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Chen, Li. "Functional and evolutionary characterization of flowering-related long non-coding RNAs." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22833.
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Genomweite Bemühungen haben eine große Anzahl langer nichtkodierender RNAs (lncRNAs) identifiziert, obwohl ihre möglichen Funktionen weitgehend rätselhaft bleiben. Hier verwendeten wir ein System zur synchronisierten Blüteninduktion in Arabidopsis, um 4106 blütenbezogene lange intergene RNAs (lincRNAs) zu identifizieren. Blütenbezogene lincRNAs sind typischerweise mit funktionellen Enhancern assoziiert, die bidirektional transkribiert werden und mit verschiedenen funktionellen Genmodulen assoziiert sind, die mit der Entwicklung von Blütenorganen zusammenhängen, die durch Koexpressionsnetzwerkanalyse aufgedeckt wurden. Die Master-regulatorischen Transkriptionsfaktoren (TFs) APETALA1 (AP1) und SEPALLATA3 (SEP3) binden an lincRNA-assoziierte Enhancer. Die Bindung dieser TFs korreliert mit der Zunahme der lincRNA-Transkription und fördert möglicherweise die Zugänglichkeit von Chromatin an Enhancern, gefolgt von der Aktivierung einer Untergruppe von Zielgenen. Darüber hinaus ist die Evolutionsdynamik von lincRNAs in Pflanzen, einschließlich nicht blühender Pflanzen, noch nicht bekannt, und das Expressionsmuster in verschiedenen Pflanzenarten war ziemlich unbekannt. Hier identifizierten wir Tausende von lincRNAs in 26 Pflanzenarten, einschließlich nicht blühender Pflanzen. Ein direkter Vergleich von lincRNAs zeigt, dass die meisten lincRNAs speziesspezifisch sind und das Expressionsmuster von lincRNAs einen hohen Transkriptionsumsatz nahe legt. Darüber hinaus zeigen konservierte lincRNAs eine aktive Regulation durch Transkriptionsfaktoren wie AP1 und SEP3. Konservierte lincRNAs zeigen eine konservierte blütenbezogene Funktionalität sowohl in der Brassicaceae- als auch in der Grasfamilie. Die Evolutionslandschaft von lincRNAs in Pflanzen liefert wichtige Einblicke in die Erhaltung und Funktionalität von lincRNAs.Genome-wide efforts have identified a large number of long non-coding RNAs (lncRNAs), although their potential functions remain largely enigmatic. Here, we used a system for synchronized floral induction in Arabidopsis to identify 4106 flower-related long intergenic RNAs (lincRNAs). Flower-related lincRNAs are typically associated with functional enhancers which are bi-directionally transcribed and are associated with diverse functional gene modules related to floral organ development revealed by co-expression network analysis. The master regulatory transcription factors (TFs) APETALA1 (AP1) and SEPALLATA3 (SEP3) bind to lincRNA-associated enhancers. The binding of these TFs is correlated with the increase in lincRNA transcription and potentially promotes chromatin accessibility at enhancers, followed by activation of a subset of target genes. Furthermore, the evolutionary dynamics of lincRNAs in plants including non-flowering plants still remain to be elusive and the expression pattern in different plant species was quite unknown. Here, we identified thousands of lincRNAs in 26 plant species including non-flowering plants, and allow us to infer sequence conserved and synteny based homolog lincRNAs, and explore conserved characteristics of lincRNAs during plants evolution. Direct comparison of lincRNAs reveals most lincRNAs are species-specific and the expression pattern of lincRNAs suggests their high evolutionary gain and loss. Moreover, conserved lincRNAs show active regulation by transcriptional factors such as AP1 and SEP3. Conserved lincRNAs demonstrate conserved flower related functionality in both the Brassicaceae and grass family. The evolutionary landscape of lincRNAs in plants provide important insights into the conservation and functionality of lincRNAs.
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Zhuang, Jimmy Jiajia. "Phenotypes and genetic mechanisms of C. elegans enhanced RNAi." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10758.
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RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.
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Neugebauer, Karla M., Inna Grishina, Anita S. Bledau, and Imke Listerman. "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III." PLOS, 2007. https://tud.qucosa.de/id/qucosa%3A27951.
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Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.
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Neugebauer, Karla M., Inna Grishina, Anita S. Bledau, and Imke Listerman. "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184076.
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Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.
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Caglio, Giulia. "RNA Polymerase II identifies enhancers in different states of activation." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/19946.
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Enhancer regulieren die Transkription ihrer Zielgene und deren Expression. Sie bieten eine Bindestelle für verschiedenste Transkriptionsfaktoren (TF) und RNA Polymerase II (RNAPII) und unterstützen die Gentranskription durch das Zustandekommen von Chromatinkontakten. Zusätzlich transkribiert RNAPII in Enhancer-Regionen kurze, non-polyadenylierte Transkripte, die man Enhancer-RNA (eRNA) nennt. Der Mechanismus der RNAPII-Rekrutierung und –Regulation an Enhancern ist bisher wenig verstanden, insbesondere wie das Vorhandensein von RNAPII-Modifikationen den Chromatinstatus, -faltung sowie die Genaktivierung beeinflusst.
In dieser Arbeit wurden verschiedene Ansätze der Enhancer-Bestimmung miteinander verglichen. Während eine klare Bestimmung des besten Ansatzes sich als komplex erwies, konnte gezeigt werden, dass die Bindung von RNAPII an regulatorische Regionen in Zusammenhang mit TF eine universelle Konstante darstellte. Weiterhin wurden der Status der Enhancer-gekoppelten RNAPII-Aktivierung und deren Transkriptionsaktivität untersucht. Als Hauptergebnis ergab sich, dass der RNAPII-Status mit der Enhancer-Aktivität und daraus folgend mit veränderter Transkriptionsaktivität korreliert ist. Weiterhin konnte gezeigt werden, dass das Vorhandensein extragenischer RNAPII ein neues Werkzeug zur Identifikation von regulatorischen Regionen ist. Erfolgreich konnten regulatorische Regionen in embryonalen Stammzellen der Maus sowie während der neuronalen Differenzierung vorhergesagt und mittels Enhancer-Aktivität in-vivo bestätigt werden. Dabei zeigte sich, dass im Laufe der der neuronalen Differenzierung extragenische RNAPII-Bindung spezifische Aktivierungsmuster aufweist: ihr Transkriptionslevel wird durch Kinasen feinmaschig reguliert und es werden verschiedene Formen maturierter RNA erzeugt.
Zusammenfassend konnte RNAPII als Werkzeug zur Identifikation und Charakterisierung regulatorischer Regionen in verschiedenen Zelltypen ausgemacht werden. Selbst mit minimalen RNAPII-Datensätzen ist es möglich, gleichzeitig regulatorische Regionen zu identifizieren als auch ihren eigenen Aktivierungsstatus sowie den ihrer kodierender Genpromotoren zu bestimmen.Enhancers regulate transcription of target genes and gene expression. They act as recruitment sites for multiple transcription factors (TFs) and RNA polymerase II (RNAPII) and favour transcription of target genes through chromatin contacts. RNAPII at enhancer regions transcribes short and mostly non-polyadenylated transcripts, called enhancer RNAs (eRNAs). The mechanisms of RNAPII recruitment and regulation at enhancers remain ill understood, in particular how signalling through RNAPII modifications may influence chromatin states, looping and gene activation. In this study, I compare enhancer lists defined with different approaches and find that their relation is very complex. However, I find that RNAPII binding co-occurs with TF binding at regulatory regions, independently of the identification approach used. I characterize the state of RNAPII activation at enhancers and its transcriptional activity. I find that RNAPII state reflects enhancer activation state and correlates with different transcriptional outputs. In addition, I demonstrate that extragenic RNAPII is a novel tool to identify regulatory regions. I successfully identified putative regulatory regions in mESC and during neuronal differentiation, with enhancer activity in vivo. Extragenic RNAPII regions have specific activation patterns during neuronal differentiation, are finely regulated at the transcriptional level by kinases and transcribe differently mature RNAs. In conclusion, I establish RNAPII as a tool to identify and characterise regulatory regions in a cell type of interest. With minimal RNAPII datasets it is possible to simultaneously identify regulatory regions, infer their state of activation, and the state of activation of coding gene promoters.
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Ribeiro, Mariana Martins 1984. "G-quadruplex formation enhances splicing efficiency of PAX9 intron 1." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290066.
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Orientadores: Sérgio Roberto Peres Line, Marcelo Rocha MarquesTexto em português e inglês
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: G-Quadruplexes são estruturas secundárias presentes nas moléculas de DNA e RNA, os quais são formados pelo empilhamento de G-quartetos (interação de quatro guaninas (G-tratos) delimitadas por ligações de hidrogênio do tipo Hoogsteen. O intron 1 do gene PAX9 humano tem um G-quadruplex formado na região localizada perto do exon 1, que é conservada entre os mamíferos placentários. Análises de Dicroísmo Circular (CD), e CD melting mostraram que estas sequências são capazes de formar estruturas quadruplex altamente estáveis. Devido à proximidade da estrutura quadruplex ao limite éxon-íntron foi utilizado um ensaio validado de splicing duplo repórter e PCR em tempo real para analisar o seu papel na eficiência de splicing. O quadruplex humano mostrou ter um papel chave na eficiência de splicing do íntron 1 do gene PAX9, já que uma mutação que aboliu a formação do quadruplex diminuiu drasticamente a eficiência de splicing. O quadruplex de rato, menos estável, mostrou menor eficiência quando comparado com sequências humanas. Além disso, o tratamento com 360A, um forte ligante que estabiliza estruturas quadruplex, aumentou ainda mais a eficiência de splicing do íntron 1 do PAX9 humano. Em conjunto estes resultados fornecem evidências de que as estruturas de G-quadruplex estão envolvidas na eficiência de splicing do intron 1 do gene PAX9
Abstract: G-Quadruplex are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e. interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex- forming region located near exon 1, which is conserved among placental mammals. Using Circular Dichroism (CD) analysis, and CD melting we showed that this region is able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary we used a validated double reporter splicing assay and real time PCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically splicing efficiency. The less stable, rat quadruplex had a less efficient splicing when comparing to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1
Doutorado
Histologia e Embriologia
Doutora em Biologia Buco-Dental
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Hughes, Amanda Dawn. "Mechanism of enhancer-dependent transcription in Escherichia coli by σâ¿-RNA polymerase." Thesis, University of York, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399583.
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Scionti, Isabella. "Epigenetic Regulation of Skeletal Muscle Differentiation." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSEN084/document.
