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Chew, Guo-Liang. "Non-Canonical Translation in Vertebrates." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467487.
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Translation is a key process during gene expression: to produce proteins, ribosomes translate the coding sequences of mRNAs. However, vertebrate genomes contain more translation potential than these annotated coding sequences: translation has been detected in many non-coding RNAs and in the non-coding regions of mRNAs. To understand the role of such translation in vertebrates, I investigated: 1) the distribution of translation in vertebrate long non-coding RNAs, and 2) the effects of translation in the 5’ leaders of vertebrate mRNAs.
To quantify and localize translation in a genome-wide manner, we produced and analyzed ribosome profiling data in zebrafish, and analyzed ribosome profiling data produced by others. The nucleotide resolution afforded by ribosome profiling allows localization of translation to individual ORFs within a transcript, while its quantitative nature enables measurement of how much translation occurs within individual ORFs.
We combined ribosome profiling with a machine-learning approach to classify lncRNAs during zebrafish development and in mouse ES cells. We found that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble that of the 5’ leaders of coding mRNAs. These results clarify the annotation of lncRNAs and suggest a potential role for translation in lncRNA regulation.
Because much of the translation in non-coding regions of mRNAs occurs within uORFs, we further examined the effects of their translation on the cognate gene expression. While much is known about the repression of individual genes by their uORFs, how uORF repressiveness varies within a genome and what underlies this variation had not been characterized. To address these questions, we analyzed transcript sequences and ribosome profiling data from human, mouse and zebrafish.
Linear modeling revealed that sequence features at both uORFs and coding sequences contribute similarly and substantially toward modulating uORF repressiveness and coding sequence translational efficiency. Strikingly, uORF sequence features are conserved in mammals, and mediate the conservation of uORF repressiveness in vertebrates. uORFs are depleted near coding sequences and have initiation contexts that diminish their translation. These observations suggest that the prevalence of vertebrate uORFs may be explained by their functional conservation as weak repressors of coding sequence translation.<br>Biology, Molecular and Cellular
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Lin, Chen-ju. "Targeting translation initiation for cancer therapy." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96981.
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The recruitment of ribosomes to the 5' end of mRNAs in eukaryotic cells is generally thought to be the rate-limiting step of translation initiation and this process is mediated by the eukaryotic initiation factor 4F complex (eIF4F). This complex consists of three subunits: eIF4E, a mRNA-cap binding protein, eIF4A, a RNA helicase and eIF4G, a large molecular scaffold that mediates the binding of mRNA to ribosomes. Deregulation of translation initiation through eIF4F activation has been widely observed in human cancers. The eIF4F complex lies downstream of key signalling pathways involved in oncogenesis (such as c-Myc and PI3K/Akt/mTOR), thus targeting translation initiation provides an attractive therapeutic approach for cancer therapy. Here, we show that c-Myc stimulates protein synthesis by up-regulating the expression and activity of not only eIF4E, but that of the other two subunits of eIF4F. In turn, this elevated eIF4F levels result in increased synthesis and function of c-Myc, establishing a positive feedforward loop. We used the Eμ-myc lymphoma mouse model to show that expression of the three eIF4F subunits is also up-regulated by c-Myc in vivo. Most importantly, we demonstrate that loss of eIF4E function using inducible and reversible RNA interference (RNAi) greatly delays the rates of c-Myc-induced lymphoma development. These data suggest that targeting eIF4E in vivo is an effective therapeutic approach. Since the assembly of eIF4F complex is regulated by mTOR signaling, the coupling of c-Myc to eIF4F is under mTOR control. In the course of a screen of inhibitors of the PI3K/Akt/mTOR signalling pathway, we found two small molecules, silibinin and the anti-depressant sertraline, both of which show anti-proliferative effects on breast cancer cells. Silibinin and sertraline effectively target eIF4F complex function by downregulating mTOR signaling. Importantly, sertraline is able to enhance the chemosensitivity of PTEN (+/-)/Eμ-Myc lymphomas to the chemotherapeutic agent doxorubicin in vivo. Thus, targeting mTOR-dependent translation initiation shows anti-cancer activity in this pre-clinical setting.<br>Il est généralement admis que le recrutement des ribosomes à l'extrémité 5' des ARN messagers (ARNm) est l'étape limitante de l'initiation de la traduction chez les eucaryotes. Cette étape est dépendante de l'activité du complexe d'initiation eIF4F qui comprend trois sous-unités: eIF4E, une protéine liant la coiffe des ARNm, eIF4A, une hélicase d'ARN et eIF4G, une grande protéine d'échafaudage dont le rôle est de coordonner la liaison du ribosome à l'ARNm. Le dérèglement du contrôle de l'initiation de la traduction suite à l'activation d'eIF4F est observé fréquemment chez les cancers humains. L'activité de ce complexe est contrôlée par plusieurs voies de signalisation clés qui sont impliquées dans la formation des tumeurs (tels que c-Myc et PI3K/Akt/mTOR). Donc, cibler l'initiation de la traduction représente une avenue attrayante pour contrer le cancer. Nous démontrons ici que l'oncogène c-Myc peut stimuler la synthèse protéique en favorisant l'expression et l'activité de non-seulement eIF4E, mais aussi des deux autres sous-unités du complexe eIF4F. En réponse à cela, les niveaux supérieurs d'eIF4F permettent une augmentation de la synthèse et donc de l'activité de c-MYC, établissant alors une boucle auto-stimulante. Nous avons utilisé le modèle de souris Eμ-myc pour démontrer que l'expression de chacune des sous-unités d'eIF4F est stimulée par c-Myc in vivo. Plus important encore, nous avons démontré que la réduction des niveaux d'eIF4E en utilisant la technique d'interférence à ARN (ARNi) de manière inductible et réversible freine considérablement le développement de lymphomes par c-Myc. Ces données suggèrent que cibler eIF4E in vivo est une approche thérapeutique viable et efficace. De plus, puisque l'assemblage d'eIF4E est contrôlé de mTOR, il en résulte donc que le couplage de c-Myc et d'eIF4F est donc aussi sous contrôle de cette voie de signalisation. Suite à un cribblage de molécules inhibitrices de la voie PI3K/Akt/mTOR, nous avons identifié deux molécules, la silibinine et l'anti-dépresseur sertraline, qui ont la propriété de bloquer la prolifération de cellules du cancer du sein. La silibinin et la sertraline inhibent efficacement l'activité du complexe eIF4F en ciblant la voie de signalisation de mTOR. Par surcroît, la sertraline accentue fortement la sensibilité des lymphomes PTEN (+/-)/Eμ-Myc à l'agent chimiothérapeutique doxorubicin in vivo. En conclusion, il appert que cibler le contrôle de la traduction par mTOR peut contrer efficacement le cancer dans ce modèle de cancer préclinique.
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Kinney, Emma. "Decoupling of HSV1 Vhs protein mRNA decay and translation stimulation." Thesis, University of Missouri - Kansas City, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=1543940.
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<p> Herpes Simplex Virus Type 1 is a member of the <i>alphaherpesvirinae </i> subfamily within the family <i>Herpesviridae</i>. This virus has both a lytic and latent cycle. Primary infection occurs when the virus enters epithelial cells around the mucosal lining of the nose and mouth. Within the epithelial cells, the virus undergoes an active lytic infection, causing an ulcerated blister, more famously known as a 'cold sore' or 'fever blister'. Once HSV enters the nearby sensory neurons the genome is transported to the neuronal cell body where its latency associated transcripts are activated and the virus remains in a dormant latent cycle until reactivation, when the virus is transported back down the axon to the epithelial cells at or near the site of initial infection. The Virion Host Shutoff protein is a tegument protein from HSV1 and acts as a ribonuclease, degrading both cellular and viral mRNAs, making the course of viral infection more efficient. A study by Saffran, Read and Smiley uncovered an unexpected new function of Vhs: stimulation of translation from some IRESs. An IRES is a section of mRNA with a high level of secondary structure, capable of inducing cap-independent translation. In similar experiments utilizing a bicistronic reporter transcript, I sought to discover whether or not these two functions of the Vhs protein could be de-coupled. Experiments involved dually transfecting HeLa cells with different Vhs mutants across a range of Vhs plasmid concentrations and the bicistronic reporter construct. Levels of reporter activity were measured from cell lysates 36 hours after transfections and provided a measurement of the control at the level of translation. As the cellular Bip IRES element was present between the cistrons, the 3' cistron provided a measure of IRES stimulation. The Results revealed examples of Vhs mutants in which the two activities had been separated. It is unknown what role IRES stimulation could play during Herpesvirus infection, although it is interesting to note that some HSV1 genes have IRES like elements within the 5' UTR. Future experiments can be done to investigate whether or not Vhs is actively recruiting transcription initiation factors to these IRES elements.</p>
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Cho, Park 1975. "The Cap-binding inhibitor of translation, d4EHP /." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111819.
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In eukaryotes, the initiation phase of protein synthesis or translation is a multi-step process that culminates in the positioning of the SOS ribosome at the initiation codon of a messenger RNA (mRNA). Recognition of the cap structure by eukaryotic initiation factor 4F (etF4F; composed of three subunits: the cap-binding protein e1F4E, the RNA-helicase eIF4A and the scaffolding protein eIF4G) facilitates this process. The ability of eIF4F to bind to the cap, as a result of the Cap:eIF4E interaction is of particular importance, as it is the major target of translational regulatory mechanism.<br>Early embryogenesis requires the activity of various maternal determinants called morphogens, whose spatial and temporal expressions are tightly regulated at the level of translation. Positional information encoded within these factors is thus important for the establishment of body polarity. For instance, in Drosophila, when maternal Caudal (Cad) and Hunchback (Hb) proteins are allowed to accumulate inappropriately in an embryo, anterior and abdominal segmentations are blocked. Hence, the precision of Cad and Hb expression domains is critical for normal development.<br>An eIF4E-related protein called eIF4E-Homologous protein (4EHP) was first described in 1998. However, the function, if any, of 4EHP in translation has been elusive, since it does not interact with any known initiation factors. In order to elucidate its biological function, the power of Drosophila genetics was used. In this thesis, I show that the Drosophila homolog of 4EHP (d4EHP) interacts with Bicoid (Bcd) and Brain tumor (Brat) proteins to inhibit the translation of maternal cad and hb mRNAs. Simultaneous interaction of d4EHP with the cap and Bcd or Brat results in mRNA circularization, which renders cad and hb mRNAs translationally inactive. This example of cap-dependent translational control that is not mediated by eIF4E defines a new paradigm for translational inhibition involving tethering of the mRNA 5' and 3' ends.
