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Xu, Ning. "Adenoviral Control of RNAi/miRNA Pathways in Human Cells." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distribution], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9387.
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Fritz, Sarah E. "Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437413252.
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Giacometti, Simone. "CBC bound proteins and RNA fate." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTS028.
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Le complexe de liaison de la coiffe des ARN (CBC) joue un rôle essentiel dans leur maturation et déclenche une variété de réactions biochimiques, via son interaction avec différents partenaires. Deux complexes, CBC-ARS2-PHAX (CBCAP), et CBC-ARS2-ZC3H18-NEXT (CBCN), ont récemment été montré comme important pour cibler les ARN vers l'export (CBCAP) ou la dégradation (CBCN). Cependant, les mécanismes par lesquels la sélection se fait pour l'une voie ou l'autre reste mystérieuse. Ainsi, une question majeure qui reste à résoudre est de savoir quand et comment ces complexes sont recrutés sur les ARN. Dans ce travail, j'ai utilisé la procédure du iCLIP (Cross-Linking and Immuno-Precipitation), afin d'identifier les cibles de ces complexes sur l'ensemble du transcriptome humain. J'ai réalisé un iCLIP sur cinq composants de CBCAP et CBCN, et j'ai comparé les résultats à ceux obtenus avec RBM7, un composant de NEXT précédemment étudié par iCLIP. Mes résultats indiquent que: (i) CBP20, ARS2, PHAX et ZC3H18 se lient près de la coiffe des ARN, tandis que RBM7 et MTR4 se lient partout; (ii) CBP20, ARS2, PHAX et ZC3H18 s'associent à un large ensemble d'ARN transcrits par l'ARN polymérase II et montrent une faible sélectivité; (iii) la liaison de ces protéines varie avec l'état de maturation des ARN, avec le CBC enrichi sur les ARN matures, tout comme ARS2/PHAX/ZC3H18 et MTR4 (bien que dans une moindre mesure), tandis que RBM7 est préférentiellement lié sur les pre-mRNAs non épissés; (iv) une liaison différentielle de RBM7 et MTR4 sur les ARN, avec RBM7 enrichi sur les introns et les PROMPTs, et MTR4 plus présent sur les ARN mature. Bien que des expériences additionnelles soient requises, nous proposons que le CBCAP et le CBCN se lient à un même ensemble d'ARN, ce qui indique à la fois une compétition entre ZC3H18 et PHAX pour la liaison à ces ARN, et l'absence de voies de routage bien déterminées qui ciblerait les ARN vers l'une ou l'autre de ces protéines. Le devenir des ARN pourrait ainsi être déterminé par d'autres caractéristiques des ARN, ou encore par des protéines additionnelles. Ces facteurs pourraient s'allier aux protéines liées à la coiffe afin de favoriser la formation du CBCAP ou du CBCN. Dans le but d’identifier des facteurs additionnels, j'ai réalisé un screen d'interaction par spectrométrie de masse après purification de ARS2 ou CBP80. Ceci a été fait dans des conditions natives ou après un cross-link des complexes à la formaldéhyde, afin de stabiliser les interactions transitoires. Ceci a permis d'identifier de nouveaux partenaires de ARS2 et de CBP80, dont la majorité sont impliqués dans l'épissage des ARN. Des expériences additionnelles seront nécessaires pour valider ces interactionsThe cap-binding complex (CBC) plays a pivotal role in post-transcriptional processing events and orchestrates a variety of metabolic pathways, through association with different interaction partners. Two CBC sub-complexes, the CBC-ARS2-PHAX (CBCAP) and the CBC-nuclear exosome targeting (NEXT) complex (CBCN), were recently shown to target capped RNA either toward export or degradation, but the mechanisms by which they can discriminate between different RNA families and route them toward different metabolic pathways still remain unclear. A major question to be answered is how and when the different CBC subcomplexes are recruited to the RNP. Here, we used an individual nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) approach to identify the transcriptome-wide targets for 5 different components of the CBCAP and CBCN complexes, and compared results to the previously analysed NEXT-component RBM7. We report that: (i) CBP20, ARS2, PHAX and ZC3H18 bind close to the cap, while RBM7 and MTR4 bind throughout the mRNA body; (ii) CBP20, ARS2, PHAX and ZC3H18 associate with a broad set of RNA polymerase II (PolII)-derived RNAs and have only mild species preferences; (iii) binding varies with the RNA maturation stage, with the CBC being highly enriched on mature mRNA, ARS2/PHAX/ZC3H18/MTR4 less so, and RMB7 preferentially bound to pre-mRNAs; (iv) MTR4 and RBM7 show different specificities, with RBM7 being highly enriched on introns and promoter upstream transcripts (PROMPTs), while MTR4 is additionally present on mature RNAs. Although more experimental work is needed to fully support our model, we propose that CBCAP and CBCN bind overlapping sets of RNAs, indicating a competition between the proteins ZC3H18 and PHAX, and the lack of a strict RNA sorting mechanism. RNA fate may therefore be determined by additional RNA features and/or by other RNA-binding proteins, which may synergize with the cap and drive the formation of one specific CBC subcomplex instead of another. In an attempt to identify yet unknown factors that may interact with cap-bound CBCAP and CBCN, we performed a protein interaction screen leveraging affinity capture-mass spectrometry (ACMS), using ARS2 and CBP80 as bait proteins. As a complementary approach, we also employed a formaldehyde-based chemical cross-linking strategy, aimed at stabilizing weak/transient interactions. Although we failed to detect any transient interactions involving the CBC, we identified several potential CBC80 and ARS2 interactors, the majority of which are involved in pre-mRNA splicing. Additional quantitative experiments are required to validate our ACMS results and confirm the existence of such protein interactions in vivo
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Hitchcock, Robert Arthur. "Epigenetic control of the kinetoplastid spliced leader RNA." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1998392041&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Wilson, Sean. "Control of messenger RNA stability in Saccharomyces cerevisiae." Thesis, University of Aberdeen, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368535.
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The effect of glucose on ribosomal protein mRNA stability was examined. The addition of glucose, to a final concentration as low as 0.01%, resulted in a dramatic, transient increase in ribosomal protein mRNA stability. It has been demonstrated that protein kinase A (PKA) activity plays an important role in the up-regulation of ribosomal protein gene transcription in response to glucose addition. PKA pathway is required for the transient stabilisation of ribosomal protein mRNA following glucose addition, mRNA half-lives were determined in a PKA mutant strain. The data indicated that this glucose effect is not dependent upon a functional Ras-cAMP-PKA signalling pathway. Previously it was shown that the glucose-induced upshift in ribosomal protein gene transcription could be triggered by the addition of exogenous cAMP. However, that dibutyryl cAMP did not act efficiently as a second messenger in the signalling pathway mediating the glucose effect, or that cAMP signalling is not required for the transient stabilisation of ribosomal protein mRNAs. The involvement of specific glucose signalling factors was also examined. The glucose sensors, Snf3p and Rgt2p, were not required for the stabilisation of the ribosomal protein mRNAs. It was noted however, that phosphorylation of glucose by any one of the three yeast hexose kinases (Hxk1p, Hxk2p or Glk1p) was required to mediate this effect.
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Brown, Justin Travis. "MRNA degradation in the control of gene expression in yeast." Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3024999.
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Carlberg, Konstantin. "In Situ RNA Quality Control : A spatial heat map of RNA integrity with single cell resolution." Thesis, KTH, Skolan för bioteknologi (BIO), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-176873.