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LSD1 et PHF2 sont des déméthylases de lysines capables de déméthyler à la fois les protéines histones qui influencent l’expression génique et les protéines non histones en affectant leurs activités ou stabilités. Des approches fonctionnelles d’inactivation de Lsd1 ou Phf2 chez la souris ont démontré l’implication de ces enzymes dans l'engagement des cellules progénitrices au cours de la différenciation. La myogenèse est l'un des exemples les mieux caractérisés sur la façon dont les cellules progénitrices se multiplient et se différencient pour former un organe fonctionnel. Elle est initiée par une expression temporelle spécifique des gènes régulateurs cibles. Parmi ces facteurs, MYOD est un régulateur clé de l'engagement dans la différenciation des cellules progénitrices musculaires. Bien que l’action de MYOD au cours de la différenciation cellulaire ait été largement étudiée, peu de chose sont connus sur les événements de remodelage de la chromatine associés à l'activation de l'expression de MyoD. Parmi les régions régulatrices de l'expression de MyoD, la région Core Enhancer (CE) qui est transcrite en ARN activateur non codant (CEeRNA) a été démontrée pour contrôler l'initiation de l'expression de MyoD au cours de l'engagement de myoblastes dans la différenciation.Nous avons identifié LSD1 et PHF2 comme des activateurs clés du CE de MyoD. L'invalidation in vitro et in vivo de LSD1 ou l'inhibition de l'activité enzymatique de LSD1 empêche le recrutement de l'ARN PolII sur le CE, empêchant l’expression du CEeRNA. D’après nos résultats, l'expression forcée du CEeRNA restaure efficacement l'expression de MyoD et la fusion myoblastique en l'absence de LSD1. De plus, PHF2 interagit avec LSD1 en régulant sa stabilité protéique.En effet, l'ablation in vitro de PHF2 entraîne une dégradation massive de LSD1 et donc une absence d'expression du CEeRNA. Cependant, toutes les modifications d'histones qui ont lieu dans la région du CE lors de l'activation de la différenciation ne peuvent pas être directement attribuées à l'activité enzymatique de LSD1 ou PHF2. Ces résultats soulèvent la question de l'identité des partenaires de LSD1 et PHF2, qui co-participeraient à l'expression du CEeRNA et donc à l'engagement des myoblastes dans la différenciation cellulaireLSD1 and PHF2 are lysine de-methylases that can de-methylate both histone proteins, influencing gene expression and non-histone proteins, affecting their activity or stability. Functional approaches using Lsd1 or Phf2 inactivation in mouse have demonstrated the involvement of these enzymes in the engagement of progenitor cells into differentiation. One of the best-characterized examples of how progenitor cells multiply and differentiate to form functional organ is myogenesis. It is initiated by the specific timing expression of the specific regulatory genes; among these factors, MYOD is a key regulator of the engagement into differentiation of muscle progenitor cells. Although the action of MYOD during muscle differentiation has been extensively studied, still little is known about the chromatin remodeling events associated with the activation of MyoD expression. Among the regulatory regions of MyoD expression, the Core Enhancer region (CE), which transcribes for a non-coding enhancer RNA (CEeRNA), has been demonstrated to control the initiation of MyoD expression during myoblast commitment. We identified LSD1 and PHF2 as key activators of the MyoD CE. In vitro and in vivo ablation of LSD1 or inhibition of LSD1 enzymatic activity impaired the recruitment of RNA PolII on the CE, resulting in a failed expression of the CEeRNA. According to our results, forced expression of the CEeRNA efficiently rescue MyoD expression and myoblast fusion in the absence of LSD1. Moreover PHF2 interacts with LSD1 regulating its protein stability. Indeed in vitro ablation of PHF2 results in a massive LSD1 degradation and thus absence of CEeRNA expression. However, all the histone modifications occurring on the CE region upon activation cannot be directly attributed to LSD1 or PHF2 enzymatic activity. These results raise the question of the identity of LSD1 and PHF2 partners, which co-participate to CEeRNA expression and thus to the engagement of myoblast cells into differentiation
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Robinson, Robert Maxwell. "Splicing signals in Caenorhabditis elegans : candidate exonic splicing enhancer motifs /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/10846.
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Donald, Claire Louisa. "Development of molecular tools to enhance understanding of antiviral RNAi in mosquitoes." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6207/.
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Mosquito-borne arboviruses are a considerable threat to human and animal health across the world. Many of them are classed as emerging or remerging pathogens and the incidence of disease for a number of serious viral infections has increased as they expand their geographical and host ranges. As with other invertebrates, mosquitoes lack the adaptive immune response present in vertebrates and instead rely on their innate immune defences to modulate viral infections. Nevertheless, in contrast to vertebrates, arboviral infections in their arthropod vector are non-pathogenic and have no cytopathic effect or detrimental impact on their survival. The response considered to be the most important for antiviral defence in mosquitoes is RNA interference (RNAi) which is a sequence-specific, RNA silencing mechanism. Most of what is known about antiviral RNAi in arthropods has been established in Drosophila as the model insect organism. These studies have benefited from an extensive range of genetic mutants, molecular tools, reporter assays and genetic profiling. The absence of these tools for use in mosquito research is a substantial deficit for arboviral studies in their natural vector system and must be rectified in order to fully understand the influence vector immunity has on virus transmission. This thesis discusses the development of a ‘molecular tool-box’ for advancing the acquisition of knowledge in this area. Efficient RNAi gene silencing and its effect on the antiviral RNAi response was established in vitro using Semliki Forest virus (SFV) as model arbovirus. This assay determined that knock-down of Argonaute-2 had the most substantial impact on virus replication compared to the knockdown of other RNAi proteins. In addition, the limited detection of virus-derived small RNAs, key molecules of the antiviral RNAi response by Northern blot analysis provides further support to previous evidence that SFV may circumvent the antiviral response by sequestering its genomic RNA, resulting in restricted access by the RNAi machinery and preventing the generation of large quantities of virus-derived small RNAs. However, some SFV-derived small RNAs are known to be produced and these have been shown to generate a pattern of ‘hot’ and ‘cold’ spots along the full-length coding sequences. This thesis has determined that this pattern is not exclusive to viral-derived dsRNA trigger molecules but is also exhibited following the treatment of mosquito cells in culture with non-viral dsRNA. This implies that all exogenous dsRNA is processed by RNAi in a similar manner. This study has also characterised the presence of an RNA-dependent RNA polymerase (RdRP) encoded by Aedes aegypti mosquitoes. RdRPs are important for the amplification and spread of the RNAi signal in other organisms such as plants and worms; however, only one study suggested the existence of one in Drosophila. Although, this project proposed the presence and transcription of a homologue of the Drosophila RdRP in the Aedes aegypti-derived Aag2 cell line, protein knockdown assays revealed that it has no effect on virus replication in vitro; suggesting that it does not function as an RdRP. Due to the lack of antibodies against the major RNAi proteins Dicer-1, Dicer-2, Argonaute-1 and Argonaute-2 in mosquitoes, these were designed and screened which allowed the identification of several candidates for the detection of the proteins in mosquito cells in culture. Further to this, recombinant forms of the RNAi initiator protein Dicer-2 and the slicer protein Argonaute-2 were successfully generated and tested in vitro using different promoters to establish their use for future temporal and spatial kinetic studies. It was concluded that of the promoters tested the most successful for the expression of these reporter constructs was the subgenomic promoter of SFV. On the other hand a second promoter, the PUb promoter, may prove more suitable in the future. Finally, this project studied the antiviral capabilities of a non-haematophagous mosquito cell line which would not come across an arboviral infection by traditional blood- feeding routes. Instead the mosquito larvae sustain their adult life stages by feeding on the larvae of other species which may be vertically infected. A cell line derived from Toxorhynchites amboinensis was characterised and was shown to carry out RNAi if induced by dsRNA suggesting that they are able to mount an antiviral response to acquired infections. This study also determined that the cell line contains an endogenous insect specific virus and, although the source of this is unknown, it adds an interesting new dimension to mosquito antiviral immunity. This thesis enhances RNAi research in Aedes mosquitoes by presenting novel molecular tools and reporter assays which will be highly valuable for facilitating future investigations. The studies performed also add to what is already understood regarding the interaction between SFV and mosquito antiviral immunity through the RNAi response.
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Wu, Connie Ph D. Massachusetts Institute of Technology. "Engineering periodic short hairpin RNA delivery systems for enhanced therapeutic efficacy." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/121821.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019Cataloged from PDF version of thesis.
Includes bibliographical references.
RNA interference (RNAi) presents a highly promising approach for cancer therapeutics via specific silencing of disease-implicated genes, but its clinical translation remains severely limited by barriers in delivering short interfering RNA (siRNA). Numerous delivery vehicles have been developed to protect siRNA from degradation, promote target cell uptake, and facilitate endosomal escape into the cytoplasm, where RNAi occurs. However, in vivo instability, low silencing efficiency, undesired toxicity, and immunogenicity remain challenges for current siRNA delivery systems, particularly as the low valency and high rigidity of siRNA make it difficult to condense into stable nanoparticles. Here we engineer the siRNA cargo to make it more amenable to stable encapsulation by using a polymeric form of siRNA, or periodic short hairpin RNA (p-shRNA), as well as design a biodegradable polycationic carrier for efficient in vivo delivery of p-shRNA.
Consisting of tens of linked siRNA repeats, p-shRNA is synthesized by the repeated action of T7 RNA polymerase around a circular DNA template. We first leverage molecular engineering design an open-ended p-shRNA structure that is efficiently processed inside cells into siRNAs, greatly enhancing its silencing potency. Furthermore, the much higher valency and flexibility of p-shRNA compared to siRNA enable more stable complexation with delivery materials. To exploit these advantages of p-shRNA, we optimize biodegradable polycations with hydrophobic regions that promote stable condensation and efficient intracellular release. Our approach unveils key design rules governing p-shRNA delivery, and we develop stabilized p-shRNA complexes that show in vivo therapeutic efficacy in a syngeneic melanoma mouse model. Finally, we extend our p-shRNA platform to act as a dual therapeutic agent, harnessing innate immune activation together with gene silencing.
By modulating the surface of the p-shRNA complexes with an anionic polypeptide, we dramatically enhance innate immune recognition of p-shRNA by pattern recognition receptors while maintaining high silencing efficiency. These dually acting complexes can target ovarian tumors in vivo and prolong survival in a syngeneic ovarian cancer mouse model. Our findings establish a potent, multifunctional RNAi platform that can potentially move RNAi therapeutics closer to clinical translation by addressing the delivery and in vivo efficacy challenges faced by current siRNA systems.
