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Artykuły w czasopismach na temat "Cellular RNA"
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Tanabe, Shihori. "Cellular Internalization And RNA Regulation Of RNA Virus." Advances In Clinical And Medical Research 1, no. 1 (May 11, 2020): 1. http://dx.doi.org/10.52793/acmr.2020.1(1)-02.
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Elfaituri, Safa, and Fatma Emaetig. "Cellular Internalization And RNA Regulation Of RNA Virus." Advances In Clinical And Medical Research 1, no. 1 (May 11, 2020): 1–11. http://dx.doi.org/10.52793/acmr.2022.3(2)-29.
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Wurtmann, Elisabeth J., and Sandra L. Wolin. "RNA under attack: Cellular handling of RNA damage." Critical Reviews in Biochemistry and Molecular Biology 44, no. 1 (February 2009): 34–49. http://dx.doi.org/10.1080/10409230802594043.
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Kretz, Markus. "TINCR, staufen1, and cellular differentiation." RNA Biology 10, no. 10 (October 2013): 1597–601. http://dx.doi.org/10.4161/rna.26249.
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Wang, Miao, Zeqian Gao, Li Pan, and Yongguang Zhang. "Cellular microRNAs and Picornaviral Infections." RNA Biology 11, no. 7 (June 12, 2014): 808–16. http://dx.doi.org/10.4161/rna.29357.
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DeRose, Victoria J. "Sensing cellular magnesium with RNA." Nature Chemical Biology 3, no. 11 (November 2007): 693–94. http://dx.doi.org/10.1038/nchembio1107-693.
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Casci, Tanita. "RNA device rewires cellular networks." Nature Reviews Molecular Cell Biology 12, no. 1 (December 8, 2010): 5. http://dx.doi.org/10.1038/nrm3034.
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Biamonti, Giuseppe, and Javier F. Caceres. "Cellular stress and RNA splicing." Trends in Biochemical Sciences 34, no. 3 (March 2009): 146–53. http://dx.doi.org/10.1016/j.tibs.2008.11.004.
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Yi, Chengqi, and Tao Pan. "Cellular Dynamics of RNA Modification." Accounts of Chemical Research 44, no. 12 (December 20, 2011): 1380–88. http://dx.doi.org/10.1021/ar200057m.
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Parlea, Lorena, Anu Puri, Wojciech Kasprzak, Eckart Bindewald, Paul Zakrevsky, Emily Satterwhite, Kenya Joseph, Kirill A. Afonin, and Bruce A. Shapiro. "Cellular Delivery of RNA Nanoparticles." ACS Combinatorial Science 18, no. 9 (August 26, 2016): 527–47. http://dx.doi.org/10.1021/acscombsci.6b00073.
Pełny tekst źródłaRozprawy doktorskie na temat "Cellular RNA"
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Hunt, Sarah Louise. "Cellular proteins required for rhinovirus RNA translation." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313880.
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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.
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Bailey, Daniel John. "Cellular proteins in picornavirus replication." Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298484.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
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The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Osborn, Maire. "Cellular RNA Targeting by Platinum (II) Anticancer Therapeutics." Thesis, University of Oregon, 2014. http://hdl.handle.net/1794/17920.
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Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis.
Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets.
This dissertation includes previously published and unpublished co-authored material.
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Brown, E. C. "Cellular proteins involved in translation of human rhinovirus RNA." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596963.
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Translation of picornavirus RNA takes place by internal initiation, determined by the presence of an internal ribosome entry site (IRES) in the 5'-untranslated region of the genomic RNA. Efficient translation from the human rhinovirus-2 (HRV-2) IRES is dependent on host cell <I>trans</I>-acting factors. These include unr, p38 and polypyrimidine tract binding protein (PTB). This thesis details the investigation into how these factors act to promote translation from the HRV-2 IRES. Unr, an RNA-binding protein with five cold-shock domains (CSDs), binds to the HRV-2 IRES and this interaction was studied by crude and then fine mapping of the binding sites of unr on the IRES. The functions of the CSDs of unr were investigated by point mutation of each of the CSDs and testing the ability of these mutants to bind the IRES and stimulate translation from it. p38, a WD-motif protein with no RNA-binding activity, was expressed using recombinant baculovirus-infected insect cells. An <I>in vivo</I> interaction between unr and p38 was demonstrated, and the effect of p38 on unr's binding to the HRV-2 IRES was tested <I>in vivo. </I>After gaining insight into the complexes of unr and p38 that form on the IRES, the function of p38 in translation from the HRV-2 IRES was demonstrated. Unr and PTB were also used as tools to compare the factor requirements of the HRV-2 and poliovirus IRESs for efficient translation. Finally, an investigation was made into the cellular role of unr, in terms of the cellular mRNAs that unr binds, and those whose translation it stimulates.