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Malina, Abba. "The therapeutic potential in eukaryotic mRNA translation." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114176.
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Inhibitors of translation have proven invaluable in delineating the overall mechanism of protein synthesis. Unlike inhibitors of prokaryotic protein synthesis, the therapeutic development of drugs that directly interfere in the eukaryotic mRNA translation for treatment of human disease has remained largely unexplored. To begin to investigate this possibility and expand the current repertoire of compounds that affect eukaryotic translation, we have undertaken several different experimental screening approaches, two of which are described below and will form the basis of this thesis. In chapter 2, we performed a multiplexed high-throughput chemical screen to identify novel inhibitors of eukaryotic protein synthesis. We identified intercalators as having unique biological properties in our assays: at high concentrations they behave like elongation inhibitors and blocked the peptidyl-transferase activity of the ribosome, while at lower concentrations they preferentially block HCV-driven, cap-independent but not cap-dependent translation. This activity appeared to be due to their ability to block the innate affinity of the HCV IRES for the 40S ribosomal subunit. Moreover, a number of intercalator-based peptide-conjugates (which can chemically "thread" through and bind strands of nucleic acid in a sequence-specific manner) were tested and one, PAC-6, was found to be a selective inhibitor of HCV-dependent initiation.In chapter 3, we performed a shRNA-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment. Using genetically-defined Arf-/-Eµ-myc lymphoma cells, pools of shRNAs targeting known factors and regulators of protein synthesis were retrovirally introduced and genomic DNA collected over the course 10 days for both vehicle and ABT-737 treated cohorts. Following deep sequencing analysis of shRNA abundance, several constructs were identified that were selectively depleted only in the presence of ABT-737. Of them, two shRNAs against the RNA/DNA helicase, DHX9, validated. Although knockdown of DHX9 was found to sensitize both mouse and human cells to ABT-737 treatment, it did so without altering Mcl-1 levels. Instead, loss of DHX9 appeared to activate a p53-dependent apoptotic program which was found to be both necessary and sufficient for the ABT-737-shDHX9 synthetic lethal interaction.<br>La compréhension du mécanisme global de la synthèse protéique a rapidement progressée en majeur partie grâce a l'utilisation d'inhibiteurs spécifiques qui bloquent ce processus. Contrairement aux inhibiteurs de la synthèse protéique procaryote, l'utilisation de molécules pouvant moduler la traduction des ARNm eucaryotes dans un but thérapeutique reste encore largement sous-évalué. Afin d'étudier cette possibilité et d'élargir le répertoire de composés chimiques pouvant interférer avec la synthèse protéique eucaryote, nous avons effectué plusieurs criblage différents. Deux d'entre eux sont décrits plus bas et formeront les fondements de cette thèse.Tel que décrit dans le chapitre 2, nous avons tout d'abord effectué un criblage à haut débit de molécules afin d'identifier de nouveaux inhibiteurs de la synthèse protéique eucaryote. Ceci nous a permis de découvrir que les molécules qui peuvent s'intercaler dans les structures en double brin des acides nucléiques possèdent des propriétés uniques d'inhibition de la traduction. En effet, à hautes concentrations, elles se comportent exactement comme des inhibiteurs de l'élongation et bloquent l'activité peptidyl-transférase des ribosomes, alors qu'à faibles concentrations, elles bloquent préférentiellement la traduction cap-indépendant sous contrôle de l'IRES de HCV sans affecter la traduction dépendante du cap. Cette activité semble être due à la capacité des molécules d'interférer avec la liaison de la sous-unité 40S à l'IRES de HCV. De plus, certaines molécules qui combinent une portion intercalatrice et une portion peptidique (connue pour pouvoir sonder et se lier spécifiquement des brins d'acides nucléiques de manière spécifique) ont été testées et une d'entre elles, nommé PAC-6, permet l'inhibition spécifique de l'initiation de la traduction sous contrôle de l'IRES de HCV.Dans le chapitre 3, nous avons effectué un criblage d'une librairie de shRNA afin d'identifier des gènes ou des voies de signalisation qui peuvent inverser la résistance de cellules à ABT-737. Des cellules de lymphomes Arf-/-Eµ-Myc génétiquement modifiées ont été infecté avec un groupe de shRNAs ciblant des gènes connus pour contrôler tous les aspects de la synthèse protéique et avons isolé l'ADN génomique de ces cellules après 10 jours de traitements avec, soit le véhicule, soit avec ABT-737. Suite à l'analyse de l'abondance relative des shRNAs par séquençage de nouvelle génération, nous en avons identifié plusieurs dont la représentation diminue sélectivement en présence d'ABT-737. Parmi ceux-ci, deux shRNAs uniques, ciblant l'hélicase à ARN/ADN DHX9 ont été identifiés et par la suite confirmés indépendamment. La diminution des niveaux de DHX9 permet la sensibilisation des cellules murines ou humaines à ABT-737 sans toutefois altérer les niveaux de Mcl-1. Plutôt, la perte de DHX9 semble activer un programme d'apoptose dépendant de p53 qui est nécessaire et suffisant pour cette interaction synthétique létale entre ABT-737 et DHX9.
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Chiappetta, Margaret Elizabeth. "Knowledge translation in action : cancer biology and systems pharmacology at the National Center for Advancing Translational Science." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50189.
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The need for novel diagnostic and therapeutic drugs with the potential to combat increasingly prevalent or particularly insidious diseases has grown in recent years. Concurrently, the issue of translating scientific knowledge from “bench to bedside” has become increasingly salient. In 2011, the U.S. National Institutes of Health created the National Center for Advancing Translational Science in an effort to remedy the recalcitrant gaps between fundamental laboratory research and late-stage clinical trial, thereby dramatically reducing the amount of time and expense needed to develop efficacious pharmaceutical prototypes for a range of emerging, re-emerging, and chronic diseases. However, the realities of pharmaceutical development are incongruous with the expectations of the lay public that even the most fundamental scientific research yield results with immediate social and commercial value. Traditional linear models of progress overlook both the epistemic nature of scientific innovation and the significance of the socio-economic supply and demand factors driving research endeavours.
The aim of this dissertation is to underline the epistemic and socio-economic characteristics of translational science – specifically in the context of research targeting novel oncology therapeutics and diagnostics – through the lens of Science and Technology Studies. In focusing on research in cancer biology funded by the National Center for Advancing Translational Science, this thesis highlights the significance of Mode 2 or “post-academic” science, and by extension the roles of interdisciplinarity and applicability, and the commodification of scientific knowledge, that arise in the process of translating scientific knowledge.<br>Arts, Faculty of<br>Graduate
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Yao, Xiaoquan. "Sequence features affecting translation initiation in eukaryotes: A bioinformatic approach." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27658.
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Sequence features play an important role in the regulation of translation initiation. This thesis focuses on the sequence features affecting eukaryotic initiation. The characteristics of 5' untranslated region in Saccharomyces cerevisiae were explored. It is found that the 40 nucleotides upstream of the start codon is the critical region for translation initiation in yeast. Moreover, this thesis attempted to solve some controversies related to the start codon context. Two key nucleotides in the start codon context are the third nucleotide upstream of the start codon (-3 site) and the nucleotide immediately following the start codon (+4 site). Two hypotheses regarding +4G (G at +4 site) in Kozak consensus, the translation initiation hypothesis and the amino acid constraint hypothesis, were tested. The relationship between the -3 and +4 sites in seven eukaryotic species does not support the translation initiation hypothesis. The amino acid usage at the position after the initiator (P1' position) compared to other positions in the coding sequences of seven eukaryotic species was examined. The result is consistent with the amino acid constraint hypothesis. In addition, this thesis explored the relationship between +4 nucleotide and translation efficiency in yeast. The result shows that +4 nucleotide is not important for translation efficiency, which does not support the translation initiation hypothesis. This work improves our current understanding of eukaryotic translation initiation process.
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Fung, Hiu Leong. "Human C7orf30 is a novel mitochondrial translation factor." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103744.
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Mitochondria generate the majority of cellular energy through oxidative phosphorlyation. The machinery of oxidative phosphroylation consists of five enzyme complexes that are located in the inner mitochondrial membrane. A small number of essential subunits in these complexes are encoded by mtDNA and synthesized on a dedicated mitochondrial translation apparatus. Defects in mitochondrial translation system cause many mitochondrial diseases, but the mechanisms that regulate mitochondrial translation remain largely unknown. We have identified an unnamed human protein C7orf30, as a possible mitochondrial translation factor. The orthologue of C7orf30 in maize is thought to be a chloroplast ribosome assembly factor. We identified human C7orf30 to be a mitochondrial protein through bioinformatics analysis and immunocytochemistry. We knocked down the expression of C7orf30 in human fibroblast using shRNA and observed a reduction in cytochrome c oxidase activity. Using a translation assay, we observed a global reduction in the synthesis of mitochondrially encoded proteins when C7orf30 was knocked down, while the transcript levels were not affected. The assembly of Complex I, III, IV and V also demonstrated defects. Sucrose density gradient analysis suggests C7orf30 interacts with 39S subunit of the mitoribosome. The assembly of the mitoribosome and the levels of 12S and 16S MT-rRNA were not affected by C7orf30 knockdown, suggesting C7orf30 is not necessary for mitochondrial ribosome assembly. We hypothesize that C7orf30 interacts with the mitoribosome and is a regulator of mitochondrial translation.<br>Les mitochondries génèrent la majorité de l'énergie cellulaire grâce à l'oxydation phosphorylative. La chaîne respiratoire responsable de ce phénomène est composée de cinq complexes enzymatiques localisés dans la membrane interne de la mitochondrie. Certaines des sous-unités essentielles de ces complexes sont codées par l'ADN mitochondrial. Leur synthèse est assurée par la mitochondrie qui possède son propre système de traduction des protéines. Les déficiences de la traduction mitochondriale sont à l'origine de nombreuses maladies et les mécanismes qui régulent le processus de traduction restent à ce jour peu élucidés. Dans cette étude, nous avons identifié chez l'homme, C7orf30, une protéine probablement impliquée dans la régulation de la traduction mitochondriale. Il existe un homologue de cette protéine chez le maïs. Une étude suggère son rôle en tant que facteur d'assemblage des ribosomes des chloroplastes. Des programmes informatiques prédisent la localisation de la protéine C7orf30 humaine dans la mitochondrie ce que nous avons confirmé par des expériences d'immunocytochimie. L'utilisation de shRNA dirigés contre C7orf30 dans des fibroblastes humains révéle d'abord une réduction de l'activité cytochrome c oxydase (complexe IV). Des expériences de traduction ex vivo montrent ensuite une réduction globale de la synthèse des protéines codées par la mitochondrie dans les cellules déficitaires en C7orf30, la transcription étant normale. L'assemblage des complexes I, III, IV et V de la chaîne respiratoire est également affecté. La séparation des protéines par gradient de sucrose suggère que C7orf30 interagit avec la sous unité 39S des ribosomes mitochondriaux. Cependant, l'assemblage et les niveaux d'expression des rRNA 12S et 16S ne sont pas affectés par la diminution de la protéine ce qui suggère qu'elle n'est pas indispensable à l'assemblage des ribosomes mitochondriaux en soit. Dans cette étude, nous émettons l'hypothèse que C7orf30 est un composant du ribosome et agit comme un régulateur de la traduction mitochondriale.