National Science Foundation Graduate Research Fellowshipgrant #1122374
Koch Institute Ludwig Center for Molecular Oncology Graduate Fellowship
Congressionally Directed Medical Research Program Ovarian Cancer Research Program Teal Innovator Award from the Department of Defense (13-1-0151)
by Connie Wu.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering
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Panaviene, Zivile Sliesaraviciute. "THE ROLE OF TOMBUSVIRUS REPLICASE PROTEINS AND RNA IN REPLICASE ASSEMBLY, REPLICATION AND RECOMBINATION." UKnowledge, 2004. http://uknowledge.uky.edu/gradschool_diss/435.
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Tombusviruses are single, positive strand RNA viruses of plants, often associated with parasitic defective interfering (DI) RNAs. Two viral- coded gene products, namely p33 and p92, are required for tombusvirus replication. The overlapping domains of p33 and p92 contain an arginine/proline-rich (RPR) RNA binding motif. In this study, the role of RPR motif and viral RNA in tombusvirus replication and recombination, as well as involvement of viral RNA in tombusvirus replicase assembly was examined. Using site-directed mutagenesis I generated a series of RPR mutants of Cucumber necrosis tombusvirus (CNV). Analysis of RPR mutants defined that wild type RPR motif, especially two of the four arginines, were required for efficient RNA binding in vitro, for replication of tombusviruses, their associated DI RNAs, subgenomic (sg)RNA synthesis and DI RNA recombination in vivo. Experiments using a two-component tombusvirus replication system showed that RPR motif is critical for functions of both p33 and p92 in replication, but its role in these proteins might not be identical. Recombination studies using a novel tombusvirus three-component system revealed that mutations in RPR motif of p33 replicase protein resulted in an altered viral RNA recombination rate. Identified DI RNA recombinants were mostly imprecise, with recombination sites clustered around a replication enchancer and an additional putative cis-acting element that might facilitate the template switching events by the tombusvirus replicase. To study the role of RNA during the assembly of functional tombusvirus replicase, recombinant CNV replicase that showed similar properties to plant-derived CNV replicase was purified from Saccharomyces cerevisiae. When in addition to p33 and p92 proteins DI RNA was co-expressed in yeast cells, the isolated replicase activity was increased ~40 fold. Further studies defined RNA motifs within two short DI RNA regions that enhanced active CNV replicase formation. In summary, this study showed that the conserved RNA binding motif of the tombusvirus replicase proteins and viral RNA are involved in replicase assembly, viral RNA replication, subgenomic RNA synthesis and RNA recombination. This data shed new light on the complex roles of the viral elements in replication, and will help future studies aimed at interfering with viral infections.
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Munroe, David. "mRNA Poly(A) tail: a 3' Enhancer of Translational Initiation: a Thesis." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/67.
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Most eukaryotic mRNAs have a sequence of polyadenylic acid [poly(A)] at their 3'-termini. Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) have yet to be clarified. Earlier results from our laboratory led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of poly(A)+, but not poly(A)-, mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include: 1) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency in vivo and in vitro; 2) correlations between the abundance and stability of PABPs and the rate of translational initiation in vivo and in vitro; and 3) the demonstration that exogenous poly(A) is a potent and specific inhibitor of the in vitro translation of poly(A)+, but not poly(A)-mRNAs.
To evaluate the hypothesis that the 3'-poly(A) tract of mRNA plays a role in translational initiation, we have constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs are translated, degraded, recruited into polysomes, and assembled into mRNPs or intermediates in the translational initiation pathway. Relative to mRNAs which are polyadenylated, we find that poly(A)- mRNAs have a reduced translational capacity which is not due to an increase in their decay rates, but is attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affects a late step in translational initiation, is distinct from the phenotype associated with cap-deficient mRNAs, and results in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibits translation from capped poly(A)+ mRNAs, but stimulates translation from capped poly(A)- mRNAs. We suggest that poly(A) is the formal equivalent of a transcriptional enhancer, i.e., that poly(A)-binding protein (PABP) bound at the 3'-end of mRNA may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5'-end.
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Caglio, Giulia [Verfasser], Ana [Gutachter] Pombo, Leonie [Gutachter] Ringrose, and Norbert [Gutachter] Hübner. "RNA Polymerase II identifies enhancers in different states of activation / Giulia Caglio ; Gutachter: Ana Pombo, Leonie Ringrose, Norbert Hübner." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1189147106/34.
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Lei, Shaohua. "A RNA Virus Reference Database (RVRD) to Enhance Virus Detection in Metagenomic Data." Thesis, Virginia Tech, 2018. http://hdl.handle.net/10919/85388.
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With the great promise that metagenomics holds in exploring virome composition and discovering novel virus species, there is a pressing demand for comprehensive and up-to-date reference databases to enhance the downstream bioinformatics analysis. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation of RNA virus genomes downloaded from the three major virus sequence databases including NCBI, ViralZone, and ViPR. To reduce viral sequence redundancy caused by multiple identical or nearly identical sequences, sequences were first clustered and all sequences except one in a cluster that have more than 98% identity to one another were removed. Other identity cutoffs were also examined, and Hepatitis C virus genomes were studied in detail as an example. Using the 98% identity cutoff, sequences obtained from ViPR were combined with the unique RNA virus references from NCBI and ViralZone to generate the final RVRD. The resulting RVRD contained 23,085 sequences, nearly 5 times the size of NCBI RNA virus reference, and had a broad coverage of RNA virus families, with significant expansion on circular ssRNA virus and pathogenic virus families. Compared to NCBI RNA virus reference in performance evaluation, using RVRD as reference database identified more RNA virus species in RNAseq data derived from wastewater samples. Moreover, using RVRD as reference database also led to the discovery of porcine rotavirus as the etiology of unexplained diarrhea observed in pigs. RVRD is publicly available for enhancing RNA virus metagenomics.Master of Science
Next-generation sequencing technology has demonstrated capability for the detection of viruses in various samples, but one challenge in bioinformatics analysis is the lack of well-curated reference databases, especially for RNA viruses. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation from the three commonly used resources: NCBI, ViralZone, and ViPR. While RVRD was managed to be comprehensive with broad coverage of RNA virus families, clustering was performed to reduce redundant sequences. The performance of RVRD was compared with NCBI RNA virus reference database using the pipeline FastViromeExplorer developed by our lab recently, the results showed that more RNA viruses were identified in several metagenomic datasets using RVRD, indicating improved performance in practice.
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Kekarainen, Tuija. "Enhanced utilisation of an infectious plant viral cDNA clone /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2001. http://epsilon.slu.se/avh/2001/91-576-5781-5.pdf.
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Yang, Qi. "Regulation of the yifK locus by multi-target small RNA GcvB in Salmonella." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00954416.
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GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK - a highly conserved locus encoding a putative amino acid transporter - by targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process. Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.
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Matsumoto, Yoshiaki. "SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer." Kyoto University, 2018. http://hdl.handle.net/2433/232117.
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Ogolla, Pauline S. "The Protein Kinase Double-stranded RNA-dependent (PKR) Enhances Protection Against Disease Cause by a Non-viral Pathogen." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1341592115.
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Cojocaru, Vlad. "Molecular motions at the 5 stem-loop of U4 snRNA: Implications for U4/U6 snRNP assembly." Doctoral thesis, [S.l.] : [s.n.], 2005. http://webdoc.sub.gwdg.de/diss/2005/cojocaru.
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Hobro, Alison J. "Structural investigations of RNA through the application of Raman, Raman optical activity and surface enhanced spectroscopies." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491481.
Full textAbstract:
RNA molecules are involved in a wide range of cellular processes from genetic coding to catalytic activities, and determination of their secondary and tertiary structures is essential for the understanding of their functions. The work presented in this thesis shows the structural information that can currently be obtained using Raman and ROA spectroscopies for nucleic acid components and RNA molecules. Raman and ROA spectra can be used to identify particular building blocks, for example, with some marker bands characteristic for mono- and tri-nucleotides. In oligonucleotides and RNA sequences, Raman is particularly sensitive to the environment of the bases, while ROA is most sensitive to the conformation of the ribose-phosphate backbone. Therefore this complementary information can be used to identify single base changes and the introduction of a bulge sequence in the EMCV IRES Domain I, as well as allow tentative identification of structural features within the Adenovirus VA RNAr. Moreover, perturbation induced changes can also be monitored using Raman spectroscopy. Results presented here show these studies can be used to identify changes in specific parts of the molecule with particular temperatures, as well as identifying particular changes in secondary and/or tertiary structure with other perturbations, such as Mg2+ concentrations. The main limitation for ROA studies of RNA is the requirement for relatively high concentrations and long data collection times which can limit the range of RNA molecules that can be studied. Surface enhanced spectroscopic techniques can reduce the sample concentration and data collection requirements through the interaction of a molecule with surface plasmons associated with a nanoscale· roughened metal surface. The second half of this thesis concentrates on the implications of surface enhancement for Raman (SERS) and ROA (SEROA) studies of RNA. The application of SERS to RNA appears to be more complex than for other analyte species because the enhancement process is strongly time dependent. This, together with the spectral profile, can be influenced by a number of factors, including the nature of the aggregating agent and the concentration of the reagents involved. The effects of changes of these variables on the spectral profile and the time dependence of SERS enhancement are presented and discussed in the context of physical changes occurring during the experiment. Overall, Raman and ROA studies of RNA have provided information about the secondary and tertiary structures of RNA molecules despite their varying conformations and transitions, a matter that can be complicated for high-resolution techniques, such as X-ray crystallography. Surface enhanced Raman studies of RNA can, with careful preparation, provide meaningful results whilst reducing sample concentration requirements. However, the processes involved in generating surface enhanced ROA spectra appear to be significantly more complex and, for nucleic acid components, SEROA has not been successfully measured.
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Chong, Vanessa. "Biotagging, a genetically encoded toolkit in the zebrafish, reveals novel non-coding RNA players during neural crest and myocardium development." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:26b7d1a0-3f03-4518-97c4-566cc5d5bf02.
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Complex multicellular organisms are composed of at least 200 cell types, which contain the same DNA "black box" of genetic information. It is the precise regime according to which they express their genes, exquisitely controlled by gene regulatory circuits, that defines their cellular identity, morphology and function. We have developed an in vivo biotinylation method that uses genetically encoded components in zebrafish, termed biotagging, for genome-wide regulatory analysis of defined embryonic cell populations. By labelling selected proteins in specific cell types, biotagging eliminates background inherent to analyses of complex embryonic environments via highly stringent biochemical procedures and targeting of specific interactions without the need for cell sorting. We utilised biotagging to characterise the in vivo translational landscape on polysomes as well as the transcriptional regulatory landscape in nuclei of migratory neural crest cells, which intermix with environing tissues during their migration. Our migratory neural crest translatome presented both known and novel players of the neural crest gene regulatory network. An in depth look into the active nuclear transcriptome uncovered a complex world of non-coding regulatory RNAs that potentially specify migratory neural crest identity and present evidence of active bidirectional transcription on regions of open chromatin that include putative cis-regulatory elements. Analysis of our transcribed cis-regulatory modules functionally links these elements to known genes that are key to migratory neural crest function and its derivatives. We also identified a novel cohort of circular RNAs enriched at regions of tandem duplicated genes. Last but not least, we recovered developmentally regulated long non-coding RNAs and transcribed transposable elements. To functionally dissect the biological roles of these factors, we have built two Ac/Ds-mediated in vivo toolkits for efficient screening of putative enhancers and for CRISPR/Cas9-based transcriptional modulation. Overall, our methods and findings present a comprehensive view of the active coding and non-coding landscapes of migratory neural crest on a genome-wide scale that refine the current regulatory architecture underlying neural crest identity.