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Todorova, Tanya (Tanya Todorova). "Function and regulation of PARP13 binding to cellular RNA." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97789.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.<br>Cataloged from PDF version of thesis. Vita.<br>Includes bibliographical references.<br>Poly(ADP-ribose) polymerase-13 (PARP13) is a member of the PARP family of proteins - enzymes that use NAD+ to synthesize a posttranslational protein modification called poly(ADP-ribose) (PAR). PARPs function in multiple cellular pathways, and recently several members of the family have been implicated in regulating various steps in RNA metabolism, from splicing to translation and decay. PARP1 3 is the best-understood RNA-regulatory PARP. Initially discovered as a host immune factor, PARP13 functions by binding viral transcripts via its four CCCH-type zinc fingers and targeting them for degradation. In the context of the immune response PARP1 3 can also inhibit the translation of its viral targets and enhance the activity of other RNA-binding viral receptors, such as RIG-1. More recently PARP13 was shown to also indirectly regulate the cellular transcriptome by inhibiting the activity of Argonaute 2 (Ago2), a member of the miRNA silencing pathway. While itself catalytically inactive, PARP13 is modified by PAR and can target Ago2 for modification by a yet unknown PARP. However, it remains unclear if RNA binding is required for this function of PARP1 3. Indeed, even though multiple viruses are known to be restricted by PARP13, cellular mRNA targets of PARP13 binding and regulation have not yet been identified. Here we show that PARP1 3 binds endogenous RNA and regulates the cellular transcriptome. We identify TRAILR4 mRNA as the first cellular target of PARP13 regulation and demonstrate that PARP13 represses TRAILR4 expression posttranscriptionally by binding to a specific region in the 3' untranslated region of the transcript and targeting it for degradation in a primarily 3'-5' decay mechanism. By inhibiting the expression of TRAILR4 - a decoy pro-survival receptor of the apoptotic ligand TRAIL, PARP1 3 regulates the cellular response to TRAIL and acts as a pro-apoptotic factor. We also examine possible mechanisms of regulation of PARP1 3 function. We identify the RNA-helicase DHX30 as a constitutive PARP1 3-interacting protein and show that the two proteins co-regulate a subset of cellular transcripts. We further demonstrate that the PAR-binding domain of PARP1 3 inhibits RNA binding, while PARP1 3 interaction with PARP5a and covalent modification with PAR appear to be mutually exclusive with RNA binding.<br>by Tanya Todorova.<br>Ph. D.
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Mullani, Nowsheen. "An RNA Signature Links Oxidative Stress To Cellular Senescence." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS560.pdf.
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Le stress oxydatif est l’une des voies menant à la sénescence cellulaire. Bien que les dommages causés par les espèces réactives de l'oxygène aux protéines et à l'ADN soient bien décrits, notre compréhension de la manière dont la transcription peut participer à l'apparition de la sénescence est encore limitée. Au niveau de la transcription, le stress oxydatif entraîne l’accumulation d’ARN promoteurs (ARNAu) et d’ARN amplificateur (ARNs), conséquence de la libération défectueuse du RNAPII de la chromatine, un phénomène connu sous le nom de RNAPII crawling. Nous avons observé que l'exploration de RNAPII était également détectée en aval d'une petite série de gènes connus pour être régulés par HP1Υ au niveau de leur terminaison. L'exploration de ce phénomène a donné un résultat inattendu, en ce sens qu'il a révélé un effet inhibiteur du peroxyde d'hydrogène sur le complexe exosome d'ARN impliqué dans la dégradation des ARN polyadénylés. Le RNAPII rampant a pour résultat la transcription de séquences d’ALU situées au voisinage des promoteurs et amplificateurs et en aval de gènes sans intron et de petites séries de gènes contenant un intron. Comme les séquences ALU contiennent des séquences A codées par le génome, elles doivent normalement être dégradées par l’exosome de l’ARN. Cependant, comme le stress oxydatif inhibe également cette activité d'ARNase, les ARNm contenant des séquences d'ALU transcrites par hasard se stabilisent et sont détectés dans le cytoplasme et même dans les fractions de polysomes. Ce phénomène peut participer à l'apparition de la réponse à l'interféron associée au stress oxydatif<br>Oxidative Stress is one of the routes leading to cellular senescence. While the damages that reactive oxygen species inflict on proteins and DNA are well described, our insight on how transcription may participate in the onset of senescence is still limited. At a transcriptional level, oxidative stress results in accumulation of promoter RNAs (uaRNAs) and enhancer RNAs (eRNAs) as a consequence of defective release of the RNAPII from the chromatin a phenomenon known as RNAPII crawling. We observed that RNAPII crawling was also detected downstream of a small series of genes known to be regulated by HP1Υ at the level of their termination. Exploring this phenomenon yielded an unexpected result in the sense that it revealed an inhibiting effect of hydrogen peroxide on the RNA exosome complex involved in degradation of polyadenylated RNAs. The crawling RNAPII results in the transcription of ALU sequences located in the neighborhood of promoters and enhancers and downstream of intron-less genes and of small series of intron-containing genes. As ALU sequences contain genome encoded A tracts, they should normally be degraded by the RNA exosome. Yet, as oxidative stress also inhibits this RNAse activity, mRNAs containing serendipitously transcribed ALU sequences get stabilized and are detected in the cytoplasm and even polysome fractions. This phenomenon may participate in the onset of the interferon response associated with oxidative stress
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Stassinopoulos, Ioannis A. "Interactions of picornavirus internal ribosome entry sites with cellular proteins." Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322940.