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Arribere, Joshua A. (Joshua Alexander). "Transcript leaders : annotation and insight into functions in translation." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/83763.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.<br>CD-ROM contains PDF of title page and .txt of tables.<br>Cataloged from PDF version of thesis. Vita.<br>Includes bibliographical references.<br>For a eukaryotic mRNA to be properly expressed, it undergoes a series of several steps, including transcription, modification, splicing, packaging, export, localization, translation, and decay. Of these steps transcription is the most extensively studied, though the remaining steps are also indispensible for proper protein production. While we understand many of these steps in biochemical detail in vitro, we have a much poorer knowledge of how they occur and are regulated for a given gene in vivo. Posttranscriptional regulation is carried out primarily through the noncoding portions of the mRNA: the Transcript Leader (TL or 5'UTR) upstream of the Open Reading Frame (ORF), and the 3'Untranslated Region (3'UTR) downstream. To understand how these regions affect post-transcriptional gene expression, it is critical to have precise annotations of the mRNA(s) produced from a gene. In Chapter 2 I describe the development of Transcript Leader Sequencing (TL-seq), a technique to annotate TLs, and demonstrate its utility in yeast. TL-seq annotations reveal interesting TL-dependent regulation, including transcription within ORFs and short TLs that are associated with translation initiation at the second AUG of the ORE. To further study the roles of TLs in translation, I develop Translation-Associated Transcript Leader Sequencing (TATL-seq). TATL-seq works by applying TL-seq across fractions of a polysome gradient, generating TL-specific translational measurements. This approach demonstrates a widespread inhibitory function for upstream AUGs (uAUGs), and that ~6% of yeast genes express multiple TL species with distinct translational activities. This demonstrates that alternative TLs are prevalent and functional even in a relatively simple eukaryote like yeast. My interest in alternative TLs prompted me to explore TL variation in mammals, where many thousands of genes are known to have alternative TLs. In Chapter 3 I enumerate the contributions of alternative mRNA processing events to alternative TLs in mice. I observe alternative TLs produced by alternative Transcription Start Sites (TSSs), and also demonstrate that alternative splicing events, such as skipped exons and alternative splice sites, contribute substantially to functional TL diversity. To facilitate the future study of alternative TLs in mammals, in Appendix I I modify TL-seq to sequence longer TL fragments and optimize TL-seq's enzymatic steps to reduce input RNA requirements. This thesis is concerned with understanding post-transcriptional mRNA expression both globally and gene-specifically. In particular, I seek to understand the role the Transcript Leader has in affecting translation and degradation of its transcript. The findings detailed here define and analyze discernable features of TLs that relate to translational properties of the downstream message. Furthermore, the techniques developed enable analyses of TLs and translation that could not be carried out with previous technologies.<br>by Joshua A. Arribere.<br>Ph.D.
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Cacan, Ercan. "Evolutionary synthetic biology: structure/function relationships within the protein translation system." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45838.
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Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life.
Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins.
In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.
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Basak, Sanjukta. "Studies of Hepatitis C virus immunology : translation and replication." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97903.
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Hepatitis C virus (HCV) has become a worldwide problem. Roughly 3% of world population are estimated to be infected with the virus, producing high rates of progressive liver disease, leading to cirrhosis and hepatocellular carcinoma. The present therapy, a combined administration of pegylated interferon-alpha (IFN) and ribavirin is costly and only successful in 50% of patients infected with HCV. It is also associated with serious side effects. Thus, there is an urgent need for better tolerated and more effective treatment modalities. A therapeutic vaccine may be the solution.<br>Recent efforts to produce efficient vaccines require not only the identification of potential viral antigens but also vaccine adjuvants or enhancers of immunity. Dendritic cells (DC) are being considered one such adjuvant for the activation of CD4+ and CD8+ T-cells. As potent antigen presenting cells, they are capable of capturing antigens, processing them into peptides, and presenting them on products of the MHC to T cells. For such reasons, peptide loading of antigens onto DCs to enhance T cell responses is becoming of increasing interest. Using cell penetrating peptides, or motifs capable of transporting cargo freely across cell membranes, we have developed a peptide based delivery system suitable for the transport of all HCV proteins into immature DCs. In our studies we demonstrated that 3.1% of immature DCs internalized the reporter cargo, eGFP. This system was then optimized to 53.81 % in target HeLa cells.<br>Another area of recent focus is the regulation of HCV translation and replication. Positive stranded viruses such as HCV use the genomic RNA as a common template for translation as well as for RNA replication, both proceeding in inverse directions. Thus, specific regulatory mechanisms must be in place in order to coordinate these two antagonistic processes. In this study, we investigated the role of HCV Core protein as a translational inhibitor and enhancer of replication. Using several transient and stable in vivo reporter assays, we showed that Core expression inhibited HCV IRES-mediated translation in trans, in a dose-dependent manner. Furthermore, HCV Core protein is able to dramatically inhibit HCV translation in the Huh7 replicon system, more so than the bicistronic reporter systems tested and subsequently increase total levels of replicon RNA by 1.5 log fold and thus, affect replication. We believe that Core may indeed be the sought regulator of translation and replication.
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Baboo, Sabyasachi. "Nuclear translation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5266f049-d576-44fd-ab26-11cf7a27f678.
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In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).
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Ying, Lanqing. "Studies on Translation Elongation and its Fidelity." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu153054014550459.
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Ivanova, Natalia. "Finding the unknowns in trans-translation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ-bib. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5756.
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Stojdl, David F. "Protein kinases and the regulation ofmRNA splicing and translation." Thesis, University of Ottawa (Canada), 2000. http://hdl.handle.net/10393/9381.
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Regulation of genetic information occurs through many intricate and varied mechanisms. We have explored two such mechanism, splicing and translation initiation, by studying two kinase families involved in these processes. The work in this thesis represents our ongoing efforts to understand the role of these proteins and the mechanisms by which they work. In the first section of this thesis, we present evidence that the Clk family of kinases are able to influence the regulatory process known as alternative splicing. In the second portion, we discuss PKR, a regulator of protein translation, and demonstrate it to be an essential mediator of antiviral defence. In the final chapter, we consider a possible application of the data from our PKR studies, and propose an oncolytic therapy based on putative defects in regulatory pathways controlling PKR and other members of the IFN pathway.
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Brasey, Ann. "Translation regulation of the Human Immunodeficiency Virus type 1." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85890.
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Viruses are among the ultimate conquerors. Even the exploits of Genghis Khan and Alexander the Great seem pale when compared to viral feats. To give but one example, over the last 50 years, the human immunodeficiency virus (HIV) has swept across six continents, now claiming several million lives yearly. Despite sustained intense research efforts, there is still no treatment to eradicate HIV infection.<br>For billions of years, viruses evolved strategies to enter and take control of organisms to generate progeny viruses. Eukaryotic cell viruses developed various means of hijacking the cellular protein synthesis machinery. Understanding these mechanisms opens a unique window of opportunity: that of eventually being able to specifically inhibit virus protein production. In this context, we investigated how HIV-1 translation is regulated. This work initially characterizes an RNA structural element in the HIV-1 leader able to directly recruit the protein synthesis machinery, i.e. an internal ribosome entry site (IRES). This element is capable of driving protein synthesis during the G2/M cell-cycle phase when cap-dependent translation is inhibited.<br>Several virus families use IRESs. IRES-dependent translation usually involves a subset of the factors implicated in cellular protein synthesis. However, toeprinting studies suggest that HIV-1 requires factors different from the canonical translation initiation factors to initiate protein synthesis. HeLa cell protein fractionation studies identified p97, an eIF4G homolog, its apoptotic cleavage product, p86, and a novel protein, ropp120, as putative HIV-1 transactivators. Further testing revealed that these proteins do not directly stimulate HIV-1 leader dependent translation. Experiments also showed that La autoantigen, another likely HIV-1 IRES transactivator candidate, does not directly stimulate HIV-1 dependent translation.<br>The last portion of this work investigates the interplay of the HIV-1 IRES with cap structures, polyA tails and the HIV-1 3'UTR region since these elements are present on the viral genomic RNA. We found that the HIV-1 leader does not synergize nor does it interfere with the translation stimulation mediated by the cap, the polyA and the HIV-1 3'UTR. Data presented herein suggest that the HIV-1 leader is an IRES able to shunt initiation complexes from the cap structure to drive protein synthesis.
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Park, Eun-Hee 1971. "Mechanisms of translation initiation of receptor tyrosine kinase Tie2." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102690.
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Angiogenesis is a process of new blood vessel formation and is the culmination of both mitogenic and tissue remodeling events, resulting in neovascularization. It is a physiological process that is required for, amongst others, normal embryonic development, female reproductive function, and wound healing. Angiogenesis is a tightly regulated process which is balanced by both positive and negative factors. However, in many disease states, including diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and several cancers, dysregulation of angiogenesis contributes to disease progression. Previous published reports have implicated the coordinated activities of at least two families of receptor tyrosine kinases (RTKs), the vascular endothelial growth factor receptor (VEGFR) and the Tie receptor families, in this process.<br>Tie2, an endothelial-specific receptor tyrosine kinase, plays an essential role in normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers indicating a role in tumor angiogenesis. The human Tie2 mRNA transcript contains an unusually long (372 nucleotides) 5' untranslated region (UTR) with five upstream open reading frames (uORFs). In this thesis, we demonstrate that the Tie2 5' UTR promotes cap-independent translation, indicating the presence of functional internal ribosome entry site (IRES). In addition, we illustrate that Tie2 IRES activity is maintained, and even slightly stimulated, under hypoxic conditions when cap-dependent protein synthesis is attenuated. We further show that the Tie2 IRES is functional during quiescence, another condition known to compromise cap-dependent translation. These results present how Tie2 mRNA is translated despite a cumbersome structured 5' UTR and how its production is secured under unfavorable environmental conditions.<br>We define experimental conditions where the Tie2 IRES is not active, allowing us to assess the contribution of cap-dependent translation to Tie2 protein synthesis. We demonstrate evidence that Tie2 mRNA can be translated via both cap-dependent scanning mechanism and internal initiation. Moreover, we show that the presence of the uORFs within the 5' UTR is inhibitory to downstream translation initiation. Our results suggest that the uORFs serve to decrease the proportion of ribosomes competent for reinitiation as they traverse the mRNA 5' UTR and thus minimizing interference with the IRES and/or mediating inefficient translation of the potent protein under normal conditions. Like many other cellular IRESes, the entire Tie2 5' UTR appears to be required for maximum IRES activity.<br>Taken together, our results underscore the complex mechanisms to control gene expression at the level of translation initiation of the Tie2 mRNA.