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Rush, Margaret. "Regulation of RNA Processing in Human Papillomavirus Type 16." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5972.
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González-Vallinas, Rostes Juan 1983. "Software development and analysis of high throughput sequencing data for genomic enhancer prediction." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/283480.
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High Throughput Sequencing technologies (HTS) are becoming the standard in genomic regulation
analysis. During my thesis I developed software for the analysis of HTS data. Through collaborations
with other research groups, I specialized in the analysis of ChIP-Seq short mapped reads. For instance,
I collaborated in the analysis of the effect of Hog1 stress induced response in Yeast and helped in the
design of a multiple promoter-alignment method using ChIP-Seq data, among other collaborations.
Making use of expertise and the software developed during this time, I analyzed ENCODE datasets in
order to detect active genomic enhancers. Genomic enhancers are regions in the genome known to
regulate transcription levels of close by or distant genes. Mechanism of activation and silencing of
enhancers is still poorly understood. Epigenomic elements, like histone modifications and transcription
factors play a critical role in enhancer activity. Modeling epigenomic signals, I predicted active and
silenced enhancers in two cell lines and studied their effect in splicing and transcription initiation.Las tecnologías High Throughput Sequencing (HTS) se están convirtiendo en el método standard de análisis de la regulación genómica. Durante mi tesis, he desarrollado software para el análisis de datos HTS. Mediante la colaboración con otros grupos de investigaci n, me he especializado ́ en el análisis de datos de ChIP-Seq. Por ejemplo, colaborado en el análisis del efecto de Hog1 en células de levadura afectadas por stress, colaboré en el diseño de un m ́ todo para el alineamiento m ́ ltiple de promotores usando datos de ChIP-Seq, entre otras colaboraciones. Usando el conocimiento y el software desarrollados durante este tiempo, analicé datos producidos por el proyecto ENCODE para detectar enhancers genómicos activos. Los enhancers son areas del genoma conocidas por regular la transcripción de genes cercanos y lejanos. Los mecanismos de activación y silenciamiento de enhancers son aún poco entendidos. Elementos epigenómicos, como las modificaciones de histonas y los factores de transcripción juegan un papel crucial en la actividad de enhancers. Construyendo un modelo con estas señales epigen ́ micas, predije enhancers activos y silenciados en dos lineas celulares y estudié su efecto sobre splicing y sobre la iniciacion de la transcripción.
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Xu, Congcong. "Optimizing the Physicochemical Properties of RNA Nanoparticles for Controllable Drug Release, Hydrophobic Drug Encapsulation and Enhanced Cancer Targeting." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1589810873310799.
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Miller, Tyler Eugene. "Identifying Novel In Vivo Epigenetic Dependencies in Glioblastoma." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1464856610.
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Creigh-Pulatmen, Tilbe. "Structural and functional characterisation of the cold-inducible RNA-binding protein CIRP and its application to enhanced recombinant protein production." Thesis, University of Kent, 2014. https://kar.kent.ac.uk/47913/.
Full textAbstract:
Expression of the RNA binding protein CIRP is up regulated in mammalian cells upon perception of mild cold shock (27-32°C), conditions that can result in enhanced recombinant protein yields from mammalian cells and improved protein folding and activity. CIRP also binds to key proteins involved in the control of mRNA translation initiation, potentially acting as a bridge between the RNA and protein synthesis machinery. CIRP has two domains, an N terminal RNA binding domain and an arginine/glycine rich C-terminal domain that is natively disordered. The N-terminal domain includes two RNA-binding sites, RNP1 and RNP2 that are conserved across many RNA binding proteins. Here, the RNA binding of the N-terminal domain of CIRP was investigated by introducing mutations into the RNP1 and RNP2 RNA binding sites and monitoring subsequent RNA binding using electromobility shift assays and NMR. These studies show that the F49 and F9 residues in these regions are important for RNA interactions. Further, NMR dynamics studies showed that the region (β2-β3 loop) just before the RNP1 sequence that includes the F49 residue has increased motion compared to the rest of the protein. Chemical shift analysis was used to map those residues in CIRP involved in RNA binding that mapped onto the RNP1 and RNP2 sites. Mutation of the F49 and F9 sites to Ala residues disrupted RNA binding as shown by NMR studies. Mutation of each residue resulted in some conformational change in the structure of the domain as determined by HSQC-NMR, particularly for the F49 residue in RNP1. The mutation of one of the phenylalanine residues affected the chemical shift of the other, confirming their proximity in space. The C-terminal is a natively disordered domain but the studies presented here suggest this plays a role in RNA binding and ligand specificity. Cell lines stably expressing CIRP were also generated to further investigate the function of CIRP and to determine binding partners. CIRP was found to interact with the translation initiation factor eIF4G, both the full length CIRP and N-CIRP molecule, suggesting that binding to 4G occurs through the N-terminal domain. The over expression of CIRP enhanced recombinant firefly luciferase expression when the luciferase mRNA contained a CIRP 3’UTR binding sequence. CIRP over-expression also enhanced growth of Chinese hamster ovary cells at 37°C but not at reduced temperature. From these studies it is hypothesised that CIRP can bind specific mRNAs and enhance their expression through its interaction with both the specific mRNA and the translation machinery via eIF4G leading to increased protein expression of the target mRNA under conditions of mild cold-stress (32°C).
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Kühnert, Julia [Verfasser], and Thomas [Akademischer Betreuer] Dobner. "SUMO-modification of the RNA-binding protein La enhances its binding to the translational start site of cyclin D1 / Julia Kühnert. Betreuer: Thomas Dobner." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2013. http://d-nb.info/1030365970/34.
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Moquet-Torcy, Gabriel. "Mécanismes transcriptionnels gouvernés par Fra-1 et Fra-2 dans les cancers du sein agressifs." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20244/document.
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Le cancer du sein est la principale cause de mortalité par cancer chez la femme. Deux des facteurs de transcription de la famille Fos, Fra-1 et Fra-2, sont surexprimés dans les cancers du sein agressifs et contribuent au phénotype tumoral en favorisant entre autres, la prolifération, la motilité et l'invasivité. De façon surprenante, les mécanismes moléculaires via lesquels Fra-1 et Fra-2 (et plus généralement le complexe transcriptionnel AP-1 dont ils sont des constituants) gouvernent la transcription de leurs gènes cibles sont quasi-inconnus. Dans ce contexte, en combinant diverses approches (immunoprécipitation de chromatine, interférence à l'ARN…), j'ai étudié les mécanismes moléculaires par lesquels Fra-1 et Fra-2 contrôlent la transcription dérégulée du gène de l'urokinase ou uPA (sérine protéase cruciale dans la progression tumorale et l'établissement de métastases) qui est l'un des nouveaux marqueurs utilisés en clinique pour la mise en place des choix thérapeutiques. Mes travaux montrent de façon originale que (i) Fra-1 et Fra-2 agissent de façon non redondante et coopèrent pour réguler l'expression d'uPA via leur fixation sur un enhancer AP-1 localisé à -1,9 kb du site d'initiation de la transcription (TSS), (ii) Fra-2 est nécessaire au recrutement de RNA Pol II au niveau de l'enhancer, tandis que Fra-1 stimule le passage de RNA Pol II de sa forme initiatrice à sa forme élongatrice et (iii) que la polymérase recrutée à l'enhancer rejoint le TSS par un mécanisme de « tracking », très rarement décrit dans la littérature, en produisant de petits ARNs non codants, bidirectionnels et instablesBreast cancer is the most frequent malignant disease among women. Two transcription factors, Fra-1 and Fra-2, belonging to the Fos family members, are overexpressed in aggressive breast cancers and contribute to the tumorigenic phenotype by favoring proliferation, motility and invasion. Surprisingly, the molecular mechanisms governed by Fra-1 and Fra-2 (and more generally by the AP-1 transcriptional complex, which they are components of) for the transcription of their target genes are still largely unknown. In this context, by combining different approaches (chromatin immunoprecipitation, RNA interference…), I studied the molecular mechanisms orchestrated by Fra-1 and Fra-2 for the expression of the urokinase (or uPA) gene (encoding a serine protease crucial for tumor progression and metastasis), which is one of the new diagnostic markers now taken into consideration for deciding therapeutic strategies. Interestingly, my results show that (i) Fra-1 and Fra-2 have non redundant functions and cooperate for the transcriptional regulation of uPA through their binding to AP-1 enhancer located 1.9 kb upstream of the transcriptional start site (TSS), (ii) Fra-2 is required for the recruitment of RNA Pol II on this enhancer while Fra-1 allows the conversion of RNA Pol II initiating form into its elongating form and (iii) enhancer-recruited RNA Pol II reaches the TSS by a tracking mechanism, mechanism very rarely described in the literature, during which it synthetizes small, unstable bidirectional, non coding RNAs
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Jamoos, Rana [Verfasser], and Goetz [Akademischer Betreuer] Reustle. "Analysis of the interaction between the helper component proteinase (HC-Pro) of Zucchini yellow mosaic virus (ZYMV) and the plant RNA methyltransferase Hua enhancer 1 (HEN1) / Rana Jamoos. Betreuer: Goetz Reustle." Hohenheim : Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim, 2012. http://d-nb.info/1027354203/34.
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Mateu, Huertas Elisabet 1983. "Insight into disease processes of fragil X premutation carriers associated pathologies : expression-profile characterization and identification of a novel pathogenic mechanisms." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/145480.
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Male premutation carriers (PM) presenting between 55-200 CGG repeats in the
Fragile X-associated (FMR1) gene are at risk to develop Fragile X Tremor/Ataxia
Syndrome (FXTAS), and females to undergo Premature Ovarian Failure (POF1).
