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Ciolli, Mattioli Camilla. "Post-transcriptional mechanisms contributing to RNA and protein localization: study of local translation and alternative 3′UTRs in induced neurons." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20702.
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Die asymmetrische Verteilung von mRNA und Proteinen innerhalb einer Zelle definiert die Polarität. Dies ermöglicht eine strikte Regulierung der Genexpression in Raum und Zeit. Ich habe in dieser Arbeit untersucht, wie das Soma und die Neuriten in induzierten Neuronen sich hinsichtlich ihres Transkriptoms und Translatoms unterscheiden. Eine räumliche ribosomale Profilanalyse ergab, dass die Hälfte des lokalen Proteoms durch die mRNA-Lokalisierung und der lokalen Translation definiert wird. Dies sind Prozesse, die durch die synergistische Aktivität von trans- und cis-agierenden Elementen durchgeführt werden. In dieser Arbeit konzentrierte ich mich auf MOV10 als trans-agierendes Element und die alternativen 3′UTRs als cis-agierende Elemente, um ihre Rolle in der Asymmetrie zu untersuchen. MOV10 ist eine RNA-Helikase, welche an vielen Aspekten des RNA-Metabolismus beteiligt ist. Mit den Methoden RIP und PAR-CLIP konnte ich zeigen, dass sowohl MOV10-Ziele als auch MOV10 selbst in den Neuriten lokalisiert sind. Aus ̈erdem ist MOV10 möglicherweise an der translationalen Repression mitinvolviert.
In der Tat konnte ich unter den MOV10-Protein-Interaktoren mehrere Proteine identifizieren, welche an der translationalen Repression beteiligt sind, wie z.Bsp. AGO2, FMR1, und TRIM71. Für die Identifizierung der cis-agierenden Elemente führte ich das "Mapping" von alternativen 3′UTRs durch. Diese Analyse zeigte mehrere Gene, die differentiell lokalisierte 3′UTR-Isoformen exprimieren. Insbesondere habe ich mich auf Cdc42 konzentriert. Ich konnte beweisen, dass die beiden Isoformen von Cdc42 auf mRNA-Ebene unterschiedlich lokalisiert sind und dass die 3′UTR der entscheidende Faktor für die mRNA- und Proteinlokalisierung ist. Darüber hinaus habe ich mehrere RBPs identifiziert, die an der Cdc42-Lokalisierung beteiligt sind. Diese Analyse zeigt, dass für die differenzierte Lokalisierung von funktional unterschiedlichen alternativen Protein-Isoformen die Verwendung von alternativen 3′UTR Isoformen als neu-entdeckter Mechanismus eine entscheidende Rolle spielt.Asymmetric distribution of mRNA and proteins inside a cell defines polarity, which allow tight regulation of gene expression in space and time. In this thesis I investigated how asymmetric distribution characterizes the somatic and neuritic compartments of in induced neurons, in terms of transcriptome and translatome. Spatial ribosome profiling analysis revealed that half of the local proteome is defined by mRNA localization and local translation. These, are processes accomplished by the synergistic activity of trans- and cis-acting elements. I focused on MOV10 as trans-acting element, and on alternative 3′UTRs as cis-elements, to investigate their role in asymmetry. MOV10 is an RNA helicase which participates to many aspects of RNA metabolism. With RIP and PAR-CLIP I showed that MOV10 targets are localized to the neurites, consistently with MOV10-neuritic localization, and that MOV10 might be involved in translational repression. Indeed, among MOV10 protein interactors, I identified several proteins involved in translational repression, i.e. AGO2, FMR1, and TRIM71. On the side of cis-elements, I performed mapping of alternative 3′UTRs. This analysis identified several genes expressing differentially localized 3′UTR isoforms. In particular, I focused on Cdc42. I showed that the two isoforms of Cdc42 are differentially localized at mRNA level, and that the 3′UTR is the driver of mRNA and protein localization. Moreover, I identified several RBPs that might be involved in Cdc42 localization. This analysis points to usage of alternative 3′UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.
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Rongo, Christopher Gabriel. "The role of RNA localization and translational regulation in Drosophila germ cell determination." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/10562.
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Vicario, Annalisa. "Analysis of the molecular mechanisms of BDNF mRNA localization and traslation in neurons." Doctoral thesis, Università degli studi di Trieste, 2010. http://hdl.handle.net/10077/3664.
