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Gil, A. "Eukaryotic messenger RNA 3'-end formation." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376921.
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Grippo, Mariangela Carnivalli. "Uso da interferencia por RNA no virus da hepatite murina tipo 3 (MHV-3)." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316862.
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Orientadores: Iscia Teresinha Lopes-Cendes, Rovilson GilioliTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A interferência do RNA (RNAi) pode ser usada como uma ferramenta eficaz no silenciamento gênico específico mediado por moléculas de dupla fita de RNA (dsRNAs). Nesse contexto possui uma variedade de aplicações biológicas, incluindo o combate a patógenos infecciosos de importância biomédica. O objetivo do estudo foi determinar a eficiência e a especificidade da técnica de RNAi em eliminar o vírus da hepatite murina tipo 3 (MIN-3) in vitro. MHVs são vírus envelopados, cujo genoma é formado por uma cadeia de RNA fita simples (+) pertecentes a família Coronaviridae. Seu genoma codifica quatro proteínas estruturais: S (proteína da espícula); M (glicoproteína da transmembrana), N (proteína do nucleocapsídeo) e E (proteína associada à membrana) . Neste trabalho foi escolhido como alvo para o silenciamento gênico a proteína N, tendo sido produzidas moléculas de dsRNA complementares a sua seqüência genômica (GenBank AF 201929). Foram obtidas duas moléculas siRNAs transcritas por T7 RNA polimerase e uma terceira molécula interferente sintetizada comercialmente. Foi observado que os siRNAs produzidos pela transcrição in vitro, induziram uma resposta antiviral não específica. Além disso demonstrou-se que este efeito foi mediado através de substâncias secretadas no meio de cultura celular, provavelmente interferons (IFNs). Este efeito foi eficientemente eliminado após tratamento dos siRNAs com fosfatase alcalina. Observou-se também que a técnica de RNAi in vitro, tendo como alvo a proteína N de MHV-3, foi um tratamento eficaz e específico na infecção viral, confirmados através de estudos fenotípicos e moleculares. Desse modo, concluímos que experiências que utilizam RNAi contra alvos virais devem ser cuidadosamente monitoradas devido aos efeitos não específicos que podem ser induzidos por moléculas de dsRNA
Abstract: RNA Interference (RNAi) can be used as a powerful tool for post transcriptional gene-silencing mediated by double stranded RNA (dsRNAs) molecules. RNAi has a variety of biological applications including the combat against pathogens of biomedical importance. The objective of our study was to determine the efficiency and specificity of this new technique in eliminating mouse hepatitis virus type 3 (MIN-3) in vitro. MIN-3 is a subtype of enveloped viroses with a large plus-stranded RNA genome belonging to the Coronavirus family. Its genome codifies four structural proteins: S (spike protein); M (membrane protein); E (transmembrane glycoprotein); N (nucleocapsid protein). In the present study we target protein N by designing and producing dsRNA molecules complementary to its genomic sequence (GenBank AF 201929). We obtained three small interfering RNAs (siRNA) by in house T7 polymerase in vitro transcription and a fourth siRNA molecule that was commercially synthetized. We identified that siRNAs produced by in vitro transcription triggered a potent and sequence-unspecificied antiviral response. In addition, we demonstrated that this antiviral effect was mediated through molecules that were secreted in medium culture, probably interferons (IFNs). This unspecific effect was efficient1y suppressed when siRNAs were treated with aIkaline phosphatase prior to in vitro experiments. We also observed that RNAi targeting the N protein ofMIN-3 was a potent and specific treatment against in vitro infection, showing significant phenotypic protection and molecular evidence of specific gene-silencing. We concluded that experiments using RNAi against viral targets, although efficient, must be carefully controlled and monitored against possible sequence-unspecific effects triggered by dsRNA molecules
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Harendza, Christopher J. "3' processing of mouse thymidylate synthase messenger RNA /." The Ohio State University, 1989. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487673114115508.
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Proutski, Vitali. "RNA secondary structure of the 3'-UTR of flaviviruses." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299156.
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Pan, Shuying. "Functional characterization of arabidopsis DXO, a5'-3' RNA exonuclease." HKBU Institutional Repository, 2019. https://repository.hkbu.edu.hk/etd_oa/680.
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RNA decay plays an essential role in the regulation of gene expression during plant development and response to environmental stimuli. The protein DXO is a 5' to 3' exonuclease that functions in RNA degradation and RNA quality control that has been studied in animals. It has not yet been identified in plants. The gene locus At4g17620 in Arabidopsis thaliana encodes a protein homolog of the mammalian DXO, termed AtDXO. Recombinantly expressed AtDXO possesses a 5'-3' RNA exonuclease activity in vitro. Loss-of-function of AtDXO in Arabidopsis generates multiple growth defects, including curled and yellowish leaves, growth retardation and limited fertility, whereas overexpression show no obvious growth phenotype. The development defect of atdxo might be attributed to aberrant RNAs, which are not degraded when AtDXO is dysfunctioning. From the RNA-Seq analysis, the transcriptome pattern of atdxo mutants shows significant disparity from wild-type. Among the differences, the defense response genes are elevated in atdxo while photosynthesis-related and plastid genesis-related genes are downregulated. The constitutive expression of defense response genes causes the autoimmune phenotypes of atdxo. This could be modulated by temperature and is partially dependent on the master immunity regulators EDS1 or NPR1. Reactive oxygen species (ROS) accumulation was also detected in the atdxo mutant, and atdxo showed insensitivity to oxidative stress imposed by paraquat. Moreover, the atdxo mutant is hypersensitive to salt stress but not sensitive to general osmotic stress. In Arabidopsis, the 5'-3' RNA decay pathway could act as a repressor of endogenous post-transcriptional gene silencing (PTGS), which is regulated by small RNAs (sRNA). The mutation of AtDXO caused productions of 24- and 25-nucleotide endogenous sRNAs. The growth defect phenotype of atdxo could not be repressed by dysfunction of the RDR6 (RNA-DEPENDENT RNA POLYMERASE 6)-dependent sRNA biogenesis pathway. These findings demonstrate that AtDXO functions as a 5'-3' exoribonuclease both in vitro and in vivo to regulate plant development and to mediate the response to environmental stresses.