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The quality of RNA is of great importance in gene expression studies. It is mostly measured using the RNA integrity number (RIN). Lately it has been shown that samples with low RIN and different fragmentation patterns could affect quality of sequencing data. For such low RIN samples a new approach has been developed by Illumina called the DV200 metric, which is the percentage of fragments >200 nucleotides. For samples with low RIN, DV200 has proved to be a better method to predict if good quality data from RNA sequencing can be generated. However, neither RIN nor DV200 provide spatial infromation on the RNA integrity. Thus, tissues with areas of heterogeneous RNA integrity, where regions of good quality RNA sequencing data could be generated from are missed. We have designed a method to spatially evaluate the RNA integrity in tissue, which we named in situ RNA QC. The method uses three probes with three different fluorophores, each bound to three specific cDNA regions synthesized from the high abundant and well conserved 18S rRNA. With the help of in-house technology from the Spatial Transcriptomics (ST) group at SciLifeLab, we enable creation of heat maps over the RNA integrity to show spatial fragmentation patterns of RNA in tissue. This could reveal the regional quality of transcripts in situ, which is crucial knowledge when selecting samples for further RNA sequencing. The assay has been tried using different tissue fixation methods in order to show a proof of concept that formalin gives shorter cDNA fragments than acetone. The generated heat-map provides a visual overview of RNA integrity in situ; hence this method could be used to select samples for sequencing by evaluation the spatial quality of RNA. For instance from fresh frozen and formalin fixated paraffin embedded (FFPE) tissue (biobanks contain large number of longterm storage FFPE samples). With this assay we will be able to determine which samples are suitable for sequencing.
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Zhang, Ze. "The control of ribosomal RNA synthesis in mammalian cells." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/350477/.
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The biogenesis of ribosomes is a fundamental process that occurs in all living cells. In mammalian cells, it is a highly complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNAs) with about 80 ribosomal proteins (RPs). More than 150 non-ribosomal proteins are involved in the processing of rRNAs. The main focus of this project is to use adult rat ventricular cardiomyocytes (ARVCs) as a model to address how mTOR complex 1 (mTORC1) and other signalling pathways regulate the synthesis of rRNAs. A new technique has been developed to monitor the synthesis of new rRNAs using 4-thiouridine (4-SU) and I have applied it in both HeLa cells and heart muscle cells to study the control of ribosome synthesis. HeLa cells were treated with different mTOR inhibitors to identify effects on the transcription and/or decay of rRNA. We analysed both the synthesis rate and the decay rate of new RNAs made by Pol I and Pol III using real-time RT-PCR. Interestingly, rapamycin not only blocked the synthesis of 18S, 28S and 5S rRNA, but also induced the decay of newly synthesized rRNAs. This demonstrates that mTORC1 regulates Pol I and Pol III transcription, as well as the decay of rRNA. In cardiomyocytes, hypertrophic agents such as phenylephrine (PE) strongly activate protein synthesis and lead to heart cell growth. The boost of protein synthesis drives the increase of cell size and leads to hypertrophy. Cardiac hypertrophy (CH) is a major risk factor for heart failure. Therefore, it is important to understand the mechanisms that how hypertrophic agents which cause the overgrowth of heart muscle increase ribosome production. Although it is known that inhibiting mTORC1 largely blocks the rapid activation of protein synthesis by PE, here it did not affect the synthesis of new 18S rRNAs. However, inhibitors of the MEK/Erk pathway and p90RSK each block the new rRNA synthesis. These data reveal that, in contrast to many other types of cell, ribosome biogenesis is controlled by MEK/ERK/p90RSK signalling, not mTORC1, in cardiomyocytes. Taken together, the data presented here may provide cues for potential valuable therapy of cardiac left ventricular hypertrophy.
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Tzelos, Thomas. "RNA interference in parasitic nematodes : from genome to control." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15906.
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Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The parasite has developed resistance to the major anthelmintic drug classes and this challenges its future control. Vaccination is a potential alternative control method since sheep are able to develop protective immunity against this parasite. Although potential vaccine candidates have been revealed, the increasing gene datasets suggest that vaccinetarget selection may be aided by screening methods such as RNAi. This is a reverse genetic mechanism that causes highly specific gene silencing which was initially described and applied to defining gene function in Caenorhabditis elegans. Nevertheless, its application was more difficult than anticipated in parasitic nematodes because of the inconsistency of the silencing effect. In the unsuccessful cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus far, there are no internal controls that indicate the activation of the pathway. Are the RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to the dsRNA exposure? The initial aim of the study was to determine potential marker genes in the RNAi pathway that could indicate the activation of the pathway in C. elegans. After the exposure to dsRNA from two target genes, the transcript levels of three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and showed that exposure to dsRNA has no effect on the transcript levels of these genes making them inappropriate markers for the activation of the RNAi pathway. The two target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently susceptible and refractory to RNAi, respectively. Another aim of the project was to develop an RNAi platform in T. circumcincta for use as a screening method for potential vaccine candidates. The targets selected for the in vitro RNAi included: five members of the Activation-associated Secreted Proteins (ASPs); a Macrophage migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene (Tci-saa-1), all of which have been associated with vaccine-induced protective immunity. The selection of the ASPs was based on a bioinformatic and transcriptomic analysis of the ASPs in T. circumcincta. The results showed successful knock-down only for three out of five ASP targets after 1 hour of soaking in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency and the target specificity of RNAi in T. circumcincta which has been observed in the past with other parasitic nematodes. Inconsistencies were also observed within the successful ASP targets with the results not being reproducible after several successful experiments. Potential reasons for the inconsistencies were examined with the duration of larval storage being a critical factor. Larvae stored for a short or long period of time were susceptible and refractory to RNAi, respectively. Experiments were also conducted to investigate how the ASPs relate to extracellular microvesicles (EMVs). These vesicles are considered to play an important role in the intercellular communication between parasites and their hosts, and thus represent potentially useful vaccine and/or drug targets. Transmission electron microscopy (TEM) confirmed that EMVs are excreted / secreted by the parasite and the proteomic analysis revealed several types of proteins within the vesicles such as: ASPs, Actins, Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free ES (Excretory / Secretory) and total ES products showed that approximately 35% of the proteins found in the vesicles could also be identified in EMV-free ES and in total ES products, whilst the remaining 65% were present only in EMVs.
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Peele, Price Jason. "Control of RNA Structure by CspA Proteins in Rhizobia." Thesis, Washington State University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10605605.
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Rhizobia are soil bacteria that can associate with some legumes and participate in symbiotic nitrogen fixation. Bacterial CspA family members are small, single stranded nucleic acid binding proteins conserved throughout all domains of life. Here, the role of CspA family proteins in the symbiotic development of Sinorhizobium meliloti with Medicago sativa (alfalfa) is investigated. Expression and genetic deletion strain analysis revealed that CspA family proteins are differentially expressed in symbiosis and contribute to symbiotic effectiveness. RNAseq analysis of native co-immunoprecipitated RNAs identified a novel interaction between several CspA family proteins and the αR14 family of small non-coding RNA (sRNAs). Whole transcriptome analysis defined transcriptional defects associated with loss of CspA function. The development of a new in vitro RNA binding assay using broccoli, a Green Fluorescent Protein (GFP) RNA mimic, is described as well as its use in defining binding specificity of CspA family proteins with synthetic and native ?R14 family sRNA structures. This work concludes that CspA family proteins interact with and influence the stability of specific RNA structures and these interactions control RNA regulated processes important for symbiotic development.
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Butsch, Melinda Sue. "Control of Retroviral Translation and Relationship to Genomic RNA Packaging." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1029510697.
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Ranji, Arnaz K. "Mechanistic insights into translational modulation of selected RNAs by RNA helicase A." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299537544.
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Soto-Rifo, Ricardo. "Translational control of HIV-1 and HIV-2 genomic RNA." Lyon, Ecole normale supérieure, 2010. http://www.theses.fr/2010ENSL0584.