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Kebache, Sem. "A role for the Nck adapter in protein translation /." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82899.
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In mammals, Nck, represented by two genes, is a 47kDa protein lacking intrinsic enzymatic function. It is composed solely of three N-terminal Src-homology 3 (SH3) domains and a single C-terminal Src-homology 2 (SH2) domain. Nck is classified as an adapter molecule that links cell surface receptors, via its SH2 domain, to downstream effectors, through its SH3 domains. Two cDNAs coding for the carboxy-terminal region of the beta subunit of the eukaryotic initiation factor 2 (eIF2beta) were isolated from a two-hybrid screen to identify new effector molecules interacting with the SH3 domains of Nck. eIF2beta is a component of the molecular complex eIF2 responsible for one of the early steps in the initiation of protein synthesis. In this thesis, I determined in vivo that the first and the third SH3 domains of Nck were both required for its interaction with eIF2beta. Furthermore, Nck and eIF2beta colocalized in an enriched ribosomal fraction. The localization of Nck in this compartment is enhanced upon insulin stimulation. I also established that Nck-1 overexpression is concomitant with an increase in protein translation. Interestingly, the effect of Nck on translation was dependent on its first and third SH3 domains originally found to mediate its interaction with eIF2beta. To elucidate how Nck modulates protein translation, I examined whether Nck maintains its positive effects under ER stress conditions where protein synthesis is normally inhibited. Interestingly, my data revealed that Nck-1 overexpression antagonized ER stress-induced inhibition of translation. Furthermore, I demonstrated that overexpression of Nck-1 prevented PERK activation, eIF2alpha phosphorylation, BiP induction and cell survival in response to ER stress treatments. This integrates Nck in the regulation of the ER unfolded protein response (UPR). I also provided evidence that the effects of Nck on the UPR involve its second SH3 domain and a phosphatase-dependent mechanis
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M'Boutchou, Marie-Noël. "Regulation of translation initiation by the elF4E-binding proteins." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80326.
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Most eukaryotic mRNAs possess a 5' cap structure which is important for their translation. The eukaryotic translation initiation factor 4E (eIF4E) binds this structure directly and is repressed by the eIF4E-binbing proteins (4E-BPs), which in turn are regulated through the FRAP/mTOR pathway by a multitude of extracellular stimuli. The focus of this thesis is to investigate the function of the 4E-BPs in the cell by the use of mouse embryonic fibroblasts (MEFs) in which both the genes for 4E-BP1 (Eif4ebp1) and 4E-BP2 (Eif4ebp2) were disrupted. We first examined the role of 4E-BPs in encephalomyocarditis virus (EMCV) infection. 35S-methionine metabolic labeling of MEF proteins revealed that 4E-BPs are not essential for EMCV replication. We then decided to identify mRNAs regulated by 4E-BPs. This was achieved by polyribosomal analysis of specific mRNAs. Our results showed that translation efficiency of c-myc and ornithine decarboxylase (ODC) mRNAs is enhanced in double knockout cells. We also tested MEFs for their sensitivity to rapamycin, a drug known to inhibit FRAP/mTOR. Fluorescence-activated cell sorting (FACS) analysis demonstrated that 4E-BPs contribute to rapamycin-induced growth arrest. Finally, we studied Eif4ebpl-/- ;Eif4ebp2-/- mice in order to test whether they show a phenotype similar to Eif4ebp1 -/- which are known to be smaller and display a lower blood glucose compared with their wild type counterparts. Surprisingly, Eif4ebp1-/-;Eif4ebp2 -/- mice were neither small nor hypoglycemic. The above results emphasize the importance of the 4E-BPs in the cell, thus encouraging further investigations in order to better understand their function.
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Yanez, Adrienne Gail. "Regulation of microRNA activity by translation initiation factors in melanoma." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11578.
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microRNAs (miRNAs) are small, noncoding RNAs that may regulate more than half of human genes, yet the molecular mechanism of miRNA-mediated repression remains obscure. Using a cell-free assay of miRNA activity, we show that miRNA-targeted mRNAs are enriched for components of the 40S, but not 60S ribosomal subunit. Additionally, a molecular toeprint of 18 nucleotides 3' relative to the start codon, consistent with nucleotide protection by 40S ribosomal subunits, is enriched on miRNA-targeted mRNAs. Our results suggest that miRNAs repress translation initiation in a cell-free system by preventing 60S ribosomal subunit joining to 40S subunits positioned at the start codon.
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Keys, Heather R. (Heather Rochelle). "A multi-level approach to understanding the regulation of translation initiation." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/106733.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.<br>Cataloged from PDF version of thesis. "September 2016."<br>Includes bibliographical references.<br>mRNA translation is an extremely complex process required for life. Translation consumes vast amounts of cellular resources, and organisms have evolved tight regulatory mechanisms to control this process, which are often deregulated in cancer and other disease states. Initiation, as the rate-limiting step in translation, is particularly well regulated. Two kinase pathways that respond to cellular stresses, the GCN2 and mTORC1 pathways, sense amino acid insufficiency to inhibit translation initiation at distinct points. GCN2 is activated in response to amino acid deprivation and inhibits formation of the ternary complex, comprising elF2, GTP, and the initiator methionyl-tRNA, which is required for recognition of the start codon. Although translation of most mRNAs is greatly suppressed when GCN2 is activated, mRNAs with certain cis elements escape inhibition. In contrast, the mTORC1 pathway is inhibited by the lack of amino acids, which ultimately results in the disruption of eIF4F, a multiprotein initiation factor complex that coordinates the recruitment of the small ribosomal subunit to the 5' end of mRNA. Like a decrease in the amount of ternary complex, disruption of eIF4F also suppresses translation of most mRNAs; however, the translation of a subset of mRNAs harboring a 5'TOP motif is even more dramatically reduced when mTORC1 is inhibited. Here we describe the translational program downstream of amino acid insufficiency, and present evidence of a novel uORF in murine ATF4 whose ribosome occupancy is regulated by the presence of amino acids. We identify the 4EBPs as the mTORC1 substrates that mediate the major effects of mTORC1 inhibition on translation of mRNAs both globally and on 5'TOP mRNAs specifically. Although we cannot mechanistically explain the dependence of 5'TOP mRNA translation on mTORC1 activity, we uncover a surprising role of the cap-proximal sequence in eIF4E recruitment. We systematically assess how the juxtacap sequence modulates eIF4E binding and translation, and present a model whereby the juxtacap sequence dictates the cap-proximal RNA secondary structure in an mRNA-context-dependent manner.<br>by Heather R. Keys.<br>Ph. D.
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Khalouei, Sam. "Translation initiation in human immunodeficiency virus type 1 (HIV-1)." Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27866.
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Translation of human immunodeficiency virus type 1 (HIV-1) mRNAs is entirely dependent on the host translation machinery. There are two prevailing hypotheses regarding the translation initiation mechanism in HIV-1; conventional cap-dependent ribosomal scanning mechanism (CDRSM) and cap-independent entry of the ribosome, usually through an internal ribosome entry site (IRES). The first mechanism makes use of the Kozak consensus sequence in locating the translation initiation codon, similar to the mechanism observed in human mRNAs. Therefore, a thorough understanding of the Kozak consensus and translation initiation in human would also shed light on the mechanism of translation initiation in HIV-1. The role of Kozak +4G site in translation initiation has been controversial, with the alternative hypothesis explaining the prevalence of +4G by invoking the observation that small amino acids, coded by G-starting codons, which are efficient for N-terminal methionine excision (NME), are preferred at the penultimate (second) position. Using two bioinformatics approaches we provide strong support for this alternative hypothesis and provide evidence contradicting the involvement of +4G in translation initiation.
One of the predictions of the CDRSM hypothesis is a high conservation of Kozak consensus sequence in different HIV-1 sequences. Our results presented here validate this prediction. The CDRSM hypothesis also predicts that there should be a selective pressure against ATG usage in optimal context in the HIV-1 5'-UTR to avoid their erroneous detection by the scanning ribosome, whereas the IRES-dependent mechanism in the presence of stable secondary structures, predicts no such selective pressure because these ATGs would be embedded in the secondary structures. Here we demonstrate this selective pressure in the HIV-1 5'-UTR which further supports the CDRSM hypothesis. Finally, we present evidence for strong site conservation in the 5'-UTR of HIV-1 sequences, which not only point to as yet unknown mechanisms of translation initiation, but also provide a mean to separate HIV-1 and human mRNAs. This implies that it is theoretically possible to design HIV-1-specific translation inhibition drugs.
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Lemay, Guy. "Study of a reovirus protein involved in viral mRNA translation." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75419.
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This thesis concerns the mechanisms responsible for preferential translation of reovirus mRNA in infected cells. The absence of a poly(A) tract at the $3 sp prime$-end is one of the structural differences that distinguishes reovirus mRNA from cellular mRNA. Addition of free poly(A) inhibits in vitro translation at the level of initiation of protein synthesis, probably by competition between free poly(A) and mRNA. Reovirus mRNA is resistant to this inhibition and this property might confer a translational advantage to the viral mRNA. Another difference between reovirus mRNA and cellular mRNA is the absence of a $5 sp prime$-cap structure on late viral mRNA. This mRNA is translated much more efficiently in lysates from infected than uninfected cells, even in the absence of inhibition of host cell protein synthesis. Addition to uninfected cell lysates of proteins from infected cells stimulates translation of late viral mRNA without effect on other mRNAs; the presence of the sigma 3 viral protein is shown to account for the stimulation of translation. Sigma 3 binds to ribosomes, probably during initiation of protein synthesis, but apparently does not interact directly with mRNA. The sigma 3 protein exists in multiple forms, differing in isoelectric point but possessing all the known properties of sigma 3. These forms possibly result from mutations in the gene encoding sigma 3. The cloned gene which encodes sigma 3 was expressed in L cells without apparent detrimental effect on the cells. Lysates prepared from these cells translate late viral mRNA with an increased efficiency. The sigma 3 protein is associated with the ribosomes in these cells even if no viral mRNA is present. It is suggested that the sigma 3 protein acts as a viral-specific initiation factor of protein synthesis.