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Egli, Christoph Mathias. "3' processing of messenger RNA in the yeast Saccharomyces cerevisiae /." [S.l.] : [s.n.], 1995. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=11109.
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Silke, Jordan. "Characterization of 16S rRNA 3’ Termini Using RNA-Seq Data." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39044.
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Optimizing the production of useful macromolecules from transgenic microorganisms is crucial to biopharmaceutical companies. Improving bacterial growth and replication depends largely on the efficiency of translation, which is rate-limited by initiation. Among the most important interactions between the mRNA translation initiation region (TIR) and the translation machinery is the association between the Shine-Dalgarno (SD) sequence in the TIR and the complementary anti-SD (aSD) sequence which is located within a short unstructured segment that includes the 3’ terminus (3’ TAIL) of the mature 16S rRNA. However, the mature 3’ TAIL has been poorly characterized in the majority of bacteria, rendering optimal SD/aSD pairing unclear in these species.
In light of this, we established a novel strategy to characterize the mature 3’ TAILs of bacterial species that leverages the availability of publically stored RNA sequencing (RNA-Seq) data. In chapter 2, we devised a RNA-Seq-based approach to successfully recover the experimentally verified 3’ TAIL in E. coli (5’-CCUCCUUA-3’) and resolve inconsistencies surrounding the identity of the 3’ TAIL in Bacillus subtilis. In chapter 3 we improve the method introduced in chapter 2 to clearly and more reliably define the 3’ TAIL termini for 13 bacterial species with available protein abundance data.
Our results reveal considerable heterogeneity in the termini of 3’ TAILs among closely related species and that sites downstream of the canonical CCUCC aSD motif are more important to initiation than previously believed. My research contributes to advance our understanding in microbial translation efficiency in two significant ways: 1) providing an RNA-Seq-based approach to characterize rRNA transcripts, and 2) elucidating optimal recognition between protein-coding genes and the rRNA translation machinery.
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Dry, Inga Ruth. "Functional analysis of viral RNA and protein-RNA interactions involved in the replication of poliovirus type 3." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409998.
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Orlandini, von Niessen Alexandra [Verfasser]. "Optimization of RNA cancer vaccines using 3' UTR sequences selected for stabilization of RNA / Alexandra Orlandini von Niessen." Mainz : Universitätsbibliothek Mainz, 2016. http://d-nb.info/1120740800/34.
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Xiong, Chen. "Enzymatic modification of DNA and RNA 3'-termini for click ligation." Thesis, University of Southampton, 2014. https://eprints.soton.ac.uk/367127/.
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Chen, Kok Hao. "Spatially Resolved, Highly Multiplexed RNA Profiling in Single Cells." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467198.
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The transcriptome of a cell contains a wealth of information about the cell’s current state and its recent history. System-wide analyses of the copy number and localization of RNAs in single cells promise to transform our understanding of many areas of biology, such as the origin and consequences of cellular heterogeneity, functions and mechanisms underlying sub-cellular localization of transcripts, and organization of cell types in tissues. Fluorescent in situ hybridization (FISH) based approaches for measuring single-cell gene expression offer high spatial resolution and single molecule sensitivity, but lack throughput in comparison with sequencing based methods.
In the first part of this thesis, we present multiplexed, error robust FISH (MERFISH) for transcriptome imaging in single cells. We labeled each cellular RNA with multiple complementary oligonucleotide probes, which contain additional readout sequences. Each RNA species is encoded with a unique combination of readout sequences. We then identified these RNAs by using successive rounds of hybridization and imaging, with a different fluorescently labeled readout probe each round. This combinatorial labeling scheme allows the number of detectable RNA species to grow exponentially with the number of hybridization rounds, but the effect of readout errors also increases. By utilizing error-robust encoding schemes similar to those common in digital electronics, we are able to match the measured sequence of on/off fluorescence signals from individual RNAs to their assigned barcodes with high efficiency and minimal error.
In the second part of this thesis, we optimize the performance of localization based super-resolution microscopy by conducting a systematic characterization of the photo-switching properties of 26 organic dyes. These properties, which include the photons per switching event, on-off duty cycle, photostability, and number of switching cycles, largely dictate the quality of super-resolution images. Our study identified the top performing dyes in each fluorescent channel, allowing us to perform super-resolution imaging in four colors.
We envision that by combining multi-color super resolution microscopy with our multiplexed RNA imaging approach, we can visualize all the RNA content, the transcriptome, of a cell in situ. The ability to perform spatially-resolved transcriptomic analysis of cells and tissues will dramatically improve our abilities to understand biology and disease.Chemistry and Chemical Biology
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Maniatis, Silas dana. "Classical Conditioning Alters Short Noncoding RNA Expression in Drosophila." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467392.
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MicroRNAs (miRNAs) and other classes of short non-coding RNAs regulate essential processes in the development and function of the nervous system. Regulation of miRNAs by neural activity has also been reported. Recently, instances of piwi interacting RNA (piRNA) and endogenous short interfering RNA (esiRNA) mediated modulation of neural physiology have been reported. To better understand the role of miRNAs and other classes of short non-coding RNAs in long term memory (LTM) formation, we have conducted high throughput sequencing on 15-35nt RNAs isolated from heads of Drosophila that have been subjected to aversive olfactory conditioning. We developed genome wide profiles of miRNA, piRNA, and esiRNA, and tested for differential expression following conditioning. We find that 5 miRNAs exhibit significant regulation in the conditioned group. We identify several esiRNA generating loci within genes required for olfactory LTM formation. Our data reveal that an intron of the multiple wing hairs (mwh) gene forms secondary structures and generates esiRNAs following conditioning from regions that correspond to lysozyme family genes located within the mwh intron. We find that piRNAs are produced in fly heads, and that a small set of piRNA generating loci mapping to LTR retrotransposons are significantly down regulated following conditioning. In addition to the well characterized classes of short non coding RNAs, we describe a set of transcripts that produce large numbers of reads with a broad size distribution from the sense strand. We find that a subset of these are regulated following treatment and contain consensus elements that may be involved in their regulation. We investigate expression of one such gene with dramatically up-regulated reads following treatment, the Drosophila beta-site APP-cleaving enzyme (dBACE), and find that increased reads reflect increased mRNA levels. Further, we find that the target of dBACE protein, drosophila β amyloid protein precursor-like (APPL), is subjected to increased cleavage following conditioning, and that dBACE is required for LTM formation, but not for learning or STM.Biology, Molecular and Cellular
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Steil, Benjamin Peter. "Protein primers and a telomerase-like mechanism of poliovirus RNA replication maintain the 3' end of the RNA genome /." Connect to abstract via ProQuest. Full text is not available online, 2008.