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2008/2009La regolazione dell’espressione genica rappresenta uno fenomeno fondamentale per garantire la sopravvivenza e la corretta funzione cellulare. In strutture complesse ed altamente specializzate come il sistema nervoso, la grande varietà morfologica e funzionale, la rapidità di interscambio di comunicazioni e di adattamento richiede un’altrettanto fine regolazione spazio-temporale dell’espressione genica. I livelli di regolazione sono molteplici e includono lo splicing alternativo, la regolazione del turnover degli mRNA, modifiche post-traduzionali ed il controllo traduzionale. La segregazione dei trascritti in diversi compartimenti subcellulari rappresenta un ulteriore meccanismo che permette di concentrare le proteine in specifici domini cellulari mediante traduzione localizzata. A partire dagli anni ’80 sono stati scoperti numerosi mRNA a livello dendritico, tra cui i trascritti codificanti Brain Derived Neurotrophic Factor (BDNF) ed il suo recettore TrkB (Tongiorgi et al., 1997). L’mRNA per BDNF si localizza nei dendriti in risposta all‘attività elettrica e al BDNF in vitro e in seguito a crisi epilettogeniche indotte in vivo (Tongiorgi et al., 1997, 2004; Righi et al., 2000). La segregazione degli mRNA nei neuriti presuppone la potenziale traduzione in loco a seguito di specifici stimoli. A tal supporto vi è la dimostrazione della presenza di fattori coinvolti nella sintesi proteica (poliribosmi, tRNA, eIFs, eEFs, marker del reticolo endoplasmatico e del golgi) alla base delle spine dendritiche (Steward and Levi,1982; Tiedge and Brosius, 1996). Le dinamiche del trasporto coinvolgono elementi in trans, le RNA binding proteins, , ed elementi in cis, riconosciuti dalle proteine di trasporto. Questi ultimi sono solitamente confinati nella regione 3’UTR (Bashirullah et al., 1998), in misura inferiore all’interno della regione codificante (CDS) (Mohr, 1999; Chiaruttini et al., 2009) e dei 5’UTR (Muslimov et al., 1997). Tra le più comuni RBPs annoveriamo CPEB, ZBP, hnRNP, Staufen, FMRP, Translin e le proteine ELAV. Ricordiamo come molte di queste proteine di trasporto degli mRNA siano in realtà repressori della traduzione, al fine di prevenirne l’espressione ectopica durante il trasporto. Le RBPs che si legano all’mRNA di BDNF non sono ancora note, tuttavia nel corso di questo studio, mediante ibridazioni in situ non radioattiva su colture ippocampali/sezioni di cervello di topo è stata scoperta un serie di elementi in cis che regolano la localizzazione del trascritto. Alla luce dei risultati ottenuti, emerge un complesso quadro di regolazione post-trascrizionale e traduzionale di BDNF. L’RNA endogeno si localizza nel compartimento dendritico distale in seguito ad attività elettrica ed applicazione di BDNF od NT-3. I segnali di trasporto sensibili sono molteplici e distribuiti in diverse regioni dell’mRNA: un segnale costitutivo a carico della CDS(riconosciuto da translin), due segnali inducibili a livello del 3’UTR short (KCL ed NT-3, riconosciuti da CPEB1 e 2, ELAV2 e 4) ed altrettanti a livello 3’UTR long (KCl e BDNF, target di ELAV e CPEB), in aggiunta ad un segnale di ritenzione all’interno della stessa regione (osservato anche in topi KO per FXR2 ed FMRP). La modulazione del trasporto del 3’UTR long è di gran lunga più finemente regolata rispetto alla variante short, e ricorda il comportamento del 3’UTR della CaMKII (Mori et al., 2000), anch’esso coinvolto nella plasticità e potenziamento sinaptici. Dal punto di vista traduzionale, la distinzione tra 3’UTR short e long è netta: per quanto riguarda il primo, il meccanismo è piuttosto lineare e viene attivato dalla cascata del glutammato/Aurora chinasi/CPEB, mentre per il secondo, scarsamente traducibile, sembra sia necessaria la compresenza di più stimoli per attivarne la corretta traduzione, suggerendo come il 3’UTR long possa rappresentare un “coincidence detector“ che viene attivato solo in particolari contesti, a traccia di una complessa attività sinaptica. Dagli studi condotti siamo stati in grado di costruire un modello che possa spiegare come un trascritto così complesso possa rispondere a diversi stimoli. La CDS contiene un segnale di trasporto costitutivo mediato da translin, che in condizioni basali viene soppresso da un elemento inibitorio all’interno del 3’UTR long. In seguito ad attivazione viene meno la repressione in modo da favorire il trasporto del trascritto mediato dai due segnali di targeting (CDS e 3’long). Per quanto riguarda i trascritti contenenti la variante short, invece, sembra non vi siano segnali di ritenzione, bensì elementi di trasporto che vengono attivati in seguito ad uno specifico stimolo extracellulare (KCL od NT3).
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Ohler, Uwe [Gutachter], Florian [Gutachter] Heyd, and Chakrabarti [Gutachter] Sutapa. "Post-transcriptional mechanisms contributing to RNA and protein localization: study of local translation and alternative 3′UTRs in induced neurons / Gutachter: Uwe Ohler, Florian Heyd, Chakrabarti Sutapa." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1199930695/34.
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Maderazo, Alan Baer. "A Study on the Cellular Localization of Factors Involved in Yeast Nonsense-Mediated mRNA Decay and their Mechanisms of Control on Nonsense mRNA Translation: a Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/105.
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Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism.
To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD.
This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.
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Eliscovich, Carolina. "Spindle-Localized CPE-Mediated Translation Controls Mediotic Chromosome Segregation." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7123.