These pathologies are caused by toxic gain of function of the premutated FMR1
mRNA. Functional alterations of several gene expression regulators provide a
detrimental mechanism underlying FMR1 PM–associated pathologies. In this thesis,
we have characterized the transcriptome alterations associated to FMR1 premutation
and further characterized the relevance of the biogenesis and activity of a small RNAs
formed by repeated CGG (sCGG) in neuronal dysfunction linked to FMR1-PM. In
blood of FMR1 premutation carriers (fXPCs) we have detected a strong deregulation
of genes enriched in FXTAS-relevant biological pathways. We have also identified a
deregulated gene (EAP1) that may underlie POF1 in female fXPCs. In addition, we
found increased levels of sCGG in different models of FMR1-PM and further
demonstrated the neurotoxic activity of sCGG through a mechanism dependent on
RNA induced silencing machinery. We propose that the activity of sCGG may
contribute to transcriptome perturbations with downstream pathogenic consequences.
Overall, we provide mechanistic insight into the disease process and further suggest
targets for FXTAS diagnosis to the myriad of phenotypes associated with FXPC.Homes portadors de la premutació (PM) en el gen associat Fràgil X (FMR1), presenten entre 55-200 repeticions de CGG, estan en risc de desenvolupar el síndrome de tremolor/atàxia associat al X fràgil (FXTAS), i les dones fallida ovàrica precoç (POF1). Aquestes malalties són causades per la funció tòxica de l'ARN missatger. Alteracions funcional de diversos reguladors de l'expressió gènica s'ha proposat com a causa subjacent a aquests trastorns. En aquesta tesi, s'han caracteritzat les alteracions associades al transcriptome de la premutació en l’FMR1 i analitzat la rellevància de la biogènesi i l'activitat d'un petit ARN format per CGG repetits (sCGG) en la disfunció neuronal relacionada amb la PM del FMR1. En sang de portadors de la premutació en l’FMR1 (fXPCs) s'ha detectat una forta desregulació de gens enriquit en vies biològiques rellevants en FXTAS. També hem identificat un gen desregulat (EAP1) que pot ser la base POF1 en dones fXPCs. A més, hem trobat un augment en els nivells de sCGG en diferents models de FMR1-PM i demostrem la seva activitat neurotòxica a través d'un mecanisme dependent en la maquinària de silenciament gènic. Proposem que l'activitat de sCGG pot contribuir a causar pertorbacions en el transcriptoma i desencadenar conseqüències patògenes.En general, oferim un nou enfoc en procés de la malaltia i un diagnòstic més exacte per la gran varietat de fenotips associats amb fXPCs.
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Mustafi, Debarshi. "Genetic Signatures of the Retina in Health and Disease." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1372776307.
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Prado, Enora. "Détection de l’ADN par spectrométrie de diffusion Raman exaltée de surface couplée à la microfluidique." Thesis, Bordeaux 1, 2011. http://www.theses.fr/2011BOR14348/document.
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Ce travail présente une méthode originale de détection et de quantification, sans étape de marquage, de la proportion de bases libres contenues dans des acides nucléiques. La spectrométrie de diffusion Raman exaltée de surface (DRES ou SERS en anglais) nous a permis d’obtenir la signature spectrale spécifique des nucléotides caractéristiques des ARN (adénosine, cytosine, guanosine et uridine), en utilisant des colloïdes d’argent comme substrat-DRES et des ajouts de MgCl2 comme agent d’agrégation. Les conditions de détection ont été optimisées pour établir un protocole de quantification de la proportion des nucléobases non-appariées par spectrométrie DRES. Les limites de détection obtenues sont de l’ordre de quelques dizaines de picomoles. L’amélioration de la reproductibilité des mesures par spectrométrie DRES passe par le contrôle précis des temps de réaction (adsorption et agrégation), qui peut être contrôlé grâce à l’utilisation de plateformes microfluidiques adaptées. Nous avons mis en œuvre deux types de plateformes microfluidiques, l’une basée sur des écoulements monophasiques et l’autre sur la génération de gouttes. Les espèces à analyser sont contenus dans les gouttes, permettant la détection in situ par spectrométrie DRES des divers nucléotidesThis work deals with the development of an original label-free method for free bases proportions detection and quantification of nucleic acids. The surface enhanced Raman spectroscopy (SERS) allowed obtaining the specific spectral signature of characteristic nucleotides of RNA (adenosine, cytosine, guanosine and uridine), using silver colloids as SERS substrate and MgCl2 addition as aggregating agent. Then, the condition detection have optimizing to establish a label-free quantification protocol of free nucleobases proportion by SERS spectroscopy. The detection limits obtained are order of few picomoles. The reproducibility improvement of SERS detection requires the precise control of time reaction (adsorption and aggregation), which could be control thanks to microfluidic chips use. We have implemented two different microfluidic chips, one based on single-phase flows and one other based on droplets generation. The analyzed species are containing in droplets, allowing in situ detection by spectroscopy SERS of various nucleotides
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Boehm, Christian Reiner. "Gene expression control for synthetic patterning of bacterial populations and plants." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267842.
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The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.
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Seridi, Loqmane. "Noncoding Elements: Evolution and Epigenetic Regulation." Diss., 2016. http://hdl.handle.net/10754/603694.
Full textAbstract:
When the human genome project was completed, it revealed a surprising result. 98% of the genome did not code for protein of which more than 50% are repeats— later known as ”Junk DNA”. However, comparative genomics unveiled that many noncoding elements are evolutionarily constrained; thus luckily to have a role in genome stability and regulation. Though, their exact functions remained largely unknown.
Several large international consortia such as the Functional Annotation of Mammalian Genomes (FANTOM) and the Encyclopedia of DNA Elements (ENCODE) were set to understand the structure and the regulation of the genome. Specifically, these endeavors aim to measure and reveal the transcribed components and functional elements of the genome. One of the most the striking findings of these efforts is that most of the genome is transcribed, including non-conserved noncoding elements and repeat elements.
Specifically, we investigated the evolution and epigenetic properties of noncoding elements.
1. We compared genomes of evolutionarily distant species and showed the ubiquity of constrained noncoding elements in metazoa.
2. By integrating multi-omic data (such as transcriptome, nucleosome profiling, histone modifications), I conducted a comprehensive analysis of epigenetic properties (chromatin states) of conserved noncoding elements in insects. We showed that those elements have distinct and protective sequence features, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix.
3. I focused on the relationship between enhancers and repetitive elements. Using Cap Analysis of Gene Expression (CAGE) and RNASeq, I compiled a full catalog of active enhancers (a class of noncoding elements) during myogenesis of human primary cells of healthy donors and donors affected by Duchenne muscular dystrophy (DMD). Comparing the two time-courses, a significant change in the epigenetic landscape in DMD was observed that lead to global dysregulation of enhancers and associated repetitive elements.
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Ferreira, Humberto Jorge Gomes. "Epigenetic regulation of non-coding RNAs in cancer." Doctoral thesis, 2017. http://hdl.handle.net/10316/31578.
Full textAbstract:
Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Biologia Celular e Molecular, apresentada à Faculdade de Farmácia da Universidade de CoimbraTumoral evolutionary process occurs through the sequential accumulation of mutations and epimutations that are responsible for cell heterogeneity and sub-clonal selection, as well as for drug resistance and patients associated mortality. Recently, diverse classes of non-coding RNAs (ncRNAs) were described to be implicated in the regulation of key players of carcinogenesis. By standard and high-throughput methods, we analyzed the epigenetic landscape of different types of cancer, uncovering cancer-related pathways, emphasizing those related to the regulation of ncRNAs. Small nucleolar RNAs (snoRNAs) guide post-transcriptional modifications of spliceosomal and ribosomal RNAs. Some members of this class of RNAs are disrupted in cancer, where modifications in ribosome biogenesis have also been implicated. We verified that SNORD123, ACA59B and U70C are transcriptionally silenced by DNA hypermethylation of the CpG Island that overlaps the promoter region of their host gene. Of particular interest, SNORD123 and ACA59B are conserved across vertebrates but they do not have a known target (orphan snoRNAs). Taking into account that these snoRNAs are expressed at least in normal colon and are epigenetically repressed in some colorectal cancer cell lines, we suggested that they can have a potential contribution to carcinogenesis. Moreover, we described the DNA hypermethylation of these three snoRNAs in leukemia samples. Piwi-interacting RNAs (piRNAs) are mainly expressed in germline cells, playing a key role in the epigenetic silence of transposons or guiding their cleavage. We reported the epigenetic transcriptional inactivation of the genes encoding the piRNA-related proteins, PIWIL1, PIWIL2, PIWIL4 and TDRD1, in both seminomas and non-seminomas. These epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation at LINE-1 loci. Importantly, recent studies had shown a similar epigenetic transcriptional disruption in other cancer types; and in non-genetic infertility syndromes, that are epidemiologically linked with testicular cancer. To better characterize the epigenetic landscape of a cancer cell, we interrogated the entire methylome in several cancer and normal samples. We first established the methylome of two acute myeloid leukemia (AML) cell lines, OCI-AML5 and OCI-AML3, the last one harboring a missense mutation in DNMT3A, present in ~20% of the AML patients. By comparison with the methylation profile of AML samples, we suggested a set of twelve candidate target loci for DNMT3A in AML, validating their transcriptional reactivation in our cell line model. Thus, the leukemogenic gene MEIS1 was actively expressed in OCI-AML3. By screening the highest-ranked differentially methylated regions that potentially regulate non-protein coding genes we described a signature of four hypomethylation-associated transcriptional reactivated ncRNAs in the DNMT3A mutant cell line, namely ENST00000413346, LOC100506585, ENST00000443490 and MIRLET7B host gene (MIRLET7BHG). We also suggested that some of the DNMT3A identified potential targets could be linked to worst prognosis observed in AML patients harboring the DNMT3A mutation, particularly MEIS1 and the host gene that carries both let-7a-3 and let-7b. These two microRNAs were previously described to be overexpressed in AML. Based on the loss of differentiation in cancer cells and expanding our study to other tissues, we interrogated the methylation profile of genomic regions known to be responsible for cell identity, namely super-enhancers. We established a correlation among tumor-related hypermethylation of super-enhancers and transcriptional silencing of the corresponding related genes. Our results showed that their methylation profile is also associated with specific cancer types. However, the methylation of the super-enhancer that regulates the host gene of let-7a-3 and let-7b tumor suppressors was linked to their silencing in both lung and breast epithelial cancers. In colorectal cancer, we described tumor-related super-enhancers undergoing hypomethylation-related transcriptional activation of the related genes, such as MYC and RNF43 oncogenes. We hypothesized that the impaired expression and binding of transcription factors could establish novel super-enhancers. We identified FOXQ1 as a probable transcription factor responsible for the loss of DNA methylation at colorectal cancer-specific super-enhancers that control MYC and RNF43. DNA methylomes highlight the epigenetic landscape that regulates the expression of key players in cancer biology. Some of these players are non-coding RNAs that should be exploited as biomarkers for cancer diagnosis, or as targets in personalized therapeutic approaches to control tumor progression and/or metastasis.