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Les virus de la immunodéficience humaine de type 1 et 2 (VIH-1 et VIH-2) sont des pathogènes qui ont un grand impact sur la santé car plus de 33 millions de personnes sont infectées dans le monde. Les mécanismes qui contrôlent les étapes post-transcriptionelles de l’ARN génomique pendant les étapes tardives du cycle réplicatif ne sont pas très connu et donc les processus moléculaires qui permettent à l’ARN génomique de s’associer aux machineries cellulaires de traduction, transport ou dégradation restent à être déterminés. Dans ces travaux, nous avons contribué à améliorer notre connaissance sur les mécanismes qui contrôlent la synthèse des protéines à partir de l’ARN génomique de VIH-1 et VIH-2. Les résultats présentés dans ces travaux montrent que la structure d’ARN TAR joue un rôle primordial dans le contrôle des interactions de l’ARN génomique et la machinerie traductionnelle de la cellule. Nous montrons des données qui suggèrent une nouvelle étape lors du cycle réplicatif du VIH-2 dans laquelle l’ARN génomique est accumulé dans des granules cytoplasmiques avec des marqueurs des granules de stress. Nous mettons en évidence un mécanisme qui permettrait à l’ARN génomique du VIH-1 d’être exporté au cytoplasme et traduit de manière efficace grâce à l’helicase à ARN DDX3Infections by Human immunodeficiency viruses type-1 and type-2 (HIV-1 and HIV-2) have an enormous impact in Human health as more than 33 million people is living with HIV/AIDS worldwide. The mechanisms controlling post-transcriptional events during the HIV life cycle have just started to capture the attention of scientists and most of the molecular processes allowing the genomic RNA to interact with the host machineries for translation, transport or decay are still obscure or in way to be determined. In this work, we contribute to the progress in the knowledge of the mechanisms controlling protein synthesis from the HIV-1 and HIV-2 genomic RNA. Results presented here provide evidence for the TAR RNA structure as a key player in controlling the interactions between the HIV-1 and HIV-2 genomic RNA with the host translational machinery. We also provide data for a new step during the HIV-2 life cycle that involves the accumulation of the genomic RNA in cytoplasmic granules containing several stress granules components. Finally, we present evidence for a potential mechanism by which nuclear export and protein synthesis are linked during the HIV-1 replication cycle. As such, we show that DEAD-box RNA helicase DDX3, previously implicated in Rev-mediated nuclear export, is absolutely required for HIV-1 genomic RNA translation. We determined the TAR structure as the viral determinant required for DDX3 function in translation. Strikingly, we also showed that DDX3 is specifically required for HIV-2 and SIV translation but not for FIV, HTLV-1, MLV or Line-1 suggesting that this function was acquired during primate lentiviruses evolution. Taken together, results obtained during this work highlight several key aspects of the HIV-1 and HIV-2 genomic RNA post-transcriptional control that may be critical for viral replication
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Smith, Richard Wilson. "RNA metabolism and the control of protein synthesis in fish." Thesis, University of Aberdeen, 1996. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU089891.
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This thesis examines the regulation of protein synthesis by pre - translational events; with particular reference to the means by which RNA is able to reduce the energetic cost of protein synthesis. Protein synthesis was measured by the application of a "flooding dose" of 3H-phenylalnine. Protein synthesis rates are then calculated from the "free intracellular" and "protein bound" specific radioactivity (dpm nmole-1 phenylalanine). A similar approach was used to investigate RNA synthesis: i.e. a flooding dose of 3H-uridine. As with protein synthesis RNA synthesis rates was assessed by the relating precursor and product (uridine nucleotide and RNA) radioactivity. Oxygen consumption was measured by monitoring the decline in partial pressure in calibrated respirometery chambers. In fish cells protein synthesis was regulated in terms of the amount (ie the "capacity" for protein synthesis) and the translational efficiency of the RNA. Translationally efficient RNA equated to RNA with an increased turnover. In order to minimise RNA production costs, rapidly synthesised RNA places more reliance on the salvage of exogenous nucleosides, as opposed to the relatively expensive alternative of intracellular synthesis. During yolk sac larval development of the African wels (Clarius gariepinus) protein synthesis rates decline whilst oxygen consumption and the amount of RNA (relative to protein) remains constant. Thus the increasing protein synthesis costs resulted from a reduction in RNA translational efficiency. This was mirrored by a declining RNA synthesis rate. Larval fish growth is primarily due to the repartitioning of yolk sac proteins since early life history stages are thought unable to sustain rapid rates of protein turnover. This pre - translational strategy optimises growth and regulates protein synthesis; whilst, at the same time maintaining the capacity for protein synthesis in anticipation of exogenous feeding.
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Bolinger, Cheryl Giles. "Study of translation control by a RNA helicase A-responsive post-transcriptional control element in Retroviridae." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1226513076.
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Hasan, Ayesha. "Global control of RNA turnover in the fission yeast Schizosaccharomyces pombe." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708613.
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Mehlhorn, Sonja Gabriele [Verfasser]. "RNA interference: Process and Application to Pest Control / Sonja Gabriele Mehlhorn." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1214887066/34.
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Sardana, Ravinder Kumar. "Structure and control of expression of ribosomal RNA genes in wheat." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305819.
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Murphy, Alexander James. "RNA and Protein Networks That Locally Control Brain Wiring During Development." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467385.
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The molecular machineries of growth cones control the formation of neural circuits in the developing brain. Although great progress has been made in elucidating axon guidance cues and their growth cone receptors, we still lack an understanding of the projection-specific RNA and protein networks in growth cones that likely control the wiring of specific circuits in vivo.
To understand how specific projection neurons make wiring decisions, I focus on callosal projection neurons (CPN), which connect the two cerebral hemispheres through the corpus callosum. I developed an approach to profile and quantify the full-depth transcriptomes and proteomes of CPN growth cones and their parent cell bodies isolated in vivo. Using this comparative approach, I uncover general patterns of RNA and protein subcellular localization, with several previously unrecognized features, that might control the wiring of specific brain circuits. First, while most transcripts are expressed at similar levels in cell bodies and growth cones, a select subset are more than 10-fold enriched in growth cones compared to cell bodies, indicating active localization of those transcripts to the growth cone. By then correlating transcriptomic and proteomic data, I characterize the spatial relationship between coding transcripts and their encoded proteins. Intriguingly, many of the growth cone-enriched transcripts are noncoding RNA with unknown function. Further, growth cones appear to have distinct ribosomes. These ribosomes lack several large subunit proteins, raising the intriguing possibility of growth cone-specific translational mechanisms for selective mRNA expression.
This approach is readily adaptable to other projection types in the brain, enabling high-throughput, quantitative investigation of RNA and protein controls over circuit development and, potentially, the regeneration of damaged circuitry. In addition, the approach is scalable to include epigenetic profiling, enabling full investigation of DNA, RNA, and protein networks that collectively coordinate brain wiring during development. The insights derived from this approach exemplify its capacity to quantify and characterize the molecular and translational mechanisms that control specific brain wiring at the subcellular level in vivo.Medical Sciences
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Ulbricht, Randi J. "Puf1p-mediated mRNA decay and combinatorial control of mRNA stability by the yeast Puf proteins." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2008. http://etd.umsl.edu/r2761.
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Lichtenberg, Stuart. "Nanomaterials for Double-Stranded RNA Delivery." UKnowledge, 2019. https://uknowledge.uky.edu/pss_etds/124.