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La progresión meiótica y el desarrollo embrionario temprano están programados, en parte, por la activación tradcuccional de mRNAs maternos como lo son los que codifican para las proteinas de ciclina B1 o mos. Estos mRNAs no son traducidos al mismo tiempo ni en el mismo lugar. Por lo contrario, su traducción está especificamente regulada por elementos de poliadenilación citoplasmática (CPEs) presentes en sus 3'UTRs. Los elementos CPEs reclutan a la proteina de unión a CPE (CPE-binding protein CPEB (Colegrove-Otero et al., 2005; de Moor et al., 2005; Mendez and Richter, 2001; Richter, 2007)). Esta proteina de unión al RNA no sólo determina cuándo y en qué medida un mRNA será activado traduccionalmente por poliadenilación citoplasmática (Mendez et al., 2000a; Mendez et al., 2000b; Mendez et al., 2002) sino que también participa, junto con el represor de la traducción Maskin, en el transporte y la localización de sus mRNAs diana hacia los sitios de localización subcelular donde su traducción ocurrirá (Huang et al., 2003; Huang and Richter, 2004). Durante el desarrollo embrionario de Xenopus, CPEB se encuentra localizada en el polo animal de los oocitos y más tarde, sobre el huso mitótico y centrosomas en el embrión (Groisman et al., 2000). Se ha demostrado que embriones de Xenopus inyectados con agentes que interrumpen la traducción dependiente de poliadenilación citoplasmática, detienen la división celular y presentan estructuras mitóticas anormales (Groisman et al., 2000). En este trabajo que derivó en mi tesis doctoral, hemos demostrado que la activación traduccional localizada en el huso mitótico de mRNAs regulados por CPEB que codifican para proteinas con una conocida función en aspectos estructurales del ciclo celular como la formación del huso mitótico y la segregación cromosómica, es esencial para completar la primera división meiótica y para la correcta segregación cromosómica en oocitos de Xenopus.
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Reynolds, Joanna Elizabeth. "Initiation of hepatitis C virus RNA translation." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264546.
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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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Lempke, Carola. "Internal initiation of translation of cardiovirus RNA." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624267.
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Donlevy, Alison. "Regulation of RNA translation by phenethyl isothiocyanate." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/362491/.
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Phenethyl isothiocyanate (PEITC) is a dietary phytochemical that has received considerable interest for its potential chemopreventive/therapeutic anti -cancer activity. PEITC inhibits cancer cell proliferation and/or survival in vitro, suppresses angiogenesis and decreases tumour growth in vivo with little toxicity. However, the mechanisms by which PEITC exerts its anti-cancer effects are not known. The goal of this project was to investigate the hypothesis that anti-cancer effects ofPEITC may involve inhibition of mRNA translation. Effects of PEITC on global mRNA translation were first studied in human MCF7 breast cancer cells using both polysome proflling and 35S-metabolic labelling experiments. PEITC caused a dose- and time-dependent inhibition of mRNA translation, which was partially reversed following removal of PEITC. Inhibition of mRNA translation was associated with decreased expression of HIFla and VEGF, two proteins that are key for pro-angiogenic responses of malignant cells, in both normoxic and hypoxic conditions, at least in part via effects on translation of HlF1A and VEGF mRNAs. Although PEITC has previously been shown to inhibit signalling via the mTORC I pathway, further analysis demonstrated that PEITC also caused a rapid increase in phosphorylation of eIF2a at SerSI which can result in inhibition of initiation of mRNA translation. Increased eIF2a phosphorylation was important for PEITCmediated inhibition of mRNA translation since mouse embryo fIbroblasts expressing nonphosphorylatable eIF2a were relatively resistant to PEITC-induced inhibition of mRNA translation compared to control cells. In addition, PEITC caused the accumulation of stress granules, which have previously been associated with translationally stalled InRNAs. To extend these results to a more clinically relevant setting, further studies were performed using cells isolated fi'om the blood of patients with chronic lymphocytic leukaemia (CLL), the most common leukaemia in the Western world. Signalling via the B-cell receptor (BCR) is known to play a major role in the development and progression of CLL, and studies first investigated how BCR stimulation altered mRNA translation in primary CLL cells. Stimulation of surface IgM (sIgM) resulted in signiflcant, but variable, increases in mRNA translation. Overall, increases in InRNA translation were higher in samples that were considered as sIgM responsive (based on previous analysis of anti-IgM-induced intracellular Ca2+ mobilisation) compared to non-responsive samples, and in samples stimulated with anti-IgM compared to anti-IgD. Anti-IgM also increased expression of MYC and MCLl, two key targets for CLL proliferation and survival, via increased transcription and mRNA translation. ,PEITC inhibited both basal and anti-IgM-induced RNA translation, whereas ibrutinib and tamatinib, inhibitors of the BCR-associated signalling kinase BTK and SYK, predominantly inhibited antiIgM- induced mRNA translation. PEITC, ibrutinib and tamatinib also decreased translation of MYC and MCLl RNAs in anti-IgM treated cells. Similar to MCF7 cells, PEITC caused a rapid increase in eIF2a phosphorylation in CLL cells. Overall, these results are consistent with the hypothesis that inhibition of mRNA translation by PEITC may contribute to its anti-cancer effects. In particular, the work has revealed the eIF2a pathway as a novel target for PEITC and uncovered new links between translation and BCR signalling in human leukaemia. Inhibition of mRNA translation, in response to PEITC or novel kinase inhibitors may play an important role in the therapeutic effects of these agents.
