fungus Fusarium solani f. sp. glycines, recently renamed Fusarium virguliforme (Fv. Due to the complexity and length of the soybean-Fusarium interaction, the molecular mechanisms underlying plant resistance and susceptibility to the pathogen are not fully understood. F. virguliforme has a very wide host range for the ability to cause root rot and a very narrow host range for the ability to cause a leaf scorch. Arabidopsis thaliana is a host for many types of phytopathogens including bacteria, fungi, viruses and nematodes. Deciphering the variations among transcript abundances (TAs of functional orthologous genes of soybean and A. thaliana involved in the interaction will provide insights into plant resistance to F. viguliforme. Results In this study, we reported the analyses of microarrays measuring TA in whole plants after A. thaliana cv 'Columbia' was challenged with fungal pathogen F. virguliforme. Infection caused significant variations in TAs. The total number of increased transcripts was nearly four times more than that of decreased transcripts in abundance. A putative resistance pathway involved in responding to the pathogen infection in A. thaliana was identified and compared to that reported in soybean. Conclusion Microarray experiments allow the interrogation of tens of thousands of transcripts simultaneously and thus, the identification of plant pathways is likely to be involved in plant resistance to Fusarial pathogens. Dissection of the set functional orthologous genes between soybean and A. thaliana enabled a broad view of the functional relationships and molecular interactions among plant genes involved in F. virguliforme resistance.

  2. Molecular cloning of a seed specific multifunctional RFO synthase/ galactosylhydrolase in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Roman eGangl

    2015-09-01

    Full Text Available Stachyose is among the raffinose family oligosaccharides one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, raffinose family oligosaccharides are indigestible for humans and can contribute to diverse abdominal disorders.In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the raffinose family oligosaccharide physiology in A. thaliana.In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970 as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 µM as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only sqPCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the raffinose family oligosaccharide physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf-accumulation under abiotic stress.

  3. RNAmutants: a web server to explore the mutational landscape of RNA secondary structures

    Science.gov (United States)

    Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

    2009-01-01

    The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics—indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/. PMID:19531740

  4. Direct measurement of the poliovirus RNA polymerase error frequency in vitro

    International Nuclear Information System (INIS)

    Ward, C.D.; Stokes, M.A.M.; Flanegan, J.B.

    1988-01-01

    The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high, depending on the specific reaction conditions. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg 2+ (pH 7.0) to 7.0 mM Mg 2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study

  5. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.; Jé gu, Teddy; Latrasse, David; Romero-Barrios, Natali; Christ, Auré lie; Benhamed, Moussa; Crespi, Martí n D.

    2014-01-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  6. A novel monopartite dsRNA virus isolated from the entomopathogenic and nematophagous fungus Purpureocillium lilacinum.

    Science.gov (United States)

    Herrero, Noemi

    2016-12-01

    Purpureocillium lilacinum is a ubiquitous saprophytic fungus commonly isolated from soils and widely known as a biological control agent against phytopathogenic nematodes and pest insects. Mycoviruses infect a wide number of fungal species, but the study of viruses infecting entomopathogenic fungi is still quite recent. In this study, a total of 86 P. lilacinum isolates collected from soil in natural and cultivated habitats throughout the Czech Republic were analyzed; 22 % of the isolates harbored double-stranded RNA (dsRNA) elements with viral characteristics. These results suggest that mycoviruses are common in P. lilacinum. One of the most common dsRNA elements detected in the survey was completely sequenced and corresponded to the 2,864-bp genome of a previously undescribed mycovirus, designated Purpureocillium lilacinum nonsegmented virus 1 (PlNV-1). Phylogenetic analysis of the RNA-dependent RNA polymerase of PlNV-1 indicated that this virus might belong to a new taxon related to the family Partitiviridae.

  7. Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

    KAUST Repository

    Ariel, Federico D.

    2014-08-01

    The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

  8. T box riboswitches in Actinobacteria: Translational regulation via novel tRNA interactions

    Science.gov (United States)

    Sherwood, Anna V.; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch. PMID:25583497

  9. Methods for RNA Analysis

    DEFF Research Database (Denmark)

    Olivarius, Signe

    of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

  10. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  11. Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench.

    Science.gov (United States)

    Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

    2017-06-01

    Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. © 2017 Beckers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. The promoter of the Arabidopsis thaliana plastocyanin gene contains a far upstream enhancer-like element involved in chloroplast-dependent expression.

    Science.gov (United States)

    Vorst, O; Kock, P; Lever, A; Weterings, B; Weisbeek, P; Smeekens, S

    1993-12-01

    Plastocyanin is part of the photosynthetic electron transport chain in the chloroplast and is encoded in the nucleus. Expression of the Arabidopsis thaliana plastocyanin gene is organ specific: high mRNA levels are observed in young green parts of the plant. Furthermore, expression is dependent on the presence of light and functional chloroplasts. When grown in the presence of norflurazon under white light conditions, resulting in the photo-oxidative destruction of the chloroplast, plastocyanin mRNA levels are strongly reduced. A -1579 to -9 promoter fragment confers light-regulated and chloroplast-dependent expression to the beta-glucuronidase reporter gene in transgenic tobacco plants. This suggests that regulation takes place at the level of transcription. A plastocyanin promoter deletion series ranging from -1579 to -121 which was also tested in tobacco, revealed the presence of a strong positive regulating element (PRE) in the -1579 to -705 region. Deletion of this part of the promoter resulted in a approximately 100-fold reduction of GUS expression as measured in mature leaves. Surprisingly, this enhancer-like element was capable of stimulating transcription from a position downstream of its reporter. Moreover, it could also activate a truncated CaMV 35S promoter. Deletion of this element coincides with the loss of chloroplast-dependency of reporter gene expression, as judged by norflurazon treatment of transgenic seedlings. So, the activity of the PRE itself might depend on the presence of functional chloroplasts.

  13. Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

    Directory of Open Access Journals (Sweden)

    Allan Olspert

    2016-06-01

    Full Text Available Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV and murine norovirus (MNV. These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

  14. An environment with strong gravitational and magnetic field alterations synergizes to promote variations in Arabidopsis thaliana callus global transcriptional state

    Data.gov (United States)

    National Aeronautics and Space Administration — Using diamagnetic levitation we have exposed A. thaliana in vitro callus cultures to five environments with different levels of effective gravity (from levitation...

  15. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-01-01

    transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time

  16. The CUP-SHAPED COTYLEDON2 and 3 genes have a post-meristematic effect on Arabidopsis thaliana phyllotaxis

    KAUST Repository

    Burian, Agata; Raczyńska-Szajgin, Magdalena; Borowska-Wykręt, Dorota; Piatek, Agnieszka Anna; Aida, Mitsuhiro; Kwiatkowska, Dorota

    2015-01-01

    Background and Aims: The arrangement of flowers in inflorescence shoots of Arabidopsis thaliana represents a regular spiral Fibonacci phyllotaxis. However, in the cuc2 cuc3 double mutant, flower pedicels are fused to the inflorescence stem

  17. Time-series of the re-establishment of desiccation tolerance by ABA in germinated Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Dias Costa, Maria; Righetti, K.; Ligterink, Wilco; Buitink, J.; Hilhorst, Henk

    2015-01-01

    Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose DT while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with abscisic acid (ABA).

  18. Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

    Science.gov (United States)

    Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

    2014-01-03

    Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome

  19. Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression.

