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Brogna, Saverio. "Nonsense-mediated mRNA reduction and pre-mRNA processing in Drosophila." Thesis, Open University, 2000. http://oro.open.ac.uk/54807/.
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Zhou, Yang. "Regulation of pre-mRNA splicing and mRNA degradation in Saccharomyces cerevisiae." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-138142.
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Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated. The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs. The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation. In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.
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SPEDALIERI, Gaetana. "Translation of Leaderless mRNAs and Structure of cspD mRNA in E. coli." Doctoral thesis, Università degli Studi di Camerino, 2010. http://hdl.handle.net/11581/401866.
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Part I: Translation of leaderless mRNA in E. coli Spedalieri G., Kashrer C., RajBhandary U.L. Leaderless mRNAs are present in all three domains of life. They do not have a 5' untranslated region (5'UTR), they start directly with the initiation codon, AUG in most cases. In Escherichia coli these mRNAs are very rare and show inefficient translation. In contrast, leaderless mRNAs are quite prevalent in archaea, where they are translated as efficiently as the ''leadered'' mRNAs containing the typical Shine-Dalgarno sequence. In leaderless translation initiation 70S ribosomes are thought to be used without a typical pre-initiation complex involving a 30S ribosomal subunit. In this study, the goal is to answer two fundamental questions: (i) Is AUG strictly required as initiation codon? (ii) Is the formylmethionyl-tRNAfMet (fMet-tRNAfMet) required as initiator tRNA? Is the amino acid ''methionine'' important? Using a chloramphenicol acetyltransferase reporter system for studying in vivo translation of leaderless mRNAs in E. coli, we have found that the initiation codon AUG is not absolutely required and the amino acid ''methionine'' plays a fundamental role. Part II: Structural and functional characterization of cspD mRNA in Escherichia coli Gaetana Spedalieri, Anna Brandi and Anna Maria Giuliodori Expression of cspD, a gene belonging to the csp family of Escherichia coli, is exclusively induced at 37°C during stationary-phase or upon glucose starvation and does not increase during cold-shock. To shed light on the mechanisms responsible for the regulation of cspD expression, I have elucidated by enzymatic and chemical probing the secondary structure of cspD mRNA as a function of temperature. The structure, which was also validated by mutagenesis, demonstrates that cspD mRNA does not undergo any temperature-dependent structural rearrangement. Functional analyses suggest that the presence of a helix occluding the initiation codon might be partly responsible for the scarce translation of cspD mRNA and that RNase III is likely involved in the regulation of cspD gene expression. Overall, these data suggest that induction of cspD, like that of many other csp gene of E. coli, is controlled also at a posttranscriptional level.
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Ruscica, Vincenzo [Verfasser]. "GIGYF recruits mRNA decay factors to repress target mRNA expression / Vincenzo Ruscica." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1225740169/34.
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Deneke, Carlus. "Theory of mRNA degradation." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2012/6199/.
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One of the central themes of biology is to understand how individual cells achieve a high fidelity in gene expression. Each cell needs to ensure accurate protein levels for its proper functioning and its capability to proliferate. Therefore, complex regulatory mechanisms have evolved in order to render the expression of each gene dependent on the expression level of (all) other genes. Regulation can occur at different stages within the framework of the central dogma of molecular biology. One very effective and relatively direct mechanism concerns the regulation of the stability of mRNAs. All organisms have evolved diverse and powerful mechanisms to achieve this. In order to better comprehend the regulation in living cells, biochemists have studied specific degradation mechanisms in detail. In addition to that, modern high-throughput techniques allow to obtain quantitative data on a global scale by parallel analysis of the decay patterns of many different mRNAs from different genes.
In previous studies, the interpretation of these mRNA decay experiments relied on a simple theoretical description based on an exponential decay. However, this does not account for the complexity of the responsible mechanisms and, as a consequence, the exponential decay is often not in agreement with the experimental decay patterns.
We have developed an improved and more general theory of mRNA degradation which provides a general framework of mRNA expression and allows describing specific degradation mechanisms. We have made an attempt to provide detailed models for the regulation in different organisms. In the yeast S. cerevisiae, different degradation pathways are known to compete and furthermore most of them rely on the biochemical modification of mRNA molecules. In bacteria such as E. coli, degradation proceeds primarily endonucleolytically, i.e. it is governed by the initial cleavage within the coding region. In addition, it is often coupled to the level of maturity and the size of the polysome of an mRNA. Both for S. cerevisiae and E. coli, our descriptions lead to a considerable improvement of the interpretation of experimental data. The general outcome is that the degradation of mRNA must be described by an age-dependent degradation rate, which can be interpreted as a consequence of molecular aging of mRNAs. Within our theory, we find adequate ways to address this much debated topic from a theoretical perspective.
The improvements of the understanding of mRNA degradation can be readily applied to further comprehend the mRNA expression under different internal or environmental conditions such as after the induction of transcription or stress application. Also, the role of mRNA decay can be assessed in the context of translation and protein synthesis.