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Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 198-225). Online version available via ProQuest Digital Dissertations.
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Lui, Wan Thomas, and 雷雲. "An analysis of the functional significance of the 3'-untranslated region of CHOP/Gadd153 messenger RNA." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hub.hku.hk/bib/B50899855.
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CHOP, or Gadd153, is a 29 kDa protein that plays a pivotal role in the mediation of cellular stress-induced cell death. The expression of the gene encoding CHOP/Gadd153 is regulated at both the transcriptional and post-transcriptional levels. Compared to transcription, the regulation of Chop gene expression at the post-transcriptional level is much less understood. In this study, the role played by the 3’-untranslated region of CHOP mRNA (3UTRChop) in mediating mRNA stability was examined. A reporter plasmid was constructed so that the mRNA expressed has 3UTRChop as its 3’-untranslated region. Partial 5’ deletions, or deletion of short internal (~30 nucleotides) sequences, or the deletion of a putative AU-rich element (ARE) within 3UTRChop, all resulted in the elevation of the steady state levels of mRNA and the encoded reporter protein, EGFP. Deletion of the ARE or sequences remote from ARE resulted in the reduced rate of mRNA degradation. Such data suggested that 3UTRChop is closely related to mRNA stability.
The effect of cellular stress on the functioning of 3UTRChop was studied by examining the change in mRNA level in cells treated with arsenic trioxide (ATO). The presence of arsenic stress stimulated a marked increase in the steady state level of not only the reporter mRNA, but also control mRNAs that did not have 3UTRChop as the 3’-untranslated region. A non-specific effect of arsenic stress on mRNA levels was therefore suggested. Consistent with the increase in the level of reporter mRNA, the expression of EGFP protein was also increased. Arsenic stress and partial deletion of 3UTRChop produce additive increase in mRNA level and EGFP protein level implying that the mRNA destabilizing function of 3UTRChop is unlikely to be stress-regulated.
The contribution of 3UTRChop in mRNA translation was then examined using reporter constructs that expressed EGFP mRNA having its original 5’ and 3’-untranslated regions replaced with 5UTRChop and 3UTRChop respectively. In the presence of wild-type 3UTRChop, the abolition of the translation repressor functions of 5UTRChop produced only mild increase in EGFP expression. However, the additional partial deletion of 3UTRChop resulted in massive increase in EGFP expression. In the presence of wild-type 5UTRChop, the partial deletion of 3UTRChop resulted in only a small increase in EGFP expression. Such data demonstrated a complementary relationship between 5UTRChop and 3UTRChop in the regulation of Chop expression in unstressed cells. EGFP mRNA having 5UTRChop and 3UTRChop as the 5’ and 3’-untranslated region respectively expressed significant EGFP protein only in the presence of ATO. The expression of EGFP was not significantly affected with swopping of 3UTRChop with another 3UTR. 3UTRChop is therefore not essential for the mediation of ATO-stimulated expression of EGFP.
The present study demonstrated that full length 3UTRChop may have constitutive mRNA destabilizing effect that is not alleviated by cellular stress. The evaluation of the relative contributions of 5UTRChop and 3UTRChop in mRNA translation suggested a model for Chop gene expression whereby the eventual protein level of Chop is determined by 5UTRChop-mediated translation as well as by 3UTRChop-mediated destabilization of mRNA template.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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Ghazal, Ghada. "Biochemical and genetic analysis of RNA processing and decay." Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/4287.
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Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.
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Deka, Pritilekha. "Structure and dynamics of proteins and RNA's involved in 3'-mRNA processsing /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8642.
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Holec, Sarah Gagliardi Dominique. "Polyadenylation and RNA degradation." Strasbourg : Université Louis Pasteur, 2008. http://eprints-scd-ulp.u-strasbg.fr:8080/987/01/HOLEC_Sarah_2008.pdf.
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Wang, Erickson Anna Fifi. "A novel sigma factor antagonist that binds to RNA polymerase." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467489.
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Sigma (σ) factors direct programs of gene expression by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP). Genes transcribed under the control of alternative σs allow cells to respond to stress and undergo developmental processes such as sporulation in the bacterium Bacillus subtilis where gene expression is controlled by a cascade of alternative σs. Much interest in RNAP function has therefore focused on σ regulation, and the mechanism of how one σ replaces another remains mysterious. While classic σ antagonists bind directly to σ to inhibit interaction with RNAP, we have identified a small protein, Fin, that binds to the RNAP β’ coiled-coil, a region that is critical for functional σ interaction with RNAP. During sporulation, fin is expressed under the control of σF, a σ factor active at early times during sporulation that is later replaced by σG. Cells deleted for fin are defective for spore formation and exhibit increased σF-directed gene transcription. We propose that Fin functions as part of a negative feedback loop to inhibit σF by competing for the β’ coiled-coil, and that this antagonism may contribute to the transition from σF to σG during sporulation.Medical Sciences
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Davis, William G. "Protein binding sites and cis-acting sequences on the West Nile Virus 3' (+) SL RNA." unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-07262007-134423/.
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Thesis (Ph. D.)--Georgia State University, 2007.Title from file title page. Margo Brinton, committee chair; W. David Wilson, Teryl Frey, committee members. Electronic text (120 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Nov. 20, 2008. Includes bibliographical references.
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Heath, Emma. "Functional analysis of def-3, a novel dynamic nuclear RNA-binding protein." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408166.
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Gaynor, James William. "Incorporation of 3-s-phosphorothiolates into oligonucleotides : changing direction for RNA interference." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507190.