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Schmier, Brad J. "The Molecular Machinery Critical to the Degradation of Cellular RNA." Scholarly Repository, 2012. http://scholarlyrepository.miami.edu/oa_dissertations/714.
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Exoribonucleases are indispensable for cellular RNA metabolism. RNA processing, end-turnover, and degradation all require the concerted action of exoribonucleases. In this thesis, two families of exoribonucleases that act in the final steps of RNA decay pathways are explored. The first of these is the RNR superfamily of processive 3’→5’ RNases with major roles in both mRNA and stable RNA degradation. The initial focus of this work is the structural and enzymatic characterization of an unusual RNR family enzyme from the radiation-resistant bacterium Deinococcus radiodurans. This enzyme is demonstrated biochemically to be an RNase II-type enzyme (DrII), based on its sensitivity to secondary structure. Analysis of the DrII X-ray structure reveals that a novel, winged-HTH domain has replaced the canonical RNA binding clamp typical of RNR family proteins. The exposed architecture of DrII’s RNA binding surface offers an explanation for the nuclease’s ability to approach within 3-5 nt of a duplex, an important mechanistic difference from the well-studied E. coli RNase II. The open, clamp architecture of DrII may have broader relevance to mechanisms of duplex RNA recognition in the RNR superfamily. RNA decay by processive exonucleases such as RNR family proteins leaves 2-5 nt nanoRNA limit products that are further degraded to mononucleotides by nanoRNases. In E. coli, the DEDD family enzyme Oligoribonuclease (ORN) executes nanoRNA decay and represents the first major family of nanoRNases, with homologs widely conserved in eubacteria and eukaryotes. The B. subtilis NanoRNase A (NrnA), a DHH family phosphoesterase, represents a second major class of nanoRNases, with broad phylogenetic distribution in organisms that lack orn homologs. The second major focus of this thesis is a structural and mechanistic study of this nanoRNase machinery. The atomic structure of the B. subtillis nanoRNase NrnA is described, and unveils a bi-lobal architecture similar to the 5’→3’ DNase RecJ, where the catalytic DHH domain is linked via a partially helical connector to the C-terminal RNA binding domain. NrnA is a highly dynamic molecule, adopting both open and closed conformations. Co-crystallization with several substrates shows that NrnA has a nanoRNA specific substrate-binding patch that offers a structural explanation for its 3’→5’ nanoRNase activity. This RNA binding site feeds substrate to the DHH active site in an orientation opposite to the 5’→3’ path proposed for RecJ. Surprisingly, NrnA also maintains a weak 5’→3’ activity on certain substrates, and thus possesses both 5’→3’ and 3’→5’ exonuclease activities. In conclusion, an overall model is presented for how DHH family exonucleaess can degrade nucleic acids from both the 5’→3’ and 3’→5’ directions. Thus, the studies described in this thesis offer both an atomic and a biochemical view of the macromolecular machinery critical to the degradation of RNA.
Książki na temat "Cellular RNA"
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Pitre, Liisa K. The application of RNA interference to cellular biotechnology. Sudbury, Ont: Laurentian University, 2003.
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Brasier, Allan R., Adolfo García-Sastre, and Stanley M. Lemon, eds. Cellular Signaling and Innate Immune Responses to RNA Virus Infections. Washington, DC, USA: ASM Press, 2008. http://dx.doi.org/10.1128/9781555815561.
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Kekkonen, Viktoria. Characterization of bacterial RNA and DNA signalling pathways that induce cellular dysfunction. Sudbury, Ont: Laurentian University, 2006.
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Post-transcriptional regulation by STAR proteins : control of RNA metabolism in development and disease. New York: Springer Science+Business Media, LLC, 2010.
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Mallick, Bibekanand. Regulatory RNAs: Basics, Methods and Applications. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.