The ultimate goal in understanding gene regulation mediated by mRNA stability will be to identify the relevance and biological function of different mechanisms. Once more quantitative data will become available, our description allows to elaborate the role of each mechanism by devising a suitable model.Ein zentrales Ziel der modernen Biologie ist es, ein umfassendes Verständnis der Genexpression zu erlangen. Die fundamentalen Prozesse sind im zentralen Dogma der Genexpression zusammengefasst: Die genetische Information wird von DNA in Boten-RNAs (mRNA) transkribiert und im Prozess der Translation von mRNA in Proteine übersetzt. Zum Erhalt ihrer Funktionalität und der Möglichkeit von Wachstum und Fortpflanzung muss in jeder Zelle und für jedes Gen die optimale Proteinkonzentration akkurat eingestellt werden. Hierzu hat jeder Organismus detaillierte Regulationsmechanismen entwickelt. Regulation kann auf allen Stufen der Genexpression erfolgen, insbesondere liefert der Abbau der mRNA-Moleküle einen effizienten und direkten Kontrollmechanismus. Daher sind in allen Lebewesen spezifische Mechanismen - die Degradationsmechanismen - entstanden, welche aktiv den Abbau befördern. Um ein besseres Verständnis von den zugrunde liegenden Prozessen zu erlangen, untersuchen Biochemiker die Degradationsmechanismen im Detail. Gleichzeitig erlauben moderne molekularbiologische Verfahren die simultane Bestimmung der Zerfallskurven von mRNA für alle untersuchten Gene einer Zelle. Aus theoretischer Perspektive wird der Zerfall der mRNA-Menge als exponentieller Zerfall mit konstanter Rate betrachtet. Diese Betrachtung dient der Interpretation der zugrunde liegenden Experimente, berücksichtigt aber nicht die fundierten Kenntnisse über die molekularen Mechanismen der Degradation. Zudem zeigen viele experimentelle Studien ein deutliches Abweichen von einem exponentiellen Zerfall. In der vorliegenden Doktorarbeit wird daher eine erweiterte theoretische Beschreibung für die Expression von mRNA-Molekülen eingeführt. Insbesondere lag der Schwerpunkt auf einer verbesserten Beschreibung des Prozesses der Degradation. Die Genexpression kann als ein stochastischer Prozess aufgefasst werden, in dem alle Einzelprozesse auf zufällig ablaufenden chemischen Reaktionen basieren. Die Beschreibung erfolgt daher im Rahmen von Methoden der stochastischen Modellierung. Die fundamentale Annahme besteht darin, dass jedes mRNA-Molekül eine zufällige Lebenszeit hat und diese Lebenszeit für jedes Gen durch eine statistische Lebenszeitverteilung gegeben ist. Ziel ist es nun, spezifische Lebenszeitverteilungen basierend auf den molekularen Degradationsmechanismen zu finden. In dieser Arbeit wurden theoretische Modelle für die Degradation in zwei verschiedenen Organismen entwickelt. Zum einen ist bekannt, dass in eukaryotischen Zellen wie dem Hefepilz S. cerevisiae mehrere Mechanismen zum Abbau der mRNA-Moleküle in Konkurrenz zueinander stehen. Zudem ist der Abbau durch mehrere geschwindigkeitsbestimmende biochemische Schritte charakterisiert. In der vorliegenden Arbeit wurden diese Feststellungen durch ein theoretisches Modell beschrieben. Eine Markow-Kette stellte sich als sehr erfolgreich heraus, um diese Komplexität in eine mathematisch-fassbare Form abzubilden. Zum anderen wird in Kolibakterien die Degradation überwiegend durch einen initialen Schnitt in der kodierenden Sequenz der mRNA eingeleitet. Des Weiteren gibt es komplexe Wechselwirkungen mit dem Prozess der Translation. Die dafür verantwortlichen Enzyme - die Ribosomen - schützen Teile der mRNA und vermindern dadurch deren Zerfall. In der vorliegenden Arbeit wurden diese Zusammenhänge im Rahmen eines weiteren spezifischen, theoretischen Modells untersucht. Beide Mechanismen konnten an experimentellen Daten verifiziert werden. Unter anderem konnten dadurch die Interpretation der Zerfallsexperimente deutlich verbessert und fundamentale Eigenschaften der mRNA-Moleküle bestimmt werden. Ein Vorteil der statistischen Herangehensweise in dieser Arbeit liegt darin, dass theoretische Konzepte für das molekulare Altern der mRNAs entwickelt werden konnten. Mit Hilfe dieser neuentwickelten Methode konnte gezeigt werden, dass sich die Komplexität der Abbaumechanismen in einem Alterungsprozess manifestiert. Dieser kann mit der Lebenserwartung von einzelnen mRNA-Molekülen beschrieben werden. In dieser Doktorarbeit wurde eine verallgemeinerte theoretische Beschreibung des Abbaus von mRNAMolek ülen entwickelt. Die zentrale Idee basiert auf der Verknüpfung von experimentellen Zerfallsmessungen mit den biochemischen Mechanismen der Degradation. In zukünftigen experimentellen Untersuchungen können die entwickelten Verfahren angewandt werden, um eine genauere Interpretation der Befunde zu ermöglichen. Insbesondere zeigt die Arbeit auf, wie verschiedene Hypothesen über den Degradationsmechanismus anhand eines geeigneten mathematischen Modells durch quantitative Experimente verifiziert oder falsifiziert werden können.
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Cumberbatch, Marcus G. "mRNA export and cancer." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/13480/.
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Sadlon, Timothy John. "Regulation of the rat 5-aminolevulinate synthase mRNA : the role of mRNA stability /." Title page, contents and summary only, 1995. http://web4.library.adelaide.edu.au/theses/09PH/09phs126.pdf.
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Swisher, Kylie. "Assembly of mRNP Complexes During Stress and Nonsense-Mediated mRNA Decay Quality Control in Saccharomyces cerevisiae." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/204068.
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In eukaryotes, mRNA is in constant flux between an actively translating state and translationally repressed states. Specifically, mRNA degradation and repression factors compete with translation factors to direct mRNAs out of translation for storage or decay. This process often leads to formation of cytoplasmic aggregates. P-bodies are granules that contain mRNA and degradation factors, suggesting they are sites of mRNA decay or storage. Stress granules form in response to stress conditions and contain mRNAs and translation factors.P-bodies and stress granules consist of mRNPs of different compositions, believed to mature and transition between the states. It is proposed that mRNAs transition between the two granules. In the work described below, we use
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Wilson, Timothy Craig. "The role of mRNA stability and Fos protein in transient c-fos mRNA accumulation." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304567.
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Brown, Cheryl Yvette. "Regulation of cytokine mRNA stabilty /." Title page, contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phb877.pdf.
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Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology and Div. of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, 1996.Copies of author's previously published works inserted. Includes bibliographical references.
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Walker, James Anthony. "Control of maternal mRNA translation." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627300.
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Assunção, Maria Saraiva Mendes. "mRNA genotyping by gold nanoprobes." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6235.
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Dissertação para obtenção do Grau de Mestre em
BiotecnologiaBionanotechnology has emerged as a field with great potential for molecular diagnose. Namely, the use of gold nanoparticles has allowed the development of molecular diagnostic methods with greater sensitivity and specificity at a fraction of the cost inherent to conventional techniques. The present work assessed the ability of gold nanoprobes to detect targets with single base differences in RNA molecules following the colorimetric non-cross-linking method. As proof-of-concept, gold nanoprobes were designed, synthesized and characterized to detect three different SNPs (c.2731C>T, c.3232A>G and c.3238G>A) in the BRCA1 gene, a gene associated with inherited breast cancer. Reference materials, susceptible to be used for the calibration of the method, were created by cloning genomic fragments amplified from biological samples containing the sequences of interest in an appropriate vector for subsequent in vitro/in vivo transcription. Initially, the ability of target recognition by the gold nanoprobes was assessed using synthetic oligonucleotides targets alone and spiked-in total RNA of Saccharomyces cerevisiae, and later using transcripts synthetized in vitro/in vivo. This study revealed the capacity of target detection up to 0.25% of complementary target/total RNA, for a final concentration of complementary target of 0.12pmol/μL. It was also possible to detect and discriminate both c.3232A>G SNP alleles using only 0.08pmol/μL of in vitro transcript. For the in vivo transcript samples the results were inconclusive.