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Roman, William. "Inhibiting translation as a novel strategy to target multiple Myeloma." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106443.
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Multiple Myeloma (MM) cell survival has been shown to depend on precise control of protein production and degradation. Disruption of protein catabolism through proteosomal or aggresomal blockade results in MM cell death. We hypothesized that inhibiting protein production would have a similarly toxic effect in MM. We explored the consequences of inhibiting mRNA translation in MM using silvestrol, a powerful inhibitor of ribosomal recruitment, which preferentially disrupts the production of certain cell regulatory and survival proteins. A panel of silvestrol-treated MM cell lines showed profound inhibition of growth and a rapid induction of apoptosis, as seen by MTT viability and AnnexinV flow cytometric assays respectively. The average IC50 in MM cells was determined to be 20nM while it was considerably higher in primary cultures of senescent fibroblasts. We show that silvestrol inhibits protein translation by inhibiting ribosome binding, decreasing polysome content and increasing 80S ribosomes. Western blot analyses show that silvestrol rapidly decreases the expression of c-Myc and non-canonical NF-KB signaling. Expression of anti-apoptotic proteins such as Mcl-1 and Bcl-2 decreased while pro-apoptotic proteins, such as BAX, increased with silvestrol treatment. In a novel transgenic mouse model of MM (vk*myc), which has been shown to behave clinically like human MM, silvestrol does not appear to be toxic and is therapeutically effective. Our results warrant clinical evaluation of silvestrol.<br>Il a été démontré que la survie des cellules cancéreuses chez les patients atteints du Myélome Multiple (MM), dépend de la régulation de la production et de la dégradation des protéines. Une perturbation du système de catabolisme des protéines, à travers un blocage proteosomale, engendre la mort de ces dernières. Nous avons donc formulé l'hypothèse que l'inhibition de la production de protéines aurait des effets toxiques similaires. Grâce à un puissant inhibiteur, le silvestrol, nous avons déterminé les conséquences liées à l'inhibition de la translation de l'ARN messager. Le silvestrol est un inhibiteur de recrutement ribosomale qui affecte de manière préférentielle certaines protéines impliquées dans la division et la survie cellulaire. Une panoplie de lignées cellulaire spécifiques au Myélome ont été susceptibles au silvestrol, ce qui s'est exprimé par une inhibition de la croissance cellulaire et une amorce rapide de l'apoptose. Cela a été observé grâce au test de viabilité MTT et par l'analyse de cytométrie en flux avec les marqueurs Annexin V et 7-AAD. La moyenne du ci50 des cellules du myélome testées a été établie à 20 nM, tandis qu'une concentration considérablement plus élevé est nécessaire pour obtenir les mêmes résultats chez les cultures primaires de fibroblaste sénescents. Nous démontrons que le silvestrol inhibe la translation des protéines en bloquant le ribosome 80S au départ du codon d'initiation de la translation. Ceci engendre une accumulation des ribosomes 80S et une décroissance du nombre de polysome par ARN messager. Nos analyses de western blot montrent que le silvestrol provoque une décroissance de l'expression de l'oncogène c-Myc et de NF-KB. De plus, l'expression de protéine anti-apoptotiques tel que Mcl-1, Bcl-Xl et Bcl-2 décroit avec un traitement de silvestrol, alors que l'expression de la protéine pro-apoptotique Bax augmente. Nos résultats montrent que le silvestrol est efficace et non toxique dans le nouveau model transgénique du MM (la souris Vk*Myc) qui est réputé pour sa similarité de caractéristiques avec le MM humain. Nous prônons une évaluation clinique du silvestrol.
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Pause, Arnim. "Mutational analysis of the mammalian translation initiation factor eIF-4A." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41744.
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eIF-4A is a eukaryotic translation initiation factor and DEAD box RNA helicase that is thought to be responsible for the melting of secondary structure in the 5$ sp prime$ untranslated region of messenger RNAs to facilitate ribosome binding. A mutational analysis of eIF-4A revealed that the ATPase A motif (AXXXXGKT) is involved in ATP binding, the ATPase B motif (DEAD) is implicated in ATP hydrolysis, the SAT region is essential for RNA unwinding, and the HRIGRXXR region is required for ATP hydrolysis-dependent RNA binding. Furthermore, defective eIF-4A mutants exhibit a strong dominant negative effect on in vitro translation of several mRNAs, including those translated by a cap-independent internal initiation mechanism. It is demonstrated that eIF-4A functions primarily as a subunit of eIF-4F, and singular eIF-4A is required to recycle through the eIF-4F during translation. Accordingly, eIF-4F appears to be required for cap-dependent and internal initiation of translation.
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Poulin, Francis. "Regulation of eukaryotic translation initiation by the eIF4E-binding proteins." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84416.
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The initiation of protein synthesis consists in the recruitment of a ribosome·initiator tRNA complex to the initiation codon of a messenger RNA. In eukaryotes, this process is facilitated by eukaryotic translation initiation factor 4E (eIF4E), which recognizes the cap structure present at the 5' end of all nuclear-transcribed mRNAs. The eIF4E-binding proteins (4E-BPs) inhibit cap-dependent translation by binding to eIF4E, and preventing ribosomal recruitment to the mRNA. The interaction of 4E-BPs with eIF4E is reversible, and it is regulated by the 4E-BPs phosphorylation state. Here I report the isolation of a third member of the 4E-BP family, 4E-BP3, and I demonstrate that it shares all the basic structural and functional characteristics of 4E-BP1 and 4E-BP2. I also analyzed the genomic locus for human 4E-BP3 (EIF4EBP3). I established that EIF4EBP3 can be fused with the gene located immediately upstream, and which encodes the human homologue of Drosophila MASK (Multiple Ankyrin Repeats Single KH domain). Interestingly, the open reading frame of the MASK-BP3 fusion transcript is different from that of 4E-BP3, indicating that the EIF4EBP3 locus encodes two different proteins. Finally, I investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice are remarkably lean, and the males' white adipose tissue contains cells exhibiting the distinctive multilocular appearance of brown adipocytes, and it expresses the uncoupling protein 1. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1) is increased in white adipose tissue from Eif4ebp1-/- mice. PGC1 is a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis. These findings demonstrate that 4E-BP1 is a novel regulator of metabolism in mammals.
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Hu, Wenqian. "The Interplay of Eukaryotic mRNA Translation and Degradation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1274900106.
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Meerovitch, Karen. "Studies on the mechanism of internal initiation of translation of poliovirus mRNA." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41178.
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Ribosome binding to cellular eukaryotic mRNAs is proposed to occur by initial attachment at or near to the mRNA 5$ sp prime$ cap structure (m$ sp7$ GpppN, where N is any nucleotide) followed by scanning until the appropriate initiator AUG is encountered. A pivotal aspect of this model is the obligatory entry of ribosomes at the 5$ sp prime$ end of the mRNA regardless if it contains a cap structure. Recent experiments demonstrated that ribosomes access certain mRNAs by internal binding to the 5$ sp prime$ untranslated region (UTR). This was most clearly demonstrated for members of the picornavirus family such as poliovirus and encephalomyocarditis virus. Further experiments suggest that other viral mRNAs and even cellular mRNAs may use similar mechanisms of ribosome binding. Some features of the poliovirus 5$ sp prime$ untranslated region and possible trans-acting factors that are involved in this mechanism were studied. A cis-acting element comprising a conserved polypyrimidine tract was critical for poliovirus RNA translation in vitro. Using a mobility shift electrophoresis assay, a fragment of the poliovirus 5$ sp prime$ UTR was found to interact with a component in a HeLa cellular extract. A 52 kDa polypeptide (p52) has been identified as part of the protein-RNA complex using an ultraviolet-light cross-linking assay. p52 is not a known initiation factor and is limiting in a reticulocyte lysate. Purification of p52 and peptide microsequencing showed that p52 is identical to the human La autoantigen (that has previously been implicated in transcription termination) and stimulates the translation of poliovirus RNA in a reticulocyte lysate. This finding will undoubtedly shed light on the mechanism of poliovirus translation, its regulation and neurovirulence.
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Wang, Yinuo J. "Functions of the conserved ribosome-bound protein Lso2 in translation and physiology." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/119979.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>The ribosome is a highly conserved macromolecular machine that carries out translation, the synthesis of proteins from mRNAs, in all domains of life. The core ribosome interacts with dozens of general translation factors that ensure accurate and efficient progression through the translation cycle. Their detailed characterization has significantly advanced our understanding of protein synthesis. However, a growing number of ribosome-associated proteins have also been discovered whose functions are less well understood. In Chapter 1, I will overview the translation cycle and describe how it is affected by nutrient availability, with a focus on functions of starvation-induced proteins that directly bind the ribosome. I will also discuss discovery approaches for expanding the study of ribosome-associated proteins. In Chapter 2, I will present the discovery and characterization of Lso2 as a conserved ribosome-bound protein required for translational recovery in budding yeast. Using quantitative mass spectrometry, we found this protein to be ribosome-associated during glucose-starved and replete growth, with moderate enrichment on translating ribosomes during starvation. Saccharomyces cerevisiae lacking Lso2 accumulate monoribosomes that are not translating normally following a shift from stationary phase to rich medium. To understand the basis of this phenotype, we used genome-wide RNA crosslinking and sequencing to determine that Lso2 binds near the A site of the ribosome tRNA channel, in a region that overlaps with the GTPase activating center, and that Lso2 also interacts with a broad spectrum of tRNAs. Consistently, Lso2 binding in the tRNA channel stabilizes ribosomal subunit association in vitro. These data, together with evidence that the accumulated ribosomes in Iso2 nulls are devoid of obvious barriers to initiation, lead to a model in which Lso2 promotes productive elongation. Finally, I show that the ribosome binding activity of Lso2 is conserved in its human ortholog, suggesting a broad importance of its molecular function. In Chapter 3, I will elaborate on the model of a function for Lso2 in elongation, propose alternative models to rationalize its effects on translation, and describe experiments for testing them. I will also describe the implications of this protein for our understanding of translation in different physiological states.<br>by Yinuo J. Wang.<br>Ph. D.
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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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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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Jaramillo, Maria. "Molecular biology and functional characterization of cap binding proteins involved in translation initiation." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35397.