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Tax, Frans. Receptor-like Kinases in Plants: From Development to Defense. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.
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Farrell, Robert E. RNA methodologies: A laboratory guide for isolation and characterization. 2nd ed. San Diego: Academic Press, 1998.
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Divan, Aysha, and Janice A. Royds. 3. RNA. Oxford University Press, 2016. http://dx.doi.org/10.1093/actrade/9780198723882.003.0003.
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The first RNA molecules to be discovered were those involved in protein synthesis, mRNA, transfer RNA (tRNA), and ribosomal RNA (rRNA). In recent years, a vast number of additional RNA molecules have been identified. ‘RNA’ explains that these are non-coding RNAs that are not involved in protein synthesis, but influence many normal cellular and disease processes by regulating gene expression. RNA interference (RNAi) as one of the main ways in which gene expression is regulated is described with applications to therapy. Classes of RNA, including long non-coding RNAs and catalytic RNAs, are explained along with RNA techniques used to study RNA molecule and gene function.
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Grimm, Dirk. Cellular RNA Interference Mechanisms. Elsevier Science & Technology Books, 2011.
Znajdź pełny tekst źródłaCzęści książek na temat "Cellular RNA"
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Parker, Henry, and Tom C. Hobman. "Cytoplasmic RNA Domains." In Cellular Domains, 429–44. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118015759.ch25.
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Gupta, Kshitij. "Cellular Delivery of siRNAs Using Bolaamphiphiles." In RNA Nanostructures, 187–205. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7138-1_12.
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Sharma, Sunny, and Karl-Dieter Entian. "Chemical Modifications of Ribosomal RNA." In Ribosome Biogenesis, 149–66. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_9.
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AbstractCellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types—methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen–Conradi syndrome and the William–Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.
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Schächner, Christopher, Philipp E. Merkl, Michael Pilsl, Katrin Schwank, Kristin Hergert, Sebastian Kruse, Philipp Milkereit, Herbert Tschochner, and Joachim Griesenbeck. "Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes." In Ribosome Biogenesis, 25–38. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_2.
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AbstractIn growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic “open” chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal “closed” chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.
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Bierhoff, Holger. "Analysis of lncRNA-Protein Interactions by RNA-Protein Pull-Down Assays and RNA Immunoprecipitation (RIP)." In Cellular Quiescence, 241–50. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7371-2_17.
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Sinkovics, Joseph G. "Viral and Cellular Proteins Interact." In RNA/DNA and Cancer, 247–50. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-22279-0_16.
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Endoh, Tamaki, and Takashi Ohtsuki. "Cellular siRNA Delivery Using TatU1A and Photo-Induced RNA Interference." In RNA Interference, 271–81. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-588-0_17.
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Yu, Qikun, Ru Zheng, Manojkumar Narayanan, and Mingxu You. "Rational Design of Allosteric Fluorogenic RNA Sensors for Cellular Imaging." In RNA Scaffolds, 141–52. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1499-0_11.
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Schultze, B., and G. Herrler. "Recognition of cellular receptors by bovine coronavirus." In Positive-Strand RNA Viruses, 451–59. Vienna: Springer Vienna, 1994. http://dx.doi.org/10.1007/978-3-7091-9326-6_44.
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Kumar Singh, Sunit, and Praveensingh B. Hajeri. "Rna Interference: From basics to Therapeutics." In Molecular and Cellular Therapeutics, 140–67. Chichester, UK: John Wiley & Sons, Ltd, 2012. http://dx.doi.org/10.1002/9781119967309.ch6.
Pełny tekst źródłaStreszczenia konferencji na temat "Cellular RNA"
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Holmes, Ian. "Cellular Automata Simulation of RNA Self-Replicators." In The 2020 Conference on Artificial Life. Cambridge, MA: MIT Press, 2020. http://dx.doi.org/10.1162/isal_a_00242.
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Belov, George. "COUPLING POLIOVIRUS RNA REPLICATION TO CELLULAR MEMBRANES." In Viruses: Discovering Big in Small. TORUS PRESS, 2019. http://dx.doi.org/10.30826/viruses-2019-12.
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Zhu, Kun Yan. "Mechanism of cellular uptake of double-stranded RNA in insects." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.94687.
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Lu, Yunxing, Xiaoyu Jian, Zhaoduo Tong, Zhenhua Wu, Shihui Qiu, Chuanjie Shen, Hao Yin, and Hongju Mao. "Integrated On-Chip Cellular Exosome Isolation and RNA Analysis Microsystem." In 2021 21st International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers). IEEE, 2021. http://dx.doi.org/10.1109/transducers50396.2021.9495727.