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The main focus of this work falls into two sections. Firstly the synthesis and hybridisation studies of RNA duplexes containing 3'-S-phosphorothiolate (3'-sp) linkages, which are prepared using traditional automated 3'-5' solid-phase phosphoramidite chemistry. Secondly, investigations into the preparation of the 3'-sp linkage using a currently untried 5'-3', or reverse, synthesis approach. Both approaches have potential use in RNA interference (RNAi). For efficient RNAi activity, there are three key points; the small-interfering RNAs (siRNAs) must maintain an A-type helix; one terminus of the duplex is required to be more thermodynamically stable; and the strand whose 5' -end is less stably paired with the opposite strand is chosen as the guide strand. It has also been shown that RNAi activity does not totally rely on the 2' -hydroxyl functionality and can tolerate 2' -deoxy modifications. In terms of the sugar conformations, the 3' -sp linkage populates the RNA-like north conformation, and hence adopts an A-type helical conformation, to a greater extent than natural RNA, even though it lacks the 2' -hydroxyl group. Incorporating of a 3' -sp modifications into the uridine tract of the RNA duplex, 5'- r(GCGU1oGCG):5'-r(CGCA1QCGC),resulted in the thermal duplex stability rising by 1- 1.2 °C per modification. Therefore, by incorporating the 3' -sp linkage into either the 5' -end of the passenger strand, or the 3' -end of the guide strand, it is anticipated that the 3' -sp linkage can act as a compatible modification in RNAi. Using traditional 3'-5' synthesis, up to five 3'-sp linkages have been incorporated into the thymidine tract of 5'-d(GCGT1QGCG),each with a coupling efficiency of -93 %. By using reverse synthesis, it was hoped that the coupling yields could be enhanced so oligomers consisting entirely of 3'-sp modifications could be prepared; these oligomers could potentially be used as siRNA alternatives. We successfully incorporated a single 3'-sp modification into a DNA oligomer, so a proof of principle has been established. The yield of the thiol-phosphoramidite coupling is obviously poor and would need significantly enhancing for this technique to become a viable alternative to the traditionaI3'-5' synthesis. Optimisation studies are currently ongoing. As a small extra investigation, the possibility of synthesising 3'-thiopurine nucleosides was investigated using an enzymatic approach via transglycosilyation reactions. Initial studies indicate that 3'-thiothymidine and its corresponding 3'-thio-deoxyribose-1aphosphate derivative are substrates for thymidine phosphorylase and purine nucleoside phosphorylase, respectively.
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Estibeiro, J. Peter. "Factors involved in 3' splice site selection in eukaryotic pre-messenger RNA." Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35213.
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The work presented in this thesis is an investigation of factors involved in 3' splice site selection. To try to determine the intrinsic strengths of 3' splice site sequences, a cis-competition assay system was used. This system was based on the large intervening sequence (IVS-2) of the rabbit beta-globin gene. Synthetic 3' splice site sequence oligonucleotides were inserted into the EcoRl restriction site, forty nine nucleotides downstream of the authentic rabbit beta-globin IVS-2 3' splice site. The oligonucleotides conformed to the established 3' splice site consensus sequence and allowed for variations within this sequence. The authentic site served as a constant reference site against which the strengths of the synthetic sites could be measured. When spliced in HeLa cells in vivo, all constructs tested were seen to choose the authentic 3' splice site over the synthetic 3' splice site under test. A series of mutageneses was carried out to try to decrease the intrinsic strength of the authentic site and/or improve the environment of the synthetic site such that the overall strengths of the two sites might be balanced. An AG→CG mutation at the authentic 3' splice site caused the synthetic 3' splice site to be activated as a cryptic site in vivo and in vitro. In this case lariat formation was mapped to an artificially created branch point within exon 3. Splicing component binding to both 3' splice sites was investigated by looking at protection of the RNA from oligonucleotide directed cleavage by RNase H. Initial protection of both 3' splice sites was independent of the final choice of site. However, branch point protection was dependent on the 3' splice site chosen. Components bound to the authentic 3' splice site could be immunoprecipitated whether that site was chosen or not. The synthetic 3' splice site was poorly precipitated even when it was chosen. This data tends to suggest that the synthetic 3' splice site directs inefficient complex assembly, and that at least partial complex assembly occurs at a 3' splice site which has been inactivated by an AG→CG mutation. Preliminary work was carried out to develop a method for the analysis of splicing component binding to either or both 3' splice sites of material within fully and partially assembled splicing complexes (spliceosomes) isolated by sucrose gradient sedimentation.
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Schumacher, Heiko Tobias [Verfasser]. "Involvements of the Plant 3'-5' Exonuclease ERL1 in Chloroplast Ribosomal RNA Biogenesis and RNA Silencing Pathways / Heiko Tobias Schumacher." Kassel : Universitätsbibliothek Kassel, 2009. http://d-nb.info/999715232/34.
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Mugnier, Marie-Ange. "RNA 3 du virus de la mosaïque de la luzerne (AIMV) obtention d'une copie cDNA complète et étude conformationnelle de la région 5' du RNA 3 de différentes souches." Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb37599900q.
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Mugnier, Marie-Ange. "Rna 3 du virus de la mosaique de la luzerne (almv) : obtention d'une copie cdna complete et etude conformationnelle de la region 5' du rna 3 de differentes souches." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13160.
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Synthese d'une copie complete d'adn complementaire qui est ensuite clone dans un vecteur d'expression pgemi. La comparaison de la sequence des cdna avec celle de l'arn 3 met en evidence une duplication dans la region 5' non codante, d'une sequence de 56 nucleotides qui constitue la difference majeure entre ces 2 sequences. La structure primaire de la region 5' non codante a ete examinee dans l'arn 3 de 3 souches du virus. Cette etude est completee par une analyse conformationnelle, en utilisant des sondes chimiques (dms) et enzymatique (v1 et s1)
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Emerman, Amy Beth. "Molecular Details and Functional Analysis of RNA Binding by ESCRT-II." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17465322.