    Science.gov (United States)

    Bertea, Cinzia M; Narayana, Ravishankar; Agliassa, Chiara; Rodgers, Christopher T; Maffei, Massimo E

    2015-11-30

    One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

  20. Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

    Science.gov (United States)

    Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

    2014-08-28

    Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

  1. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

  2. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-09-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

  3. Biological effects and oxidative stress responses in Arabidopsis thaliana following exposure to uranium and copper

    Energy Technology Data Exchange (ETDEWEB)

    Horemans, N.; Saenen, E.; Vandenhove, H.; Vanhoudt, N.; Wannijn, J.; Nauts, R. [Belgian Nuclear Research Centre SCK-CEN (Belgium); Vangronsveld, J.; Cuypers, A. [Hasselt University (Belgium)

    2014-07-01

    Since organisms are almost always exposed to multiple stressors, it is important to investigate the toxicity effects in plants in a multiple stressor context to provide a more realistic estimate of environmental risks. Therefore, we evaluated the toxicity of U and Cu individually and in combination. Arabidopsis thaliana plants were exposed during 3 days to 25 μM U, 2.5 μM Cu or 12.5 μM U + 1.25 μM Cu at pH 7.5. The concentrations of U and Cu administered to the plants were the derived EC30 values for plant growth reduction based on the single-dose response curves. When plants were exposed to U or Cu, this resulted in an increased metal concentration in both roots and shoots as compared to the control plants. The increased Cu content of the plant, led to a significant decrease in leaf and root growth, while U exposure did not affect plant growth. By exposing plants to both metals, it seems that Cu interferes with the translocation of U from the roots to the leaves. In addition, U interferes with the Cu uptake, since less Cu was found in the roots than expected. This can probably be explained by the fact that U causes a shift in the Cu speciation in our medium. As such, the percentage of the bioavailable fractions of Cu decreased, while the percentage Cu-EDTA (i.e. non-available fraction) increased. No effects on plant growth were observed by exposing plants to U+Cu. Exposing plants to heavy metals can lead to an increased production of reactive oxygen species (ROS). After U or Cu exposure, the transcript levels of different ROS-producing enzymes (NADPH oxidases and lipoxygenases (LOX)) were up-regulated in the roots. Furthermore, exposing plants to U+Cu resulted in a 20-fold increase in the expression of LOX1 as compared to the single stressor conditions. This possibly indicates an enhanced ROS production. In addition, the increased expression of LOX1 can also indicate an increased production of oxylipins, important molecules in inter-organ signalling. In the

  4. Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

    Science.gov (United States)

    Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

    2014-09-22

    Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Allelopathic Effects of Plant-Derived Aerosol Smoke on Seed Germination of Arabidopsis thaliana (L.) Heynh

    International Nuclear Information System (INIS)

    Pennacchio, M.; Jefferson, L.V.; Havens, K.

    2007-01-01

    The role that plant-derived smoke plays in promoting seed germination is well documented, but little is known about its ability to inhibit seed germination. To better understand this phenomenon, we tested the effects of eight aerosol smoke treatments on the Columbia-3 ecotype of non dormant Arabidopsis thaliana (L.) Heynh. seeds. Our results revealed that aerosol smoke significantly inhibits germination when seeds were exposed to prolonged periods of aerosol smoke. Short durations of smoke treatments significantly promoted the rate of germination of A. thaliana seed. We briefly discuss this dual regulation of smoke and its possible impact on conservation and restoration practices. We also propose that plant-derived smoke may be another vehicle by which allelo chemicals can be introduced into the environment.

  6. Toxicity and transfer of CuO Nanoparticles on Arabidopsis thaliana

    Science.gov (United States)

    Zhao, Shilin; Dai, Yanhui; Xu, Lina

    2018-02-01

    CuO engineered nanoparticles (ENPs) are widely used in commercial applications. With increasing CuO ENPs production, CuO ENPs are likely to present in the environment and cause a potential threaten to ecosystem. In this work, Arabidopsis thaliana (Bay-0) was chosen to take the toxic experiment after exposed to CuO ENPs (0, 20, and 50 mg/L) and Cu2+ (0.15 mg/L). And the copper content of shoots at 50 mg/L CuO ENPs was about 20 times of control, indicating that CuO ENPs could be absorbed into Arabidopsis thaliana seedlings and transfered from root to shoot in a certain way.

  7. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Bajaj, Mamta; Moriyama, Hideaki

    2007-01-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V M of 1.8 Å 3 Da −1

  8. Strictly NO3- Nutrition Alleviates Iron Deficiency Chlorosis in Arabidopsis thaliana Plants

    Directory of Open Access Journals (Sweden)

    Najoua Msilini

    2014-03-01

    Full Text Available The effects of NO3- nutrition on iron deficiency responses were investigated in Arabidopsis thaliana. Plants were grown with or without 5 µM Fe, and with NO3- alone or a mixture of NO3- and NH4+. The results indicated that, NO3- nutrition induced higher dry matter production, regardless the Fe concentration. Fe deficiency reduced growth activity, photosynthetic pigment concentration and Fe content of plants, whatever the N forms. This decrease was more pronounced in plants grown with mixed N source; those plants presented the highest EL and MDA and anthocyanin contents compared to plants grown under Fe sufficient conditions. In iron free-solutions, with NO3- as the sole nitrogen source, enhanced FC-R activity in the roots was observed. However, in the presence of NH4+, plants displayed some decrease in in FC-R and PEPC activities. The presence of NH4+ modified typical Fe stress responses in Arabidopsis thaliana plants.

  9. Genome-wide association study of Arabidopsis thaliana leaf microbial community.

    Science.gov (United States)

    Horton, Matthew W; Bodenhausen, Natacha; Beilsmith, Kathleen; Meng, Dazhe; Muegge, Brian D; Subramanian, Sathish; Vetter, M Madlen; Vilhjálmsson, Bjarni J; Nordborg, Magnus; Gordon, Jeffrey I; Bergelson, Joy

    2014-11-10

    Identifying the factors that influence the outcome of host-microbial interactions is critical to protecting biodiversity, minimizing agricultural losses and improving human health. A few genes that determine symbiosis or resistance to infectious disease have been identified in model species, but a comprehensive examination of how a host genotype influences the structure of its microbial community is lacking. Here we report the results of a field experiment with the model plant Arabidopsis thaliana to identify the fungi and bacteria that colonize its leaves and the host loci that influence the microbe numbers. The composition of this community differs among accessions of A. thaliana. Genome-wide association studies (GWAS) suggest that plant loci responsible for defense and cell wall integrity affect variation in this community. Furthermore, species richness in the bacterial community is shaped by host genetic variation, notably at loci that also influence the reproduction of viruses, trichome branching and morphogenesis.

  10. Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

    Science.gov (United States)

    Kawaguchi, Kosuke; Yurimoto, Hiroya; Oku, Masahide; Sakai, Yasuyoshi

    2011-01-01

    The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels. PMID:21966472

  11. Natural genetic variation in Arabidopsis thaliana defense metabolism genes modulates field fitness.

    Science.gov (United States)

    Kerwin, Rachel; Feusier, Julie; Corwin, Jason; Rubin, Matthew; Lin, Catherine; Muok, Alise; Larson, Brandon; Li, Baohua; Joseph, Bindu; Francisco, Marta; Copeland, Daniel; Weinig, Cynthia; Kliebenstein, Daniel J

    2015-04-13

    Natural populations persist in complex environments, where biotic stressors, such as pathogen and insect communities, fluctuate temporally and spatially. These shifting biotic pressures generate heterogeneous selective forces that can maintain standing natural variation within a species. To directly test if genes containing causal variation for the Arabidopsis thaliana defensive compounds, glucosinolates (GSL) control field fitness and are therefore subject to natural selection, we conducted a multi-year field trial using lines that vary in only specific causal genes. Interestingly, we found that variation in these naturally polymorphic GSL genes affected fitness in each of our environments but the pattern fluctuated such that highly fit genotypes in one trial displayed lower fitness in another and that no GSL genotype or genotypes consistently out-performed the others. This was true both across locations and within the same location across years. These results indicate that environmental heterogeneity may contribute to the maintenance of GSL variation observed within Arabidopsis thaliana.

