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Yu, Yi-Hsin Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Role of the RNAi pathway in influenza a virus infected mammalian cells." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41545.
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The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.
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Aitken, Amelia. "Blocking the RNA Interference Pathway Improves Oncolytic Virus Therapy." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36821.
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Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.
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Alagia, Adele. "Modulation of the RNAi pathway by chemically modified siRNA molecules." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/379307.
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To direct post-transcriptionally gene silencing, RNAi machinery exploits the formation of base pairs between the loaded guide strand and the complementary mRNA. The Ago2 protein is the “slicer” effector of the RISC and drives the endonucleolytic cleavage only when the siRNA guide strand is full paired with its RNA counterpart. Ago2 is able to incorporate a duplex siRNA molecule, unwinds the double helix and holds one strand while discarding the other. Ago2 bearing only the guide strand is defined “active” and can guide multiple cleavage reactions against the complementary mRNAs. Structural insights into Ago2 assembly process have speculated that early interactions between the siRNA and the Ago2 relies on specific recognition by the PAZ domain. Thus, proper PAZ domain recognition contributes to the specific and productive incorporation of siRNAs into the Ago2. Interactions between PAZ domain and siRNA molecule are essentially asymmetric. The guide strand with its 2-nt 3’overhang is involved in the majority of the contacts between the PAZ pocket and the siRNA, whereas the passenger strand interacts only with its 5’ end residue. In principle, overhang modifications (i.e. 2’-deoxy units) were just introduced to protect the RNA duplex integrity. Only after the understanding of the Ago2 architecture, overhang modifications were also harnessed to improve the siRNA potency and specificity. The comprehension of the PAZ lodging/dislodging motion during the formation of binary (Ago2 + guide) and ternary complex (Ago2 + guide + mRNA) pointed out the importance of adequate affinity between the guide overhang and the PAZ cleft during the Ago2 multi-turnover cleavage process. Affinity analysis on PAZ/siRNA overhang complex has proved the influence of the overhang presence for efficient binding. SiRNA duplexes with shorter overhang (1-nt) or blunt end have respectively highlighted 85-fold and >5000-fold reduced affinity. Hence, taking advantage of more efficient interactions between the PAZ pocket and the strand bearing the unpaired di-nucleotides structure, structural asymmetric siRNA molecules bearing only the antisense overhang were successful employed to bias the RISC strand selection. Moreover, competition between siRNAs, resulting in preferential incorporation of one siRNA type into the RISC machinery, is influenced by the distinct loading kinetics of siRNA molecules. Thus, the knockdown ability of siRNA mixtures is often compromised due to competition between siRNAs. It also has been reported that the simultaneous transfection of two or more siRNAs causes reduced silencing activity of one siRNA species whereas the potency of the other siRNAs were not affected. Even if siRNA competition effects are essentially produced by the interactions with the Ago2 protein, up to now, no available data about a specific Ago2 domain involvement into the siRNA competition have been described. We have been hypothesized that the PAZ domain, playing an important role in the first steps of the strand loading could be specifically involved in the siRNA competition. Given this background we are questioning how the di-nucleotide unpaired structure can influence the siRNA silencing efficiency and specificity. To explore the structural hallmarks critical for the PAZ pocket interaction, we modified the siRNA overhangs with several modifications. In detail, 2 units of β-L-nucleosides (mirror image L-Thymidine), 2’-deoxyribitol, GNA (glycerol nucleic acids)-Thymine and acyclic L-threoninol were introduced at overhang level and the silencing potency (IC50) was measured. Such modifications may provide fundamental clues on structural prerequisite needed for the PAZ recognition and strand loading into the Ago2.Para dirigir el silenciamiento génico post-transcripcional, la maquinaria de RNAi explota la formación de pares de bases entre la hebra guía cargado y el ARNm complementario. La proteína Ago2 (Argonauta 2) es la "máquina de cortar" del complejo RISC y dirige la rotura endonucleolítica sólo cuando la hebra guía del siRNA está completamente