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Iwakawa, Hirooki. "Translation and RNA replication mechanisms of Dianthovirus." Kyoto University, 2010. http://hdl.handle.net/2433/120491.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第15448号
農博第1833号
新制||農||982(附属図書館)
学位論文||H22||N4547(農学部図書室)
27926
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 奥野 哲郎, 教授 遠藤 隆, 教授 荒木 崇
学位規則第4条第1項該当
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Saini, Harleen. "Intron and Small RNA Localization in Mammalian Neurons." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1044.
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RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.
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Johnston, Julie Catherine. "In vitro translation of cucumber necrosis virus RNA." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.
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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.Land and Food Systems, Faculty of
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Starmer, Joshua Mr. "What can RNA hybrids tell us about translation?" NCSU, 2006. http://www.lib.ncsu.edu/theses/available/etd-10202006-155443/.
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Molecular biologists have been observing interactions between messenger RNA (mRNA) molecules and other non-coding RNA molecules for quite some time. Here I revisit some of the classical hybridizations between the 16S ribosomal RNA (rRNA) and mRNA during initiation, as well as investigate the interactions between small interfering RNA (siRNA) molecules and mRNA. In reviewing rRNA-mRNA interactions, I observed that the majority of both bacterial and eukaryote genes can bind at the start codon. This novel result lead to a method for improving genome annotation as well as a new theory of translation initiation. The examination of siRNA-mRNA interactions lead to new criteria for predicting an siRNA's efficacy.
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Howell, Michael Terence. "The mechanism of initiation of poliovirus RNA translation." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386094.
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Borman, Andrew Mark. "Internal initiation of translation of human rhinovirus RNA." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281908.
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Qin, Daoming. "Role of 16S Ribosomal RNA in Translation Initiation." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299007063.
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Leipuvienė, Ramunė. "Frameshifting as a tool in analysis of transfer RNA modification and translation /." Umeå : Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-302.
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Daughenbaugh, Katie Finney. "The role of VPg in translation of calicivirus RNA." Diss., Montana State University, 2005. http://etd.lib.montana.edu/etd/2005/daughenbaugh/DaughenbaughK1205.pdf.
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Méthot, Nathalie. "RNA and protein interactions by eIF4B during translation initiation." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40401.
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One of the most enduring questions pertaining to eukaryotic translation initiation is how the 40S ribosomal subunit recognizes and binds at or near the cap-structure of mRNAs. Eukaryotic initiation factor 4B (eIF4B) is one of the factors that are required for this step of protein synthesis. eIF4B stimulates the RNA helicase activity of eIF4A and eIF4F, melting RNA secondary structure in the 5$ sp prime$ untranslated region (UTR), and is thus believed to contribute to ribosome binding by creating an area of single-stranded RNA accessible to the 40S ribosomal subunit We studied the mechanism of action of eIF4B by initiating a structure-function analysis of this factor. An RNA binding site, located in the carboxy-terminal end between amino acids 367 and 423, was found essential for non-specific RNA binding and eIF4A helicase stimulation. This region is distinct and independent from the canonical RNA Recognition Motif (RRM) located near the amino-terminus. The latter plays no role in non-specific RNA binding and has little impact on the eIF4A helicase stimulation. A self-association region located between residues 213-312 was identified. This segment is rich in aspartic acid, arginine, tyrosine and glycine (DRYG) residues, and can self-associate independently from other regions of eIF4B. The DRYG domain also interacts directly with the p170 subunit of eIF3. Finally, iterative in vivo RNA selection demonstrated that the eIF4B RRM is functional and binds specifically to RNA stem-loop structures. The RRM also associates with 18S rRNA. eIF4B possesses two independent RNA binding sites and associates with two different RNA molecules simultaneously. We conclude that eIF4B is organized into three distinct domains: the carboxy-terminal eIF4A RNA helicase stimulatory domain, the DRYG dimerization and eIF3 p170 interaction domain, and the RRM. Furthermore, two additional mechanisms by which eIF4B could stimulate ribosome binding to the mRNA are now apparent. eIF4B may target t
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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 trans-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 in vivo interaction between unr and p38 was demonstrated, and the effect of p38 on unr's binding to the HRV-2 IRES was tested in vivo. 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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Deumer, Claudia D. "RNA-binding proteins in yeast mitochondria." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2002. http://nbn-resolving.de/urn:nbn:de:swb:14-1035897639531-83407.
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This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.
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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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Doyle, Olivia E. "Cajal bodies sites of RNA polymer II localization and modification /." Available to US Hopkins community, 2003. http://wwwlib.umi.com/dissertations/dlnow/3080652.
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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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Dienstbier, Martin. "Recognition of mRNA localization signals in Drosophila development." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608472.
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Bourdeau-Julien, Isabelle. "ALS-associated RNA-binding protein FUS and mRNA translation regulation." Master's thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/68742.