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Many RNAs show distinct localization patterns in cells with enrichment at particular subcellular sites or organelles. RNA localization is a highly conserved process that both spatially and temporally controls gene expression. A common mechanism to selectively sort RNAs within the cell involves recognition of cis-acting sequences on the RNA by trans-acting RNA-binding proteins. Recently, the ESCRT-II complex was identified as a novel trans-acting factor required for the localization of bicoid mRNA in Drosophila oocytes. ESCRT-II was previously uncharacterized as an RNA-binding complex but has a well-established role in multivesicular body formation and receptor downregulation. Recent studies have revealed links between endosomes and RNA regulatory pathways, and the dual roles of ESCRT-II in both cellular processes suggest that it will be an important factor to better understand as an RNA-binding complex. However, bicoid is the only identified direct RNA target of ESCRT-II, and whether ESCRT-II’s role in RNA localization is conserved in other organisms is unclear.
Here we report that the role of ESCRT-II in RNA regulation is conserved in Xenopus eggs. We found that ESCRT-II interacts with hundreds of RNAs in Xenopus eggs, and we characterized the molecular details of this interaction. Using a UV-crosslinking approach, we show that ESCRT-II binds directly to RNA through the subunit Vps25. Furthermore, by performing CLIP-seq, we found that ESCRT-II recognizes a polypurine motif. Selective binding of the polypurine motif through Vps25 can be recapitulated in vitro by multiple binding assays using purified components. Furthermore, ESCRT-II interacts with a subset of RNAs that are enriched on the mitotic spindle, and we provide preliminary evidence that ESCRT-II may be involved in localizing RNAs to the mitotic spindle. Consistent with previous reports, we found that ESCRT-II localizes to the centrosome in Xenopus tissue culture cells and to exogenous centrosomes added to egg extract. Our results suggest that the role of ESCRT-II in RNA regulation is conserved and shed light on an unexpected link between the cellular systems that control endosomal sorting and RNA localization.Medical Sciences
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Murphy, Alexander James. "RNA and Protein Networks That Locally Control Brain Wiring During Development." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467385.
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The molecular machineries of growth cones control the formation of neural circuits in the developing brain. Although great progress has been made in elucidating axon guidance cues and their growth cone receptors, we still lack an understanding of the projection-specific RNA and protein networks in growth cones that likely control the wiring of specific circuits in vivo.
To understand how specific projection neurons make wiring decisions, I focus on callosal projection neurons (CPN), which connect the two cerebral hemispheres through the corpus callosum. I developed an approach to profile and quantify the full-depth transcriptomes and proteomes of CPN growth cones and their parent cell bodies isolated in vivo. Using this comparative approach, I uncover general patterns of RNA and protein subcellular localization, with several previously unrecognized features, that might control the wiring of specific brain circuits. First, while most transcripts are expressed at similar levels in cell bodies and growth cones, a select subset are more than 10-fold enriched in growth cones compared to cell bodies, indicating active localization of those transcripts to the growth cone. By then correlating transcriptomic and proteomic data, I characterize the spatial relationship between coding transcripts and their encoded proteins. Intriguingly, many of the growth cone-enriched transcripts are noncoding RNA with unknown function. Further, growth cones appear to have distinct ribosomes. These ribosomes lack several large subunit proteins, raising the intriguing possibility of growth cone-specific translational mechanisms for selective mRNA expression.
This approach is readily adaptable to other projection types in the brain, enabling high-throughput, quantitative investigation of RNA and protein controls over circuit development and, potentially, the regeneration of damaged circuitry. In addition, the approach is scalable to include epigenetic profiling, enabling full investigation of DNA, RNA, and protein networks that collectively coordinate brain wiring during development. The insights derived from this approach exemplify its capacity to quantify and characterize the molecular and translational mechanisms that control specific brain wiring at the subcellular level in vivo.Medical Sciences
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張韻怡 and Wan-yee Ana Cheong. "MicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193411.
Full textAbstract:
MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins.
Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates.
Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3.
Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts.
Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation.published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy
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Elbahesh, Husni. "Functional Analysis of the Murine Oligoadenylate Synthetase 1b (Oas1b)." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/biology_theses/3.
Full textAbstract:
The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.
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Bamford, Anona Isabelle. "Interactions between cytotoxic effector cells and bovine parainfluenza type 3 virus." Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241326.
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Chambers, A. "RNA 3' cleavage and polyadenylation in oocytes, eggs and embryos of Xenopus laevis." Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380275.
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Albino, Christine D. "RNA-protein interactions within the 3'untranslated region (3'UTR) of the human multidrug resistance type 1 (MDR1) mRNA." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0020/MQ54160.pdf.
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Pankraz, Alexander. "Analyse der 3' nicht translatierten Region von BVDV CP7." Giessen VVB Laufersweiler, 2007. http://d-nb.info/989047369/34.
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Humphrey, Timothy C. "3' end formation of yeast and mammalian transcripts produced by RNA polymerase II." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293452.
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Williams, Heike. "Untersuchungen zur Genexpressionshemmung durch Doppelstrang-RNA-Interferenz in Larven 3 von Lucilia cuprina." Giessen : VVB Laufersweiler, 2008. http://d-nb.info/990218074/34.
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Broadbent, Kate Mariel. "The regulatory capacity of long non-coding RNA in Plasmodium falciparum malaria." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13065005.
Full textAbstract:
The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale.
First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA.
Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures.
Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.
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Durbin, Ann M. "Nucleotide Modifications of RNA Suppress RIG-I Antiviral Signaling by Unique Mechanisms." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493466.
Full textAbstract:
In order to counter pathogen infection while preventing autoimmune responses, the human innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling. Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade or suppress immune signaling; however, the precise mechanisms are not understood. Defining these mechanisms is relevant toward understanding the biology of immunity as well as the applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I (retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of RNA, but also the presence/absence of modified nucleotides. In the present study, we use a model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different nucleotide modifications, both natural and synthetic, share a common mechanism of innate immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified nucleotides may have evolved to effect a common result of interrupting innate immune signaling responses to “self” RNA. Our findings hold implications for understanding the co-evolution of the innate immune response and RNA modification pathways across domains of life, as well as for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides that may have utility in the design of therapeutic RNAs.Medical Sciences
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Dalgleish, Gillian Denise. "Localisation signals within the c-myc and c-fos 3'untranslated regions." Thesis, University of Aberdeen, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.481826.