  12. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, Mamta [School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States); Moriyama, Hideaki, E-mail: hmoriyama2@unl.edu [Department of Chemistry, e-Toxicology and Biotechnology, University of Nebraska-Lincoln, Hamilton Hall, Lincoln, Nebraska 68588-0304 (United States); School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States)

    2007-05-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V{sub M} of 1.8 Å{sup 3} Da{sup −1}.

  13. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.; Marondedze, Claudius

    2015-01-01

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  14. Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana.

    Science.gov (United States)

    Yunus, Ian Sofian; Cazenave-Gassiot, Amaury; Liu, Yu-Chi; Lin, Ying-Chen; Wenk, Markus R; Nakamura, Yuki

    2015-01-01

    Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.

  15. Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Lu, Lu; Nan, Jie; Mi, Wei; Wei, Chun-Hong; Li, Lan-Fen; Li, Yi

    2010-01-01

    Tubulin-folding cofactor A from A. thaliana has been crystallized and preliminarily analyzed using X-ray diffraction. Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the β-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289 K. The crystal diffracted to 1.6 Å resolution using synchrotron radiation and belonged to space group I4 1 , with unit-cell parameters a = 55.0, b = 55.0, c = 67.4 Å

  16. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  17. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    Science.gov (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  18. Comparative radioresistance of chronically irradiated populations of Arabidopsis thaliana (L.) Heynh

    International Nuclear Information System (INIS)

    Dineva, S.B.; Abramov, V.I.; Shevchenko, V.A.

    1994-01-01

    The radioresistance of seeds of populations of Arabidopsis thaliana (L.) Heynh. growing for 5 years in the regions with different levels of radioactive contamination within 30 km zone of Chernobyl NPP was studied. The analysis of comparative radiosensitivity by root test was performed. It has been shown that plants from arabidopsis population growing under chronic irradiation did not gain an increased radioresistance. The data obtained shown that they are more radiosensitive

  19. Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system

    Czech Academy of Sciences Publication Activity Database

    Maršík, Petr; Šíša, Miroslav; Lacina, O.; Moťková, Kateřina; Langhansová, Lenka; Rezek, Jan; Vaněk, Tomáš

    2017-01-01

    Roč. 220, JAN (2017), s. 383-392 ISSN 0269-7491 R&D Projects: GA ČR(CZ) GA14-22593S Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * Ibuprofen * Metabolism * Plant cells * Sequestration Subject RIV: CE - Biochemistry OBOR OECD: Plant sciences, botany Impact factor: 5.099, year: 2016

  20. Analyse fonctionnelle de la protéine WSCP chez Arabidopsis thaliana

    OpenAIRE

    Boex-Fontvieille , Edouard

    2010-01-01

    Class II WSCP proteins (Watersoluble Chlorophyll binding Proteins) are soluble proteins that interact with chlorophyll and its derivatives in Brassicaceae. These proteins belong to the protease inhibitor family and they are induced by abiotic stress under light conditions. To date, their functions in plants are not well documented. The aim of this study is to characterize the physiological function of a WSCP protein in model plants Arabidopsis thaliana. We demonstrated initially that theWSCP ...

  1. The instability of the flax element LIS-1 in transgenic Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bastaki NK

    2015-05-01

    Full Text Available Nasmah K Bastaki, Christopher A Cullis Department of Biology, Case Western Reserve University, Cleveland, OH, USA Background: The LIS-1 is an element that appears as a site-specific insertion event in some flax lines in response to certain growth conditions and can be transmitted to subsequent generations. The origin of LIS-1 in the flax genome is uncertain. One possibility is that since LIS-1 does not exist intact in the progenitor line, it is assembled from small sequences found scattered throughout the genome, and that, under stressful growth conditions, induction occurs and these sequences are rearranged and assembled to form the intact LIS-1 element. It is unknown whether the intact LIS-1 element would remain stably integrated in other plant species or if it would be destabilized from their genome. Results: In this study, Agrobacterium-mediated plant transformation via floral dipping was used to transform different accessions of the Columbia ecotype of Arabidopsis thaliana, with either LIS-1 or the target site into which LIS-1 integrates. The stability and the inheritance patterns of both elements were followed in subsequent generations. Our results indicate that, in the different transformed accessions, the target site of LIS-1 remains stable in the T1 and T2 generations. However, LIS-1 is not found intact in any transformed A. thaliana plants. Instead, it goes through multiple fragmentation events, which seem to be genotype dependent. In the process, the region originally flanking LIS-1 in the T-DNA construct can be converted to the same sequence found at the target site in flax, followed by complete excision of all the flax DNA in the construct. Conclusion: These results demonstrate that the processes by which LIS-1 is produced in flax are also present in A. thaliana because both plants are capable of destabilizing the intact LIS-1 element.Keywords: flax (Linum usitatissimum, Arabidopsis thaliana, plant transformation, Linum insertion

  2. ML3: a novel regulator of herbivory-induced responses in Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Fridborg, I.; Johansson, A.; Lagensjo, J.; Leelarasamee, N.; Floková, Kristýna; Tarkowská, Danuše; Meijer, J.; Bejai, S.

    2013-01-01

    Roč. 64, č. 4 (2013), s. 935-948 ISSN 0022-0957 R&D Projects: GA AV ČR KAN200380801 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Arabidopsis thaliana * herbivory * jasmonic acid Subject RIV: EC - Immunology Impact factor: 5.794, year: 2013

  3. Round-shape gold nanoparticles: effect of particle size and concentration on Arabidopsis thaliana root growth

    Czech Academy of Sciences Publication Activity Database

    Siegel, J.; Záruba, K.; Švorčík, V.; Kroumanová, Kristýna; Burketová, Lenka; Martinec, Jan

    2018-01-01

    Roč. 13, APR 10 (2018), č. článku 95. ISSN 1556-276X R&D Projects: GA ČR GA17-10907S; GA ČR(CZ) GBP108/12/G108 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * Concentration * Gold nanoparticles * Root growth * Size Subject RIV: JJ - Other Materials OBOR OECD: Plant sciences, botany Impact factor: 2.833, year: 2016

  4. The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

    Science.gov (United States)

    Ravi, Maruthachalam; Kwong, Pak N; Menorca, Ron M G; Valencia, Joel T; Ramahi, Joseph S; Stewart, Jodi L; Tran, Robert K; Sundaresan, Venkatesan; Comai, Luca; Chan, Simon W-L

    2010-10-01

    Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

  5. Isoflavonoids are present in Arabidopsis thaliana despite the absence of any homologue to known isoflavonoid synthases

    Czech Academy of Sciences Publication Activity Database

    Lapčík, O.; Honys, David; Koblovská, R.; Macková, Z.; Vítková, M.; Klejdus, B.

    2006-01-01

    Roč. 44, 2-3 (2006), s. 106-114 ISSN 0981-9428 R&D Projects: GA ČR GA525/03/0352; GA AV ČR KJB6038409 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arabidopsis thaliana * Brassicaceae * HPLC-MS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.847, year: 2006

  6. Mass spectrometry-based metabolomic fingerprinting for screening cold tolerance in Arabidopsis thaliana accessions

    Czech Academy of Sciences Publication Activity Database

    Václavík, L.; Mishra, Anamika; Mishra, Kumud; Hajslova, J.