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The cap structure, m7GpppX (where X is any nucleotide), is present at the 5' end of all nuclear encoded mRNAs. Previous studies demonstrated that this structure facilitates the unwinding of mRNA 5' secondary structure through the action of eukaryotic initiation factors eIF-4A, eIF-4B and eIF-4F. eIF-4F contains three subunits of which one, eIF-4E, contains the cap binding site. To investigate the possible regulation of eIF-4E, an initiation factor whose limiting nature and activity have been implicated in the regulation of translation and cell growth, several murine cDNAs were isolated and characterized. Multiple mRNAs were shown to exist for eIF-4E, which are generated by differential polyadenylation. In addition, tissue-specific differences in eIF-4E mRNA expression and utilization of polyadenylation sites were demonstrated. To elucidate the mechanism of RNA unwinding, the interaction of eIF-4A, eIF-4B and eIF-4F with RNA was studied by examining RNA-complex formation in relation to unwinding activity. eIF-4F and eIF-4B form stable complexes with duplex RNA. These complexes dissociate in the presence of ATP, concomitant with the RNA unwinding, suggesting that they represent intermediates in the unwinding process of mRNA. An RNA unwinding assay was used to compare the RNA helicase activities of initiation factors from different species. Yeast eIF-4A was able to effectively substitute for mammalian eIF-4A in combination with mammalian eIF-4B in RNA unwinding, whereas wheat germ eIF-4A was able to partially substitute. Mammalian eIF-4A was able to substitute in the wheat germ RNA unwinding system. These results suggest that initiation factor requirements for RNA unwinding are largely similar in mammals, plants and yeast.
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Jaramillo, Maria. "Molecular biology and functional characterization of cap binding proteins involved in translation initiation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1992. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ50193.pdf.
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Kershner, Leah. "RACK1 regulates point contact formation and local translation in neuronal growth cones." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1524159362714285.
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Cale, Stephanie. "Doc of bacteriophage P1 is an enzyme that inhibits translation and phosphorylates a protein target." Thesis, The University of Alabama in Huntsville, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=1570494.
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<p> Doc induces cell death by inhibiting translation; however, the mechanism of Doc-induced cell death and the cellular target of the toxin were unknown. One theory suggested that Doc inhibits translation elongation by binding directly to the 30S ribosomal subunit. Later evidence showed catalytic activity in distant homologs of Doc. These homologs contain a Fic-domain that has been shown to modify target GTPases by AMPylation and phosphocholination. Therefore, [<sup>35</sup>S] – Met, α[<sup>32</sup>P] – ATP, and γ[<sup>32</sup>P] – ATP were used in conjunction with an S30 extract to confirm that Doc inhibits translation, to assess the mechanism of modification, and to identify the modified target. The results showed that Doc is an enzyme that inhibits translation and phosphorylates a protein target.</p>
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Livingstone, Mark. "A nuclear role for the eukaryotic translation initiation factor 4E-binding proteins." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97024.
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The regulation of mRNA translation is crucially important in determining which cellular proteins are produced in response to intracellular and extracellular cues. The resulting collection of functional proteins determines which physiological processes a given cell will carry out; therefore, the deregulation of protein production is strongly implicated in diseases, such as cancer, in which cells fail to appropriately respond to stimuli. The mammalian target of rapamycin(mTOR) signaling pathway, which links amino acid, growth factor, and energy availability to mRNA translation resulting in cellular growth and proliferation, is frequently deregulated in cancer and is an active target for drug discovery. Among the effectors of mTOR signaling are the eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which when phosphorylated bymTOR release the mRNA 5'-cap protein eIF4E to promote translation of progrowth/proliferation mRNAs. While previous biochemical fractionation experiments have suggested that 4E-BP1 is exclusively cytoplasmic, we show using immunoassays that this protein is also present in the nuclei of mammalian cells, where it sequesters eIF4E upon mTOR inhibition. This nuclear eIF4E accumulation is useful as a biomarker for mTOR signaling, as we use it as the read-out for a chemical genetic screen for novel mTOR pathway inhibitors. This discovery of a nuclear eIF4E:4E-BP1 complex opens the door to the potential for4E-BPs to impact nuclear RNA processing events. Evidence for and against iv such a nucleus-specific function for 4E-BPs is evaluated and the nuclear function of 4E-BPs is assessed experimentally.<br>La régulation de la traduction des ARN messagers (ARNm) est d'une importance cruciale afin de contrôler quelles protéines sont produites en réponseaux signaux intra- et extracellulaires. La collection de protéines fonctionnelles quien résulte détermine quels processus physiologiques seront effectués par la cellule. Conséquemment, la dérégulation du contrôle traductionnel est fortement impliquée dans plusieurs pathologies, incluant le cancer, ceci dû au fait que les cellules ne répondent pas de manière appropriée aux stimuli qu'elles reçoivent. Une voie de signalisation impliquée dans la croissance et la prolifération cellulaire qui est souvent dérégulée dans les cancers, la voie de la cible mammifère de la rapamycine (mTOR), et qui intègre la disponibilité en acides aminés, facteurs de croissance et énergie avec la traduction des ARNm, est une cible pharmacologique préférentielle. Parmi les effecteurs de la voie mTOR, on retrouve les protéines s'associant au facteur d'initiation de la traduction eIF4E, les 4E-BP, qui lorsqu'elles sont phosphorylées relâchent la protéine liant la coiffe5' des ARNm, eIF4E, promouvant ainsi la traduction des ARNm encodant des protéines impliquées dans la croissance et la prolifération. En contraste avec les résultats de fractionnement subcellulaires reportés précédemment dans la littérature suggérant que 4E-BP1 est une protéine exclusivement cytoplasmique, nous montrons ici, par essais immunologique, que cette protéine est également v résidente du noyau des cellules mammifères où elle séquestre eIF4E suivant l'inhibition de mTOR. Cette accumulation nucléaire de eIF4E est un biomarqueur de choix que nous avons utilisé comme lecture du niveau d'activité de la voie mTOR lors d'un criblage chimio-génétique entrepris dans le but de trouver de nouveaux inhibiteurs de la voie mTOR. Cette découverte d'un complexe nucléaire eIF4E :4E-BP1 à ouvert la porte à une possible fonction des 4E-BP dans certains processus nucléaires. Les évidences en faveur et en opposition àun tel rôle nucléaire spécifique des 4E-BPs sont évaluées et la fonction nucléaire des 4E-BPs est testée expérimentalement.
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Rajakumar, Arjuna. "Characterizing the anti-tumor effects of inhibiting translation initiation in glioblastoma multiforme." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121345.
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Many genetic aberrations contribute to the aggressive phenotype of Glioblastoma Multiforme (GBM), a heterogenous tumor which consists of four major subtypes with distinct molecular characteristics. The differential activity of oncogenic and tumor suppressive networks between these subtypes makes targeted therapy difficult as not all subtypes will be affected to the same degree. As well, the genetic complexity of GBM allows for compensatory signaling to sustain malignancy and resist apoptosis. During the last decade evidence has accumulated about the importance of mRNA translation initiation in tumorigenesis. Over 10 translation initiation factors are implicated in oncogenesis, acting as either proto-oncogenes or tumor suppressors, the most well described being the eIF4F initiation complex, consisting of eIF4E, eIF4G and eIF4A. Increased complex formation results in mRNA discrimination, leading to an increased translation of a subset of "weak" oncogenic mRNAs with highly structured 5'UTRs. Thus, inhibition of translation initiation can simultaneously target many oncogenic pathways. However it is unclear what role translation initiation plays in GBM biology, and whether its inhibition may have anti-neoplastic properties. In this study, we used 4EGI-1 and silvestrol, small-molecule inhibitors of eIF4E and eIF4A respectively, to target translation initiation in three GBM cell lines U87, U251N and the more aggressive U87ΔEGFR. Here, we show in U87 cells that 4EGI-1 blocks the eIF4E/eIF4G interaction which is necessary for eIF4F complex formation. This coincides with a reduction in cell proliferation and survival, in a dose and time-dependent manner, with <1% of U87 and U251N cells and 17.5% of U87∆EGFR cells remaining after a 96 hour treatment with 80µM 4EGI-1. There is marked induction of apoptosis at 48 hours, with increased annexin V+/7AAD- staining and Capsase-9/PARP cleavage, accompanied by a decrease in the protein expression of several oncogenes, with highly structured 5'UTRs, involved in proliferation and survival such as c-Myc, Cyclin D-1, Bcl-2, Bcl-xL, Mcl-1 and Survivin. Additionally 4EGI-1 inhibited the migration of U87 and U87∆EGFR cells in response to chemotactic stimuli, as well as anchorage –independent growth of all three cell lines. Silvestrol showed similar effects as 4EGI-1 in inhibiting cell proliferation, survival, oncogenic protein expression and inducing apoptosis, although at nanomolar concentrations and at earlier time points, indicating superior pharmacokinetics.Our results demonstrate the sensitivity of GBM towards inhibition of translation initiation and its ability to induce a robust apoptotic response. This further highlights translation regulation control as a strong force in cancer therapy, particularly in GBM where therapeutic options are limited.<br>Un grand nombre d'aberrations génétiques contribuent au phénotype virulent du glioblastome multiforme (GBM), un cancer hétérogène en quatre sous-types principaux qui présentant des caractéristiques moléculaires distinctes. L'activité différentielle des réseaux suppressifs oncogènes et tumoraux complique le traitement ciblé puisque il n'aura pas la même effet sur chacun des sous-types. De même, la complexité génétique du GBM permet une signalisation compensatrice favorisant l'entretien de la tumeur et une résistance à l'apoptose.Au cours de la dernière décennie, plusieurs données probantes ont été recueillies portant sur l'importance de l'initiation de la traduction des ARNm dans la tumorigenèse. Plus de 10 facteurs d'initiation de la traduction, agissant comme proto-oncogènes ou suppresseurs de tumeur, interviennent dans l'oncogenèse. De ces facteurs, le complexe d'initiation eIF4F, qui est composé de l'eIF4E, de l'eIF4G et de l'eIF4A est le mieux décrit. La formation accrue de complexes entraîne une discrimination de l'ARNm, ce qui augmente la traduction d'un sous-groupe d'ARNm oncogènes « faibles » dotés de séquences 5' non traduites (5'UTR) très structurées. Par conséquent, l'inhibition de l'initiation de la traduction peut simultanément cibler de nombreuses voies oncogènes. Toutefois, le rôle de l'initiation de la traduction dans la biologie du GBM est incertain tout comme les propriétés anti-néoplastique que pourraient entraîner son inhibition. Lors de cette étude, nous avons utilisé le 4EGI-1 et le silvestrol, inhibiteurs des molécules eIF4E et eIF4A, respectivement, afin de cibler l'initiation de la traduction dans les trois lignées cellulaires du GBM, soit U87, U251N et la plus virulente U87ΔEGFR. Ici, nous montrons que le 4EGI-1 inhibe l'intéraction eIF4E/eIF4G qui est essentielle à la formation du complexe eIF4F dans les cellules U87. Cette inhibition entraîne une diminution de la prolifération et de la survie cellulaires en fonction de la dose et du temps traitement. Moins de 1 % des cellules U87 et U251N et de 17,5% des cellules U87∆EGFR étant toujours présentes après un traitement de 4EGI-1 à 80 µM d'une durée de 96 heures. Une induction marquée de l'apoptose est observée après 48 heures, ainsi qu'une augmentation du marquage à de même qu'une l'annexine V+/7AAD et le clivage de la capsase-9/PARP. Ceci est s'accompagné d'une diminution de l'expression des protéines de plusieurs oncogènes dotés de 5'UTR très structurées, qui sont impliquées dans la prolifération et la survie, tels que c-Myc, cycline D-1, Bcl-2, Bcl-xL, Mcl-1 et survivine. De plus, le 4EGI-1 inhibe la migration des cellules U87 et U87∆EGFR en réponse aux stimuli chimioattractifs ainsi que la prolifération des trois lignées cellulaires dans un milieu en suspension solide. Les effets du silvestrol sont similaires à ceux de 4EGI-1 à l'égard de l'inhibition de la prolifération cellulaire, de la survie, de l'expression des protéines oncogènes et de l'induction de l'apoptose, bien qu'à des concentrations nanomolaires et à des points d'analyse différent, ce qui indique une pharmacocinétique supérieure.Nos résultats montrent la sensibilité du GBM en ce qui concerne l'inhibition de l'initiation de la traduction et sa capacité de déclencher une réponse apoptique solide. Ceci corrobore que la maîtrise de la régulation de la traduction constitue un élément puissant du traitement du cancer, particulièrement du GBM, où les options thérapeutiques sont limitées.