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Des mutations dans plusieurs gènes ont été liés à la sclérose latérale amyotrophique (SLA),en particulier dans celui codant pour la protéine Fused in Sarcoma (FUS). Les mutations sont retrouvées dans la partie codant pour le signal de localisation nucléaire, rendant la protéine anormalement abondante dans le cytoplasme. Combiné à d’autres observations, ça suggère qu’un gain de fonction toxique de FUS dans le cytoplasme serait à l’origine de la neurodégénérescence. La SLA est une maladie neurodégénérative qui affecte les neurones moteurs et cause une paralysie progressive. Les mécanismes moléculaires causant la maladies ont toujours inconnus. Une des pistes serait la perturbation de la traduction locale desARNm, qui permet aux synapses de répondre rapidement et indépendamment du corps cellulaire. Une traduction locale insuffisante pour soutenir l’activité synaptique à long terme mènerait à la perte des synapses et à la neurodégénérescence. Mon objectif est donc de déterminer le rôle de FUS dans régulation de la traduction des ARNm en caractérisant son interaction avec les composantes traductionnelles et d’évaluer sa fonction dans une condition reproduisant les caractéristiques de la SLA. J’ai montré que FUS s’associe aux polyribosomes inactifs, ce qui suggère que FUS jouerait un rôle dans la régulation de la traduction des ARNm en interagissant avec le cœur de la traduction. Il est également possible d’observer une augmentation de la présence de FUS dans le cytoplasme et de son interaction avec les polyribosomes suite à une inhibition de la traduction par mTOR, suggérant son rôle de régulateur négatif. De plus, les mutations liées à la SLA amplifient la fonction inhibitrice de FUS en rendant FUS cytoplasmique et en réduisant la synthèse des protéines. Mes résultats montrent que la protéine FUS aurait un rôle d’inhibiteur de la traduction quand celle-ci est cytoplasmique. Par conséquent, l’augmentation de la présence de FUS dans le cytoplasme dans la SLA entrainerait une inhibition de la traduction importante, à un niveau insuffisant pour soutenir l’activité synaptique.Mutations in several genes have been linked to amyotrophic lateral sclerosis (ALS),particularly in the gene coding for the Fused in Sarcoma protein (FUS). Those mutations are found in the part encoding for the nuclear localization signal, making the protein abnormallyabundant in the cytoplasm. Combined with other observations, it suggests that a toxic gainof function of FUS in the cytoplasm would be the cause of the neurodegeneration. ALS is a neurodegenerative disease that affects motor neurons and causes progressive paralysis. The molecular mechanisms causing the disease are still unknown. One of the hypotheses is the disruption of local translation of mRNAs, which allows synapses to respond quickly and independently from the cell body. Insufficient local translation to support long-term synapticactivity would lead to synaptic loss and neurodegeneration. Thereby, the objective of mystudy is to determine the role of FUS in the regulation of mRNA translation by characterizing its interaction with translational components and evaluate its function in an ALS-linked condition. I have shown that FUS is associated with stalled polyribosomes, which suggests that it plays a role in regulating mRNA translation by interacting with the core of translation.There is also an increase in the presence of FUS in the cytoplasm and in its interaction with polyribosomes following inhibition of translation through mTOR, suggesting its role as anegative regulator. In addition, ALS-related mutations amplify FUS inhibitory function bymaking FUS cytoplasmic and reducing protein synthesis. My results show that the FUSprotein would have a role as a translation inhibitor when it is cytoplasmic. There fore, increasing the presence of FUS in the cytoplasm in ALS would result in significant translation inhibition, at a level insufficient to support synaptic activity.
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Yoon, Young J. "Role of Xenopus staufen and kinesin I in vegetal RNA localization /." View online version; access limited to Brown University users, 2005. http://wwwlib.umi.com/dissertations/fullcit/3174705.
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Halsell, Susan Richardson Lipshitz Howard D. Lipshitz Howard D. "Expression and localization of Hsp83 RNA in the early Drosophila embryo /." Diss., Pasadena, Calif. : California Institute of Technology, 1995. http://resolver.caltech.edu/CaltechETD:etd-10092007-075248.
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RIBEIRO, GABRIELA CASTELO BRANCO. "MACHINE TRANSLATION EVALUATION FOR THE SOFTWARE LOCALIZATION INDUSTRY: A CASE STUDY." PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2006. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=9105@1.
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COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOREste estudo foi motivado pela utilização, ainda em caráter experimental, de um tradutor automático por uma empresa multinacional de localização de software. A fim de contribuir para essa iniciativa pioneira no país, propomos uma avaliação do programa, enfocando as implicações da utilização desta tecnologia no processo de localização de software. Empregamos a taxonomia proposta pelo FEMTI (Framework for the Evaluation of Machine Translation in ISLE), desenvolvida especialmente para a avaliação de tradução automática, com base nas normas ISO/IEC de qualidade de software. São considerados aspectos operacionais, como a integração do sistema de tradução automática às ferramentas de memória de tradução, bem como questões relacionadas à linguagem. O corpus utilizado para a avaliação foi um manual de usuário de um telefone celular. Além dos problemas lingüísticos recorrentes na maioria das ferramentas de tradução automática disponíveis atualmente, são analisados os desvios relacionados à tradução da interface com o usuário, mais especificamente aos menus do telefone celular. Esses desvios são discutidos dentro das categorias pertinentes da taxonomia do FEMTI e, sempre que possível, foram sugeridas soluções. Para complementar a análise lingüística, apresentamos outros três estudos realizados para o português. Nossos resultados indicam que o sistema pode ser bem-sucedido neste mercado em função principalmente da delimitação do domínio e da adoção dos procedimentos impostos pelo processo de localização. Esse sucesso depende da integração do tradutor automático às memórias de tradução e de investimentos relativamente pequenos na atualização dos recursos lingüísticos (regras gramaticais e dicionários) para refletir as características próprias do domínio e do tipo de texto.