    2013-01-01

    Roč. 405, č. 8 (2013), s. 2671-2683 ISSN 1618-2642 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0073; GA MŠk OC08055 Institutional support: RVO:67179843 Keywords : cold tolerance * Arabidopsis thaliana * metabolomic fingerprinting * LC-MS * DART-MS * chemometric analysis Subject RIV: EH - Ecology, Behaviour Impact factor: 3.578, year: 2013

  7. Madeiran Arabidopsis thaliana reveals ancient long-range colonization and clarifies demography in Eurasia.

    Science.gov (United States)

    Fulgione, Andrea; Koornneef, Maarten; Roux, Fabrice; Hermisson, Joachim; Hancock, Angela M

    2017-12-05

    The study of model organisms on islands may shed light on rare long-range dispersal events, uncover signatures of local evolutionary processes, and inform demographic inference on the mainland. Here, we sequenced the genomes of Arabidopsis thaliana samples from the oceanic island of Madeira. These samples include the most diverged worldwide, likely a result of long isolation on the island. We infer that colonisation of Madeira happened between 70 and 85 kya, consistent with a propagule dispersal model (of size > =10), or with an ecological window of opportunity. This represents a clear example of a natural long-range dispersal event in A. thaliana. Long-term effective population size on the island, rather than the founder effect, had the greatest impact on levels of diversity, and rates of coalescence. Our results uncover a selective sweep signature on the ancestral haplotype of a known translocation in Eurasia, as well as the possible importance of the low phosphorous availability in volcanic soils, and altitude, in shaping early adaptations to the island conditions. Madeiran genomes, sheltered from the complexities of continental demography, help illuminate ancient demographic events in Eurasia. Our data support a model in which two separate lineages of A. thaliana, one originating in Africa and the other from the Caucasus expanded and met in Iberia, resulting in a secondary contact zone there. While previous studies inferred that the westward expansion of A. thaliana coincided with the spread of human agriculture, our results suggest it happened much earlier (20-40 kya). © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. Kontrolle der Expression des UNUSUAL FLORAL ORGANS (UFO) Gens in Arabidopsis thaliana

    OpenAIRE

    Hobe, Martin

    2004-01-01

    Die vorliegende Arbeit befaßt sich mit der Kontrolle des Expressionsmusters des UNUSUAL FLORAL ORGANS (UFO) Gens von Arabidopsis thaliana. UFO wird im Sproß- und Blütenmeristemen aller Entwicklungsstadien der Pflanze exprimiert. In Blütenmeristemen agiert UFO als Kofaktor von LEAFY (LFY) bei der Aktivierung der Organidentitätsgene des zweiten und dritten Wirtels. UFO stellt also einen generellen Faktor der Musterbildung in Meristemen dar. Um regulatorische Gene, die die Expression von UFO bee...

  9. PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Mozgová, Iva; Munoz-Viana, R.; Hennig, L.

    2017-01-01

    Roč. 13, č. 1 (2017), s. 1-27, č. článku e1006562. ISSN 1553-7404 R&D Projects: GA ČR(CZ) GJ16-08423Y; GA MŠk(CZ) LO1416 Institutional support: RVO:61388971 Keywords : PRC2 * SE formation * Arabidopsis thaliana Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 6.100, year: 2016

  10. Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus.

    Directory of Open Access Journals (Sweden)

    Nemanja Vukašinović

    Full Text Available Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370 locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.

  11. Chlorophyll fluorescence emission can screen cold tolerance of cold acclimated Arabidopsis thaliana accessions

    Czech Academy of Sciences Publication Activity Database

    Mishra, Anamika; Heyer, A. G.; Mishra, Kumud

    2014-01-01

    Roč. 10, č. 38 (2014) ISSN 1746-4811 R&D Projects: GA MŠk EE2.3.20.0246; GA MŠk 7E12047 Institutional support: RVO:67179843 Keywords : high-throughput screening * chlorophyll a fluorescence transients * cold tolerance * cold acclimation * whole plant * Arabidopsis thaliana Subject RIV: EH - Ecology, Behaviour Impact factor: 3.102, year: 2014

  12. Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus

    Czech Academy of Sciences Publication Activity Database

    Vukašinović, Nemanja; Cvrčková, F.; Eliáš, M.; Cole, R.; Fowler, J.E.; Žárský, Viktor; Synek, Lukáš

    2014-01-01

    Roč. 9, č. 4 (2014) E-ISSN 1932-6203 R&D Projects: GA ČR GPP501/11/P853; GA ČR(CZ) GAP305/11/1629 Grant - others:GA MŠk ME10033 Institutional support: RVO:61389030 Keywords : WHOLE-GENOME * ARABIDOPSIS-THALIANA * RECENT SEGMENTAL DUPLICATIONS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  13. In silico Identifikation von Abszisinsaeure-regulierten Genen in Arabidopsis thaliana (L.) Heynh.

    OpenAIRE

    Gómez-Porras, Judith Lucia

    2006-01-01

    Abscisic acid (ABA) is a major plant hormone that plays an important role during plant growth and development. During vegetative growth ABA mediates (in part) responses to various environmental stresses such as cold, drought and high salinity. The response triggered by ABA includes changes in the transcript level of genes involved in stress tolerance. The aim of this project was the In silico identification of genes putatively regulated by ABA in A. thaliana. In silico predictions were combin...

  14. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  15. The AraGWAS Catalog: a curated and standardized Arabidopsis thaliana GWAS catalog

    Science.gov (United States)

    Togninalli, Matteo; Seren, Ümit; Meng, Dazhe; Fitz, Joffrey; Nordborg, Magnus; Weigel, Detlef

    2018-01-01

    Abstract The abundance of high-quality genotype and phenotype data for the model organism Arabidopsis thaliana enables scientists to study the genetic architecture of many complex traits at an unprecedented level of detail using genome-wide association studies (GWAS). GWAS have been a great success in A. thaliana and many SNP-trait associations have been published. With the AraGWAS Catalog (https://aragwas.1001genomes.org) we provide a publicly available, manually curated and standardized GWAS catalog for all publicly available phenotypes from the central A. thaliana phenotype repository, AraPheno. All GWAS have been recomputed on the latest imputed genotype release of the 1001 Genomes Consortium using a standardized GWAS pipeline to ensure comparability between results. The catalog includes currently 167 phenotypes and more than 222 000 SNP-trait associations with P < 10−4, of which 3887 are significantly associated using permutation-based thresholds. The AraGWAS Catalog can be accessed via a modern web-interface and provides various features to easily access, download and visualize the results and summary statistics across GWAS. PMID:29059333

  16. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  17. Phenotypic effects of salt and heat stress over three generations in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Léonie Suter

    Full Text Available Current and predicted environmental change will force many organisms to adapt to novel conditions, especially sessile organisms such as plants. It is therefore important to better understand how plants react to environmental stress and to what extent genotypes differ in such responses. It has been proposed that adaptation to novel conditions could be facilitated by heritable epigenetic changes induced by environmental stress, independent of genetic variation. Here we assessed phenotypic effects of heat and salt stress within and across three generations using four highly inbred Arabidopsis thaliana genotypes (Col, Cvi, Ler and Sha. Salt stress generally decreased fitness, but genotypes were differently affected, suggesting that susceptibility of A. thaliana to salt stress varies among genotypes. Heat stress at an early rosette stage had less detrimental effects but accelerated flowering in three out of four accessions. Additionally, we found three different modes of transgenerational effects on phenotypes, all harboring the potential of being adaptive: heat stress in previous generations induced faster rosette growth in Sha, both under heat and control conditions, resembling a tracking response, while in Cvi, the phenotypic variance of several traits increased, resembling diversified bet-hedging. Salt stress experienced in earlier generations altered plant architecture of Sha under salt but not control conditions, similar to transgenerational phenotypic plasticity. However, transgenerational phenotypic effects depended on the type of stress as well as on genotype, suggesting that such effects may not be a general response leading to adaptation to novel environmental conditions in A. thaliana.