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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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McLeod, Tina Louise. "Investigating methods of visualising translation in Schizosaccharomyces pombe." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/7105/.
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Gene expression is compartmentalised in eukaryotes due to the nuclear envelope separating the nuclear processes of transcription and pre-mRNA processing from cytoplasmic translation. While ribosomes are synthesised in the nucleus, it is understood that a number of mechanisms keep them inactive until they reach the cytoplasm, where they mature to become translation-competent. However, this consensus view is being challenged by a growing body of evidence in support of nuclear translation. A newly developed technique, known as ribopuromycylation (RPM), had reported the presence of puromycin-bound nascent peptides on immobilised ribosomes in the nuclei of human cells. I investigated whether this method could be used, combined with chromatin immunoprecipitation, to determine whether nuclear ribosomes can cotranscriptionally translate nascent transcripts in Schizosaccharomyces pombe. Surprisingly, I discovered that, in contrast to that reported in the original study, immobilising ribosomes with translation elongation inhibitors does not lead to retention of puromycylated peptides on ribosomes in either S. pombe, Drosophila melanogaster or HeLa cells. However, I show here preliminary data which suggest that despite puromycylated peptides being released from the ribosome, puromycin immunostaining might still be used to visualise the sub cellular localisation of ribosomes inS. pombe, along with other approaches which I also describe.
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Ghosh, Arnab. "Coevolution of Ribosomes and The Translational Apparatus: The Structure and Function of Eukaryotic Ribosomal Protein uS7 from Yeast, Saccharomyces cerevisiae." Cleveland State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=csu1435159279.
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Herdy, Barbara. "The role of eukaryotic translation initation factor 4E (eIF4E)regulation during viral infection." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86803.
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Translation of mRNA into protein is a fundamental process and requires tight regulation. Primary control occurs at the initiation step. A critical protein for this regulation is the eukaryotic translation initiation factor 4E (eIF4E), which binds to the 5´ cap structure found on all nuclear transcribed mRNAs. This interaction initiates translation by assembling the eIF4F complex on the mRNA, which subsequently recruits the ribosome. The function of eIF4E is regulated in two ways, by the 4E binding proteins (4E-BPs), which disrupt the eIF4F complex and secondly by eIF4E phosphorylation on serine 209. However, the consequences of eIF4E phosphorylation are not clearly understood.<br>Cells continuously encounter pathogens including viruses. Lacking their own metabolic machinery, viruses rely on the translational apparatus of the host to produce their proteins. During many viral infections reprogramming of the cellular translation system occurs, favoring translation of viral mRNAs. Altering the eIF4F complex is one means by which reprogramming occurs. Picornaviruses initiate translation of their mRNAs independent of eIF4E through an internal ribosomal entry site (IRES). Work in this thesis demonstrates that eIF4E still impacts picornavirus mRNA translation, but only when viral mRNAs compete with cellular RNAs. IRES mediated translation was strongly enhanced when eIF4E abundance was decreased in an in vitro system by addition of 4E-BP or in vivo by siRNA knock down. Decreased eIF4E abundance reduced cap-dependent translation, but stimulated IRES mediated translation.<br>Viral invasion is immediately recognized by the cell and accordingly, several defense mechanisms are activated. One of them entails secretion of type I interferon. This thesis shows that phosphorylation of eIF4E on serine 209 impedes type I interferon production. Cells carrying an altered eIF4E phosphorylation site, impairing eIF4E phosphorylation, secreted elevated levels of type I interferon upon stimulation with synthetic RNA. Consequently, these cells were more protected against infection with interferon sensitive viruses. In conclusion, regulation of eIF4E is important for picornavirus infection and plays a key role in generating a potent antiviral response.<br>La traduction des ARN messagers (ARNm) en protéines est un processus essentiel qui requiert une étroite régulation. Le principal contrôle a lieu à l'étape de l'initiation. Une protéine essentielle pour cette régulation est le facteur d'initiation de la traduction 4E (eIF4E), qui se lie à la structure de la coiffe présente en 5' de tous les ARNm transcrits par le noyau. Cette interaction initie la traduction par l'assemblage du complexe eIF4F sur l'ARNm, ce qui permet le recrutement des ribosomes. La fonction d'eIF4E est régulée d'une part par les protéines se liant à 4E (4E-BPs) qui dissocient le complexe eIF4F, et d'autre part par la phosphorylation d'eIF4E sur la sérine 209. Toutefois, les conséquences de cette dernière ne sont pas clairement comprises.<br>Les cellules rencontrent constamment des pathogènes, dont des virus. Ces derniers étant dépourvue de machinerie métabolique propre, ils se doivent d'utiliser l'appareil traductionnel de l'hôte pour exprimer leurs protéines. De nombreuses infections virales résultent en un changement du système de traduction cellulaire favorisant la traduction de protéines à partir des ARNm viraux. L'altération du complexe eIF4F est l'un des moyens utilisés lors de cette reprogrammation. Les Picornavirus initie la traduction de leurs ARNm indépendamment d'eIF4E par le biais d'un site d'entrée interne des ribosomes (IRES). Le travail effectué lors de cette thèse démontre qu'eIF4E peut toujours influencer la traduction des ARNm des picornavirus, mais uniquement lorsque ceux-ci entrent en compétition avec les ARN cellulaires. La traduction à partir de l'IRES est accrue lorsque la quantité d'eIF4E disponible est réduite dans un système in vitro par addition de 4E-BP, ou in vivo par un ARN interférant. La diminution des niveaux d'eIF4E réduit la traduction dépendante de la coiffe, mais stimule la traduction par l'IRES.<br>Les infections virales sont reconnues immédiatement par les cellules et engendrent l'activation de plusieurs mécanismes de défense. L'un d'entre eux implique la sécrétion d'interféron de type I. Nous avons démontré que la phosphorylation d'eIF4E sur la sérine 209 entrave cette production d'interféron de type I. Des cellules dont le site de phosphorylation d'eIF4E a été altéré pour empêcher sa phosphorylation, produisent des niveaux plus élevés d'interféron de type I suite à une stimulation par des ARN synthétiques. En conséquence, elles sont mieux protégées contre les infections virales provenant de virus sensibles aux interférons. En conclusion, la régulation d'eIF4E est importante lors des infections par les picornavirus et elle joue un rôle essentiel dans la genèse d'une réponse virale efficace.
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Bidinosti, Michael Anthony. "Identification and characterisations of a novel posttranslational modification of translation repressor 4E-BP2." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86551.
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In eukaryotes, control of protein synthesis or translation is critical for maintenance of cellular function and adaptation to environmental stimuli or stresses. The entire process of producing a functional protein molecule from an mRNA template is elaborately controlled and involves several integrated phases. It is the initiation phase of recruiting the protein synthesis machinery to an mRNA that is rate-limiting for translation. Importantly, a major regulatory mechanism of translation initiation is performed by the eIF4E-binding proteins (4E-BPs). These small proteins interact with the mRNA 5' cap-binding protein, eIF4E, and inhibit translation by preventing it from forming a complex that promotes translation. This is accomplished by the competition of the 4E-BPs with eIF4G for association to eIF4E. This competition is determined by the degree of stimulus-induced 4E-BP phosphorylation: whereas hypophophorylated 4E-BPs bind tightly to eIF4E, hyperphosphorylation causes their dissociation from eIF4E and permits translation. In the nervous system, translational control is obligatory for learning and memory. 4E-BP2, the predominant mammalian 4E-BP in the brain, is required to ensure normal functioning of translation-dependent memory processes. This thesis describes the identification of asparagine deamidation as a novel posttranslational modification of 4E-BP2 in the brain. Deamidation is the spontaneous conversion of asparagines to aspartates. Deamidated 4E-BP2 exhibits increased binding to the mammalian Target of Rapamycin (mTOR)-binding protein, Raptor. Furthemore, 4E-BP2 deamidation, which occurs during postnatal development, alters neuronal activity. It is conceivable that this modification of 4E-BP2 compensates for its attenuated phosphorylation in the brain. 4E-BP2 is also identified here, by virtue of its propensity to deamidate, as a novel substrate for the enzyme Protein L-Isoaspartyl Methyltransferase (PIMT). As a whole, this thesis describes a posttransl<br>Le contrôle de la synthèse protéique ou traduction chez les eucaryotes est d'une importance capitale dans le maintien de l'homéostasie cellulaire et dans l'adaptation aux stimuli et stress environnementaux. La production de protéines fonctionnelles à partir d'ARN messagers est soumise à un fin contrôle et consiste en une succession d'étapes intégrées. C'est au cours de la première étape de la traduction, l'initiation, que la machinerie traductionnelle est recrutée au niveau de l'ARN messager. L'initiation est l'étape limitante du processus de synthèse. Au coeur du processus de régulation de l'initiation de la traduction se trouve les facteurs liant eIF4E, les 4E-BP. Ces protéines de faible poids moléculaire interagissent avec le facteur eIF4E qui lie le 5'-cap des ARN messagers et inhibe ainsi la traduction en empêchant eIF4E de participer à la formation du complexe d'initiation. Cette inhibition est causée par la compétition entre les 4E-BP et eIF4G pour lier eIF4E. Le niveau de compétition est déterminé par le degré de phosphorylation de 4E-BP induit par les stimuli extracellulaire. Les formes hypophosphorylées de 4E-BP lient eIF4E avec une forte affinité, tandis que les formes hyperphosphorylées relâchent eIF4E, ce qui stimule la traduction. Dans le système nerveux, le contrôle de la traduction est nécessaire pour l'apprentissage et la mémoire. 4E-BP2, le facteur liant eIF4E dont l'abondance est prédominante dans le système nerveux des mammifères, est requis afin d'assurer un fonctionnement normal des processus de mémoire qui dépendent de la traduction. Cette thèse décrit la déamidation d'asparagine en tant que nouvelle modification post-traductionnelle du facteur 4E-BP2 dans le cerveau. Cet événement de déamidation consiste en la conversion spontanée de résidu asparagine en résidu acide aspartique. La forme déamidée de 4E-BP2 présente une affinité accrue pour la protéine Raptor, un facteur associé au comp
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Cotnoir-White, David. "NMR study of the interaction between PABP and the translation down-regulator Paip2." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101712.