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Schreieck, Amelie. "Role of the RNA polymerase II C-terminal domain in transcription termination and function of Spt5 in 3' RNA-processing factor recruitment." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-164544.
Full textAbstract:
Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II). This process is tightly regulated and coupled to RNA processing. Many transcription and RNA processing factors are recruited to Pol II via its conserved C-terminal domain (CTD) containing 27 heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 in Saccharomyces cerevisiae. These repeats can be differentially phosphorylated during the transcription cycle serving as a code for interacting factors. During transcription initiation, Ser5 is phosphorylated at the 5’-end of genes and this phosphorylation is required for RNA capping enzyme binding. During transcription elongation, the Pol II CTD becomes phosphorylated at Tyr1 and Ser2 and binds the elongation factor Spt5. Spt5 also contains a repetitive C-terminal region (CTR) required for cotranscriptional recruitment of proteins. At the 3’-end of genes, Ser2-phosphorylated Pol II associates with the cleavage and polyadenylation factor (CPF) and is dephosphorylated at Tyr1 residues.
This work shows that CPF is a Pol II CTD phosphatase and that its subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, Glc7 activity is required for normal Tyr1 dephosphorylation at the polyadenylation (pA) site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing and transcription termination are coupled via CPF-dependent Pol II Tyr1 dephosphorylation. Additionally, 19 kinases were tested for activity on Tyr1 in yeast by selective inhibition or knock-out in vivo. However, none of the candidates was identified as the Tyr1 kinase. Possibly this enzyme is an atypical kinase not known to be involved in transcription so far.
Furthermore, this work reports a new role of the Spt5 CTR in recruitment of RNA 3’-end processing factors. The results show that the Spt5 CTR as well as RNA is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3’-end of yeast genes. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 base pairs downstream of the pA site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at Ser2 of the CTD. Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo.
In summary, we characterized two new aspects of transcription and RNA processing regulation by two different C-terminal repetitive protein domains. CTD Tyr1 phosphorylation, which is removed by Glc7, regulates termination factor recruitment by masking their binding site, the Spt5 CTR is involved in recruitment of CFI. Both results greatly contribute to a more detailed understanding of the mechanisms involved in transcription termination and RNA
3’-end processing.
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Pankraz, Alexander. "Analyse der 3' nicht translatierten Region von BVDV CP7 /." Gießen : VVB Laufersweiler, 2008. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016535375&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Jajoo, Rishi Har. "Experiments and Analysis of Mitochondrial DNA Segregation, Yeast Polarization and RNA Polymerase Dynamics." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463961.
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This thesis presents work done in three different topics in biology with a combination of experiments, mathematical modeling and quantitative data analysis.
All cellular materials are partitioned between daughters at cell division, but by different mechanisms and with different accuracy. Many macromolecules partition in proportion to cytoplasmic volume with significant errors at low numbers, while chromosomes accurately push half the copies to each cell regardless of volume. Little is known about organelles, but in Schizosaccharomyces pombe the mitochondria are pushed to the cell poles by the spindle, along with the chromosomes. Here we find that mitochondria spatially re-equilibrate just before division, and that the mitochondrial volume and DNA-containing nucleoids instead segregate in proportion to the cytoplasm inherited by each daughter. However, in contrast to other macromolecules, nucleoid partitioning errors are still strongly suppressed. This is ensured by control at two levels: mitochondrial volume is actively distributed throughout a cell in a process involving the microtubule associated protein Mmb1p, and nucleoids are spaced out in semi-regular arrays within mitochondria. The data also suggest that nucleoid replication control is passive and Poisson, and that low concentration noise is achieved by accurate segregation rather than corrective feedback control.
Patterning in organisms is both beautiful and functional, but we have limited understanding of the general principles that cause patterns to emerge from isotropic initial conditions. Alan Turing proposed a minimal set of conditions that would allow reaction-diffusion systems to spontaneously form stable patterns. Since then many biological systems have been hypothesized to be of the Turing type, but with limited evidence that they actually rely on this mechanism. One of the conditions of Turing's theory is that reactions must have non-linear sensitivities (cooperativity) in their positive feedback cycles. We use a minimal model of yeast bud site selection to show that the amount of cooperativity achievable is naturally limited by the kinetic parameters achievable by proteins and the type of reaction mechanisms commonly used to achieve cooperativity. Further, higher levels of cooperativity necessarily cause pattern formation to slow down, likely causing a fitness defect for the organism. Then, because cooperativity is also used in many other biology systems, we show examples in TF-promoter binding and cooperative ligand binding where the trade-off between cooperativity speed of response is also present. These considerations bring into question how biological systems respond to these trade-offs. We then explore a conceptual model for yeast budding where an additional actin-based positive feedback loop uses a time-delay, not cooperativity, to ensure that yeast picks a single bud site.
RNA polymerase II (RNAP) pauses and backtracks during transcription elongation to regulate gene expression, control transcriptional efficiency and ensure transcriptional accuracy. After a pause, RNAP often backtracks and, in the presence of TFIIS, cleaves off the 3’ end of nascent RNA, allowing productive transcription to restart. However, the precise determinants of RNAP pausing, backtracking, and restarting remain unclear. Native elongating transcript sequencing (NET-seq) visualizes RNA polymerase pausing and backtracking with nucleotide resolution. Applying NET-seq to wild-type S. cerevisiae gives locations where cleavage occurs after backtracking. In contrast, NET-seq data from a strain where TFIIS is deleted reveal the primary points of pausing. Using these data, we show that RNAP pausing is largely controlled by the relative strength of the RNA:DNA and DNA:DNA hybrids in and downstream of the transcription bubble. In addition, backtracking is likely determined by the stacking interactions between the 3’-end of nascent RNA and Tyr769 of the Rpb2 subunit of RNAP. Though other factors beyond structural energetics of the transcription elongation complex certainly play a role in RNAP backtracking, this work suggests that the ubiquity of RNAP backtracking is largely controlled by sequence elements.Systems Biology
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Silva, Amanda Gabrielle da. "Isolamento de aptâmeros ligantes à sequência 3'-UTR do RNA do vírus da dengue." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/5135.