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Paulo, Jorge Fernando Ferreira de Sousa. "mRNA mistranslation in Saccharomyces cerevisiae." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/7775.
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Mestrado em Biologia Molecular e CelularThe genetic code is defined as a series of biochemical reactions that establish the cellular rules that translate DNA into protein information. It was established more than 3.5 billion years ago and it is one of the most conserved features of life. Over the years, several alterations to the standard genetic code and codon ambiguities have been discovered in both prokaryotes and eukaryotes, suggesting that the genetic code is flexible. However, the molecular mechanisms of evolution of the standard genetic code and the cellular role(s) of codon ambiguity are not understood. In this thesis we have engineered codon ambiguity in the eukaryotic model Sacharomyces cerevisiae to clarify its cellular consequences. As expected, such ambiguity had a strong negative impact on growth rate, viability and protein aggregation, indicating that it affects fitness negatively. However, it also created important selective advantages in certain environmental conditions, suggesting that it has the capacity to increase adaptation potential under environmental variable conditions. The overall negative impact of genetic code ambiguity on protein aggregation and cell viability, suggest that codon ambiguity may have catastrophic consequences in multicellular organisms. In particular in tissues with low cell turnover rate, namely in the brain. This hypothesis is supported by the recent discovery of a mutation in the mouse alanyl-tRNA synthetase which creates ambiguity at alanine codons and results in rapid loss of Purking neurons, neurodegeneration and premature death. Therefore, genetic code ambiguity can have both, negative or positive outcomes, depending on cell type and environmental conditions.
O código genético pode ser definido como uma série de reacções bioquímicas que estabelecem as regras pelas quais as sequências nucleotídicas do material genético são traduzidas em proteínas. Apresenta um elevado grau de conservação e estima-se que tenha tido a sua origem há mais de 3.5 mil milhões de anos. Ao longo dos últimos anos foram identificadas várias alterações ao código genético em procariotas e eucariotas e foram identificados codões ambíguos, sugerindo que o código genético é flexível. Contudo, os mecanismos de evolução das alterações ao código genético são mal conhecidos e a função da ambiguidade de codões é totalmente desconhecida. Nesta tese criámos codões ambíguos no organismo modelo Saccharomyces cerevisiae e estudámos os fenótipos resultantes de tal ambiguidade. Os resultados mostram que, tal como seria expectável, a ambiguidade do código genético afecta negativamente o crescimento, viabilidade celular e induz a produção de agregados proteicos em S. cerevisiae. Contudo, tal ambiguidade também resultou em variabilidade fenótipica, sendo alguns dos fenótipos vantajosos em determinados condições ambientais. Ou seja, os nossos dados mostram que a ambiguidade do código genético afecta negativamente a capacidade competitiva de S. cerevisiae em meio rico em nutrientes, mas aumenta a sua capacidade adaptativa em condições ambientais variáveis. Os efeitos negativos da ambiguidade do código genético, nomeadamente a agregação de proteínas, sugerem que tal ambiguidade poderá ser catastrófica em organismos multicelulares em que a taxa de renovação celular é baixa. Esta hipótese é suportada pela recente descoberta de uma mutação na alaniltRNA sintetase do ratinho que induz ambiguidade em codões de alanina e resulta numa forte perda de neurónios de Purkinge, neurodegeneração e morte prematura. Ou seja, a ambiguidade do código genético pode ter consequências negativas ou positivas dependendo do tipo de células e das condições ambientais.
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Caponigro, Giordano Michael. "mRNA decay in Saccharomyces cerevisiae." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/187472.
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mRNA decay is an important step in the control of gene expression. To study mRNA degradation I have exploited the genetic, biochemical, and molecular tools available in Saccharomyces cerevisiae. These studies provided insight into the signals within individual transcripts which specify their half-lives, the various mechanisms by which mRNAs are degraded, and the trans-acting factors which both perform and control nucleolytic events. I identified a 65 nucleotide segment from the coding region of the unstable MATɑl mRNA which was capable of targeting both the MATɑl and stable PGKI transcripts for rapid degradation. This "instability element" was divided into two parts, one located in the first 33, and the second in the latter 32, nucleotides. The first part could be functionally replaced by different mRNA sequences containing rare codons, and while unable to promote mRNA decay by itself, enhanced degradation mediated by the second part. I determined that the MATɑl Instability Element (MIE) targets mRNAs for rapid degradation by increasing the rates of two nucleolytic steps in a pathway of mRNA decay common to several stable and unstable yeast transcripts. The initial step in this pathway is shortening of the poly(A) tail of an mRNA. Subsequently, mRNAs are decapped, after which the transcript body is degraded in a 5' to 3' exonucleolytic manner. The MIE promotes decay of the MATɑl mRNA through an increase in its decapping rate. In contrast, PGKI mRNA decay was stimulated through an increase in its rate of deadenylation. The observation that the poly(A) tail must be removed prior to mRNA decapping suggests that the poly(A) tail inhibits decapping. I determined that the major poly(A)binding protein (Pablp) is required for the inhibition of decapping mediated by the poly(A) tail. Pablp is also required for normal deadenylation rates. Pablp therefore affects mRNA decapping and deadenylation, the two rate determining steps in a common pathway of mRNA decay. Determining how Pablp, and additional trans-acting factors, exert influence over both decapping and deadenylation will provide a greater understanding of the basis of differential rates of mRNA degradation.
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Dunckley, Travis Lee. "mRNA decapping in Saccharomyces cerevisiae." Diss., The University of Arizona, 2000. http://hdl.handle.net/10150/289165.