O processo evolutivo de um tumor é feito através da acumulação sequencial de mutações e epimutações, sendo estas responsáveis pela heterogeneidade celular e por uma seleção sub-clonal, bem como pela resistência dos doentes a fármacos e mortalidade associada. Recentemente, diversas classes de RNAs não-codificantes (ncRNAs) foram implicadas na regulação de elementos-chave da carcinogénese. Através de métodos standard e de high-throughput, analisámos o perfil epigenético de diferentes tipos de cancro, encontrando vias transcripcionais alteradas, dando especial ênfase às vias relacionadas com a regulação dos ncRNAs. Os small nucleolar RNAs (snoRNAs) dirigem modificações pós-transcripcionais de RNAs spliceossomais e ribossomais. Alguns membros desta classe de RNAs estão desregulados em cancro, onde modificações na biogénese do ribossoma também têm sido implicadas. Verificámos que os snoRNAs SNORD123, ACA59B e U70C estão silenciados transcripcionalmente por um aumento da metilação do DNA da ilha CpG sobreposta à região promotora do seu gene hospedeiro. É de destacar que os SNORD123 e ACA59B estão conservadas em vertebrados, mas não têm um alvo conhecido (snoRNAs órfãos). Tendo em conta que estes são expressos em cólon saudável e que estão epigeneticamente silenciados em algumas linhas celulares de cancro colorrectal, sugerimos que também podem contribuir para o processo de carcinogénese. Além disso, o facto de termos detectado um aumento de metilação do DNA correspondente a estes três snoRNAs em amostras de leucemia, reforça a nossa teoria. Os piwi-interacting RNAs (piRNAs) são expressos principalmente em células germinativas, desempenhando um papel fundamental no silenciamento epigenético de transposons ou dirigindo a sua clivagem. Os nossos resultados demonstram o silenciamento epigenético da transcripção dos genes que codificam para as proteínas relacionadas com os piRNAs, nomeadamente PIWIL1, PIWIL2, PIWIL4 e TDRD1, tanto em seminomas como em não-seminomas. Estas lesões epigenéticas ocorrem num contexto de baixos níveis de piRNAs e de perda de metilação do DNA nas regiões correspondentes ao LINE-1. De forma semelhante, estudos recentes descrevem o silenciamento epigenético da transcrição noutros tipos de cancro; e em síndromes não genéticos de infertilidade, neste caso epidemiologicamente associados ao cancro de testículo. De maneira a caracterizar melhor o perfil epigenético de uma célula cancerígena, estudámos o metiloma de vários tipos de cancro e de amostras de tecido normal. Em primeiro lugar, estabelecemos o metiloma de duas linhas celulares de leucemia mieloide aguda (AML), OCI-AML5 e OCI-AML3, a última das quais tem uma mutação missense no gene DNMT3A, estando presente em ~ 20% dos doentes com AML. Por comparação com o perfil de metilação do DNA de amostras de AML, sugerimos um conjunto de doze regiões alvo para a DNMT3A na AML, validando a reativação da sua transcrição no modelo de linhas celulares. Deste modo, vimos que o gene leucemogénico MEIS1 se expressa ativamente em OCI-AML3. Analisando as regiões com maiores diferenças a nível de metilação de DNA e que pudessem potencialmente regular genes não codificantes para proteínas, detetámos a existência de quatro ncRNAs, ENST00000413346, LOC100506585, ENST00000443490 e MIRLET7BHG, associados a uma reativação transcripcional devido à perda de metilação do DNA na linha celular portadora da mutação no gene DNMT3A. Sugerimos que alguns dos potenciais alvos da DNMT3A, poderiam estar relacionados com pior prognóstico em pacientes com AML que são portadores da mutação no gene DNMT3A, especialmente MEIS1 e o gene hospedeiro que alberga let-7a-3 e let-7b, tendo já sido descrito que estes microRNAs exibem maior expressão em AML. Tendo presente a perda de diferenciação das células cancerígenas e querendo expandir o nosso estudo a outros tecidos, investigámos o perfil de metilação de regiões descritas como responsáveis pela identidade celular, os super-enhancers. No contexto tumoral encontrámos uma correlação entre o aumento de metilação do DNA dos super-enhancers e o silenciamento transcripcional dos genes correspondentes. Apesar do perfil de metilação dos super-enhancers ser específico do tipo de cancro, a metilação do super-enhancer que regula o gene hospedeiro dos supressores tumorais let-7a-3 e let-7b foi associada ao seu silenciamento, tanto em cancro de pulmão como em cancro de mama, ambos epiteliais. No caso do cancro colorrectal, descrevemos super-enhancers submetidos a uma perda de metilação associada à ativação transcripcional dos oncogenes MYC e RNF43. Estabelecemos ainda a teoria de que a expressão e ligação desreguladas de fatores de transcripção poderiam promover a formação de novos super-enhancers. Identificámos FOXQ1 como um provável fator de transcripção, responsável pela perda de metilação dos super-enhancers específicos de cancro colorrectal e que controlam MYC e RNF43. Os metilomas de DNA destacam o perfil epigenético que regula a expressão de elementos-chave na biologia do cancro. Alguns destes elementos são ncRNAs que deveriam ser explorados como biomarcadores para diagnóstico de cancro e também como alvos em estratégias de terapia personalizada, com vista ao controlo da progressão tumoral e/ou das metástases.
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Chen, Yaqiong. "Studies on RNA Regulation: From Enhancer RNAs to RBBP6 Isoform3." Thesis, 2019. https://doi.org/10.7916/d8-e3p7-st32.
Full textAbstract:
This dissertation contains two separate yet interconnected pieces of work, which shed light on the complicated RNA regulatory mechanism. The first part, as the main focus of the thesis, characterizes a large pool of human polyadenylated enhancer RNA under deficient nuclear surveillance conditions, and investigates their metabolism mechanisms. The second part elucidates the dynamic localization mechanism of RBBP6 isoform3, which inhibits pre-mRNA 3’ processing by completing with RBBP6 isoform1.
Despite being composed of approximately 3 billion base pairs, only 1 to 2% of the human genome codes for proteins. The non-coding DNA regions can however function as transcription units and generate non-coding RNAs such as enhancer-derived RNAs, or eRNAs, that play crucial roles in gene expression regulation, cell differentiation, development, and diseases. Previous studies have suggested that most eRNAs are transcribed by RNA polymerase II (RNAP II), but not polyadenylated. In Chapter 3, I identify a large fraction of polyadenylated enhancer RNAs under deficient nuclear surveillance conditions via genome-wide analyses, and explore their biogenesis and degradation mechanisms. I find that the Integrator complex plays an important role in polyadenylated eRNA biogenesis, and that their exosome-dependent degradation requires two cofactor complexes containing the RNA helicase Mtr4: the PAXT/PPC complex and the NEXT complex. Additionally, the canonical poly(A) polymerases PAP-α and PAP-γ play a major role in the 3’ end processing of pA+ eRNA. Finally, I show that under deficient nuclear surveillance conditions, pA+ eRNAs accumulate in the cytoplasm and associate with polysomes, suggesting that at least some might have translation potential.
I also contributed to the discovery of two novel complexes both containing the RNA helicase Mtr4, which is a master player of the nuclear surveillance system. Mtr4 and ZFC3H1 form the PAXT/PPC complex, which facilitates the turnover of polyadenylated nuclear RNAs, including prematurely terminated RNAs (ptRNAs), upstream antisense RNAs (uaRNAs), and eRNAs (see the paper in Appendix II). Mtr4 also associates with NRDE2 to form a complex, functioning in the DNA damage response pathway (see the paper in Appendix III). These works provide additional insights into the complexity and significance of the RNA helicase Mtr4.
In the second part of the thesis, presented in Chapter 4, I studied a polyadenylation factor known as Retinoblastoma-binding protein 6 (RBBP6). RBBP6 was initially identified as a large multidomain protein, interacting with tumor suppressors p53 and Rb. Later, its diverse roles were uncovered in cell cycle progression, apoptosis, nucleic acid metabolism, differentiation, and mRNA processing. RBBP6 protein has four isoforms, among which the shortest isoform, iso3, has only one domain: the DWNN (Domain With No Name) domain. The DWNN domain displays high similarities with ubiquitin, implying its function as a novel ubiquitin-like modifier. However, I show that the DWNN domain is actually not a ubiquitin-like modifier, but is itself ubiquitinated. Moreover, the monoubiquitylation of iso3 can facilitate its localization at chromatin. Additionally, I find that the C-terminal tail of iso3 also plays a role in iso3 chromatin localization, presumably by interacting with other factors of the polyadenylation machinery. Pulldown experiments of iso3 followed by mass spectrometry identified Importin7 as an iso3-interacting factor that assists its cytoplasmic retention. Our results identified novel mechanisms for the dynamic localization of RBBP6 iso3, which shed light on the role of iso3 in mRNA 3’ processing and disease.
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Tu, Yeuh-Hua, and 杜岳華. "Identification of cell states using super-enhancer RNA." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/s4grmk.
Full textAbstract:
碩士國立陽明大學
生物醫學資訊研究所
104
Genome-wide expression analysis with next-generation sequencing techniques has helped us discover numerous facts and improve our understandings about gene regulation. There still remain unknowns in development of cancers, dynamic response of cell against environment changes. Many evidences have implied that enhancers and epigenome play important roles in gene regulation, especially in cell response to environmental changes. Remarkably, genes nearby super-enhancers are important transcription factors in development. The term super-enhancer has been used to describe a group of putative enhancers in close genomic proximity with unusually high levels of mediator binding. Here, we propose to define super-enhancer RNA as highly expressed enhancer RNAs (eRNA) transcribed from a cluster of localized genomic region. Using the cap analysis of gene expression sequencing data from FANTOM5, we systematically explored the eRNA and mRNA landscapes in hundreds of different cell types with responses to various environments. First, we observed positive correlation between eRNA abundance and mRNA expression levels of the proximal genes, implying eRNAs as effective indicator of the enhancer activity. Similarly, super-enhancer RNA and the proximal gene expression was highly correlated. Second, we applied non-negative matrix factorization (NMF) on super-enhancer RNA profiles in two scenarios. Applying NMF on all available profiles, we found that different cell types were well classified. Again, with NMF on individual time-course profiles of a single cell-type, they were clustered into several states showing progressive patterns. We further investigated the enriched biological functions of the involved proximal genes in each pattern, and found their potent association with the corresponding developmental process. Last, we utilized the identified super-enhancer RNAs as features to classify different cell types and found that they performed better than typical eRNAs. In conclusion, we have demonstrated that the proposed super-enhancer RNA can act as a good alternative, without complicated measurements of histone modifications, for identifying important regulatory elements for cell type specification and identifying dynamic cell states.