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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Considering potential molecular methods for diagnostic and/or therapeutic purpose of infectious human diseases, such as dengue, highlights the systematic evolution of ligands by exponential enrichment, known as SELEX. In this method, ligands to a target are exponentially enriched generating aptamers of DNA or RNA with high specificity, being considered as artificial antibodies. In this work, we aimed to realize the isolation of aptamers binding to 3'-UTR (untranslated region) of the RNA from dengue virus (DENV), because present important conformational structures that act as functional elements into RNA necessary in the infectious process. Total RNA of C6/36 infected cells was extracted, submitted to reverse transcription reaction to obtain viral cDNA (serotypes 2 and 3) and amplified by symmetric and/or asymmetric PCR technique to produce the 3’-UTR from DENV as target, generating RNA-like by containing deoxyuridine triphosphate (dUTP) and biotin in the 5’ region of the target. The random library of RNA ligands was obtained by sonication of a pool from human genomic DNA used in three successive PCR, and in the last reaction was introduced the promoter of T7 RNA polymerase, presenting fragments ranging from 80 to 600-bp. Eight rounds of selection were performed between the target and the library by using paramagnetic particles coated with streptavidin. For each round, after the incubation, the non-ligands were removed by using magnetic platform, and the ligands were eluted with NaOH. The eluted ligands were precipitated, submitted to RT-PCR and transcription in vitro, completing one round of selection. For analysis of variability of ligands, the product obtained from the eighth round was cloned and fourteen clones were randomly selected for amplification. The results demonstrated that the aptamers presented sizes with estimated molecular weights varying from 80 to 100-bp. These data indicated the viability of aptamers isolation against conformational elements present in the 3'-UTR of RNA from dengue virus, which may contribute to future research focusing on prevention and/or control of the disease.
Dentre os potenciais métodos moleculares para o diagnóstico e/ou terapêutica de doenças que acometem a saúde humana, tais como a dengue, destaca-se a seleção de ligantes pela utilização da química combinatória, conhecida como SELEX. Nesse método, os ligantes a um alvo são enriquecidos exponencialmente tendo como produto final a obtenção de aptâmeros de DNA ou RNA com elevada especificidade, sendo considerados como anticorpos artificiais. No presente trabalho foi realizado o isolamento de aptâmeros de RNA ligantes à extremidade 3’-UTR (untranslated region) do RNA do vírus da dengue (DENV), por apresentar elementos de RNA conformacionais e funcionais importantes no processo infeccioso. A partir do RNA extraído de células C6/36 infectadas foi feita a transcrição reversa (RT) para produção de cDNA viral (sorotipos 2 e 3) e amplificação por PCR simétrica e/ou assimétrica para a produção do alvo 3’-UTR do DENV, na forma de RNA-like por conter desoxiuridina trifosfatada (dUTP) e biotina na extremidade 5’. A biblioteca randômica de ligantes de RNA foi obtida por sonicação de um pool de DNA genômico humano utilizado como alvo em três PCRs sucessivas sendo que na última reação foi introduzido o promotor da T7 RNA polimerase e cujos fragmentos variaram de 80 a 600-pb. Foram realizados oito rounds de seleção entre alvo e biblioteca utilizando partículas paramagnéticas revestidas com estreptavidina. A cada round, após o período de incubação, os oligonucleotideos não ligantes foram removidos com o auxílio de plataforma magnética, e os ligantes foram eluídos com NaOH. Os ligantes eluídos foram precipitados e submetidos à RT-PCR e transcrição in vitro, finalizando um round de seleção. Para verificar a variabilidade de ligantes, o produto do oitavo round foi clonado e 14 clones foram selecionados aleatoriamente para amplificação. Os resultados demonstraram que os aptâmeros isolados possuem tamanhos distintos, com pesos moleculares estimados variando de 80 a 100-pb. Os dados aqui obtidos indicaram a viabilidade do processo de isolamento de aptâmeros para elementos conformacionais presentes na extremidade 3’-UTR do RNA do vírus da dengue os quais poderão contribuir para pesquisas futuras com foco na prevenção e/ou controle da doença.
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Guidi, Mònica. "Micro RNA-Mediated regulation of the full-length and truncated isoforms of human neurotrophic tyrosine kinase receptor type 3 (NTRK 3)." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7114.
Full textAbstract:
Neurotrophins and their receptors are key molecules in the development of thenervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor
NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive
form (150 kDa) and a truncated non-catalytic form (50 kDa). The two
variants show different 3'UTR regions, indicating that they might be differentially
regulated at the post-transcriptional level. In this work we explore how
microRNAs take part in the regulation of full-length and truncated NTRK3,
demonstrating that the two isoforms are targeted by different sets of microRNAs.
We analyze the physiological consequences of the overexpression of some of the
regulating microRNAs in human neuroblastoma cells. Finally, we provide
preliminary evidence for a possible involvement of miR-124 - a microRNA with no
putative target site in either NTRK3 isoform - in the control of the alternative
spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.
Las neurotrofinas y sus receptores constituyen una familia de factores cruciales
para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función
principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual
se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo
tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas
no comparten la misma región 3'UTR, lo que sugiere la existencia de una
regulación postranscripcional diferente. En el presente trabajo se ha explorado
como los microRNAs intervienen en la regulación de NTRK3, demostrando que las
dos isoformas son reguladas por diferentes miRNAs. Se han analizado las
consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando
células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del
microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la
regulación de represor de splicing PTBP1.
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Luo, Weifei. "The coupling of transcription termination by RNA polymerase II to MRNA 3' end processing in Saccharomyces cerevisiae /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
Find full textAbstract:
Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Forbes, Kevin Patrick. "Characterization of plant polyadenylation transacting factors--factors that modify poly(A)polymerse activity." Lexington, Ky. : [University of Kentucky Libraries], 2005. http://lib.uky.edu/ETD/ukyplph2005d00278/etd.pdf.
Full textAbstract:
Thesis (Ph. D.)--University of Kentucky, 2005.Title from document title page (viewed on November 7, 2005). Document formatted into pages; contains vi, 135 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 113-133).
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Wells, Christopher D. "Formation of a Termination-Resistant RNA Polymerase Complex: Studies on the Phage 82 Q Protein." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845405.