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The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body. The products of the DCP1 and DCP2 genes are required for mRNA decapping. DCP1 encodes a conserved mRNA decapping enzyme. Dcp2p is a highly conserved protein that is required for the activation of Dcp1p. The Dcp2p contains a functional Muff motif that is required for its decapping function, suggesting that Dcp2p encodes a pyrophosphatase. These results suggest that Dcp2p hydrolyzes a specific pyrophosphate bond that either directly activates Dcp1p or removes a specific inhibitor of Dcp1p. In addition to Dcp2p, several additional proteins were identified that influence mRNA decapping. Edc1p and Edc2p are related proteins whose overexpression suppressed conditional mutations in dcp1 and dcp2, respectively. The Edc1 protein interacts in vivo with Dcp1p and Dcp2p. Based on similar genetic data for EDC1 and EDC2, the Edc2p also likely interacts directly with the mRNA decapping machinery. Edc1p and Edc2p may function to activate transitions in the decapping complex that lead to the Dcp2p-dependent activation of Dcp1p. The SBP1 protein was identified as an overexpression suppressor of a conditional dcp2 allele, termed dcp2-7. SBP1 overexpression also suppressed a conditional allele of the decapping enzyme (dcp1-2). In addition, the sbp1Delta was found to partially suppress the decapping defect of the dcp2-7 allele. This suggests that SBP1, which is a highly conserved RNA binding protein related to nucleolin, may influence the assembly or organization of the mRNP. Lastly, loss of function mutations in the previously uncharacterized IDC1 gene were shown to stimulate decapping in the presence of the dcp2-7 mutation. This suggests that the wild-type Idc1p inhibits mRNA decapping. Interestingly, the idc1 mutations described here represent the only known loss of function mRNA decapping suppressors that are not known to influence the rate of translation initiation, suggesting a more direct role for Idc1p in the inhibition of Dcp2p function. Combined, these results indicate that mRNA decapping is a highly controlled process involving the intricate and coordinated function of multiple proteins, in addition to the Dcp1p decapping enzyme.
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Ulbricht, Randi J. "Puf1p-mediated mRNA decay and combinatorial control of mRNA stability by the yeast Puf proteins." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2008. http://etd.umsl.edu/r2761.
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Bäjen, Carlo [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition / Carlo Bäjen. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1110749554/34.
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Stephen, Susan. "mRNA, microtubules and motor proteins : investigations into mRNA translocation along nutritive tubes of an hemipteran insect." Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312416.
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Sullivan, Kelly Daniel Marzluff William F. "Cotranscriptional processing of histone pre-mRNA." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,2870.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Jun. 4, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine.
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Poon, Leo L. M. "The polyadenylation of influenza virus mRNA." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312548.
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Siskoglou, Anastasios. "mRNA splicing in early mammalian embryos." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445092/.
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The overall aim of the experiments presented in this thesis is to investigate the onset and regulation of mRNA splicing in early mammalian development. To investigate splicing efficiency in single mouse embryos, we have constructed splicing probes that exploit the properties of fluorescent proteins. A DsRed Express-intron-EGFP construct would report splicing by the expression of the fusion DsRed-EGFP protein, when injected in mouse embryos. In the absence of splicing the vector yields only DsRed. The ability of the probe to monitor splicing was confirmed by compromising the Ul small nuclear ribonucleoprotein or by mutation of the intron's 5' splice site. Furthermore, I show that inhibiting Ul-mediated splicing at the onset of development inhibits development beyond the 2-cell stage. Following the characterization of the splicing probe, the development of nuclear speckles was investigated during early mouse development by determining the nuclear localisation of the splicing factor SC35. These experiments showed a developmental change in nuclear organisation during oocyte maturation and through embryo development. We could also show that splicing takes place as early as the 1 -cell stage embryos in the absence of nuclear speckles. Finally, the role of an SR protein, SRp38, was also investigated. SRp38 has been reported to be a potent splicing inhibitor when dephosphorylated in mitotic cells or under heat stress. SRp38 was present during all stages of the early mouse development. Oocytes and embryos were found to be sensitive to the levels of SRp38. Expression of SRp38-EGFP or depletion of SRp38 using morpholinos caused an arrest of oocytes at the GV stage and embryos at the 2-cell stage. The splicing reporter was used to reveal that these effects were independent of splicing suggesting a novel role for SRp38 in cell biology. These studies provide a novel approach to monitoring splicing activity in single living cells and that splicing activity is necessary for the onset of early mammalian development.
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Kajala, Kaisa. "An mRNA blueprint for C₄ photosynthesis." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608836.
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Alqara, Yazan Ali. "Protein Interactions in mRNA Methylation Complexes." ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/honors_theses/28.
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Experiments were performed to test sequence and structural specific interactions of proteins with a conserved RNA modification enzyme, which is known as Ime4 in yeast and Mettl3 in mammals. Ime4 methylates N6-adenosine bases on mRNA molecules. The goal of this project is to gain direct insights into how novel proteins interact with Ime4 to form the methyltranferase (MTase) complex and to identify proteins that are essential for Ime4 activity. It has been recognized that there are two proteins that interact within the Ime4 complex, which are known as Mum2 (a cytoplasmic protein essential for meiotic DNA replication within yeast) and Slz1 (a transcription factor). We hypothesize that the N-terminal domain of Ime4 is the location of binding of the aforementioned proteins in this complex. Similarly, we tested whether the human ortholog of Ime4 (Mettl3) forms an analogous complex that includes an ortholog of Mum2, known as WTAP, and its binding partner WT1. The major approaches include in vivo genetic assays in yeast to test protein-protein interactions and the use of recombinant DNA technology to construct fusion genes/deletions. The results demonstrate that Mum2 interacts with a specific, non-conserved region in the Ime4 N-terminal domain. Furthermore, we discovered a new binding partner, Ygl036w, which also interacts with Ime4. Currently, several experiments are being carried out with the Mettl3 complex and its hypothesized protein binding partners to assess the interactions of this complex.
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Gioldasi, Ioanna. "Translation initiation on feline calicivirus mRNA." Thesis, University of Surrey, 2003. http://epubs.surrey.ac.uk/844326/.
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Caliciviruses are single-stranded positive-sense RNA viruses. The viral genome is polyadenylated and 7-8 kb in length. Non-structural polypeptide coding sequences are located in the 5' region of the genome whereas structural polypeptide coding sequences are located at the 3' end. In addition the replication cycle involves the synthesis of at least one 3' co-terminal subgenomic RNA. The most important feature of calicivirus mRNA is the lack of a "cap" structure at the 5' end. The viral mRNA bears a 10-15 IcDa VPg protein linked to both genomic and sub-genomic RNAs. The lack of a 'cap' structure suggests that calicivirus mRNA is translated by a cap-independent mechanism. The aim of this project was to investigate the mechanism of translation initiation on feline calicivirus (FCV) mRNA. This was achieved by examining (i) the proposed role for the 15 IcDa VPg protein, as a 'cap analogue', and (ii) the interactions of cellular proteins to the 5' and 3' ends of the FCV genome. Firstly, expressed FCV VPg was purified and used to raise antisera in rabbits. The antisera were subsequently used to analyse proteins from FCV-infected CRFK cells. Secondly, pull-down and ELISA-based binding assays suggested interaction of recombinant FCV VPg with the canonical initiation factor eIF4E. Thirdly, studies of the interactions between cellular proteins and the 5' and 3' terminal ends of the FCV genome by UV cross-linking and oligo(dT) RNA-protein binding assays were conducted. Results suggested interaction of the 5' end of the FCV genome with proteins eIF4A, polypyrimidine tract-binding protein (PTB) and La, and eIF4A with the 3' end of the FCV genome. Based on the results of this work, this thesis proposes a model of the interactions between the FCV genome, VPg and cellular proteins in translation initiation.