  18. Hydroxymethylated Dioxobilins in Senescent Arabidopsis thaliana Leaves: Sign of a Puzzling Biosynthetic Intermezzo of Chlorophyll Breakdown.

    Science.gov (United States)

    Süssenbacher, Iris; Kreutz, Christoph R; Christ, Bastien; Hörtensteiner, Stefan; Kräutler, Bernhard

    2015-08-10

    1-Formyl-19-oxobilin-type tetrapyrroles are characteristic, abundant products of chlorophyll breakdown in senescent leaves. However, in some leaves, 1,19-dioxobilin-type chlorophyll catabolites (DCCs) lacking the formyl group accumulate instead. A P450 enzyme was identified in in vitro studies that removed the formyl group of a primary fluorescent chlorophyll catabolite (pFCC) and generated fluorescent DCCs. These DCCs are precursors of isomeric nonfluorescent DCCs (NDCCs). Here, we report a structural investigation of the NDCCs in senescent leaves of wild-type Arabidopsis thaliana. Four new NDCCs were characterized, two of which carried a stereoselectively added hydroxymethyl group. Such formal DCC hydroxymethylations were previously found in DCCs in leaves of a mutant of A. thaliana. They are now indicated to be a feature of chlorophyll breakdown in A. thaliana, associated with the specific in vivo deformylation of pFCC en route to NDCCs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Human Norovirus and Its Surrogates Induce Plant Immune Response in Arabidopsis thaliana and Lactuca sativa.

    Science.gov (United States)

    Markland, Sarah M; Bais, Harsh; Kniel, Kalmia E

    2017-08-01

    Human norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways was measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants that were inoculated with Murine Norovirus-1, Tulane Virus, human norovirus GII.4, or Hank's Balanced Salt Solution (control). Genes involving both the SA and JA pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, as well as controls. Studies, including gene expression of SA- and JA-deficient A. thaliana mutant lines, suggest that the JA pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the preharvest environment.

  20. The structure of the nucleoprotein binding domain of lyssavirus phosphoprotein reveals a structural relationship between the N-RNA binding domains of Rhabdoviridae and Paramyxoviridae.

    Science.gov (United States)

    Delmas, Olivier; Assenberg, Rene; Grimes, Jonathan M; Bourhy, Hervé

    2010-01-01

    The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.

  1. Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

    Directory of Open Access Journals (Sweden)

    Rajeev K. Varshney

    2017-04-01

    Full Text Available Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA mediated defense in plants. Small RNAs (sRNAs have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL, Argonaute (AGO, and RNA dependent RNA polymerase (RDR constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

  2. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  3. Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Ira eDeveson

    2013-04-01

    Full Text Available Plant and animal microRNA (miRNA pathways share many analogous components, the ARGONAUTE (AGO proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2 and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1, which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta.

  4. JNSViewer-A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures.

    Science.gov (United States)

    Shi, Jieming; Li, Xi; Dong, Min; Graham, Mitchell; Yadav, Nehul; Liang, Chun

    2017-01-01

    Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome) were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html.

  5. JNSViewer—A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures

    Science.gov (United States)

    Dong, Min; Graham, Mitchell; Yadav, Nehul

    2017-01-01

    Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome) were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html. PMID:28582416

  6. JNSViewer-A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures.

    Directory of Open Access Journals (Sweden)

    Jieming Shi

    Full Text Available Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html.

  7. Assembling RNA Nanoparticles.

    Science.gov (United States)

    Xiao, Shou-Jun

    2017-01-01

    RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

  8. Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.

    Science.gov (United States)

    Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

    2011-12-02

    The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.

  9. Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5.

    Science.gov (United States)

    Louber, Jade; Brunel, Joanna; Uchikawa, Emiko; Cusack, Stephen; Gerlier, Denis

    2015-07-28

    The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5'-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5-2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial. A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged. The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response

  10. The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2 [v1; ref status: indexed, http://f1000r.es/201

    Directory of Open Access Journals (Sweden)

    Benjamin K Dickerman

    2013-10-01

    Full Text Available The primary function of the dsRNA binding protein (dsRBP PACT/RAX is to activate the dsRNA dependent protein kinase PKR in response to stress signals.  Additionally, it has been identified as a component of the small RNA processing pathway.  A role for PACT/RAX in this pathway represents an important interplay between two modes of post-transcriptional gene regulation.  The function of PACT/RAX in this context is poorly understood.  Thus, additional models are required to clarify the mechanism by which PACT/RAX functions.  In this study, Drosophila melanogaster was employed to identify functionally orthologous dsRNA-binding proteins.  Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis.  Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.

  11. The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification.

    Science.gov (United States)

    Zhang, Chi; Montgomery, Taiowa A; Fischer, Sylvia E J; Garcia, Susana M D A; Riedel, Christian G; Fahlgren, Noah; Sullivan, Christopher M; Carrington, James C; Ruvkun, Gary

    2012-05-22

    In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Shapes of interacting RNA complexes

    DEFF Research Database (Denmark)

    Fu, Benjamin Mingming; Reidys, Christian

    2014-01-01

    Shapes of interacting RNA complexes are studied using a filtration via their topological genus. A shape of an RNA complex is obtained by (iteratively) collapsing stacks and eliminating hairpin loops.This shape-projection preserves the topological core of the RNA complex and for fixed topological...... genus there are only finitely many such shapes. Our main result is a new bijection that relates the shapes of RNA complexes with shapes of RNA structures. This allows to compute the shape polynomial of RNA complexes via the shape polynomial of RNA structures. We furthermore present a linear time uniform...... sampling algorithm for shapes of RNA complexes of fixed topological genus....

  13. Remote Network Access (RNA)

    National Research Council Canada - National Science Library

    2002-01-01

    .... Remote Network Access (RNA) includes or is associated with all communication devices/software, firewalls, intrusion detection systems and virus protection applications to ensure security of the OIG, DoD, Network from remote...

  14. RNA/PNA Approach

    Indian Academy of Sciences (India)

    In this approach we want to develop structural analogue of the leader that might have higher affinity towards the Phosphoprotein, but would impair the dimerization process and viral leader RNA binding.

  15. Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

    Directory of Open Access Journals (Sweden)

    Bianca Genenncher

    2018-02-01

    Full Text Available The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5 RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

  16. Comparative evolution history of SINEs in Arabidopsis thaliana and Brassica oleracea: evidence for a high rate of SINE loss.

    Science.gov (United States)

    Lenoir, A; Pélissier, T; Bousquet-Antonelli, C; Deragon, J M

    2005-01-01

    Brassica oleracea and Arabidopsis thaliana belong to the Brassicaceae(Cruciferae) family and diverged 16 to 19 million years ago. Although the genome size of B. oleracea (approximately 600 million base pairs) is more than four times that of A. thaliana (approximately 130 million base pairs), their gene content is believed to be very similar with more than 85% sequence identity in the coding region. Therefore, this important difference in genome size is likely to reflect a different rate of non-coding DNA accumulation. Transposable elements (TEs) constitute a major fraction of non-coding DNA in plant species. A different rate in TE accumulation between two closely related species can result in significant genome size variations in a short evolutionary period. Short interspersed elements (SINEs) are non-autonomous retroposons that have invaded the genome of most eukaryote species. Several SINE families are present in B. oleracea and A. thaliana and we found that two of them (called RathE1 and RathE2) are present in both species. In this study, the tempo of evolution of RathE1 and RathE2 SINE families in both species was compared. We observed that most B. oleracea RathE2 SINEs are "young" (close to the consensus sequence) and abundant while elements from this family are more degenerated and much less abundant in A. thaliana. However, the situation is different for the RathE1 SINE family for which the youngest elements are found in A. thaliana. Surprisingly, no SINE was found to occupy the same (orthologous) genomic locus in both species suggesting that either these SINE families were not amplified at a significant rate in the common ancestor of the two species or that older elements were lost and only the recent (lineage-specific) insertions remain. To test this latter hypothesis, loci containing a recently inserted SINE in the A. thaliana col-0 ecotype were selected and characterized in several other A. thaliana ecotypes. In addition to the expected SINE containing

  17. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  18. Diverse evolutionary trajectories for small RNA biogenesis genes in the oomycete genus Phytophthora

    Directory of Open Access Journals (Sweden)

    Stephanie eBollmann

    2016-03-01

    Full Text Available Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL, and RNA-dependent RNA polymerase (RDR through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.