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RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA polyplexes are equally as effective as naked dsRNA for gene knockdown on a concentration basis, these materials are assimilated into cells in a manner independent of dsRNA specific transport proteins. The mechanism of uptake is likely clathrin-mediated endocytosis. In addition, I identify a significant and yet unreported side-effect associated with chitosan exposure, the dysregulation of a major myosin isoform. Next, I have determined the efficacy of chitosan/dsRNA polyplex nanoparticles under different environmental conditions. The presence of inorganic ions (phosphate and nitrate) at realistic environmental concentrations does not alter the efficacy of the nanoparticles for gene knockdown, nor do they inhibit knockdown by naked dsRNA. These conditions did not cause any significant changes to the hydrodynamic diameter or zeta potential of the particles themselves between treatments. By contrast, a pH higher than six and the presence of natural organic matter significantly reduce the efficacy of the nanomaterials at gene knockdown but leave knockdown by naked dsRNA unaffected. Though some changes in polyplex size are observed in the pH treatments, these changes are comparatively small, and particles remain well within the size that can be ingested by C. elegans. At pH 8, the charge of the particles is effectively neutral. Similarly, concentrations of natural organic matter >2.5 mg/L cause a charge reversal of the particles, from strongly cationic to strongly anionic. Large aggregates are also visible in each of these treatments. Lastly, I characterize the efficacy of a suite of different polymer and solid core nanomaterials for dsRNA delivery, similar to the above. Poly-L-lysine, poly-L-arginine, Ge-doped imogolite, and poly-L-arginine-citrate coated Au nanoparticles all fail to cause any appreciable knockdown in the same C. elegans reporter system. Uptake of the polymers was exceedingly poor, and though the Au particles appear to have been ingested, there is no evidence of significant gene knockdown. Furthermore, poly-L-arginine caused significant injury to the mouthparts of C. elegans exposed to these materials. Layered double-hydroxide nanoparticles were successful at gene knockdown, and appear to function slightly better than naked dsRNA alone, and were translocated in C. elegans in a similar fashion to naked dsRNA. Taken together, these findings aid in the development of safe and effective RNAi biological control agents.
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Chen, Augustine, and n/a. "Translational control mechanisms used by the human Hepatitis B virus : an upstream open reading frame modulates expression of the pregenomic RNA." University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20080130.123000.
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The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis.
However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5' leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated.
Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression.
To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG.
Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.
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Séguin, Béatrice. "Control of HIV-1 RNA metabolism, the role of splice sites and intron sequences in unspliced viral RNA subcellular distribution." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0002/MQ40793.pdf.
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Kaufman, Ethan Joshua. "MicroRNA control of excretory cell development in C. elegans." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609988.
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Kew, Chun [Verfasser], and Adam [Gutachter] Antebi. "Control of Innate Immunity by RNA Metabolism / Chun Kew ; Gutachter: Adam Antebi." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1180601556/34.
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Santos, Viviane. "Riqueza e alterações morfofisiológicas associadas à infecção por vírus de RNA de fita dupla no fungo entomopatogênico Metarhizium anisopliae." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11146/tde-26042013-162222/.
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O Brasil é o país líder no uso do fungo entomopatogênico Metarhizium anisopliae para o controle pragas agrícolas. Pouco se conhece sobre a diversidade e o impacto dos micovírus em M. anisopliae sensu stricto. Este trabalho mostra a riqueza dos micovírus associados a isolados de M. anisopliae da coleção de entomopatógenos da Universidade de São Paulo, usinas sucroalcooleiras e produtos microbianos à base do entomopatógeno. RNAfd foram encontrados em 55% dos 36 isolados de Metarhizium e apresentaram 16 diferentes padrões eletroforéticos consistindo de 3 a 18 bandas de RNAfd em gel de poliacrilamida. RNAfd não foram detectados em nenhum dos produtos comerciais utilizados no presente estudo. Os diferentes padrões de vírus encontrados nos isolados aqui estudados, aparentemente, não têm relação com os locais onde foram coletados. O inibidor de síntese protéica ciclohexamida não foi eficiente na eliminação dos vírus de RNAfd em nenhum dos isolados de fungos testados. Colônias dos isolados de M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 e M. anisopliae CTC F8 foram curadas por meio do cultivo monoconidial ou isolamento de ponta de hifas. Alguns segmentos do isolado M. anisopliae PL26 foram perdidos após o subcultivo de ponta das hifas. Colônias de M. anisopliae ESALQ PL26 obtidas por meio do cultivo monoconidial ou subcultivo de ponta de hifas apresentaram grande variabilidade morfológica, no entanto, essa variação não foi correlacionada com a presença de micovírus. Colônias isogênicas de M. anisopliae ESALQ 1256 mostraram diferenças no crescimento, na produção de conídios e na virulência, no entanto, essas diferenças não foram associadas à presença de RNAfd. Não foram observadas diferenças na tolerância aos raios ultravioleta e ao calor entre as colônias de M. anisopliae ESALQ 1256, com e sem vírus de RNAfd. Colônias oriundas de setores formados no isolado M. anisopliae PL26 produziram menor quantidade de conídios e apresentaram menor quantidade de vírus RNAfd em comparação com as colônias oriundas de outras regiões da colônias originais com características normais. A repicagem sucessiva dos isolados de M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 e CTC F15, infectados por diferentes vírus de RNAfd, nos meios de cultura BDA, SDAY e meio de arroz, bem como a passagem dos fungos em larvas de Tenebrio molitor, em geral, não afetaram a replicação dos micovírus.Brazil is the leading country in the use of the entomopathogenic fungus Metarhizium anisopliae against agricultural pests. Little is known about the diversity and the impact of mycovirus in M. anisopliae sensu stricto. This study shows the richness of mycovirus associated with M. anisopliae isolates from the collections of entomopathogens of the University of São Paulo, from sugar-alcohol factories and microbial based products. dsRNA were found in 55% of the 36 Metarhizium isolates and showed 16 different electrophoretic patterns consisting of 3 to 18 dsRNA bands in polyacrylamide gels. dsRNA was not detected in any of the commercial products used in this study. The different viral patterns found in the isolates studied here apparently have no relation to the locations where they were collected. The inhibitor of protein synthesis cycloheximide culture was efficient in eradicating dsRNA virus from fungi. Colonies of isolates of M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 and M. anisopliae F8 were cured by monoconidial or by hyphal tip isolation of. Some segments of the M. anisopliae PL26 isolate were lost following hyphal tip subculture. Colonies both from monoconidial culture and hyphal tip subculture of M. anisopliae ESALQ PL26 showed great morphological variability; however, this variation was not correlated with the presence of mycoviruses. Isogenic colonies of M. anisopliae ESALQ 1256 showed differences in growth, conidia production and virulence; however, these differences were not associated to the presence of the dsRNA. No difference was observed regarding tolerance to ultraviolet rays and heat among the colonies of M. anisopliae ESALQ 1256, with and without the dsRNA virus. Sectors formed in the isolate M. anisopliae PL26 produced a smaller number of conidia and fewer dsRNA virus in comparison to the original colonies with normal characteristics. The repeated subculture of isolates of M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 and CTC F15, infected by different virus of dsRNA, in the PDA, SDAY culture media and in rice, as well as the fungi passage in larvae of Tenebrio molitor, in general, did not affect the replication of the mycovirus.
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Costa, Pedro Rafael [UNESP]. "Modelo cinético estocástico para a transcrição considerando colisões entre as moléculas de RNA polimerase." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/92463.
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A transcrição realizada pela RNA polimerase (RNAP) é um processo cuidadosamente controlado no desenvolvimento e na manutenção das funções vitais dos organismos. O desenvolvimento de novas técnicas e equipamentos para seu estudo, como as técnicas de pinça ótica ou magnética, a microscopia de força atômica e a fiuorescência de molécula única, complementaram os resultados dos estudos bioquímicos tradicionais e nos levaram a um maior entendimento do processo. A ocorrência de pausas em sítios específicos durante o alongamento, já observadas na década de 80, passou a ser estudada com maior interesse devido a sua importância biológica: acredita-se que essas pausas assegurem que a transcrição e a tradução ocorram simultaneamente em bactérias, permitam o dobramento correto das estruturas secundárias e terciárias do R A, facilitem a ligação de reguladores de alongamento e precedam a etapa de terminação transcricional. Modelos teóricos baseados na estabilidade termodinâmica do complexo de alongamento transcricional foram bem sucedidos na previsão da cinética do alongamento. Seus resultados indicaram que a RNAP pode ser vista como um motor molecular e sua motilidade possui características do modelo de catraca browniana. Entretanto, esses modelos consideram a presença de apenas uma polimerase realizando a transcrição. Experimentos recentes mostraram que a ocorrência de colisões entre essas enzimas durante a transcrição múltipla de um mesmo gene altera seu comportamento. Baseados nesses resultados, propomos a generalização de um dos modelos estocásticos que consideram a sequência molde para o estudo desse fenômeno. Em nossa aproximação, colisões entre as moléculas RNAP modificam a taxa de ocorrência da transcrição. A implementação do modelo foi realizada em...