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Januschke, Jens. "MRNA localization in the Drosophila oocyte." Paris 7, 2005. http://www.theses.fr/2005PA077101.
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Alves, Lysangela Ronalte. "Caracterização funcional de complexos mRNA-proteínas." reponame:Repositório Institucional da FIOCRUZ, 2010. https://www.arca.fiocruz.br/handle/icict/6242.
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Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil
Em tripanossomatídeos a regulação da expressão gênica ocorre principalmente em nível pós-transcricional. Acredita-se que a estabilidade do mRNA e o acesso aos polissomos sejam fortemente regulados, permitindo ao Trypanosoma cruzi uma rápida adaptação à diferentes condições ambientais as quais está exposto durante seu ciclo de vida. A regulação pós-transcricional requer uma associação entre mRNAs e determinadas proteínas formando complexos ribonucleoprotéicos (mRNPs). Nosso objetivo foi investigar a dinâmica de associação entre os mRNAs e proteínas, isolando e caracterizando proteínas e complexos protéicos ligados a mRNAs poliA+ das frações polissomal e pós-polissomal de epimastigotas em fase exponencial de crescimento e epimastigotas sujeitos a estresse nutricional. As amostras obtidas foram analisadas por espectrometria de massas (LC-MS/MS) e posteriormente comparadas. Nós identificamos 542 proteínas e dentre essas 24 estavam presentes em todas as frações analisadas, enquanto que outras eram exclusivas de uma fração específica: epimastigota frações polissomal (0,37%) e pós-polissomal (2,95%); estresse frações polissomal (13,8%) e pós-polissomal (40,78%). Proteínas sabidamente envolvidas com metabolismo de mRNA foram identificadas, sendo que esse resultado é importante para confirmar a confiabilidade da nossa técnica de isolamento das mRNPs. Essa abordagem em larga escala possibilitou uma análise mais completa da composição dos mRNPs e a dinâmica durante o estresse nutricional em T. cruzi. A partir dos dados obtidos nós selecionamos 6 proteínas para caracterização: fator de elongação 1-alfa (EF1-), proteína de ligação a RNA com domínio dedo de zinco (ZF-211.70), proteína de ligação a RNA com domínio cold shock (CD-33.60), prostaglandina F 2 alfa sintase (PF2 S), prostaglandina F sintase (PFS) e uma proteína hipotética com domínio de fator de replicação A (Hip -11.150). Os anticorpos contra as proteínas EF1-, ZF-211.70, PF2S e PFS apresentaram reatividade específica com uma proteína única de tamanho esperado, já as proteínas CD-33.60 e Hip-11.150 apresentaram um reatividade baixa ou inexistente em extratos de epimastigotas e por isso não foram utilizadas para os ensaios posteriores. Ensaios de imunofluorescência, sedimentação de polissomos em gradiente de sacarose e expressão ao longo do ciclo de vida nos permitiu uma caracterização inicial das proteínas selecionadas, etapas importantes para aprofundarmos o estudo na regulação de expressão gênica em T. cruzi.
Gene regulation is mainly posttranscriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Posttranscriptional regulation requires the association between mRNAs and some proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poli(T) beads to isolate and characterize proteins and protein complexes bound to poli -A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC -MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and postpolysomal (2.95%) fractions; stress polysomal (13.8%) and postpolysomal (40.78% ) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi. From the results obtained we selected six proteins for characterization: elongation factor 1-alpha (EF1-), zinc finger RNA binding protein (ZF-211.70), RNA binding protein with a cold-shock domain (CD-33.60), prostaglandin F 2 alfa synthase (PF2S), prostaglandin F synthase (PFS) and a hypothetical protein with a replication factor domain (Hip-11.150). The antibodies produced against EF1-, ZF-211.70, PF2S and PFS recognized a specific protein of expected size in epimastigote protein extracts; however, the CD-33.60 and Hip-11.150 antibodies did not recognized a specific protein and they were not used for further experiments. Immunofluorescence assays, polysome profile in sucrose density gradient and the expression pattern through the parasite life cyle with the selected proteins allowed us a preliminary characterization and further studies will help to elucidate the posttranscriptional regulation and the formation of RNA regulons in T. cruzi.
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Steimer, Sarah Reath. "Investigation of novel ribosomal recognition sites in Escherichia coli noncanonical mRNAs containing multiple start codons." Miami University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1461847705.
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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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Uhlitz, Florian Sören. "ERK signal duration decoding by mRNA dynamics." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20005.
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Der RAF-MEK-ERK-Signalweg steuert grundlegende, oftmals entgegengesetzte zelluläre Prozesse wie die Proliferation und Apoptose von Zellen. Die Dauer des vermittelten Signals wurde als entscheidener Faktor für die Steuerung dieser Prozesse identifiziert. Es ist jedoch nicht eindeutig geklärt, wie die verschiedenen früh und spät reagierenden Genexpressionsmodule kurze und lange Signale unterscheiden können und durch welche kinetischen Merkmale ihre Antwortzeit bestimmt wird. In der vorliegenden Arbeit wurden sowohl Proteinphosphorylierungsdaten als auch Genexpressionsdaten aus HEK293-Zellen gewonnen, die ein induzierbares Konstrukt des Proto-Onkogens RAF tragen. Hierbei wurde ein neues Genexpressionsmodul identifiziert, dass sich aus sofort induzierten aber spät antwortenden Genen zusammensetzt. Es unterscheidet sich in der Genexpressionsdynamik und Genfunktion von anderen Modulen, und wurde mit Hilfe mathematischer Modellierung experimenteller Daten identifiziert. Es wurde festgestellt, dass diese Gene aufgrund von langen Halbwertszeiten der vermitteltenden mRNA in der Lage sind spät auf das eingehende Signal zu reagieren und die Dauer des Signals in die Amplitude der Genantwort zu übersetzen. Trotz der langsamen Akkumulation und damit späten Antwortzeit, konnte aufgrund einer GC-reichen Promoterstruktur zunächst vermutet und mit Hilfe eines Markerverfahrens bestätigt werden, dass die Transkription dieser Gene instantan mit Beginn der ERK-Aktivierung startet. Eine vergleichende Analyse zeigte, dass das Prinzip der Signaldauer-Entschlüsselung in PC12-Zellen und MCF7-Zellen, zwei paradigmatischen Zellsystemen für die ERK-Signaldauer, konserviert ist. Insgesamt deuten die Ergebnisse der Untersuchung darauf hin, dass das neu identifizierte Genexpressionsmodul der Entschlüsselung der ERK-Signaldauer dient und das mRNA Halbwertszeiten sowohl hierfür, als auch für die zeitliche Abfolge der Genantwort eine entscheidende Rolle spielen.The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals and what features govern their timing. Both protein phosphorylation and gene expression time course data was obtained from HEK293 cells carrying an inducible construct of the proto-oncogene RAF. A new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function was identified by mathematical modelling. It was found that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis showed that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, the findings of this study indicate that ILGs decode ERK signal duration and that both decoding capacity and gene expression timing are governed by mRNA half-life.