This study was motivated by the trial implementation of a machine translation engine by a multinational software localization company. In order to contribute to this innovative experiment in the Brazilian market, we evaluate the engine, focusing on the implications of its implementation in the software localization industry. We use the FEMTI (Framework for the Evaluation of Machine Translation in ISLE) taxonomy, which is based on the ISO/IEC guidelines for software evaluation. Operational aspects, such as the engine s integration with translation memory tools, are taken into consideration, as well as language issues. Our evaluation is based on the machine translated version of a mobile phone user guide. In addition to the language problems common to most machine translation engines currently available, we analyze issues related to the user interface, particularly to the phone menus. These problems are discussed as examples of each related FEMTI topic and we suggest solutions whenever possible. To add to our language evaluation, we present three other studies dedicated to Portuguese. Our results indicate the engine can be successful in this industry mainly in terms of domain restriction and localization workflow procedures. Its success depends on its integration to translation memory tools and requires relatively little investment in updating the language resources (rules and dictionaries) to reflect the language characteristics specific to domain and text type.
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Kumari, Sunita. "Role of RNA G-quadruplexes within 5' UTRs in translation regulation." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612262.
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Swift, Michael A. "Identification of RNA-binding proteins associated with H19 lncRNA translation avoidance." Scholarship @ Claremont, 2016. http://scholarship.claremont.edu/cmc_theses/1302.
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High throughput RNA-sequencing data has revealed a cellular transcriptome that represents a much larger part of the genome than was thought even 10 years ago. The newfound transcripts are not translated to protein and a few are known to have essential functions regulating gene expression. However, most non-coding RNAs in the transcriptome have no identified function or mechanism of action. H19 is a long non-coding RNA (lncRNA) that looks much like an mRNA with a 5’ methyl cap and a polyA tail, but it is not translated. H19 is associated with cell proliferation and most human cancers. The mechanisms governing H19’s role in these processes is unknown, but we wondered how translation avoidance is involved. Exciting preliminary results from the Weinberg lab show that H19 co-sediments with the 40S ribosomal subunit, suggesting the RNA is associated with cytoplasmic proteins. We hypothesized that RNA binding proteins may be mediating H19’s ability to avoid translation despite its having the characteristics of mRNA. Therefore, we are working to identify these RNA binding proteins associated with H19 by adapting a recently reported RNA antisense purification method.
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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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Leen, Eoin. "Structural insights into the transcription and translation of murine norovirus RNA." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11173.
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Noroviruses are positive-sense single-stranded RNA viruses which, in humans, cause rapid onset diarrhoea and vomiting. There is an estimated 21 million cases of noroviral gastroenteritis in the United States per year. The work in this thesis has focused on two noroviral proteins, the viral protein genome-linked (VPg), and the viral RNA dependent RNA polymerase (NS7pol). The VPg protein is covalently linked to the 5' end of the noroviral genome and is a key component of translation and replication initiation in noroviruses. Using Nuclear magnetic resonance spectroscopy (NMR) we have analysed the murine norovirus (MNV) and a human norovirus VPg (Lordsdale virus (LDV)) proteins. The VPg protein of both viruses has a small structured helical core with extensive N and C-terminal flexible regions. We has also determined the structure of the MNV NS7pol as well as two high fidelity mutants (P72S and E75S) using X-ray crystallography. The NS7pol protein has a very similar “right drinking hand” structure to other RNA dependent RNA polymerases. The fidelity mutants were structurally identical to the wild type protein but had subtle changes in local hydrogen bonding networks. This is consistent with similar studies performed with picornaviral polymerases. In addition we used NMR and surface plasmon resonance to characterise the interaction of the MNV VPg protein with the NS7pol protein. The interaction between full length VPg and MNV NS7pol is weak, (KD ~160 μM). In addition the NS7pol interacts with both the full length VPg and the core domain of this protein.
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SARAWANEEYARUK, SIRIRUK. "Studies on Cap-Independent Translation and RNA Replication Mechanisms of Dianthovirus." Kyoto University, 2010. http://hdl.handle.net/2433/131903.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第15733号
農博第1845号
新制||農||985(附属図書館)
学位論文||H22||N4468(農学部図書室)
28278
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 奥野 哲郎, 教授 遠藤 隆, 教授 左子 芳彦
学位規則第4条第1項該当
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Karakasiliotis, Ioannis. "Analysis of RNA-protein interactions involved in calicivirus translation and replication." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1304.