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Liu, Chenliang, Yuan Zhu, and Houwang Zhang. "Cellular Similarity based Imputation for Single cell RNA Sequencing Data." In ICBBT '21: 2021 13th International Conference on Bioinformatics and Biomedical Technology. New York, NY, USA: ACM, 2021. http://dx.doi.org/10.1145/3473258.3473269.
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Chen, Cheng, Houqiang Li, Xiaobo Zhou, and Stephen Wong. "GRAPH CUT BASED ACTIVE CONTOUR FOR AUTOMATED CELLULAR IMAGE SEGMENTATION IN HIGH THROUGHPUT RNA INTERFACE (RNAi) SCREENING." In 2007 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro. IEEE, 2007. http://dx.doi.org/10.1109/isbi.2007.356790.
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Ghalali, Aram, Konrad H. Stopsack, James M. Rice, Liangzhe Wang, Shulin Wu, Chin Lee Wu, Bruce Zetter, and Michael S. Rogers. "Abstract 1822: RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-1822.
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Mackintosh, Carlos, Sergiy Konovalov, and Ivan Garcia-Bassets. "Abstract 2358: Dissecting the cellular response to cisplatin from RNA transcription to translation." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-2358.
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Hassan, H. J., A. Leonardi, C. Chelucci, R. Guerriero, P. M. Mannucci, and C. Peschle. "EXPRESSION IN ONTOGENESIS OF HUMAN BLOOD COAGULATION FACTORS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644610.
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We have analyzed the expression of several blood coagulation factors (IX, VIII, X, fibrinogen chains) and inhibitors (antithrombin III, protein C) in human embryonic and fetal livers, obtained from legal abortions at 6-11 week post-conception. The age was established by morphologic staging and particularly crown-rump lenght measurement.Total cellular RNA was isolated from partially purified hepatocytes or total liver homogenate using the guanidine isothiocyanate method. Poly(A)+ RNA was selected by oligodT cellulose chromatography. The size and the number of the embryonic and fetal transcripts are equivalent to those observed in adult liver, as evaluated by Northern blot analysis of total or poly(A)+ RNA hybridized to human cDNA probes.The level of coagulation factor transcripts in embryonic and fetal liver was evaluated by dot hybridization of total RNA (0.5-10 ug), as compared to RNA extracted from normal adult liver biopsies. The expression of blood coagulation factors in embryos is generally reduced for all factors, but at a different degree. In 5-11 wk liver, the level of factor IX is 5-10% of that observed in adults, while fibrinogen, protein C, antithrombin III RNA level rises from 25 to 50% and factor X is expressed at a level comparable to that observed in adult liver.We conclude that during these stages of development blood coagulation factors are expressed according to three different time, curves, possibly due to the effect of different types of regulatory mechanisms.
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Mizikova, Ivana, Maria Hurskainen, David Cook, Chanéle Cyr-Depauw, Flore Lesage, Noora Andresson, Emmi Helle, et al. "Single cell RNA analysis of cellular niche in normal and impaired late lung development." In ERS International Congress 2020 abstracts. European Respiratory Society, 2020. http://dx.doi.org/10.1183/13993003.congress-2020.5242.
Pełny tekst źródłaRaporty organizacyjne na temat "Cellular RNA"
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Schuster, Gadi, and David Stern. Integrated Studies of Chloroplast Ribonucleases. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7697125.bard.
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Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.
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Lers, Amnon, and Pamela J. Green. Analysis of Small RNAs Associated with Plant Senescence. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7593393.bard.
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Senescence is an agriculturally significant process due to its negative impact to crop yield and postharvest quality. The genetic regulatory systems controlling senescence induction and progress respond to both developmental and environmental stress signals and involve numerous gene expression changes. Knowledge about the key molecular factors which control senescence is very limited. MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, responses to environmental stresses, and senescence. The long-term goal of this work is to elucidate roles of small RNAs associated with plant senescence. The hypothesis underlying this research is that miRNA-mediated regulation makes important contributions to the senescence process in plants. Specific, original research objectives included: 1) Profiling of small RNAs from senescing plants; 2) Data Analysis and public access via a user-friendly web interface; 3) Validation of senescence-associated miRNAs and target RNAs; 4) Development of transgenic plants for functional analysis of miRNAs in Arabidopsis. Major revisions made in the research compared to the original work plan included 1) Exclusion of the planned work with tomato as recommended by the BARD review panel; 2) Performing miRNA study also in senescing Arabidopsis siliques, in addition to senescing leaves. To identify senescenceregulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel Analysis of RNA Ends (PARE) libraries, which enable the large-scale examination of miRNA-guided cleavage products, were also constructed and sequenced, resulting in over 750 million genome-matched sequences. These massive datasets lead to the identification of new miRNAs, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence. Experiments were initiated for functional analysis of specific senescence-associated miRNAs and respective target genes. Transgenic Arabidopsis plants were generated in which miR408, found in this study to be significantly induced in leaf senescence, was over-expressed either constitutively or under a senescence-specific promoter. These plants are currently being characterized for any altered phenotypes. In addition T-DNA knock out mutants for various target genes identified in this research are being analyzed. This work provides insights about specific miRNAs that contribute to leaf and silique senescence. The knowledge generated may suggest new strategies to monitor and alter the progression of senescence in crops for agricultural improvement.