The transcription of the information encoded within the DNA to the RNA molecule is exquisitely controlled during the development of the organisms and to its vital functions and has as the protagonist the RNA polymerase enzyme (RNAP). The development of single-molecule techniques, such as the magnetic and optical tweezers, atomic-force microscopy and single-molecule uorescence, increased our understanding of the process, complementing traditional biochemical studies. The non-homogeneity of the RNAP movement due to the occurrence of \pauses at speci c sites during elongation was revealed using electrophoresis gels. It is believed that these pauses ensure concurrency between transcription and translation in bacteria, allow the correct folding of RNA secondary and tertiary structures, facilitate the binding of regulating factors during elongation and preceding the transcriptional termination step. Theoretical models have been proposed to explain and predict the RNAP kinetics during the polymerization. Models based on the thermodynamic stability of the transcription elongation complex recover much of the kinetics and indicate that its movement has a Brownian ratchet mechanism. However, experiments showed that if more than one RNAP molecule initiate from the same promoter, their behavior changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAP molecules and predicts their cooperative behavior during multi-round transcription. The model generalizes a stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica and compared the results of the single and the multiple-molecule transcription with experimental... (Complete abstract click electronic access below)
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Costa, Pedro Rafael. "Modelo cinético estocástico para a transcrição considerando colisões entre as moléculas de RNA polimerase /." Botucatu, 2012. http://hdl.handle.net/11449/92463.
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Orientador: Ney LemkeBanca: Scheila de Ávila e Silva
Banca: José Luiz Rybarczyk Filho
Resumo: A transcrição realizada pela RNA polimerase (RNAP) é um processo cuidadosamente controlado no desenvolvimento e na manutenção das funções vitais dos organismos. O desenvolvimento de novas técnicas e equipamentos para seu estudo, como as técnicas de pinça ótica ou magnética, a microscopia de força atômica e a fiuorescência de molécula única, complementaram os resultados dos estudos bioquímicos tradicionais e nos levaram a um maior entendimento do processo. A ocorrência de "pausas" em sítios específicos durante o alongamento, já observadas na década de 80, passou a ser estudada com maior interesse devido a sua importância biológica: acredita-se que essas pausas assegurem que a transcrição e a tradução ocorram simultaneamente em bactérias, permitam o dobramento correto das estruturas secundárias e terciárias do R A, facilitem a ligação de reguladores de alongamento e precedam a etapa de terminação transcricional. Modelos teóricos baseados na estabilidade termodinâmica do complexo de alongamento transcricional foram bem sucedidos na previsão da cinética do alongamento. Seus resultados indicaram que a RNAP pode ser vista como um motor molecular e sua motilidade possui características do modelo de catraca browniana. Entretanto, esses modelos consideram a presença de apenas uma polimerase realizando a transcrição. Experimentos recentes mostraram que a ocorrência de colisões entre essas enzimas durante a transcrição múltipla de um mesmo gene altera seu comportamento. Baseados nesses resultados, propomos a generalização de um dos modelos estocásticos que consideram a sequência molde para o estudo desse fenômeno. Em nossa aproximação, colisões entre as moléculas RNAP modificam a taxa de ocorrência da transcrição. A implementação do modelo foi realizada em ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The transcription of the information encoded within the DNA to the RNA molecule is exquisitely controlled during the development of the organisms and to its vital functions and has as the protagonist the RNA polymerase enzyme (RNAP). The development of single-molecule techniques, such as the magnetic and optical tweezers, atomic-force microscopy and single-molecule uorescence, increased our understanding of the process, complementing traditional biochemical studies. The non-homogeneity of the RNAP movement due to the occurrence of \pauses" at speci c sites during elongation was revealed using electrophoresis gels. It is believed that these pauses ensure concurrency between transcription and translation in bacteria, allow the correct folding of RNA secondary and tertiary structures, facilitate the binding of regulating factors during elongation and preceding the transcriptional termination step. Theoretical models have been proposed to explain and predict the RNAP kinetics during the polymerization. Models based on the thermodynamic stability of the transcription elongation complex recover much of the kinetics and indicate that its movement has a Brownian ratchet mechanism. However, experiments showed that if more than one RNAP molecule initiate from the same promoter, their behavior changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAP molecules and predicts their cooperative behavior during multi-round transcription. The model generalizes a stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica and compared the results of the single and the multiple-molecule transcription with experimental... (Complete abstract click electronic access below)
Mestre
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Kemp, Alain. "Regulation of developmental differentation control by changes in tRNA decoding efficiency in yeast." Thesis, University of Aberdeen, 2011. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=166224.
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The yeast Saccharomyces cerevisiae decodes CAG codons using tRNAGlnCUG, encoded by the single-copy gene SUP70. On rich medium, the sup70-65 and sup70-33, alleles induce pseudohyphal growth, ordinarily a response to nitrogen limitation. sup70-65 tRNA is additionally a UAG stop codon suppressor. Characterisation of sup70 diploids revealed that they form pseudohyphal-like structures on both rich and N-deprived liquid, but not solid medium. Unlike canonical pseudohyphae, which bud in a unipolar fashion and are RAS2Val19 stimulated, sup70 pseudohyphal cells budded in a bipolar fashion that was RAS2Val19–resistant. Site-directed mutants of sup70-65 and sup70-33 that restored base complementarity in the mutant tRNA stems partially complimented the pseudohyphal phenotype and revealed that structural rigidity, rather than sequence identity, was the key phenotypic driver. A library of sup70 mutants, screened for novel nonsense UAG suppressor alleles, indentified several novel sites in the anticodon stem that induce tRNA distortion and UAG recognition. None of the new alleles induced formation of pseudohyphae. The sup70-65 pseudohyphal phenotype was weakly complemented through overexpression of the isoacceptor tRNAGlnUUG, which can inefficiently decode CAG via 3rd base wobble pairing, inferring pseudohyphal growth can be triggered by inefficient translation of CAG codons. Supporting this observation, tRNA Northern blotting revealed that all pseudohyphae-inducing sup70 mutants have reduced overall levels of tRNAGlnCUG, as well as reduced levels of tRNAGlnCUG charging. A systemic comparison of wild-type and sup70-65 proteomes using stable isotope labelling with amino acids revealed that 28 proteins showed significant changes in expression in the mutant, including 4 with known roles in control of bud size and/or budding pattern. This study revealed that pseudohyphae-inducing sup70 mutations compromise tRNAGlnCUG structure and amino acid charging and thus slow translation of cognate CAG codons, probably down-regulating the expression of a yet-to-be identified gene affecting the control of pseudohyphal differentiation.
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Pizzinga, Mariavittoria. "Granules of translation factor mRNAs and their potential role in the localisation of the translation machinery to regions of polarised growth." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/granules-of-translation-factor-mrnas-and-their-potential-role-in-the-localisation-of-the-translation-machinery-to-regions-of-polarised-growth(9cb42e69-3c8c-4f10-b79f-ba8261be4430).html.