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The interaction of host-cell nucleic acid-binding proteins with the genomes of positive-stranded RNA viruses is known to play a role in the translation and replication of many viruses. To date, however, the characterisation of similar interactions with the genomes of members of the Caliciviridae family has been limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine norovirus (MNV) have been used as model systems to identify and characterise the role of host-cell factors that interact with the viral RNA and RNA structures that regulate virus replication. It was demonstrated that RNA-binding proteins such as polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and La protein interact with the extremities of MNV and FCV genomic and subgenomic RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved in the switch between translation and replication during the late stages of the infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus replication takes place. Furthermore, using the MNV reverse-genetics system, disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter and an RNA structure at the 3' end completely inhibited virus replication. Restoration of infectivity by repair mutations in the subgenomic promoter region and the recovery of viruses that contained repressor mutations within the disrupted structures, in both the subgenomic promoter region and the 3' end, confirmed a functional role for these RNA secondary structures. Overall this study has yielded new insights into the role of RNA structures and RNA-protein interactions in the calicivirus life cycle.
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Krech, Sabine. "Translation of the genomic RNA of hepatitis A virus in vitro /." [S.l : s.n.], 1988. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Nashchekin, Dmitri. "A Y-box protein/RNA helicase complex links mRNP assembly on the gene to mRNA translation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-811-8/.
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Presnyak, Vladimir. "Effects of Codon Usage on mRNA Translation and Decay." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1427387336.
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Gillespie, Doreen. "The role of homeless in RNA transport and localization during Drosophila oogenesis /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/10305.
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Zimmermann, C. "Regulation of mRNA localization, stability and local translation in sympathetic neuron axons." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1405619/.
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Polarity is dependent on the asymmetric distribution of proteins, which can be especially challenging in neurons as the nucleus, where transcription takes place, can be far away from the final location of the protein. One essential mechanism by which protein compartmentalization can be achieved is through the targeting of specific mRNA transcripts to specific cellular regions where they are locally translated. Neurotrophin 3 (NT3) is secreted by blood vessels and is required for the initial growth of proximal sympathetic neuron axons along the vasculature. NGF on the other hand, is secreted by the sympathetic target tissue and is required for sympathetic neuron survival and the final innervation of target organs. Although both neurotrophins act through the common receptor TrkA, NGF supports TrkA internalisation and retrograde signalling from distal axons to cell bodies to regulate gene expression, whereas NT3 can only induce a local signalling response in axons and is unable to support retrograde TrkA signalling. I performed a Digital Gene Expression Tag Profiling of axonal and cell body mRNA obtained from rat superior cervical ganglia cells maintained with NT3 with the aim of identifying mRNAs localized in subcellular compartments and to understand the control of mRNA stability and local protein translation in axons. I found that several hundred transcripts are significantly enriched in axons of sympathetic neurons when compared to cell bodies. Rapid Amplification of cDNA Ends (RACE) analysis of untranslated regions (UTRs) of candidate genes showed that at least some of the transcripts enriched in axons possess different UTRs. The most abundant transcript in axons was SCG10 (or Stmn2), a regulator of microtubule dynamics. Furthermore, I identified a member of the Hu protein family as an RNA binding protein that interacts with the UTR of SCG10 and will discuss its potential role in neurotrophin-dependent regulation of SCG10 mRNA stability and translation.
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Cridge, Andrew Graham, and n/a. "Characterization of the eukaryotic translation termination sequence element." University of Otago. Department of Biochemistry, 2005. http://adt.otago.ac.nz./public/adt-NZDU20060804.095722.
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Termination of protein synthesis occurs in response to the translocation of a stop codon (UAA, UAG or UGA) into the A site of the ribosome. Unlike sense codons, stop signals in the mRNA are recognized by two classes of specialized proteins called release factors (RFs): the class I or decoding RF, which recognizes the stop codon and promotes peptidyl-tRNA hydrolysis and class II RF, a G-protein that promotes the dissociation of the decoding RF from the ribosome. The discovery that stop codons are decoded by a protein factor rather than a specific tRNA opened up the possibility that the signal for termination of protein synthesis might extend beyond the stop codon itself. Biochemical and genetic experiments in prokaryotes confirmed that bias in nucleotide usage around stop codons correlates with translation termination efficiency. The objective of the current investigation was to define the eukaryotic termination signal by determining the bias in the nucleotide sequence surrounding eukaryotic stop codons and to identify whether this was a determinant of translation termination efficiency.
Bioinformatic analysis of five diverse eukaryotic genomes was undertaken to identify potential eukaryotic translation termination signal elements. Significant nucleotide bias was identified both 5� and 3� of the stop codon in all the genomes investigated. Correlations were identified between nucleotide bias and gene expression levels, and between nucleotide bias and natural recoding sites predicting that nucleotides 5� and 3� of the stop codon affect termination efficiency. These correlations were common to all organisms investigated and suggested the existence of a eukaryotic termination signal.
Termination signals identified from the bioinformatic analysis were assayed to determine the efficiency of termination in an in vitro dual luciferase reporter assay. Results indicated that nucleotides both 5� and 3� of the stop codon could significantly alter termination signal efficiency, although readthrough did not vary by greater than 1%. The effect of nucleotides 3� to the stop codon on termination efficiency was investigated further in mammalian cultured cells using the dual luciferase reporter assay. Results showed a significant relationship between the identity of these nucleotides and observed termination efficiencies with nucleotides at positions +4 and +8 giving the strongest correlation. Termination sequence elements of the form UGA CUN NCN mediated up to 5% readthrough in cultured cells.