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Lapidot, Moshe, and Vitaly Citovsky. molecular mechanism for the Tomato yellow leaf curl virus resistance at the ty-5 locus. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7604274.bard.
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Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2
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Porat, Ron, Gregory T. McCollum, Amnon Lers, and Charles L. Guy. Identification and characterization of genes involved in the acquisition of chilling tolerance in citrus fruit. United States Department of Agriculture, December 2007. http://dx.doi.org/10.32747/2007.7587727.bard.
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Citrus, like many other tropical and subtropical fruit are sensitive to chilling temperatures. However, application of a pre-storage temperature conditioning (CD) treatment at 16°C for 7 d or of a hot water brushing (HWB) treatment at 60°C for 20 sec remarkably enhances chilling tolerance and reduces the development of chilling injuries (CI) upon storage at 5°C. In the current research, we proposed to identify and characterize grapefruit genes that are induced by CD, and may contribute to the acquisition of fruit chilling tolerance, by two different molecular approaches: cDNA array analysis and PCR cDNA subtraction. In addition, following the recent development and commercialization of the new Affymetrix Citrus Genome Array, we further performed genome-wide transcript profiling analysis following exposure to CD and chilling treatments. To conduct the cDNA array analysis, we constructed cDNA libraries from the peel tissue of CD- and HWB-treated grapefruit, and performed an EST sequencing project including sequencing of 3,456 cDNAs from each library. Based on the obtained sequence information, we chose 70 stress-responsive and chilling-related genes and spotted them on nylon membranes. Following hybridization the constructed cDNA arrays with RNA probes from control and CD-treated fruit and detailed confirmations by RT-PCR analysis, we found that six genes: lipid-transfer protein, metallothionein-like protein, catalase, GTP-binding protein, Lea5, and stress-responsive zinc finger protein, showed higher transcript levels in flavedo of conditioned than in non-conditioned fruit stored at 5 ᵒC. The transcript levels of another four genes: galactinol synthase, ACC oxidase, temperature-induced lipocalin, and chilling-inducible oxygenase, increased only in control untreated fruit but not in chilling-tolerant CD-treated fruit. By PCR cDNA subtraction analysis we identified 17 new chilling-responsive and HWB- and CD-induced genes. Overall, characterization of the expression patterns of these genes as well as of 11 more stress-related genes by RNA gel blot hybridizations revealed that the HWB treatment activated mainly the expression of stress-related genes(HSP19-I, HSP19-II, dehydrin, universal stress protein, EIN2, 1,3;4-β-D-glucanase, and SOD), whereas the CD treatment activated mainly the expression of lipid modification enzymes, including fatty acid disaturase2 (FAD2) and lipid transfer protein (LTP). Genome wide transcriptional profiling analysis using the newly developed Affymetrix Citrus GeneChip® microarray (including 30,171 citrus probe sets) revealed the identification of three different chilling-related regulons: 1,345 probe sets were significantly affected by chilling in both control and CD-treated fruits (chilling-response regulon), 509 probe sets were unique to the CD-treated fruits (chilling tolerance regulon), and 417 probe sets were unique to the chilling-sensitive control fruits (chilling stress regulon). Overall, exposure to chilling led to expression governed arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defense, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced various adaptation processes, such as changes in the expression levels of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.
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Epel, Bernard L., Roger N. Beachy, A. Katz, G. Kotlinzky, M. Erlanger, A. Yahalom, M. Erlanger, and J. Szecsi. Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria. United States Department of Agriculture, September 1999. http://dx.doi.org/10.32747/1999.7573996.bard.