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Tai, Pei-Syuan, and 戴珮瑄. "Super-enhancer RNA associated gene co-expression networks in breast cancer cell." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/97463040147748309936.
Full textAbstract:
碩士國立陽明大學
生物醫學資訊研究所
105
Super-enhancers are enhancer-dense regions found near genes that play key roles in determining cellular and functional identity through regulation of gene xpression. It has been found that active enhancers generate bidirectional transcripts called enhancer RNAs. The time-course Cap Analysis of Gene Expression (CAGE) has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. Here, we have performed an integrative analysis on the CAGE time-course datasets of MCF-7 breast cancer cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We first constructed their gene co-expression networks using Weighted Gene Co-expression Network Analysis (WGCNA) and observed several co-expression gene modules. Second, we applied non-negative matrix factorization (NMF) that can divide the super-enhancer RNA expression profiles into different states corresponding to the stimulated cell state transitions along the time course. By comparison of the genomic proximity of the top super-enhancer RNAs in different states with the co-expression gene modules, we identified the active modules for each decomposed cell state. Last, we performed enrichment analyses to elucidate their associated biological functions and to identify the plausible pioneering transcription factors for each cell state transition. Our analysis results revealed a clue of the key regulatory factors for cellular changes of breast cancer cells stimulated by ErbB receptors.
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"Identification and Functional Characterization of Novel Long Non-coding RNAs Regulated by Enhancer of Zest Homolog 2 in Hepatocellular Carcinoma." 2016. http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292181.
Full textAbstract:
背景及目的：EZH2是PRC2的核心單元，其作用是抑制下游基因的轉錄。EZH2在肝細胞癌(HCC)中過度表達，因此有必要研究其在致癌網絡中的作用機理。受EZH2調控的蛋白質編碼基因研究較多而其他因子例如長鏈非編碼RNA(lncRNAs)研究較少。lncRNAs在組織及癌症中特異性表達，表明了其在癌症中起到了關鍵作用。lncRNAs在HCC腫瘤中表達異常，對導致其表達失調的機制所知甚少。之前有研究EZH2和lncRNAs的相互作用，但受EZH2調控的lncRNAs則鮮有報道。因此，我們的目標就是鑒定受EZH2調控的lncRNAs并探索它們在HCC中的作用。方法：我們用EZH2抑制劑DZnep處理了Hep3B細胞，然後對全基因組的lncRNAs進行定量分析。并測量受DZnep處理后上調以及下調最明顯的lncRNAs在HCC細胞以及臨床樣本中的表達，進一步篩選出候選lncRNAs。通過敲降及過表達來研究生物功能。基因芯片微陣列分析揭示了候選lncRNAs的下游相關基因以及信號通路。同時，我們鑒定并研究了這些lncRNAs的結合蛋白。
結果：兩種受EZH2調控的lncRNA被鑒定出來并分別命名為DN2和DN5。DN2和DN5在HCC臨床樣本及多數的HCC細胞系中表達量顯著下降。細胞功能學實驗證明，抑制DN2和DN5表达,可以促進細胞生長，細胞存活能力，轉化能力以及体内腫瘤形成能力。在Hep3B和Huh7細胞中過表達DN2或DN5時，上述能力被有效抑制。实验结果表明,DN2和DN5具有抑癌基因的生物功能。EZH2可以結合到DN2和DN5的啟動子上并招募其他單元誘導異染色質的形成，抑制DN2和DN5的轉錄表達。基因芯片微陣列分析結果認為DN2主要與p53和MAPK 信號通路相關。而DN5則涉及到p53，HIF-1α和MAPK信號通路。RNA pull down 和RNA免疫沉澱反應發現DN2可以與蛋白質IGF2BP1和 HIST1H1C結合。而DN5則與IGF2BP1和STAU1結合。DN2和DN5都能與IGF2BP1結合並且抑制IGF2的表達以及促進DDIT3的表達。DN2與HIST1H1C共同作用可以提高DDIT3的表達。DN5和STAU1結合可以調控PFKFB4的表達水平。
結論：我們鑒定了兩種受EZH2調控的抑癌lncRNA，DN2和DN5。DN2主要與p53和MAPK 信號通路相關。而DN5則可能涉及到p53，HIF-1α和MAPK信號通路。DN2和DN5與IGF2BP1結合后可以抑制IGF2表達并提高DDIT3的表達水平。DN2和HIST1H1C結合可以提高DDIT3的表達水平。DN5與STAU1作用抑制了PFKFB4的表達。
Background and Aims: Enhancer of zest homolog 2 (EZH2), which is a core component of polycomb repressive complex 2 (PRC2), acts as a transcriptional repressor of target genes. In Hepatocellular Carcinoma (HCC), EZH2 is elevated aberrantly. The complete oncogenic network of EZH2 needs to be revealed. Previous reports focused on the protein-coding genes regulated by EZH2. The effects of EZH2 on other important factors such as long non-coding RNAs (lncRNAs) left largely unexplored. LncRNAs are expressed in tissue and cancer-specific manners that suggest their potential crucial regulatory roles in cancer biology. LncRNA profiling of HCC tumor and normal tissues indicated that numerous lncRNAs expressed differently in cancer cells. The underlying mechanisms for their dysregulation are poorly understood. Previous researches were focused on the interaction between EZH2 and lncRNAs, but dysregulation of lncRNAs controlled by EZH2 remains unexplored in HCC. Therefore, we aimed to identify novel lncRNAs aberrantly altered by EZH2 and their contribution to HCC progression.
Methods: Global lncRNA profiling was performed in Hep3B cells treated with EZH2 inhibitor DZnep. The most upregulated and downregulated lncRNAs with DZnep treatment were measured in HCC cell lines and human HCC tissues. Candidate lncRNAs were knocked down and overexpressed to study their biological roles in HCC. We used global mRNA profiling to discover down-target genes and signaling pathways related to candidate lncRNAs. We identified the binding proteins of candidate lncRNAs.
Results: Two lncRNAs, DN2 and DN5, which were regulated by EZH2 in HCC cell lines were identified. We found that RNA levels of DN2 and DN5 were decreased in HCC clinical samples and in most HCC cell lines. Cell growth, cell survivability, and transformation and tumor formation abilities in vivo were promoted when DN2 and DN5 were inhibited. During overexpression of DN2 and DN5, cell proliferation, cell survival and transformation and tumor formation abilities were suppressed in Hep3B and Huh7 cells. Functional assays indicated that DN2 and DN5 may play a role as tumor suppressors. EZH2 suppressed expression of DN2 and DN5 by binding to their promoters and recruiting silencing partners, thus promoting heterochromatin formation. Gene chip expression microarray data indicated that DN2 was involved in the p53 and MAPK pathways, and DN5 was associated with the p53, HIF-1α and MAPK pathways. RNA pull-down assay and RNA immunoprecipitation assay indicated that DN2 bound with IGF2BP1 and HIST1H1C, and DN5 interacted with IGF2BP1 and STAU1. DN2 and DN5 interacted with IGF2BP2 to inhibit IGF2 expression and promote DDIT3 transcription. DN2 may also interact with HIST1H1C to stimulate DDIT3 expression. In addition, we found that PFKFB4 to be the downstream target gene of DN5 and STAU1.
Conclusion：We identified two tumor suppressors lncRNAs, DN2 and DN5, that were suppressed by EZH2. DN2 was involved in the p53 and MAPK pathways, and DN5 was associated with the p53, HIF-1α and MAPK pathways. DN2 and DN5 interacted with protein IGF2BP1 to repress IGF2 translation and then activated DDIT3 expression. DN2 also combined with HIST1H1C to increase DDIT3 levels. DN5 cooperated with STAU1 to suppress PFKFB4 expression.
Xu, Feiyue.
Thesis Ph.D. Chinese University of Hong Kong 2016.
Includes bibliographical references (leaves ).
Abstracts also in Chinese.
Title from PDF title page (viewed on …).
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
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Rahman, Samir. "Single molecule characterization of the roles of long non-coding RNAs in eukaryotic transcription regulation." Thèse, 2017. http://hdl.handle.net/1866/19322.
Full textAbstract:
Récemment, des analyses dans divers organismes eucaryotes ont révélé que l'ensemble
du génome est transcrit et produit en plus des ARNs messagers, une grande variété d’ARNs
non codants de différentes longueurs. Les ARNs non codants de plus de 200 nucleotides,
classés comme longs ARNs non codants (LARNnc), représentent la classe la plus abondante
de transcripts non codants. Les études des fonctions des LARNnc suggèrent que beaucoup
d'entre eux seraient impliqués dans la régulation de la transcription. L'objectif de ma thèse de
doctorat était d'élucider les mécanismes de la régulation transcriptionnelle médiée par des
LARNnc dans différents systèmes eucaryotes.
Dans mon premier projet, j'ai étudié le rôle d'un long ARN non codant antisens dans la
régulation transcriptionnelle du gène PHO84, codant un transporteur de phosphate à haute
affinité, chez S. cerevisiae. Des études antérieures ont montré que la suppression d’une
proteine de l’exosome Rrp6 entraîne une augmentation de l'expression antisens et la répression
de PHO84. Il a été suggéré que la perte de Rrp6 entraîne une stabilisation antisens au locus
PHO84, entraînant le recrutement de l'histone de-acétylase Hda1 et la répression de PHO84.
Cependant, le mécanisme par lequel Rrp6p régule la transcription de PHO84 n’était pas
connu. En combinant des méthodes à l’échelle de cellule unique, des approches biochimiques
et génétiques, nous avons montré que les niveaux d'ARN antisens sont régulés principalement
lors de l'élongation par le complexe Nrd1-Nab3-Sen1, qui nécessite Rrp6 pour un recrutement
efficace à l`extrémité 3`de PHO84. De plus, nous révélons l'expression anticorrelé du sens et
de l'antisens, En résumé, nos données suggèrent que la transcription antisens régule le seuil
d'activation du promoteur PHO84.
Dans mon second projet, j'ai étudié les rôles des ARNs dérivés des amplificateurs
(ARNa) dans la regulation de la transcription. En utilisant les cellules de cancer du sein MCF7
comme système modèle, nous avons cherché à déterminer comment les ARNa induits par
l'oestrogène (E2) participent à la régulation de la transcription médiée par le recepteur
d’oestrogène (ERα) au niveau de l'allèle unique. À l'aide de l’hybridation fluorescente à
l’échelle de molécule unique (smFISH), nous avons révélé qu`après induction d'E2, les ARNa
sont induits avec une cinétique similaire à celle des ARNm cibles, sont localisés
exclusivement dans le noyau, principalement associés à la chromatine, et sont moins
abondants que les ARNm. De manière surprenante, nous avons constaté que les ARNa sont
rarement co-transcrits avec leurs loci cibles, indiquant que la transcription active des gènes ne
nécessite pas la synthèse continue ou l'accumulation d'ARNa sur l'amplificateur. En outre, en
utilisant des mesures de la distance à sous-diffraction, nous avons démontré que la cotranscription
des ARNa et des ARNm se produit rarement dans une boucle amplificateurpromoteur.