  19. The first phlebo-like virus infecting plants: a case study on the adaptation of negative-stranded RNA viruses to new hosts.

    Science.gov (United States)

    Navarro, Beatriz; Minutolo, Maria; De Stradis, Angelo; Palmisano, Francesco; Alioto, Daniela; Di Serio, Francesco

    2018-05-01

    A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  20. RNA sequence determinants of a coupled termination-reinitiation strategy for downstream open reading frame translation in Helminthosporium victoriae virus 190S and other victoriviruses (Family Totiviridae).

    Science.gov (United States)

    Li, Hua; Havens, Wendy M; Nibert, Max L; Ghabrial, Said A

    2011-07-01

    The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.

  1. Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D.

    Science.gov (United States)

    Newburn, Laura R; White, K Andrew

    2017-04-15

    Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3' untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3' to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed. IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional

  2. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

    2009-01-01

    to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  3. Broad distribution spectrum from Gaussian to power law appears in stochastic variations in RNA-seq data.

    Science.gov (United States)

    Awazu, Akinori; Tanabe, Takahiro; Kamitani, Mari; Tezuka, Ayumi; Nagano, Atsushi J

    2018-05-29

    Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions. In many recent transcriptome analyses based on RNA sequencing (RNA-seq), variations in gene expression levels among replicates were assumed to follow a negative binomial distribution, although the physiological basis of this assumption remains unclear. In this study, RNA-seq data were obtained from Arabidopsis thaliana under eight conditions (21-27 replicates), and the characteristics of gene-dependent empirical probability density function (ePDF) profiles of gene expression levels were analyzed. For A. thaliana and Saccharomyces cerevisiae, various types of ePDF of gene expression levels were obtained that were classified as Gaussian, power law-like containing a long tail, or intermediate. These ePDF profiles were well fitted with a Gauss-power mixing distribution function derived from a simple model of a stochastic transcriptional network containing a feedback loop. The fitting function suggested that gene expression levels with long-tailed ePDFs would be strongly influenced by feedback regulation. Furthermore, the features of gene expression levels are correlated with their functions, with the levels of essential genes tending to follow a Gaussian-like ePDF while those of genes encoding nucleic acid-binding proteins and transcription factors exhibit long-tailed ePDF.

  4. Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

    Science.gov (United States)

    Hajjaran, Homa; Mahdi, Maryam; Mohebali, Mehdi; Samimi-Rad, Katayoun; Ataei-Pirkooh, Angila; Kazemi-Rad, Elham; Naddaf, Saied Reza; Raoofian, Reza

    2016-12-01

    Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

  5. 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

  6. Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection.

    Science.gov (United States)

    Barah, Pankaj; Winge, Per; Kusnierczyk, Anna; Tran, Diem Hong; Bones, Atle M

    2013-01-01

    Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth) during insect Brevicoryne brassicae (B. brassicae henceforth) and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth) attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and

  7. Reconstruction and analysis of nutrient-induced phosphorylation networks in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Guangyou eDuan

    2013-12-01

    Full Text Available Elucidating the dynamics of molecular processes in living organisms in response to external perturbations is a central goal in modern systems biology. We investigated the dynamics of protein phosphorylation events in Arabidopsis thaliana exposed to changing nutrient conditions. Phosphopeptide expression levels were detected at five consecutive time points over a time interval of 30 minutes after nutrient resupply following prior starvation. The three tested inorganic, ionic nutrients NH4+, NO3-, PO43- elicited similar phosphosignaling responses that were distinguishable from those invoked by the sugars mannitol, sucrose. When embedded in the protein-protein interaction network of Arabidopsis thaliana, phosphoproteins were found to exhibit a higher degree compared to average proteins. Based on the time-series data, we reconstructed a network of regulatory interactions mediated by phosphorylation. The performance of different network inference methods was evaluated by the observed likelihood of physical interactions within and across different subcellular compartments and based on gene ontology semantic similarity. The dynamic phosphorylation network was then reconstructed using a Pearson correlation method with added directionality based on partial variance differences. The topology of the inferred integrated network corresponds to an information dissemination architecture, in which the phosphorylation signal is passed on to an increasing number of phosphoproteins stratified into an initiation, processing, and effector layer. Specific phosphorylation peptide motifs associated with the distinct layers were identified indicating the action of layer-specific kinases. Despite the limited temporal resolution, combined with information on subcellular location, the available time-series data proved useful for reconstructing the dynamics of the molecular signaling cascade in response to nutrient stress conditions in the plant Arabidopsis thaliana.

  8. Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

    Science.gov (United States)

    Sammons, R Douglas; You, Jinsong; Qi, Youlin; Flasinski, Stanislaw; Kavanaugh, Christina; Washam, Jeannie; Ostrander, Elizabeth; Wang, Dafu; Heck, Greg

    2018-05-01

    Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T 102 I, P 106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS K i ∼ 0.5 μM) was engineered to express a variant AtEPSPS-T 102 I, P 106 A (TIPA K i = 150 μM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC 50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha -1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

  9. Introduction of tri-antennary N-glycans in Arabidopsis thaliana plants.

    Science.gov (United States)

    Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Weterings, Koen

    2012-04-01

    Because the pathway for protein synthesis is largely conserved between plants and animals, plants provide an attractive platform for the cost effective and flexible production of biopharmaceuticals. However, there are some differences in glycosylation between plants and humans that need to be considered before plants can be used as an efficient expression platform. In the presented research the human genes encoding α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) were introduced in the fast cycling model plant Arabidopsis thaliana to synthesize tri-antennary N-glycans. The GnT-IV and -V enzymes were targeted to the Golgi apparatus with plant-specific localization signals. The experiments were performed both in a wild type background, as well as in plants lacking β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) activity. Glycan analysis of endogenous proteins in the transgenic lines using CE-LIF showed that tri-antennary N-glycans could be produced in the XylT/FucT deficient line, while these structures were not found in the wild type background. Since β-N-acetylhexosaminidases, that remove terminal GlcNAcs, are active in A. thaliana plants, the specificity of these enzymes for different GlcNAc linkages was tested. The results showed that there is no pronounced preference of the A. thaliana hexosaminidases for human-type GlcNAc-linkages. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Michael D W Griffin

    Full Text Available In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS and dihydrodipicolinate reductase (DHDPR catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2 has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S-lysine. Structural studies of At-DHDPS2 show (S-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2 has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

  11. Comparative metabolic profiling of Haberlea rhodopensis, Thellungiella halophyla, and Arabidopsis thaliana exposed to low temperature

    Directory of Open Access Journals (Sweden)