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Morgado, Ana Sofia João. "Analysis of the intranuclear life of nonsense transcripts." Doctoral thesis, Faculdade de Ciências e Tecnologia, 2013. http://hdl.handle.net/10362/8798.
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Dissertação para obtenção do Grau de Doutor em
Biologia, Especialidade de Biologia MolecularNonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. Therefore, we hypothesized that human β-globin transcripts sensitive to NMD could have a singular subcellular localization and processing state in mammalian cells nuclei. To determine if PTCs could influence nuclear events, we have established mouse erythroleukemia (MEL) cell lines stably transfected with wild-type or PTC-containing human β-globin genes. Subsequently, we analyzed the accumulation of NMD-competent β-globin transcripts versus wild-type counterparts using two different approaches: visualization of transcripts localization by fluorescence in situ hybridization (FISH); and quantification of pre-mRNA steady-state levels by ribonuclease protection assays (RPA) and reverse transcription-coupled quantitative polymerase chain reaction (RT-qPCR). FISH analysis shows that MEL cells stably expressing PTC-containing β-globin transcripts present a marked tendency to display an abnormal speckled-like pattern of localization in the nucleus. However, in addition to the presence of the PTC, other effectors may act on the β-globin transcripts localization, as some wild-type β-globin MEL cells presented this abnormal FISH phenotype as well. On the other hand, our analyses by RPA and RT-qPCR clearly show that β- -globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA. Conversely, in non-erythroid HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Half-life analysis of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. In conclusion, our set of data highlights potential nuclear pathways that induce a selective downregulation of PTC-containing β-globin pre-mRNA in MEL cells, albeit not affecting their stability or splicing effectiveness. These specialized nuclear pathways, which may act in concert with the general NMD mechanism, might discriminate the NMD-sensitive transcripts as abnormal in a promoter- and/or cell line-specific manner, probably to obtain optimal NMD activity.
Fundação para a Ciência e Tecnologia - (SFRH/BD/31920/2006); financial support [Centro de Investigação em Genética Molecular Humana (CIGMH) and Center for Biodiversity, Functional and Integrative Genomics (BioFIG)]
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Hessle, Viktoria. "Characterization of RNA exosome in Insect Cells : Role in mRNA Surveillance." Doctoral thesis, Stockholms universitet, Institutionen för molekylärbiologi och funktionsgenomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-52127.
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The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.
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Nashchekin, Dmitri. "A Y-box protein/RNA helicase complex links mRNP assembly on the gene to mRNA translation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-811-8/.
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Trinh, Tat To. "REGULATION OF DROSOPHILA mRNA STABILITY BY DEADENYLATION ELEMENTS AND miRNAs." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1436441986.
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SALADINO, Patrizia. "ROLE OF RNA BINDING PROTEIN IN THE NERVE CELL DIFFERENTIATION." Doctoral thesis, Università degli Studi di Palermo, 2014. http://hdl.handle.net/10447/91214.
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Synthesis of H1˚ and H3.3 histone proteins, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs), we have been searching for RBPs involved in the post-transcriptional regulation of the H1˚ and H3.3 genes. Previously, we reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). We showed that LPI, as well as PEP-19, can bind H1˚ RNA. Since PEP19 and LPI contain a calmodulin binding domain, we also investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1˚ RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. Pep-19/calmodulin high affinity binding has been demonstrated by Biolayer interferometry (BLI). This finding suggests that calcium/calmodulin may have a role in controlling H1˚ mRNA metabolism in the developing brain. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step.
Furthermore, we found that both LPI and PEP-19 can compete for H1˚ RNA binding with PIPPin (also known as CSD-C2), another RBP previously discovered in our laboratory. PIPPin/CSD-C2 binds with high specificity to the mRNAs encoding H1° and H3.3 histone variants, undergoes thyroid hormone-dependent SUMOylation, and has been recently demonstrated to interact with other RBPs. PIPPin belongs to the CSD-containing class of RBPs, also called Y-box proteins, that play a key role in controlling the recruitment of mRNAs to the translational machinery, in response to environmental cues, both in development and in differentiated cells.
Another aspect of this study was to confirm histone mRNAs-PIPPin interactions and to describe binding properties through streptavidin-biotin conjugation method, by BLI.
We report the data obtained in the case of H3.3 and H1° mRNA-PIPPin interactions, and the specific affinity constant for these bindings. In order to identify RNA portions involved in binding, we used different RNA probes for H3.3 and H1°. In summary, we were able to confirm that PIPPin binds H3.3 and H1° mRNA with very high affinity.
Searching for other RBPs, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of CSD-C2. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70, whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70.
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Shan, Xiu. "Investigation of mRNA oxidation in Alzheimer's disease." Connect to this title online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1117205039.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xvii, 161 p.; also includes graphics (some col.) Includes bibliographical references (p. 144-161). Available online via OhioLINK's ETD Center
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García, Beyaert Marina 1983. "Molecular mechanisms of msl2 mRNA translational regulation." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/663873.