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The subcellular localisation of mRNA is a widespread mechanism to determine the fate of mRNAs in eukaryotes. Translationally repressed mRNAs localise to P-bodies and stress granules where their decay and storage, respectively, are directed. In a study from the Ashe lab, specific mRNAs were identified to localise, in actively growing S. cerevisiae, to cytoplasmic granules that do not seem to be related to P-bodies or stress granules but appear to be associated with active translation (Lui et al., 2014).It is possible that this might represent a strategy to co-regulate the expression of proteins from the same pathway. In the work of this thesis, microscopy techniques to visualise RNAs in live cells were used to extend the localisation analysis to several mRNAs encoding translation factors. The investigated transcripts were all found to localise to mostly one or two cytoplasmic granules per cell and would sometimes overlap with other transcripts, suggesting that each granule contains a mixture of mRNAs. Granules tend to migrate to the bud tip and may provide the daughter cell with a "start-up kit" of transcripts essential for rapid growth. A similar pattern can be observed in yeast cells growing undergoing filamentous growth, with granules harbouring translation factor transcripts often found in the apical quarter of the elongated cell. Although the mechanism by which the granules form and their protein composition are not yet known, high-throughput genetic screens performed as part of this work offer some insight into factors that might be involved in granule assembly and proteins that partially overlap with the granules. We propose that granules containing translation factor mRNAs might be functioning as a specialised factory for the translation machinery and are possibly being directed to the point in the cell where the rhythm of protein production is highest.
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Kabnick, Karen Stephanie. "A novel approach for the study of sequences that control cytoplasmic RNA stability." Thesis, Massachusetts Institute of Technology, 1987. https://hdl.handle.net/1721.1/121909.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1987.Bibliography: leaves 190-202.
by Karen Stephanie Kabnick.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1987.
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Stanoszek, Lauren M. "Biospecimen RNA Quality Control in Reverse-Transcription, Quantitative PCR (RT-qPCR) Clinical Tests." University of Toledo Health Science Campus / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=mco1430323793.
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Aung-Htut, May Thandar Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Characterisation of Escherichia coli GTPase Der reveals previously unknown regulation by RNA." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41840.
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GTPases are found in all domains of life and are highly conserved. In eukaryotes, they serve as signalling molecules for many cellular processes. However, the prokaryotic GTPases play a very different role and are found to be associated with ribosome function. Among the 11 conserved GTPases, Der is the most interesting in prokaryotes. It possesses a unique structure with two GTPase domains (G-Domains) tethered by a variable length acidic linker and a carboxyl terminal KH-like domain. The exact function of Der is still under investigation and most of the data suggest that it is important for 50S ribosomal assembly or stability. In order to investigate the function of Escherichia coli Der (Ec-Der), expression plasmids for wild-type and mutated proteins were created and the proteins were successfully expressed. The expression of the mutant protein that lacked G-Domain 1 was toxic to the cells and it was found that some large ribosomal proteins were missing from the ribosomes of these cells. In addition, other macromolecular complexes such as the GroEL/GroES chaperonin appeared not to be assembled under these conditions. The activities of both wild-type and mutated proteins were also tested and found to be dependent on potassium ions (K+), which enhanced nucleotide binding. Additionally, intra-molecular control over nucleotide binding and release was also observed for Ec-Der. The in vitro selection of RNA aptamers with nanomolar affinity for Ec-Der produced aptamers that contained short variable sequences. These aptamers affected the growth of the E. coli cells and caused a change in cellular morphology that had been noted previously during Ec-Der over-expression. Ec-Der showed high affinity (nM) to both selected RNA and the unselected RNA library. The activity of Ec-Der and Era was inhibited in the presence of any sequence of RNA that has the length of greater than 16 nucleotides. RNA was also cross-linked to Ec-Der in the presence of GTP, but not GDP, suggesting that RNA was a regulator of the Ec-Der GTPase cycle. Based on these results, it is speculated that Ec-Der might be involved in more than one function. It may be acting at the level of the membrane (based on cellular morphology reported here and by Hwang and Inouye 2001) and may also take part in processes related to ribosome function. Regulation of protein activity by RNA length has not been predicted or described and this may represent a novel mean of regulation of the Era subfamily of GTPases.
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Roberts, Tiffiney Marie. "Redundant structural motifs in a unique retroviral posttranscriptional control element mediate a novel mechanism of translational enhancement." Columbus, Ohio : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1066927045.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xix, 165 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Kathleen Boris-Lawrie, Dept. of Molecular, Cellular and Developmental Biology. Includes bibliographical references (p. 148-165).
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Bardella, Daniela Zardini. "Silenciamento gênico via RNAi visando o controle da broca da cana-de-açúcar (Diatraea saccharalis)." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-01022016-172023/.
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A cana-de-açúcar (Saccharum spp.) é uma importante cultura na produção de alimentos e energia. Várias espécies de insetos podem causar sérios prejuízos econômicos à cultura da cana-de-açúcar. A broca da cana-de-açúcar (Diatraea saccharalis) é a praga de maior relevância por estar amplamente distribuída nas regiões canavieiras. O silenciamento gênico por RNA de interferência (RNAi) se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia. Uma de suas aplicações é no controle de insetos-praga como uma alternativa de alta eficiência, especificidade e reduzido impacto ambiental. A ingestão de moléculas de RNA dupla fita (dsRNA) com identidade a regiões de genes essenciais de insetos-praga pode resultar no silenciamento destes genes, levando a fenótipos deficientes. Neste contexto, o presente trabalho teve como objetivo buscar genes alvos para o silenciamento com potencial para impedir o desenvolvimento normal da D. saccharalis e estabelecer uma forma de entrega do dsRNA eficiente para o teste de genes, visando assim validar o uso da técnica para a espécie. Por meio da clonagem de regiões de genes ortólogos já utilizados como alvo de silencimento em outras espécies de insetos (V-ATPase A, Receptor de Ecdisona e Arginina Kinase), e de genes com função específica identificadas após a caracterização do transcritoma de D. saccharalis (Juvenile Hormone Epoxide Hydrolase, Neverland e Quitina Sintase) foram conduzidos ensaios de RNAi. Foram realizados ensaios de dose resposta para o gene V-ATPaseem lagartas neonatas, onde a concentração selecionada por causar melhor redução na expressão do gene alvo foi de 2,5 µg µL-1. Esta concentração foi então utilizada em ensaios de alimentação para os outros genes. Os genes V-ATPase A, receptor de Ecdisona, Arginina Kinase, Juvenile Hormone Epoxide Hydrolase e Quitina Sintase apresentaram redução significativa no número de transcritos em larvas, demonstrando a viabilidade do uso de RNAi em D. saccharalisneonatas. O gene Neverland não demonstrou redução no acúmulo de transcritos nas condições trabalhadas. O gene GFP inicialmente utilizado como controle negativo apresentou variação na expressão de genes alvo, sendo desconsiderado como bom controle para D. saccharalis. O silenciamento dos genes alvo requer quantidades elevadas de dsRNA, superiores aos obtidos por transcrição in vitro, o que limita a viabilidade de ensaios com maiores replicatas e para determinar efeitos biológico. Alternativas de produção de dsRNA devem ser avaliadas para viabilizar a seleção de genes alvo efetivosSugarcane (Saccharum spp.) is an important crop for the production of food and bioenergy. Many insect species can cause economic losses in sugarcane. The sugarcane borer (Diatraea saccharalis) is the most important sugarcane pest, because it occurs in all production regions. Gene silencing by RNA interference (RNAi) rapidly became a widely investigated approach, adopted in various aspects of biology. One of the potential applications of RNAi is agricultural pest control, as an alternative with high efficiency, specificity and reduced environmental impact. The ingestion of double-stranded RNA (dsRNA) molecules with identity to regions of essential genes of the insect-pest can result in the target gene knock-down and, consequently, to deficient phenotypes. In the present work, target genes with the potential to affect the normal development of D. saccharaliswere searched, together with an efficient dsRNA delivery approach to test the target-genes to validate the use of the RNAi in D. saccharalis. Based on degenerated primers, expressed orthologous genes previously tested in other insect species (V-ATPase A, Ecdisone Receptor, and Arginine Kinase) were cloned,whilegenes with specific function (Juvenile Hormone Epoxide Hydrolase, Neverland, and Chitin Synthase) were identified from an in-house assembled transcriptome of D. saccharalis and cloned. A dose-response assay was conducted using the V-ATPase gene region delivered by droplets to neonate larvae, and the 2.5 µg µL-1dsRNA concentration was selected for further tests. This concentration was then used to deliver the other genes. The dsRNA version from the genes V-ATPase A, Ecdisone Receptor, Arginine Kinase, Juvenile Hormone Epoxide Hydrolase and Chitin Synthaseexhibited a significant reduction in the accumulation of transcripts, indicating the viability of RNAi to D. saccharalis in 1st instar larvae. The Neverland gene was not silenced by RNAi in the used conditions. The dsRNA of the Green Fluorescent Protein gene, used as negative control appeared to affectother gene targets. Target gene silencing require large amounts of dsRNA, more than what is achievable by in vitro transcription, which limits the viability to conduct large assays with more replicates and to determine biological effects. Alternatives to produce dsRNA need to be evaluated to enable the selection of effective target genes
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Loomis, Wendy Pulkkinen. "Translational control of messenger RNA processing in the F1845 fimbrial operon of Escherichia coli /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11507.