    Maria eBenina

    2013-12-01

    Full Text Available Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea’s remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4°C and subsequent return to optimal temperatures was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive A. thaliana. The effect of the low temperature treatment in the three species was confirmed by gene expression of low-temperature- and dehydration-inducible genes. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21°C. H. rhodopensis had significantly more raffinose, melibiose, trehalose, myo-inositol, sorbitol, and galactinol than the other two species. A. thaliana had the highest levels of putrescine and fumarate, while T. halophila had much higher levels of several amino acids, including alanine, asparagine, beta-alanine, histidine, isoleucine, phenylalanine, serine, threonine, and valine. In addition, the three species responded differently to the low temperature treatment and the subsequent recovery, especially with regard to the sugar metabolism. Chilling induced accumulation of maltose in Haberlea and raffinose in A. thaliana, but raffinose levels in low temperature exposed Arabidopsis were still much lower than these in unstressed Haberlea. While all species accumulated sucrose during chilling, that accumulation was transient in Haberlea and Arabidopsis but sustained in T. halophila after the return to optimal temperature. In T. halophila, the levels of proline and hydroxyproline drastically increased upon recovery. Collectively, these results show inherent. differences in the metabolomes under the ambient temperature and the strategies to respond to low

  12. Burkholderia phytofirmans PsJN reduces damages to freezing temperature in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Fan eSU

    2015-10-01

    Full Text Available Several plant growth-promoting rhizobacteria (PGPR are known to improve plant tolerance to multiple stresses, including low temperatures. However, mechanisms underlying this protection are still poorly understood. The aim of this study was to evaluate the role of the endophytic PGPR, Burkholderia phytofirmans strain PsJN (Bp PsJN, on Arabidopsis thaliana cold tolerance using photosynthesis parameters as physiological markers.Under standard conditions, our results indicated that Bp PsJN inoculation led to growth promotion of Arabidopsis plants without significant modification on photosynthesis parameters and chloroplast organization. However, bacterial colonization induced a cell wall strengthening in the mesophyllImpact of inoculation modes (either on seeds or by soil irrigation and their effects overnight at 0, -1 or -3°C, were investigated by following photosystem II (PSII activity and gas exchanges. Following low temperatures stress, a decrease of photosynthesis parameters was observed. In addition, during three consecutive nights or days at -1°C, PSII activity was monitored. Pigment contents, RuBisCO protein abundance, expression of several genes including RbcS, RbcL, CBF1, CBF2, CBF3, ICE1, COR15a, and COR78 were evaluated at the end of exposure. To assess the impact of the bacteria on cell ultrastructure under low temperatures, microscopic observations were achieved. Results indicated that freezing treatment induced significant changes in PSII activity as early as the first cold day, whereas the same impact on PSII activity was observed only during the third cold night. The significant effects conferred by PsJN were differential accumulation of pigments, and reduced expression of RbcL and COR78. Microscopical observations showed an alteration/disorganization in A. thaliana leaf mesophyll cells independently of the freezing treatments. The presence of bacteria during the three successive nights or days did not significantly improved A

  13. Enhancement of Chlorogenic Acid Production in Hairy Roots of Platycodon grandiflorum by Over-Expression of An Arabidopsis thaliana Transcription Factor AtPAP1

    Directory of Open Access Journals (Sweden)

    Pham Anh Tuan

    2014-08-01

    Full Text Available To improve the production of chlorogenic acid (CGA in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1 using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.

  14. Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana.

    Science.gov (United States)

    Reimegård, Johan; Kundu, Snehangshu; Pendle, Ali; Irish, Vivian F; Shaw, Peter; Nakayama, Naomi; Sundström, Jens F; Emanuelsson, Olof

    2017-04-07

    Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. The Simbox Experiment with Arabidopsis Thaliana Cell Cultures: Hardware-Tests and First Resutls from the German-Chinese satellite Mission Shenzhou 8

    Science.gov (United States)

    Fengler, Svenja; Neef, Maren; Ecke, Margret; Hampp, Ruediger

    2013-02-01

    The Simbox experiment was the first joint German-Chinese space project. In this context Arabidopsis thaliana cell cultures were exposed to microgravity for a 17-day period. To carry out a successful space mission, diverse hardware tests were performed in advance. Due to the limited oxygen supply inside the hardware units, cells were fixed after 5 days under microgravity conditions. As a control, samples were exposed in an on-board 1g reference centrifuge. To investigate the space effect, a ground-based study was performed with the same hardware and identical experimental procedures. As we were able to obtain high quality RNA from the RNAlater quenched samples, we used the Affymetrix Arabidopsis genome array for a transcriptome analysis. Our experiment aimed at the identification of plant genes that were differentially expressed after long-term exposure to microgravity. Pair-wise comparison of flight samples with 1g controls revealed the largest differences between space 1g and ground 1g controls.

  16. Arabidopsis thaliana plants expressing Rift Valley fever virus antigens: Mice exhibit systemic immune responses as the result of oral administration of the transgenic plants.

    Science.gov (United States)

    Kalbina, Irina; Lagerqvist, Nina; Moiane, Bélisario; Ahlm, Clas; Andersson, Sören; Strid, Åke; Falk, Kerstin I

    2016-11-01

    The zoonotic Rift Valley fever virus affects livestock and humans in Africa and on the Arabian Peninsula. The economic impact of this pathogen due to livestock losses, as well as its relevance to public health, underscores the importance of developing effective and easily distributed vaccines. Vaccines that can be delivered orally are of particular interest. Here, we report the expression in transformed plants (Arabidopsis thaliana) of Rift Valley fever virus antigens. The antigens used in this study were the N protein and a deletion mutant of the Gn glycoprotein. Transformed lines were analysed for specific mRNA and protein content by RT-PCR and Western blotting, respectively. Furthermore, the plant-expressed antigens were evaluated for their immunogenicity in mice fed the transgenic plants. After oral intake of fresh transgenic plant material, a proportion of the mice elicited specific IgG antibody responses, as compared to the control animals that were fed wild-type plants and of which none sero-converted. Thus, we show that transgenic plants can be readily used to express and produce Rift Valley Fever virus proteins, and that the plants are immunogenic when given orally to mice. These are promising findings and provide a basis for further studies on edible plant vaccines against the Rift Valley fever virus. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis.

    Science.gov (United States)

    Iwasaki, T; Yamaguchi-Shinozaki, K; Shinozaki, K

    1995-05-20

    In Arabidopsis thaliana, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) but the gene does not include any sequence corresponding to the consensus ABA-responsive element (ABRE), RYACGTGGYR, in its promoter region. The cis-regulatory region of the rd22 promoter was identified by monitoring the expression of beta-glucuronidase (GUS) activity in leaves of transgenic tobacco plants transformed with chimeric gene fusions constructed between 5'-deleted promoters of rd22 and the coding region of the GUS reporter gene. A 67-bp nucleotide fragment corresponding to positions -207 to -141 of the rd22 promoter conferred responsiveness to dehydration and ABA on a non-responsive promoter. The 67-bp fragment contains the sequences of the recognition sites for some transcription factors, such as MYC, MYB, and GT-1. The fact that accumulation of rd22 mRNA requires protein synthesis raises the possibility that the expression of rd22 might be regulated by one of these trans-acting protein factors whose de novo synthesis is induced by dehydration or ABA. Although the structure of the RD22 protein is very similar to that of a non-storage seed protein, USP, of Vicia faba, the expression of the GUS gene driven by the rd22 promoter in non-stressed transgenic Arabidopsis plants was found mainly in flowers and bolted stems rather than in seeds.

  18. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and ph...