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The poly(A) binding protein (PABP) is an essential translation factor that enhances protein synthesis and protects mRNAs from degradation. PABP is composed of four RNA recognition motifs (RRM) at its N-terminal and a peptide-interacting C-terminal domain. PABP-interacting proteins 1 (Paip1) and 2 (Paip2) were found to respectively stimulate or inhibit translation. Both Paip 1 and 2 contain two PABP interacting regions (PAM1 and PAM2). In this study, NMR was used to elucidate the mechanism by which Paip2 down-regulates translation through disruption of PABP's binding to poly(A) RNA. A fragment consisting of PABP RRM 2 and 3 was isotopically labeled and backbone NMR assignments were determined. From NMR titration with PABP RRM2-3 and the Paip2 PAM1 region, we determined that Paip2 binds in a positively charged cleft on the beta-sheet surface of RRM2-3 which is evolutionarily conserved. These results suggest a mechanism of how Paip2 displaces the mRNAs from PABP leading to translation inhibition.
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Islas-Osuna, Maria A. "Genetic analysis of the Cbp1-COB mRNA interaction and the role of Cbp1 in translation of COB RNAs." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/279945.
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Mitochondria are the organelles where respiration occurs. The yeast mitochondrial genome encodes only 8 proteins, therefore the organelle depends on the nuclear genome for many proteins required in different steps of mitochondrial gene expression. Regulation of mRNA stability, processing and translation are important steps in gene expression within the mitochondrion. Cbp1, a protein encoded by the nuclear gene CBP1, is required specifically for stabilization of precursor and mature cytochrome b (COB) RNA, which is encoded in the mitochondrial genome. Previous work identified a cis-element, CCG, in the 5' untranslated region (UTR) of COB, that is critical for the Cbp1-dependent stability of COB mRNA. Mutation of any single nucleotide resulting in an A̲CG, CA̲G or CCU̲ triplet causes destabilization of COB mRNA and concomitant loss of respiratory capability. In the present study, suppressors were selected in the CCU strain in an effort to define important sites in Cbp1 for protection of COB mRNA. The mitochondrial mutant strain CCU is conditional; it grows slowly at 25°C but does not grow at 18°C or 33°C on the non-fermentable carbon source glycerol. Twelve dominant suppressors in CBP1 were obtained. They define two main groups, based on the pattern of growth on glycerol at different temperatures. The CBP1-encoded suppressors make strains containing the mitochondrial CCU̲ mutation respiratory competent at 33°C by allowing accumulation of mature COB mRNA. Suppressors that map to the carboxyl half of Cbp1, such as S289G, S330R, Q358K, Q358R, L489W, K532M, D533Y and I638M, rescue the temperature-sensitive (ts) phenotype caused by the CCU̲ mutation. The suppressors that map to the amino half of Cbp1, such as K205R, E241G, I249T, N281D and I293L, rescue the ts phenotype to a lesser extent than the suppressors that map to the carboxyl half of Cbp1. These results suggest that the two halves of Cbp1 have different functions in the processing and stability of COB transcripts. The hypothesis that Cbp1 has a role in translation of cytochrome b (COB) mRNA was not testable previously, since disruption of CBP1 results in instability and degradation of COB mRNA. In a Δpet127 strain, COB precursor mRNA is not processed to the mature 5' site and thus accumulates to levels equivalent to that of the wild-type mature mRNA (Wiesenberger and Fox, 1997). Null alleles of pet127 were selected as suppressors of A̲CG and CCU̲ mutations in COB. COB precursor mRNA levels in these strains were similar to the Δpet127 strain with a wild-type mitochondrial genome (Chen et al., 1999). In the present study, the effect of deleting CBP1 in a Δpet127 strain was measured. Strain Δcbp1 Δpet127 accumulated no mature COB mRNA but high levels of COB precursor mRNA. The levels of precursor mRNA in the Δcbp1 Δpet127 strain were 3-fold higher than in wild-type strain and approaching 60% of wild-type mature levels (wild-type COB precursor is 18% of mature COB mRNA). Absolutely no apocytochrome b protein was detected in the Δcbp1 Δpet127 strain. This result suggests that Cbp1 is required for translation of the COB message. Future experiments to determine the role of Cbp1 in the translation of COB mRNA are described.
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Silver, Stanley. "Glutamine Synthetase: Isolation of Isoforms, Poly (A) +RNA and In Vitro Translation." TopSCHOLAR®, 1987. https://digitalcommons.wku.edu/theses/2852.
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Two forms of glutamine synthetase (GS) have been isolated from three-day old etiolated soybean hypocotyls by ammonium sulfate precipitation and DEAE-cellulose chromatography. The GS isoforms were eluted from a 30 ml DEAE-cellulose column using 0.14 M KC1 and 0.175 M KC1 and from a 1.0 ml DEAE-cellulose column using 0.08 M KC1 and 0.14 M KC1. The two isoforms of soybean GS have a molecular weight of about 392,000 daltons as indicated by in gel enzyme assays and staining of protein bands.
The poly(A)+RNA specific for GS synthesis was isolated using phenol and chloroform followed by Hybond mAP paper. Translation was performed using a wheatgerm cell-free lysate. Following the application of the in vitro translated products to a 1 ml DEAE-cellulose column, GS was successfully eluted at 0.14 M KC1.
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Su, Yang Ph D. Massachusetts Institute of Technology. "Disassembly of electron transport chain complexes drives macrophage TLR responses by reprogramming metabolism and translation." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/127139.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, May, 2020<br>Cataloged from the official PDF of thesis.<br>Includes bibliographical references.<br>Metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis is a key feature of inflammatory response in macrophages, but how this switch occurs in response to inflammatory signals and how it precisely contributes to macrophage function is still obscure. Here we show that stimulation of macrophages through Toll-like receptors (TLR) disrupts the assembly of mitochondrial electron transfer chain (ETC) complexes I-V, leading to the metabolic switch by inhibiting OxPhos and activating HIF-1[alpha]-dependent glycolysis. Disassembly of ETC complexes influences the global metabolic status of macrophages not only by inducing glycolysis but also largely by inducing the reprogramming of cellular translational capacity via mTORC1 and ATF4, leading to enhanced global translation rate, cell growth, and production of inflammatory cytokines. Inhibition of OxPhos via myeloid-specific knockout of OPA1, which stimulates ETC complex assembly, exacerbates sepsis in mice while inhibition of mTORC1 reverses this effect. These findings reveal that disassembly of ETC complexes underlies macrophage metabolic switch and inflammatory responses and may be a conserved pathway to reprogram cellular anabolism and function.<br>by Yang Su.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology
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Hagerty, James Robert. "Developmental Regulation of Translation in Parasitic Flatworms." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1623424469091568.
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Brubaker, Sky William. "Identification of an Antiviral Signaling Variant Demonstrates Immune Regulation Through Alternative Translation." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070055.
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Innate immune signaling pathways initiate host defenses against viral pathogens. Receptors specific for viral nucleic acids activate these pathways culminating in cell-to-cell communication and/or cell death. In mammals, this cell- to-cell communication is achieved through the production of interferons and pro- inflammatory cytokines, which activate antiviral defenses in uninfected neighboring cells and instruct adaptive immune responses. The production of these signaling molecules is essential for the defense against viral infection, but must also be tightly regulated to prevent unnecessary inflammation. As an antiviral defense, cell death is also an effective mechanism to limit viral replication and spread but comes with the cost of tissue damage and inflammation. Therefore, regulating these antiviral responses is critical for controlling the spread of infection as well as preventing unnecessary pathologies related to excessive signaling. Hundreds of genes are involved in controlling these immune responses and a wide variety of mechanisms are utilized to regulate them. One mechanism to regulate gene function is the generation of protein variants through alternative translation. While polycistronic transcripts are a common feature of bacterial and viral gene expression, the process of alternative translation as a means to regulate gene function is not a feature generally attributed to mammalian mRNA. This dissertation describes a novel regulator of antiviral signaling that is generated through alternative translation. Expression of the transcript encoding the antiviral adaptor protein, MAVS, results in the production of two proteins: the full-length MAVS adaptor and a truncated variant, miniMAVS. Production of these proteins is in part regulated by cis-acting elements that control translation initiation. Production of miniMAVS regulates antiviral signaling by limiting interferon production induced by full-length MAVS, whereas both MAVS variants positively regulate cell death. To identify other examples of alternative translation in mammalian cells a genome-wide ribosomal profiling technique was used to generate a candidate list of antiviral truncation variants. This dissertation therefore demonstrates that protein variants generated through alternative translation of polycistronic mRNAs can be an effective mechanism for immune regulation and may be more common than previously understood.
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Sham, Mai Har. "Regulation of transcription and translation of phloem proteins in cucurbitaceae during differentiation." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329169.
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Chen, Menglin. "Studies of Translation Elongation and its Relationship to Transcription Elongation in Bacteria." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586468314499281.
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