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Jones, Steven Tarran. "The role of the #beta# subunit of E. coli DNA-dependant RNA polymerase in stringent control." Thesis, University of Nottingham, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.232905.
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Jenkins, Adam. "Analysis of anopheline mosquito behavior and identification of vector control targets in the post-genomic era." Thesis, Boston College, 2015. http://hdl.handle.net/2345/bc-ir:104489.
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Thesis advisor: Marc A.T. MuskavitchThe protozoan Plasmodium falciparum, the mosquito-borne pathogen that causes human malaria, remains one of the most difficult infectious parasites to combat and control. Campaigns against malaria eradication have succeeded, in most instances, at the level of vector control, rather than from initiatives that have attempted to decrease malaria burden by targeting parasites. The rapid evolution and spread of insecticide-resistant mosquitoes is threatening our ability to combat vectors and control malaria. Therefore, the development, procurement and distribution of new methods of vector control are paramount. Two aspects of vector biology that can be exploited toward these ends are vector behaviors and vector-specific insecticide targets. In this thesis, I describe three aspects of vector biology with potential for the development of improved means of vector control: photopreference behavior, long non-coding RNA (lncRNA) targets and epigenetic gene ensemble targets. My studies of photopreference have revealed that specific mosquito species within the genus Anopheles, An. gambiae and An. stephensi, exhibit different photopreference behaviors, and that each gender of mosquito in these species exhibits distinct light-dependent resting behaviors. These inter-specific behavioral differences may be affected by differing numbers of long-wavelength sensing Opsin genes in each species, and my findings regarding species-specific photopreferences suggest that some behavioral interventions may need to be tailored for specific vector mosquito species. Based on the advancement of next-generation sequencing technologies and the generation by others of assembled genomes of many anopheline mosquito species, I have identified a comprehensive set of approximately 3,000 lncRNAs and find that RNA secondary structures are notably conserved within the gambiae species complex. As lncRNAs and epigenetic modifiers cooperate to modulate epigenetic regulation, I have also analyzed the conservation of epigenetic gene ensembles across a number of anopheline species, based on identification of homologous epigenetic ensemble genes in An. gambiae compared to Drosophila melanogaster. Further analyses of these ensembles illustrate that these epigenetic genes are highly stable among many anopheline species, in that I detect only eight gene family expansion or contraction events among 169 epigenetic ensemble genes within a set of 12 anopheline species. My hope is that my findings will enable deeper investigations of many behavioral and epigenetic processes in Anopheles gambiae and other anopheline vector mosquitoes and thereby enable the development of new, more effective means of vector and malaria control
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Machado, Madson Cruz. "Sintonia RNA-RBF para o Projeto Online de Sistemas de Controle Adaptativo." Universidade Federal do Maranhão, 2017. http://tedebc.ufma.br:8080/jspui/handle/tede/1744.
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The need to increase industrial productivity coupled with quality and low cost requirements has generated a demand for the development of high performance controllers. Motivated by this demand, we presented in this work models, algorithms and a methodology for the online project of high-performance control systems. The models have characteristics of adaptability through adaptive control system architectures. The models developed were based on artificial neural networks of radial basis function type, for the online project of model reference adaptive control systems associated with the of sliding modes control. The algorithms and the embedded system developed for the online project were evaluated for tracking mobile targets, in this case, the solar radiation. The control system has the objective of keeping the surface of the photovoltaic module perpendicular to the solar radiation, in this way the energy generated by the module will be as high as possible. The process consists of a photovoltaic panel coupled in a structure that rotates around an axis parallel to the earth’s surface, positioning the panel in order to capture the highest solar radiation as function of its displacement throughout the day.
A necessidade de aumentar a produtividade industrial, associada com os requisitos de qualidade e baixo custo, gerou uma demanda para o desenvolvimento de controladores de alto desempenho. Motivado por esta demanda, apresentou-se neste trabalho modelos, algoritmos e uma metodologia para o projeto online de sistemas de controle de alto desempenho. Os modelos apresentam características de adaptabilidade por meio de arquiteturas de sistemas de controle adaptativo. O desenvolvimento de modelos, baseia-se em redes neurais artificiais (RNA), do tipo função de base radial (RBF, radial basis function), para o projeto online de sistemas de controle adaptativo do tipo modelo de referência associado com o controle de modos deslizantes (SMC, sliding mode control). Os algoritmos e o sistema embarcado desenvolvidos para o projeto online são avaliados para o rastreamento de alvos móveis, neste caso, o rastreamento da radiação solar. O sistema de controle tem o objetivo de manter a superfície do módulo fotovoltaico perpendicular à radiação solar, pois dessa forma a energia gerada pelo módulo será a maior possível. O processo consiste de um painel fotovoltaico acoplado em uma estrutura que gira em torno de um eixo paralelo à superfície da terra, posicionando o painel de forma a capturar a maior radiação solar em função de seu deslocamento ao longo do dia.
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Bing, Xin Y. "Mechanistic Basis for Control of Early Embryonic Development by a 5’ tRNA Fragment." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1035.
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Ancestral environmental conditions can instruct offspring development, although the mechanism(s) underlying such transgenerational epigenetic inheritance is unclear. In murine models focused on paternal dietary effects, we and others have identified tRNA fragments (tRFs) in mature sperm as potential carriers of epigenetic information. In our search for molecular targets of specific tRFs, we observed that altering the level of 5’-tRF Glycine-GCC (tRF-GG) in mouse embryonic stem cells (mESCs) and preimplantation embryos modulates the expression of the endogenous retrovirus MERV-L and genes regulated by MERV-L. Intriguingly, transient derepression of MERV-L is associated with totipotency of two-cell stage embryos and a subset of two-cell-like mESCs.
Here, I reveal the mechanistic basis for tRF-GG regulation of MERV-L. I show that tRF-GG supports the production of numerous small nuclear RNAs associated with the Cajal body, in mouse and human embryonic stem cells. In particular, tRF-GG modulates the levels of U7 snRNA to ensure an adequate supply of histone proteins. This in turn safeguards heterochromatin-mediated transcriptional repression of MERV-L elements. Importantly, tRF-GG effects on histone mRNA levels, activity of a histone 3’UTR reporter, and expression of MERV-L associated transcripts can all be suppressed by appropriate manipulation of U7 RNA levels. I also show that hnRNPF and H bind directly to tRF-GG, and display a stark overlap of in vivo functions to tRF-GG. Together, this data uncovers a conserved mechanism for a 5’ tRNA fragment in the fine-tuning of a regulatory cascade to modulate global chromatin organization during pre-implantation development.