De plus, nous avons révélé que la transcription basale d'ARNa n'exige pas ERα ou
l'histone méthyltransférase MLL1 qui active l'amplificateur par la mono-méthylation H3K4.
Dans l'ensemble, nos résultats ont montré que les ARNa peuvent jouer un rôle lors de
l'activation du promoteur, mais ne sont pas nécessaires pour maintenir la transcription de
l'ARNm ou pour stabiliser les interactions amplificateur-promoteur.Transcription is the initial step in gene expression and is subject to extensive regulation. Recently, analyses in diverse eukaryotes have revealed that in addition to protein coding genes, transcription occurs throughout the noncoding genome, producing non-coding RNAs of various lengths. Non-coding RNAs longer than 200 nucleotides, classified as long non-coding RNAs (lncRNAs), represent the most abundant class of non-coding transcripts, whose functions however are poorly understood. Recent studies suggest that many lncRNAs might have roles in transcription regulation. The goal of my PhD thesis was to elucidate the mechanisms of lncRNA mediated transcription regulation in different eukaryotic systems. For my first project, I investigated the role of an antisense long noncoding RNA in transcription regulation of the high-affinity phosphate transporter gene PHO84 in the unicellular eukaryote S. cerevisiae. Previous studies showed that deletion of the nuclear exosome component Rrp6 results in increased antisense expression and repression of PHO84. It was suggested that the loss of Rrp6 results in antisense stabilization at the PHO84 locus, leading to recruitment of the histone de-acetylase Hda1 and repression of PHO84. However, most of the mechanistic details of how Rrp6p functions in regulating PHO84 transcription were not understood. Combining single cell methods with biochemical and genetic approaches, we showed that antisense RNA levels are regulated primarily during transcriptional elongation by the Nrd1-Nab3-Sen1 complex, which requires Rrp6 for efficient recruitment to the 3’end of PHO84. Furthermore, we reveal anti-correlated expression of sense and antisense, which have distinct modes of transcription. In summary, our data suggest a model whereby antisense transcriptional read-through into the PHO84 promoter regulates the activation threshold of the gene. For my second project, I investigated the roles of enhancer derived RNAs (eRNAs). eRNAs are lncRNAs transcribed from enhancers that have been suggested to regulate transcription through different mechanisms, including enhancer-promoter looping, RNA polymerase elongation, and chromatin remodeling. However, no coherent model of eRNA function has yet emerged. Using MCF7 breast cancer cells as a model system, we sought to determine how estrogen (E2) induced eRNAs participate in estrogen receptor alpha (ERα) mediated transcription regulation at the single allele level. Using single molecule fluorescent in situ hybridization (smFISH), we revealed that upon E2 induction eRNAs are induced with similar kinetics as target mRNAs, but are localized exclusively in the nucleus, mostly chromatin associated, and are less abundant than mRNAs. Surprisingly, we found that eRNAs are rarely co-transcribed with their target loci, indicating that active gene transcription does not require the continuous synthesis or accumulation of eRNAs at the enhancer. Furthermore, using sub-diffraction-limit distance measurements, we demonstrated that co-transcription of eRNAs and mRNAs rarely occurs within a closed enhancer-promoter loop. Moreover, we revealed that basal eRNA transcription does not require ERα or the histone methyltransferase MLL1, which activates the enhancer through H3K4 mono-methylation. Altogether, our findings showed that eRNAs may play a role during promoter activation, but are not required to sustain mRNA transcription or stabilize enhancer-promoter looping interactions.
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Liu, Chun-Cheng, and 劉俊成. "An enhanced functional pathway annotation for RNA-seq differentially expressed gene clusters." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/ffct5z.
Full textAbstract:
碩士國立臺灣海洋大學
資訊工程學系
105
Biological pathway enrichment analysis is mainly applied to interpret correlated behaviors of activated gene clusters. In traditional approaches, significant pathways were highlighted based on hypergeometric distribution statistics and calculated corresponding P-values. However, two important factors are ignored for enrichment analysis, including fold-change levels of gene expression and gene locations on biological pathways. In addition, several reports have shown that noncoding RNAs could inhibit/activate target genes and seriously impact the results of over-representation analysis. Hence, in this study, we provided an alternative approach to enhance functional gene annotations. Different fold-change level, gene locations, and non-coding RNA associated genes will be considered simultaneously. By considering these additional factors, the ranking of significant P-values would be rearranged and several important and associated biological pathways could be successfully identified. To demonstrate superior performance, we used two experimental RNA-seq datasets as samples, including Birc5a and HIF2α knocked down in zebrafish during embryogenesis. Regarding Birc5a knock-down experiments, two biological pathways of Sphingolipid metabolism and Herpes simplex infection were additionally identified, for HIF2α knock-down experiments, four missed biological pathways could be re-identified including Ribosome biogenesis in eukaryotes, Proteasome, Purine metabolism, and Complement and coagulation cascades. Thus, a comprehensive enrichment analysis for discovering significant biological pathways could be overwhelmingly retrieved and it would provide integrated and suitable annotations for further biological experiments.
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王奕杰. "Enhancement of promoter activity of a rice RNA-binding protein gene by enhancer and intron." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/13445140356307618843.
Full textAbstract:
碩士國立屏東科技大學
生物科技研究所
91
Rice is grown worldwide as one of the most important staple food crops. Recently, expression of foreign proteins in rice by transgenic approaches has been actively pursued for future food security as well as for added values of rice. Promoters play a very significant role in controlling gene expression and protein accumulation during plant growth. It has been an important issue to identify promoters conferring high level expression of foreign proteins in transgenic rice. A1 gene encodes a rice glycine-rich RNA binding protein. A1(1800) promoter with 1800-bp length has been isolated. The A1(1800) promoter confers foreign gene expression in cultured rice suspension cells and seedlings. Deletion analysis has showed that deletion of the A1(1800) promoter to 540 bp, designated as the A1(540) promoter, maintained promoter activity similar to that of the full length A1(1800) promoter. To enhance activity of the A1(1800) and A1(540) promoters, the Ubiquitin intron（Ubi(In)）, Actin intron（Act(In)）, A1 intron (A1(In)) and the sugar response sequence（SRS）ofαAmy3 were inserted into the A1(1800) and A1(540) promoters. Luciferase gene was fused downstream of the modified promoters as a reporter. These constructs were delivered into rice embryos by particle bombardment and Agrobacterium-mediated transformation. The results from transient expression assays were consistent with those from stable transformation analysis. Luciferase activity was significantly higher when the A1(1800) and A1(540) promoters contained Ubi(In), Act(In) and A1(In), with the Ubi(In) conferring the highest activity. Insertion of SRS into the A1(1800) and A1(540) promoters also enhanced luciferase activity in a sugar-dependent manner. The enhancement was dependent on the position of SRS inserted relative to the TATA box within the A1(1800) promoter and the copy number of SRS. Our studies demonstrated that introns and SRS insertions significantly increase the activity of A1(1800) and A1(540) promoters. Future studies will be to determine whether SRS and introns confer temporal and spatial enhancemant of the A1(1800) promoter activity in transgenic rice.
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Nagarajan, Sankari. "Role of BRD4 and histone acetylation in estrogen receptor-positive breast cancers." Thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0023-9615-4.
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"Exploring Growth Essential Genes in E. Coli using Synthetic Small RNA to Enhance Production of Phenylalanine." Master's thesis, 2016. http://hdl.handle.net/2286/R.I.38412.
Full textAbstract:
abstract: Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.Dissertation/Thesis
Masters Thesis Chemical Engineering 2016
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He, Bicheng. "The role of Tc-foxQ2 in the central brain development in Tribolium castaneum." Doctoral thesis, 2018. http://hdl.handle.net/11858/00-1735-0000-002E-E56C-9.
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Löffler, Dennis, Katja Brocke-Heidrich, Gabriele Pfeifer, Claudia Stocsits, Jörg Hackermüller, Antje K. Kretzschmar, Renate Burger, et al. "Interleukin-6-dependent survival of multiple myeloma cells involves the Stat3-mediated induction of micro-RNA-21 through a highly conserved enhancer." 2007. https://ul.qucosa.de/id/qucosa%3A32128.
Full textAbstract:
Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.
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Boyne, J. R., B. R. Jackson, A. Taylor, S. A. Macnab, and A. Whitehouse. "Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs." 2010. http://hdl.handle.net/10454/5870.
Full textAbstract:
Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.
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Lin, Hang, and 林涵. "Engineering cytokinin-related genes to enhance cytokinin level and using RNA interference-based gene silencing of CASBENE SYNTHASE 1 for seed detoxification in Jatropha curcas L." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/26030320614148540826.
Full textAbstract:
碩士國立臺灣大學
植物科學研究所
102
Jatropha curcas L. is one of the best candidates for biodiesel production owing to high oil content of seeds. However, there are several disadvantages, such as low seed yields and phorbol esters toxicity of seed cake for feedstuff use. Previous studies indicated that cytokinin spraying was able to promote cell division, repressed apical dominance and led to increase seed yield of J. curcas. Our preliminary study revealed that exogenous application of 6-benzyladenine on inflorescence caused phenotypical changes of numerous flowers and higher seed yield. Previous studies have shown that ISOPENTENYLTRANSFERASE and CYTOKININ OXIDASE were two genes playing the major roles in controlling endogenous cytokinin level and seed yield in plants. In order to increase endogenous cytokinin level in J. curcas, we have constructed CKX5-RNAi and 35S::JcIPT1 vectors based on the spatial expression profile of JcIPTs and JcCKXs and transformed to J. curcas mediated by A. tumefaciens. Data revealed the JcIPT1 expression level in 35S::JcIPT1 transgenic line 15 is much higher than empty-vector transformed J. curcas. The 35S::JcIPT1 transgenic line 1 has higher cytokinin level than empty-vector transformed J. curcas. In parallel, the seed detoxification by engineering JcCAS1-RNAi is in progress. Casbene synthases (CASs) catalyze the first step of phorbol ester (PE) biosynthesis. PEs are the main toxic compounds in J. curcas. Our spatial expression profile of JcCAS1 indicated that JcCAS1 had higher expression level in roots and germination seeds but did not express in leaves. The expression level of JcCAS1 increased towards fruit maturity. Thus far two transgenic lines were obtained.