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Regulation of msl2 translation is a key step in the modulation of X-chromosome dosage compensation. MSL2 is the limiting subunit of the dosage compensation complex, an assembly that promotes hyper-transcription of the single male X-chromosome to equalize expression of X-linked genes between males and females. In females, dosage compensation must be repressed for viability, and this is achieved in large part by translational repression of msl2 mRNA. The female-specific protein Sex-lethal (SXL) binds to both untranslated regions (UTRs) of the msl2 transcript to inhibit two steps of translation initiation: SXL bound to the 3’ recruits the co-factor UNR and inhibits ribosomal recruitment; SXL bound to the 5’ UTR inhibits ribosomal scanning by promoting the recognition of an upstream AUG. In the lab, we recently found that eIF3d is a target of the 3’UTR repressor complex. In this thesis, we show that eIF3d can be recruited to the mRNA even in the absence of a cap structure by virtue of its binding to msl2 5’ UTR. Our results suggest that recruitment of this factor to the mRNA by multiple routes may sensitize translation of msl2 to inhibition of eIF3d. In addition, we have identified residues of SXL important for 5’ UTR, but not 3’ UTR, -mediated repression. Analysis of SXL variants with mutations in these residues has led us to identify two factors as likely mediators of inhibition via the 5’ UTR. Interestingly, the location of these factors in the ribosome allows us to propose an integrated model for translational repression that explains coordinated inhibition of ribosome recruitment and scanning by SXL.La regulación de la traducción de msl2 es un paso crucial en la modulación de la compensación de la dosis del cromosoma X. MSL2 es la subunidad limitante del complejo de compensación de dosis génica, un complejo que promueve la hiper-transcripción del único cromosoma X en machos para igualar la expresión de sus genes a la de hembras. La viabilidad de las hembras require que la compensación de dosis esté reprimida, y esto se consigue en gran parte por la represión de la traducción del ARNm que codifica para MSL2. La proteína específica de hembras Sex-lethal (SXL) se une a ambas regiones no traducidas (UTRs) del mensajero para inhibir dos etapas del inicio de la traducción: SXL unido al 3’ UTR recluta al co-factor UNR e inhibe el reclutamiento del ribosoma; SXL unido al 5’ UTR inhibe el escaneo del ribosoma al promover el reconocimiento de un uAUG. En el laboratorio, identificamos recientemente eIF3d como una diana del complejo represor unido al 3’ UTR. En esta tesis mostramos que eIF3d puede ser reclutado al ARNm incluso en ausencia del cap, gracias a su unión al 5’ UTR. Nuestros resultados sugieren que el reclutamiento de este factor al ARNm a través de varias rutas sensibiliza la traducción de msl2 a la inhibición de eIF3d. Además, hemos identificado resíduos de SXL importantes para la represión mediada por el 5’ pero no por el 3’ UTR. El análisis de variantes de SXL con mutaciones en estos resíduos nos ha permitido identificar dos factores como posibles mediadores de la inhibición por el 5’ UTR. Curiosamente, la localización de estos factores en el ribosoma nos permite proponer un modelo integrado para la inhibición coordinada del reclutamiento y escaneo del ribosoma por SXL.
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Segger-Junius, Mailin. "Charakterisierung der dendritischen Lokalisierung der Shank1-mRNA." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969985665.
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Gonchar, Oksana. "Interconnections between transcription and pre-mRNA splicing." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/38288.
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Eukaryotic gene expression involves many processes some of which are transcription and pre-mRNA splicing. It has been shown that the majority of splicing events are functionally or physically coupled to RNA polymerase II transcription machinery. This suggests that transcription and splicing processes might influence one another. For example, a number of studies have implicated snRNPs, in particular U1 snRNP, in transcription initiation and elongation. In this work, interconnections between transcription and splicing were tested using an in vitro RNA polymerase II transcription/splicing assays (Pol II-TSRs). The results obtained in this study showed that inhibition of U1 but not U2 or U6 snRNPs led to a major reduction in transcript levels using different DNA templates. However, interference with initiation was excluded because it was found that this effect was the result of reduced RNA stability. Moreover, similar results were observed both with transcription by T7 RNA polymerase and with purified transcripts added to the extract. These results allow to conclude that the U1 snRNP has a novel function in protecting RNA from degradation. Further investigations showed that the RNA is protected by the U1 snRNP against 5’ exonucleases and 3’ exonucleases and possibly endonucleases. It was found that the presence of 5’ splice site (5’SS) is necessary for RNA stability. These data suggest that the U1 snRNP through a direct interaction with the 5’SS of the pre-mRNA protects it from degradation and only U1 snRNP but not the active spliceosome is required for RNA stability. It was observed that under splicing conditions, the RNA level of a transcript lacking the intronic consensus 5’SS but having it at the 3’ end was significantly higher compared to that for a transcript lacking any consensus 5’SSs, suggesting that the U1 snRNP protects transcripts from degradation along their length. It is proposed that rapid binding of the U1 snRNPs to the nascent transcripts induces the assembly of RNA binding proteins that protect the RNA. Interestingly, any transcripts tested regardless of the presence of the consensus 5’SSs were stable in the reaction with ongoing transcription, suggesting that Pol II transcription has an additional effect on RNA stability.
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Jacobs, Jonathan L. "mRNA suicide destabilization by programmed ribosomal frameshifting /." College Park, Md. : University of Maryland, 2006. http://hdl.handle.net/1903/3412.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2006.Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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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.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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Causton, Helen Claire. "Mechanisms of mRNA decay in Escherichia coli." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334899.
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Edwards, John Michael. "Binding interactions of the mRNA regulator CELF1." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13453/.
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CELF1 is an RNA binding protein with regulatory roles in translation, alternative splicing and mRNA degradation. This protein is of particular interest as its upregulation is believed to be involved in the pathogenesis of type 1 myotonic dystrophy. CELF1 functions by binding to a specific sequence in the 3’ untranslated region of its target mRNAs. This sequence has been termed the “EDEN” motif, but the exact requirements for binding of CELF1 were not well defined. In this study we therefore aimed to determine the sequence requirements for an RNA substrate to form a high affinity interaction with CELF1, and characterise the structure of the resulting complex. The CELF1 protein is composed of three structured RNA recognition motifs separated by flexible linkers. Our strategy was to investigate the RNA binding properties of each domain in isolation, and then the requirements for tandem binding of the domains in order to build up the complete “EDEN” motif capable of forming a high affinity complex with the wild type protein. This has been accomplished using NMR spectroscopy to map the chemical shift perturbations in each domain on binding to a range of RNA substrates. ITC was also used to investigate the binding affinities of each domain, and the enhancement of affinity when domains bind in tandem. By these methods we have refined the sequence requirements for simultaneous binding of all domains of CELF1, and designed RNA substrates which will bind with higher affinity than any previously reported. We have also shown the potential involvement of RNA secondary structure in forming the CELF1 binding site, and identified two possible examples of this in natural mRNA targets. CELF1 binding triggers deadenylation of its target mRNAs and this is suspected to be via a mechanism involving recruitment of poly (A) ribonuclease. These two proteins have been shown to interact, but no structural information was available to show which domains were interacting, or whether CELF1 was capable of forming a ternary complex with both RNA and poly(A) ribonuclease. Since the ribonuclease exists as a 146 kDa dimer, the complex of it with CELF1 was an ambitious target for NMR. In this study we demonstrate that high resolution NMR data can be acquired on this key regulatory complex. Using this we go on to confirm the interaction between these two proteins, and that the domains involved in binding suggest a ternary complex is possible.