Investigations into the underlying mechanisms that were responsible for the variation in termination efficiency were also undertaken. Co-transfection of specific suppressor tRNAs enhanced but did not change the pattern of observed termination efficiency, indicating that the mechanisms mediated by the termination signal element was not mediated through suppressor tRNA binding. Alignments of 18S rRNA sequences indicated potential extensive interactions between the rRNA and the mRNA termination signal element. Experiments that assessed the effect of eRF1 levels on termination at inefficient termination signals in vitro revealed that increased levels of eRF1 could improve termination efficiency.
These results indicate that, as in prokaryotes, specific nucleotides beyond the stop codon modulate translation termination efficiency in eukaryotes, and that the translation termination signal should be considered a sequence element.
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Crandall, Jacob N. "Ribosomal RNA Mutations that Inhibit the Activity of Transfer-Messenger RNA of Stalled Ribosomes." Diss., CLICK HERE for online access, 2010. http://contentdm.lib.byu.edu/ETD/image/etd3535.pdf.
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Gill, Kirsty. "Elucidating the molecular mechanism that determines the specific localisation of gurken mRNA during Drosophila development." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:01e61a3d-b29f-4766-b8c8-81552e5cd11d.
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mRNA localisation is a widely used mechanism for achieving temporal-spatial restriction of protein expression and is essential during development to establish cell polarity. mRNA localisation is particularly well studied in the Drosophila egg chamber where gurken mRNA is localised to the dorsal-anterior corner of the oocyte in a Dynein-dependent process that establishes the anterior-posterior and dorsal-ventral axes of the future embryo. An RNA stem-loop called the gurken localisation signal is necessary and sufficient to drive gurken localisation through interactions with a specific complement of protein factors. However, the exact RNA sequence and structural features required to promote each stage of gurken localisation are unknown. Using a live-cell injection assay I have dissected regions of the mRNA signal that are responsible for driving gurken transport and anchoring through their association with Egalitarian, Me31B and Squid proteins. I show the structure of an AU-rich stem and a purine stack are essential for gurken transport, and demonstrate that the size of the internal loop between these stems is important. These features of the localisation signal are essential for recruitment of Egalitarian, which links the mRNA to the Dynein transport machinery. I also show that these mRNA sequence and structural elements are present in several other Dynein-transported mRNAs. The bulge at the distal end of the gurken localisation signal is important for anchoring grk at the dorsal-anterior of the oocyte, possibly through Squid binding, and the proximal third of the signal is essential for recruitment of the translation component Me31B. These studies indicate that the role of the gurken localisation signal in controlling gurken transport, anchoring and translation can be mapped to distinct regions of the signal and provide insights into how the signal carries out these numerous functions at a molecular level. Determining the molecular interactions involved in mRNA localisation improves our understanding of how specificity is generated to direct different mRNAs to distinct regions of the cell to restrict protein expression.
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Bilali, Loubna. "Localization Training: Towards an Industry-based Requirements-Gathering Model." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532649023272877.
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Leipuviene, Ramune. "Frameshifting as a tool in analysis of transfer RNA modification and translation." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknat- och Medfak), 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-302.
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Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.
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Fred, Rikard G. "The Role of RNA Binding Proteins in Insulin Messenger Stability and Translation." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130234.
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Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and translation are still largely obscure, the work presented in this thesis therefore aimed to further investigate the role of insulin mRNA binding proteins in the control of insulin mRNA break-down and utilization for insulin biosynthesis. To clarify how glucose regulates insulin mRNA stability and translation we studied the correlation between polypyrimidine tract binding protein (PTB) gene expression and insulin mRNA levels. It was found that an increase in PTB mRNA and protein levels is paralleled by an increase in insulin mRNA levels. It was also found that PTB binds to the 5’-untranslated region of the insulin mRNA and that insulin mRNA can be translated through a cap-independent mechanism in human islets of Langerhans, possibly due to the interaction with PTB. Further it was discovered that the suppressed insulin biosynthesis in human islets during glucotoxicity is partly due to an induction of the microRNA miR-133a. This induction leads to decreased levels of PTB and insulin biosynthesis rates in human islets. Finally, we were able to identify two proteins, hnRNP U and TIAR, that bind specifically to the insulin mRNA in vitro, and show that the stability and translation of insulin mRNA is oppositely affected by these proteins. In conclusion, insulin producing cells seem to be able to regulate insulin messenger stability and translation by a control mechanism in which the binding of specific proteins to the insulin messenger dictates the outcome. A better understanding of the events leading to insulin production may in the future aid in optimal diagnosis and treatment of type 2 diabetes.
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Subhan, Syed Abdus. "Dissecting the initiation factors required for translation initiation on feline calicivirus RNA." Thesis, University of Surrey, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543924.
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Chuang, Ray-Yuan. "Requirement of a putative RNA helicase, ded1p, for translation in saccharomyces cerevisiae /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487948158625166.
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Sartor, Francesca. "Regulation of translation initiation and RNA decay is important for neuronal differentiation." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=.
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