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The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, and studied the intra- and intercellular targeting mechanism of MP during the infection cycle. Most of the goals we set for ourselves were met. The Israeli PI and collaborators (Oparka et al., 1999) demonstrated that Pd permeability is under developmental control, that Pd in sink tissues indiscriminately traffic proteins of sizes of up to 50 kDa and that during the sink to source transition there is a substantial decrease in Pd permeability. It was shown that companion cells in source phloem tissue export proteins which traffic in phloem and which unload in sink tissue and move cell to cell. The TAU group employing MP:GFP as a fluorescence probe for optimized the procedure for Pd isolation. At least two proteins kinases found to be associated with Pd isolated from source leaves of N. benthamiana, one being a calcium dependent protein kinase. A number of proteins were microsequenced and identified. Polyclonal antibodies were generated against proteins in a purified Pd fraction. A T-7 phage display library was created and used to "biopan" for Pd genes using these antibodies. Selected isolates are being sequenced. The TAU group also examined whether the subcellular targeting of MP:GFP was dependent on processes that occurred only in the presence of the virus or whether targeting was a property indigenous to MP. Mutant non-functional movement proteins were also employed to study partial reactions. Subcellular targeting and movement were shown to be properties indigenous to MP and that these processes do not require other viral elements. The data also suggest post-translational modification of MP is required before the MP can move cell to cell. The USA group monitored the development of the infection and local movement of TMV in N. benthamiana, using viral constructs expressing GFP either fused to the MP of TMV or expressing GFP as a free protein. The fusion protein and/or the free GFP were expressed from either the movement protein subgenomic promoter or from the subgenomic promoter of the coat protein. Observations supported the hypothesis that expression from the cp sgp is regulated differently than expression from the mp sgp (Szecsi et al., 1999). Using immunocytochemistry and electron microscopy, it was determined that paired wall-appressed bodies behind the leading edge of the fluorescent ring induced by TMV-(mp)-MP:GFP contain MP:GFP and the viral replicase. These data suggest that viral spread may be a consequence of the replication process. Observation point out that expression of proteins from the mp sgp is temporary regulated, and degradation of the proteins occurs rapidly or more slowly, depending on protein stability. It is suggested that the MP contains an external degradation signal that contributes to rapid degradation of the protein even if expressed from the constitutive cp sgp. Experiments conducted to determine whether the degradation of GFP and MP:GFP was regulated at the protein or RNA level, indicated that regulation was at the protein level. RNA accumulation in infected protoplast was not always in correlation with protein accumulation, indicating that other mechanisms together with RNA production determine the final intensity and stability of the fluorescent proteins.
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Ullman, Diane, James Moyer, Benjamin Raccah, Abed Gera, Meir Klein, and Jacob Cohen. Tospoviruses Infecting Bulb Crops: Evolution, Diversity, Vector Specificity and Control. United States Department of Agriculture, September 2002. http://dx.doi.org/10.32747/2002.7695847.bard.
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Objectives. The overall goal of the proposed research was to develop a mechanistic understanding of tospovirus evolution, diversity and vector specificity that could be applied to development of novel methods for limiting virus establishment and spread. Our specific objectives were: 1) To characterize newly intercepted tospoviruses in onion, Hippeastrum and other bulb crops and compare them with the known tomato spotted wilt virus (TSWV) and its isolates; 2) To characterize intra- and interspecific variation in the virus transmission by thrips of the new and distinct tospoviruses. and, 3) To determine the basis of vector specificity using biological, cellular and molecular approaches. Background. New tospoviruses infecting bulb crops were detected in Israel and the US in the mid-90s. Their plant host ranges and relationships with thrips vectors showed they differed from the type member of the Tospovirus genus, tomato spotted wilt virus (TSWV). Outbreaks of these new viruses caused serious crop losses in both countries, and in agricultural and ornamental crops elsewhere. In the realm of plant infecting viruses, the tospoviruses (genus: Tospovirus , family: Bunyaviridae ) are among the most aggressive emerging viruses. Tospoviruses are transmitted by several species of thrips in a persistent, propagative fashion and the relationships between the viruses and their thrips vectors are often specific. With the emergence of new tospoviruses, new thrips vector/tospovirus relationships have also arisen and vector specificities have changed. There is known specificity between thrips vector species and particular tospoviruses, although the cellular and molecular bases for this specificity have been elusive. Major conclusions, solutions and achievements. We demonstrated that a new tospovirus, iris yellow spot virus (IYSV) caused "straw bleaching" in onion (Allium cepa) and lisianthus necrosis in lisianthus (Eustoma russellianum). Characterization of virus isolates revealed genetic diversity among US, Brazilian, Dutch and Israeli isolates. IYSV was not seed transmitted, and in Israel, was not located in bulbs of infected plants. In the US, infected plants were generated from infected bulbs. The relationship between IYSV and Thrips tabaci was shown to be specific. Frankliniella occidentalis, the primary vector of many other tospoviruses, did not transmit IYSV isolates in Israel or the US. Furthermore, 1': tabaci populations varied in their transmission ability. Transmission was correlated to IYSV presence in thrips salivary glands. In Israel, surveys in onion fields revealed that the onion thrips, Thrips tabaci Lindeman was the predominant species and that its incidence was strongly related to that of IYSV infection. In contrast, in the U.S., T. tabaci and F. occidentalis were present in high numbers during the times sampled. In Israel, insecticides reduced onion thrips population and caused a significant yield increase. In the US, a genetic marker system that differentiates non-thrips transmissible isolates from thrips transmissible isolate demonstrated the importance of the M RNA to thrips transmission of tospoviruses. In addition, a symbiotic Erwinia was discovered in thrips and was shown to cause significant artifacts in certain types of virus binding experiments. Implications, scientific and agricultural. Rapid emergence of distinct tospoviruses and new vector relationships is profoundly important to global agriculture. We advanced the understanding of IYSV in bulb crops and its relationships with thrips vector species. The knowledge gained provided growers with new strategies for control and new tools for studying the importance of particular viral proteins in thrips specificity and transmission efficiency.