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Due to their early discovery, relative simplicity, and well-defined function, the Q antiterminator proteins of bacteriophage lambda and related lambdoid phages serve as a model for understanding how the elongation and termination behavior of RNA polymerase (RNAP) can be modified. Q initially engages E. coli RNAP that has paused immediately downstream of the phage late gene promoter PR', making contacts with both the RNAP and a DNA element embedded within PR' (the Q Binding Element, or QBE). Following successful engagement, Q remains associated with RNAP as it escapes the pause, rendering the elongation complex (EC) resistant to termination signals that otherwise prevent transcription of the phage late genes. Although Q can modify RNAP on its own, it is assisted by the essential E. coli elongation factor NusA, which facilitates Q engagement and is thought to persist as a stable component of the Q-modified EC.
In this thesis, I describe a series of studies directed towards achieving a better understanding of the formation of the Q-modified EC. Focusing on the Q protein from phage 82 (82Q), I first describe an investigation of the effect of position on the ability of Q to engage the paused EC (Chapter 2). We developed an in vitro transcription assay that allowed us to compare Q engagement at the native promoter-proximal position and a novel promoter-distal position. We found that the relevance of EC positioning was unveiled by addition of the NusA protein, which stimulates Q-dependent terminator read-through when 82Q engages the promoter-proximal EC and inhibits it when 82Q engages the promoter-distal EC. Furthermore, by shortening the long nascent RNA of the promoter-distal EC, we demonstrate that nascent RNA length determines the effect of NusA on 82Q engagement. These results suggest that one function of the promoter-proximal pause is to trap the EC at a nascent RNA length that permits functional cooperation between NusA and Q.
In Chapter 3, I describe a genetic approach that allowed us to rapidly identify, counterscreen, and categorize 82Q mutants. We identified two mutant classes based on the results of our analyses, one that could no longer bind the QBE and a second that was unable to modify the RNAP. An attempt to predict the 82Q structure using homology modeling produced a high-confidence structural model of 82Q (residues 82-209) based on the crystal structure of a portion of the lambdaQ protein (residues 62-206), despite relatively low sequence identity. This structural model revealed a striking structural overlap between the positions of 82Q mutations and the positions of lambdaQ mutations with analogous phenotypes, strengthening our confidence in both our 82Q mutant classification and the 82Q homology model.Medical Sciences
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Zechner, Kerstin. "3' end processing and RNA polymerase II transcription termination in protein coding genes in the nematode C. elegans." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.564397.
Full textAbstract:
In all organisms studied so far, the recognition of a functional poly(A) site is essential for RNA polymerase II termination at the end of nearly all genes transcribed by this enzyme (Whitelaw and Proudfoot, 1986; Guo et al., 1995; Birse et al. 1997). A number of eukaryotes have some of their genes organised in polycistronic structures which resemble bacterial operons (Davis and Hodgson, 1997; Ganot et al., 2004; Spieth et al. 1993), and in C. elegans, approximately 20% of all genes are contained within these operon-like structures (Blumenthal et al., 2002). Here, functional poly(A) sites will be synthesised and recognised by RNA polymerase II at the end of each gene within the operon, however termination of the polymerase only occurs at the final gene of the polycistronic transcription unit In these studies, we analyse the halting of RN A polymerase II transcription at the end of monocistronic genes and furthermore observe how premature RNA polymerase II termination is prevented during polycistronic transcription in the nematode C. elegans. We predominantly make use of reverse transcriptase PCR-based techniques to examine these mechanisms. We show that a large increase in pre-mRNAs stretching into the 3' flank of genes can be detected in worms depleted of the riboexonuclease XRN-2, indicating that this enzyme may have a possible role in RNA pol II termination and 3' end formation in C. elegans. Furthermore, we provide evidence that the polymerase can read into telomeric structures in the nematode. Also, we demonstrate that an RNAi-mediated knockdown of the UI-70K subunit of the UI snRNP causes a drop in polycistronic transcripts, providing a link between cis- splicing and the prevention of premature RNA polymerase II termination at operon-internal poly(A) sites. Finally, we illustrate that operon-internal poly(A) sites are capable of directing efficient 3' end formation outside of a polycistronic background. Together, these findings provide valuable insights into the mechanisms involved in directing or preventing premature RNA polymerase II transcription termination at C. elegans poly(A) sites.
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Liu, Peng. "Regulation of cIAP1 mRNA Stability Through Its 3’ UTR by the RNA-Binding Protein HuR." Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26009.
Full textAbstract:
The RNA-binding protein HuR is involved in numerous aspects of the RNA life-cycle. It is known for its ability to stabilize AU-Rich Element (ARE)-containing transcripts in the cytoplasm. The transcript of cIAP1, an important protein involved both in the regulation of apoptosis and NF-κB signaling, contains four such AREs, raising the question of whether HuR can modulate the stability of cIAP1 mRNA. First, using C2C12 cells, we observed a positive correlation between cIAP1 mRNA levels and HuR cytoplasmic localization. We then show that knockdown of HuR in U2OS cells results in a decrease in steady-state cIAP1 mRNA levels through destabilization of the cIAP1 mRNA. Furthermore, we are able to show in vitro that HuR binds directly to the second of the four AREs in the 3’ UTR. The direct link between the binding of HuR to the second ARE and its effect on cIAP1 mRNA stability remains to be shown.
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McCulley, Anna. "Primer selection of E. coli tRNALys,3 by human immunodeficiency virus type-1." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/mcculley.pdf.
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Davis, William G. "Protein Binding Sites and Cis-acting Sequences on the West Nile Virus 3' (+) SL RNA." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/biology_diss/40.
Full textAbstract:
RNase footprinting and nitrocellulose filter-binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3’(+) stem loop (SL) RNA of West Nile virus (WNV) (2). Base substitutions in the major eEF1A binding site or adjacent areas of the 3’(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative affect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3’ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, mutations that increased the efficiency of eEF1A binding to the 3’ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3’ SL facilitates viral minus-strand initiation. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3’ end of the genome and the RC. eEF1A bound with similar efficiency to the 3’ terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue RC in infected cells. These results suggest that eEF1A plays a similar role in the RNA replication of all flaviviruses.