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Wootton, Lucie Jan. "Investigation Nonsense mediated mRNA decay in Arabidopsis." Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494252.
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Ahmed, Atique U. "Targeting cancer through tumor-selective mRNA stabilization." Thesis, Open University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441144.
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Parisi, D. "Analysis of mRNA 3' tagging pathway components." Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3021109/.
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Merkin, Jason Jay. "The evolution of mRNA splicing in mammals." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/89945.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Title as it appears in MIT commencement exercises program, June 6, 2014: The evolution of mRNA splicing in mammalian tissues Cataloged from PDF version of thesis.
Includes bibliographical references (pages 167-172).
In this thesis, I describe investigations into the evolution of splicing in mammals. I first investigate a small class of alternative splicing events, tandem splice sites, and show how they are used to introduce and remove coding sequence in a species-specific manner. I then describe the generation and analysis of a large RNA-seq dataset from 9 matched tissues in 5 species, with the aim to investigate the evolution of splicing in mammals. I first investigate the evolution of exons that predate the most ancient divergence of species studied, finding that their splicing is frequently poorly conserved. For a subset of these exons, I identify unique regulatory properties and provide evidence linking alternative splicing to phosphorylation potential of proteins. I then consider sources of novel exons, in these species. I use these and other published data to identify one way in which splicing of novel exons impacts the biology of the cell. I also present evidence implicating genomic indels in exon creation and splicing variation.
by Jason Jay Merkin.
Ph. D.
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Agarwala, Sudeep D. "Regulation of yeast development by mRNA methylation." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/72616.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
The internal methylation of mRNA post-transcriptionally is an essential component of the mRNA editing machinery in virtually every eukaryotic system. Despite this ubiquity, little is known about the relevance, consequences or machinery involved in this process. The recent demonstration of this modification in the brewers' yeast, Saccharomyces cerevisiae, has allowed the study of this modification using the vast array of genetic and biochemical tools available in the organism. In the second chapter of this thesis, we show that diploid cells of the yeast Saccharomyces cerevisiae experiencing nutrient limitation undergo a restriction of cellular potential and commitment in which the cells cease vegetative, mitotic growth and commit to meiosis. We show that the period prior to commitment can be divided further into two distinct phases: an early stage of initial starvation followed by a commitment to differentiation. Cells that are in the initial starvation phase revert to yeast-form mitotic growth if shifted to nutrientrich conditions. Cells that are in the commitment to differentiation phase are incapable of returning to yeast-form growth if shifted to nutrient-rich medium, but instead synchronously engage in pseudo-hyphal budding-a nutrient foraging response. Co-ordination of meiosis and PH development in the commitment to differentiation phase is regulated by mRNA methylation. We dissect this mRNA methylation upon nutrient starvation in the third chapter of this thesis. We identify Ime4, Mum2 and Slz1 as the components of a protein complex that catalyzes mRNA methylation in yeast. These components are necessary for m⁶A accumulation during nutrient starvation; mutation of any one of these components results in defects in meiotic and PH development. Furthermore, we find that ectopic expression of these components under nutrient-rich conditions is sufficient to catalyze this methylation of mRNA. Finally, we provide evidence that this modification is necessary for the activation of translation of genes under starvation conditions. These findings provide evidence for a method of finetuning translation under nutrient-stress conditions. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of posttranscriptional RNA methylation in regulating these decisions.
by Sudeep D. Agarwala.
Ph.D.
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Belaya, Katsiaryna. "Mechanisms of mRNA localisation during Drosophila oogenesis." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612350.
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Fennessy, Michael Joseph. "Investigation of mRNA sequences in ulcerative colitis." Thesis, University of Leicester, 1992. http://hdl.handle.net/2381/35294.
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After decades of research into the epidemiology, microbiology and immunology of ulcerative colitis (UC) the aetiology has still not been elucidated. An alternative approach is to compare gene expression in mucosa affected by UC with that of normal mucosa. Differentially expressed genes may elucidate the cells and processes involved in the disease. Such genes may be reliable markers of disease which facilitate diagnosis and provide an avenue for further research. In this thesis mRNA expression in normal and UC mucosa was compared by screening a cDNA library prepared from UC mucosa with cDNA probes with the aim of identifying abnormally expressed mRNAs. cDNA probes, including subtracted probes, were prepared from normal mucosa and mucosa affected by quiescent and active colitis. Differentially hybridising clones were screened with a spleen cDNA probe which failed to eliminate immunological sequences. With the exception of one sequence that could not be identified, all differentially hybridising clones were either immunoglobulin or mitochondrial genes. K and light-chain and IgG heavy-chain were all overexpressed in active disease. Using in situ hybridisation, the signal was localised in the lamina propria. IgA heavy-chain could be either overexpressed or underexpressed in UC, possibly reflecting the degree of ulceration of the epithelium, beneath which the IgA plasma cells were localised. Mitochondrial sequences were overexpressed in quiescent colitis but underexpressed in active disease. Using in situ hybridisation, signals were localised in the crypt bases probably representing proliferating cells. However, RNA dot blot hybridisation was unable to confirm that mitochondrial sequences and the unidentified sequence were consistently abnormally expressed in other cases of UC. Finally, enriched cDNA was generated by repeated subtraction with appendix mRNA/cDNA and amplified by the polymerase chain reaction. Analysis of the enriched cDNA demonstrated substantial loss of immunoglobulin sequences. In conclusion, the only informative sequences were immunoglobulin sequences which confirmed previous findings. Screening of immunoglobulin-subtracted cDNA may overcome this problem.
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Li, Weikuan Schneller Stewart W. "Seeking mRNA methylation inhibitors as antiviral agents." Auburn, Ala, 2008. http://hdl.handle.net/10415/1540.
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