  19. A Shortest-Path-Based Method for the Analysis and Prediction of Fruit-Related Genes in Arabidopsis thaliana.

    Science.gov (United States)

    Zhu, Liucun; Zhang, Yu-Hang; Su, Fangchu; Chen, Lei; Huang, Tao; Cai, Yu-Dong

    2016-01-01

    Biologically, fruits are defined as seed-bearing reproductive structures in angiosperms that develop from the ovary. The fertilization, development and maturation of fruits are crucial for plant reproduction and are precisely regulated by intrinsic genetic regulatory factors. In this study, we used Arabidopsis thaliana as a model organism and attempted to identify novel genes related to fruit-associated biological processes. Specifically, using validated genes, we applied a shortest-path-based method to identify several novel genes in a large network constructed using the protein-protein interactions observed in Arabidopsis thaliana. The described analyses indicate that several of the discovered genes are associated with fruit fertilization, development and maturation in Arabidopsis thaliana.

  20. Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

    Science.gov (United States)

    Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

    2016-07-01

    The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

  1. A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis

    DEFF Research Database (Denmark)

    Ohnuma, Takayuki; Numata, Tomoyuki; Osawa, Takuo

    2011-01-01

    Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA......, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common...

  2. ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

  3. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Hoff, Tine; Frandsen, Gitte Inselmann; Rocher, Anne

    1998-01-01

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde...... oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis....

  4. Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds

    LENUS (Irish Health Repository)

    McKeown, Peter C

    2011-08-12

    Abstract Background Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. Results cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was

  5. Radiosensitivity of Arabidopsis thaliana L. in condition of influence of low ionizing radiation doses

    International Nuclear Information System (INIS)

    Shershunova, V.I.

    2000-01-01

    Arabidopsis thaliana is a convenient genetic object. This work represents the date of laboratory experiments concerning research of influence of chronic γ-irradiation on plants of arabidopsis at rosette stage (short stemmed mutant Lansberg Erecta). The findings contribute to the high sensitivity of rosette stage of arabidopsis to irradiation by γ-rays in low doses (0.67-10.0 cGy). It is shown in depressing effects of ionising radiation on growth, development, vitality and bearing of plants, but also in hightened output morphological anomalies of plants and embryonic lethalities in pods. (authors)

  6. The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    DANIELA C URBINA

    2006-01-01

    Full Text Available Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 µM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism

  7. Blue and Green Light-Induced Phototropism in Arabidopsis thaliana and Lactuca sativa L. Seedlings 1

    Science.gov (United States)

    Steinitz, Benjamin; Ren, Zhangling; Poff, Kenneth L.

    1985-01-01

    Exposure time-response curves for blue and green light-induced phototropic bending in hypocotyls of Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. seedlings are presented. These seedlings show significant phototropic sensitivity up to 540 to 550 nanometers. Since wave-lengths longer than 560 nanometers do not induce phototropic bending, it is suggested that the response to 510 to 550 nanometers light is mediated by the specific blue light photoreceptor of phototropism. We advise care in the use of green `safelights' for studies of phototropism. PMID:16664021

  8. Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds

    Directory of Open Access Journals (Sweden)

    Wennblom Trevor J

    2011-08-01

    Full Text Available Abstract Background Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. Results cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination. We identified these MEGs by developing a bioinformatics tool (GenFrag which can directly determine the identities of transcript-derived fragments from (i their size and (ii which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1

  9. Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice.

    Science.gov (United States)

    Wang, Ningning; Zhang, Di; Wang, Zhenhui; Xun, Hongwei; Ma, Jian; Wang, Hui; Huang, Wei; Liu, Ying; Lin, Xiuyun; Li, Ning; Ou, Xiufang; Zhang, Chunyu; Wang, Ming-Bo; Liu, Bao

    2014-06-30

    Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown. We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.

  10. Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.

    Directory of Open Access Journals (Sweden)

    Luis Apolonia

    2015-01-01

    Full Text Available The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G and APOBEC3F (A3F, act as potent human immunodeficiency virus type-1 (HIV-1 restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.

  11. An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

    Science.gov (United States)

    Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

    2017-01-01

    We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L

  12. A ribosome without RNA

    Directory of Open Access Journals (Sweden)

    Harold S Bernhardt

    2015-11-01

    Full Text Available It was Francis Crick who first asked why the ribosome contains so much RNA, and discussed the implications of this for the direct flow of genetic information from DNA to protein. Remarkable advances in our understanding of the ribosome and protein synthesis, including the recent publication of two mammalian mitochondrial ribosome structures, have shed new light on this intriguing aspect of evolution in molecular biology. We examine here whether RNA is indispensable for coded protein synthesis, or whether an all-protein ‘ribosome’ (or ‘synthosome’ might be possible, with a protein enzyme catalyzing peptide synthesis, and release factor-like protein adaptors able to read a message composed of deoxyribonucleotides. We also compare the RNA world hypothesis with the alternative ‘proteins first’ hypothesis in terms of their different understandings of the evolution of the ribosome, and whether this might have been preceded by an ancestral form of nonribosomal peptide synthesis catalyzed by protein enzymes.

  13. Pyrite footprinting of RNA

    International Nuclear Information System (INIS)

    Schlatterer, Jörg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

    2012-01-01

    Highlights: ► RNA structure is mapped by pyrite mediated · OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High · OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ( · OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS 2 ) can produce sufficient · OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to · OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated · OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

  14. RNA Regulation of Estrogen

    Science.gov (United States)

    2010-08-01

    Berglund, Rodger Voelker, Paul Barber and Julien Diegel 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING...estrogen  receptors  [reviewed  in  (3,  4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA...Mog myelin oligodendrocyte glycoprotein 6.06 207115_x_at mbtd1 mbt domain containing 1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at

  15. RNA Regulation by Estrogen

    Science.gov (United States)

    2011-08-01

    Julien Diegel, Amy Mahady, and Micah Bodner 5e. TASK NUMBER E-Mail: aberglund@molbio.uoregon.edu 5f. WORK UNIT NUMBER 7. PERFORMING...4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA  processing  events  such  as  splicing...1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at NOTCH4 Notch homolog 4 (Drosophila) 6.06 211203_s_at Cntn1 contactin 1 6.06 220689_at

  16. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.......Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

  17. RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

    Directory of Open Access Journals (Sweden)

    Andronescu Mirela

    2008-08-01

    Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

  18. Sensitivity of two ecotypes of Arabidopsis Thaliana (Cvi and Te) towards UV-B irradiation

    International Nuclear Information System (INIS)

    Velichkova, M.; Stanoeva, D.; Popova, A.

    2013-01-01

    he susceptibility of Arabidopsis thaliana towards the detrimental effect of UV-B irradiation was investigated using two ecotypes, Cvi and Te. The effect of UV-B treatment on primary photosynthetic reactions - energy interaction between the main pigment-protein complexes and oxygen evolution, was evaluated at low (4 0 C) and at room (22 0 C) temperature. UV-B-induced alterations of investigated photosynthetic reactions are better expressed at 22 0 C than at 4 0 C for Cvi. For Te ecotype the energy interaction was suppressed to higher extent at 22 0 C, while oxygen evolving activity was affected similarly at both temperatures. At low and room temperature, the energy interaction in the complex PSII-core antenna is affected stronger by UV-B treatment than the energy distribution between both photosystems, as revealed by fluorescence ratios of 77 K spectra. The results presented indicate that the Arabidopsis thaliana ecotype Cvi (Cape Verde Islands) is less affected by UV-B irradiation in respect to the investigated primary photosynthetic reactions than the ecotype Te (Finland)

  19. Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Rao, M.V.; Paliyath, G.; Ormrod, D.P.

    1996-01-01

    Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O 3 ) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O 3 -induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O 3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O 3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O 3 , enhanced the activation oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O 3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O 3 , UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity. 10 figs., 4 tabs

  20. Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

    Science.gov (United States)

    Zhao, Yu; Liu, Xuehe; Xu, Ziyan; Yang, Hui; Li, Jixi

    2017-07-22

    Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4 IKD ). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4 IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4 IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4 IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4 IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana. Copyright © 2017 Elsevier Inc. All rights reserved.