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Ghanim, Murad, Joe Cicero, Judith K. Brown, and Henryk Czosnek. Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels. United States Department of Agriculture, February 2010. http://dx.doi.org/10.32747/2010.7592654.bard.
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Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.
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Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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Sadot, Einat, Christopher Staiger, and Mohamad Abu-Abied. Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7592652.bard.
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In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.
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Olszewski, Neil, and David Weiss. Role of Serine/Threonine O-GlcNAc Modifications in Signaling Networks. United States Department of Agriculture, September 2010. http://dx.doi.org/10.32747/2010.7696544.bard.
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Significant evidence suggests that serine/threonine-O-linked N-acetyl glucosamine0-(GlcNAc) modifications play a central role in the regulation of plant signaling networks. Forexample, mutations in SPINDLY,) SPY (an O-GlcNAc transferase,) OGT (promote gibberellin GA) (signal transduction and inhibit cytokinin responses. In addition, mutating both Arabidopsis OGTsSEC (and SPY) causes embryo lethality. The long-term goal of this research is to elucidate the mechanism by which Arabidopsis OGTs regulate signaling networks. This project investigated the mechanisms of O-GlcNAc regulation of cytokinin and gibberellin signaling, identified additional processes regulated by this modification and investigated the regulation of SEC activity. Although SPY is a nucleocytoplasmic protein, its site of action and targets were unknown. Severalstudies suggested that SPY acted in the nucleus where it modified nuclear components such as the DELLA proteins. Using chimeric GFP-SPY fused to a nuclear-export signal or to a nuclear-import signal, we showed that cytosolic, but not nuclear SPY, regulated cytokinin and GA signaling. We also obtained evidence suggesting that GA and SPY affect cytokinin signaling via a DELLA-independent pathway. Although SEC and SPY were believed to have overlapping functions, the role of SEC in cytokinin and GA signaling was unclear. The role of SEC in cytokinin and GA responses was investigated by partially suppressing SPY expression in secplants using a synthetic Spymicro RNA miR(SPY). The possible contribution of SEC to the regulation of GA and cytokinin signaling wastest by determining the resistance of the miR spy secplants to the GA biosynthesis inhibitor paclobutrazol and to cytokinin. We found that the transgenic plants were resistant to paclobutrazol and to cytokinin, butonlyata level similar to spy. Moreover, expressing SEC under the 35S promoter in spy mutant did not complement the spy mutation. Therefore, we believe that SEC does not act with SPY to regulate GA or cytokinin responses. The cellular targets of Spy are largely unknown. We identified the transcription factor TCP15 in a two-hybrid screen for SPY-interacting proteins and showed that both TCP15 and its closely homolog TCP14 were O-GlcNAc modified by bacterially-produced SEC. The significance of the interaction between SPY and these TCPs was examined by over-expressing the minwild-type and spy-4plants. Overexpression of TCP14 or TCP15 in wild-type background produced phenotypes typical of plants with increased cytokinin and reduced GA signaling. TCP14 overexpression phenotypes were strongly suppressed in the spy background, suggesting that TCP14 and TCP15 affect cytokinin and GA signaling and that SPY activates them. In agreement with this hypothesis, we created a tcp14tcp15 double mutant and found that it has defects similar to spyplants. In animals, O-GlcNAc modification is proposed to regulate the activity of the nuclear pore. Therefore, after discovering that SEC modified a nucleoporinNUP) (that also interacts with SPY, we performed genetic experiments exploring the relationship between NUPs and SPY nupspy double mutants exhibited phenotypes consistent with SPY and NUPs functioning in common processes and nupseeds were resistant to GA biosynthesis inhibitors. All eukaryotic OGTs have a TPR domain. Deletion studies with bacterially-expressed SEC demonstrated SEC'sTPR domain inhibits SEC enzymatic activity. Since the TPR domain interacts with other proteins, we propose that regulatory proteins regulate OGT activity by binding and modulating the inhibitory activity of the TPR domain.