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Luhtala, Natalie. "LSM1 AND RNY1: CLUES IN THE SEARCH FOR HOW RNA METABOLIC PATHWAYS CONTROL CANCER." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/204114.
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Carcinogenesis requires numerous alterations to gene expression to evade normal controls on cellular growth, invasion, and immortality. Traditionally, these changes have been examined in the context of deregulated transcriptional control of oncogenes and tumor suppressors. However, in recent years, research has revealed that processes outside of transcription such as RNA splicing, translation, and decay are also deregulated in cancers, sustaining tumorigenic potential. This dissertation details our investigation into the cellular functions of two RNA metabolic proteins whose human orthologs are deregulated in tumors: a putative oncogene, Lsm1, and a putative tumor suppressor, Rny1. Herein, we reveal interesting functions for these proteins that might provide insight into their roles in carcinogenesis. First, we demonstrate a role for Lsm1 over-expression in altered splicing through depletion of U6 snRNA levels. Second, we clarify the mechanism for Rny1's activity against RNA substrates and identify cis regions required for its non-catalytic role in growth inhibition. Overall, this knowledge expands our understanding of how RNA metabolism might be deregulated in cancer and could provide novel pathways to target for synthetic lethal responses in cancers with altered expression of these proteins.
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Black, E. J. "Studies on the use of antisense RNA to control gene expression in Nicotiana tabacum." Thesis, Imperial College London, 1987. http://hdl.handle.net/10044/1/38236.
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McFarland, Matthew R. "Transfer RNAs as regulatory agents in the translational control of gene expression." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=231855.
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Translational efficiency is dictated in part by the availability of charged transfer RNA. Depletion of aminoacylated tRNAs (e.g. during recombinant protein expression) can increase translational errors and associated stress responses. Here, the role of tRNAs as regulators of gene expression was explored through development of synthetic, tRNA-regulated gene circuits, and through an investigation of the impact of tRNA aminoacylation on endogenous gene expression. Synthetic gene circuits initially explored the use of dominant negative alleles of the release factor eRF1 to modulate stop codon readthrough and translationally regulate gene expression. Mutant eRF1 proteins exhibited only a six-fold stimulatory effect on stop codon readthrough. The dominant negative phenotype was rescued partially by overexpression of eRF1, but not eRF3. Ultimately the severity of growth inhibition by these eRF1 alleles limited their utility in synthetic gene circuit design. A novel synthetic circuit was then implemented that utilised TetR interaction with a TetR-inducing peptide in order to control the expression of a suppressor tRNA, and thus a luciferase reporter gene. Using a parameterised mathematical model, the promoter configuration of the circuit was successfully optimised, allowing suppressor tRNAs to regulate the production of luciferase in both feedforward and positive feedback modes of operation. The effects of charged tRNA levels on the global translation network were dissected by regulating the S.cerevisiae glutamine tRNA synthetase gene GLN4 using a tet-off doxycyclineregulated promoter. tRNA synthetase depletion caused the activation of the Gcn4 amino acid starvation response due to accumulation of uncharged glutamine tRNAs. Doxycycline GLN4 shut-off caused increased amino acid production, and decreased ribosome biosynthesis at the transcriptomic and proteomic level, and further physiological changes proposed to result from compromised translation of glutamine-rich regulatory proteins. tRNA overexpression in the GLN4 depletion strain successfully caused altered competition between different isoacceptor tRNA types for their cognate synthetase resource. Together, these results support a growing understanding of tRNA as a key modulator of translation and gene expression in synthetic and natural systems.
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Sharma, Amit. "Functional control of HIV-1 post-transcriptional gene expression by host cell factors." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1330658547.
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Hull, Stacey Lynn. "Identification and Characterization of New and Distinct Functional Roles of Posttranscriptional Control Elements in Cytoplasmic Expression of Retroviral RNA." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1038349636.
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Bronicki, Lucas M. "Characterization of Multiple Exon 1 Variants and Neuron-specific Transcriptional Control of Mammalian HuD." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23682.
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The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models.
Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD.
Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.
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Thakur, Noopur. "Long-range Control of Gene Expression by Imprinting Control Regions During Development and Neoplasia." Doctoral thesis, Uppsala universitet, Zoologisk utvecklingsbiologi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5725.
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Genomic imprinting is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin specific manner. Most of the imprinted genes are located in clusters. Genetic evidences suggest that genes in imprinted clusters are regulated by Imprinting Control Regions (ICRs). To elucidate the mechanisms by which the imprinting is maintained in clusters, we have chosen a well characterized cluster at the distal end of mouse chromosome 7. This cluster contains 15 imprinted genes and they have been shown to be regulated by H19 and Kcnq1 ICRs. The mouse H19 ICR, which is shown to have a chromatin insulator function, is implicated in the regulation of H19 and Igf2 genes by interacting with the CTCF protein. It has been documented that CTCF is also involved in the maintenance of differential methylation at the ICR. In this investigation we demonstrated that CTCF maintained differential methylation is lost when we subjected the ICR containing episomal plasmids to de novo methylation machinery of the human choriocarcinoma cell line, JEG3, suggesting that the H19 ICR looses its methylation privilege property under neoplastic conditions. The Kcnq1 ICR has been implicated in the regulation of 11 imprinted genes. The Kcnq1 ICR is methylated on the active maternal allele but unmethylated on the inactive paternal allele and overlaps an oppositely oriented and paternally expressed gene known as Kcnq1ot1. In this investigation, we documented that the Kcnq1 ICR controls the imprinting of neighboring genes by behaving as a bidirectional silencer and that this function is regulated by antisense RNA Kcnq1ot1. Furthermore, we have documented that duration of antisense transcription plays a critical role in the antisense RNA- mediated silencing. In conclusion, this thesis provides more insights into the complex mechanistic aspects by which ICRs, control imprinting of genes in clusters during development and neoplasia.
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Stevenson, Michael Andrew. "RNA interference as a tool to control plant parasitic nematode infestation in key plant crops." Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.677278.
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The work presented in this thesis documents studies on the impact of RNA interference (RNAi) on potential control target genes and how this alters the phenotype of the pre-parasitic stage of the plant parasitic nematodes (PPN), Globodera pallida and Meloidogyne incognita. A wide range of targets were selected across all the main tissue types within the worms (hypodermis, muscle, nervous system, subventral gland, dorsal gland) and significant knockdown was achieved (using short-interfering [si]RNAs) in all tissue types except the dorsal gland. Overall, the data strongly support the utility of PPN J2s as a model for functional genomics studies using RNAi. Further work was carried out on selected targets including the neuropeptide-encoding genes, Gp-flp-30 and Gp-flp-31 which are thought to be only expressed in PPN species. These targets showed reduced target transcript levels following RNAi, however not to the same degree as most of the previously targeted genes; their silencing did not induce a significant phenotype suggesting they are involved in functions other than normallocomotion/chemotaxis. We also targeted Gp-flp-21 and its putative receptor encoding gene Gp-flp-21 R which in Caenorhabditis elegans modulate sociality. Silencing either gene did not alter PPN motility but did disrupt positive chemotaxis, consistent with a role in chemosensation and consistent with the FLP-21 R being the FLP-21 receptor. Finally, the work reported here has shown that silencing Gp-ace-2, which encodes an acetylcholinesterase, results in complete paralysis of the parasites and prevents them from infecting their host plant and thus complete their life cycle. These data provide functional validation for a variety of PPN control targets.
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Hoyos, Mona [Verfasser], and Kai [Akademischer Betreuer] Papenfort. "Principles of RNA-based gene expression control in Vibrio cholerae / Mona Hoyos ; Betreuer: Kai Papenfort." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1227188471/34.
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Sweet, Thomas Jeffrey. "New Insights Into the Relationship Between Messenger RNA Translation and Degradation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1321298653.
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