apareada con su homóloga de ARN. Ago2 es capaz de incorporar una molécula de dúplex de siRNA, desenrolla la doble hélice y mantiene una hebra mientras se descarta la otra cadena. Ago2 cargada con la hebra guía se define "activa" y puede guiar múltiples reacciones de escisión contra los ARNm complementarios. El análisis estructural del proceso de ensamblaje de Ago2 ha llevado a la conclusión de que las primeras interacciones entre el siRNA y la proteina Ago2 se basa en el reconocimiento específico por el dominio PAZ. Por lo tanto, el correcto reconocimiento de dominio PAZ contribuye a la incorporación específica y productiva de los siRNAs en el Ago2. La hebra guía con su extremo 3' protuberante que tiene 2-nt está implicada en la mayoría de los contactos entre la cavidad presente en el dominio PAZ. En principio, las modificaciones en los extremos protuberantes se introdujeron para proteger la integridad del dúplex de ARN. Sólo después de la comprensión de la arquitectura de Ago2, se pensó en la utilización de las modificaciones en los extremos protuberantes para mejorar la potencia y especificidad de los siRNAs. Para explorar las características estructurales críticas para la interacción entre la cavidad PAZ, modificamos los extremos protuberantes de los siRNA con varias modificaciones. Específicamente, 2 unidades de un beta-L-nucleósido como la L-timidina (imagen especular de la timidina), de 2'-desoxiribitol, de GNA (glycerol nucleic acids)- timina y del derivado acíclico L-treoninol se introdujeron a los extremos protuberantes y se midió la potencia de silenciamiento (IC50). Tales modificaciones pueden proporcionar pistas fundamentales sobre el requisito estructural necesario para el reconocimiento y carga de la cadena del dominio PAZ de Ago2.
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Nord, Dianna M. "Knockdown of the Yes-associated Protein 1 pathway provides a basis for targeted therapy to treat infantile hemangioma." Thesis, Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/53736.
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Hemangioma is a type of tumor commonly found in infants that is characterized by heavy vascularization and a disfiguring appearance. Hemangioma, though benign, can sometimes proliferate and be threatening to infants. Current treatments for infantile hemangioma include surgical removal as well as the use of topical and oral medication. However, current therapies are often ineffective at treating lesions and are commonly accompanied by dangerous side effects, creating the need for a new, safer treatment. This study targets the Yes-Associated Protein-1 (YAP-1), which has been described as an oncogene, by use of an interfering RNA technique in attempts to mediate tumor growth and progression. Western blotting of treatment and control BEND3 murine cells reveals that YAP-1 is knocked-down in treatment groups which have been infected with shYAP-1 siRNA genes. By successfully knocking down the YAP-1 protein, the potential for developing a novel targeted therapy for infantile hemangioma has been established.
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Roy, Matthew Stephen. "Development and application of a high-throughput RNAi screen to reveal novel components of the DNA sensing pathway." Thesis, Harvard University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3567049.
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The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures.
Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called 'Interferon Stimulatory DNA' or 'ISD') remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA.
Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA-binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins.
We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA.
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Kingham, Guy L. "Screening for inhibitors of and novel proteins within the homologous recombination DNA repair pathway." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:e2988d0b-c6d4-42a8-aef9-f320a13d6391.
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The homologous recombination (HR) pathway of DNA repair is essential for the faithful repair of double-stranded DNA breaks (DSBs) in all organisms and as such helps maintain genomic stability. Furthermore, HR is instrumental in the cellular response to exogenous DNA damaging agents such as those used in the clinic for chemo- and radiotherapy. HR in humans is a complex, incompletely understood process involving numerous stages and diverse biochemical activities. Advancing our knowledge of the HR pathway in humans aids the understanding of how chemo- and radiotherapies act and may be used to develop novel therapeutic strategies. Recent studies have identified inhibition of HR as one of the mechanisms via which a number of recently developed chemotherapeutics have their effect. Accordingly, the clinical potential of HR inhibitors is under investigation. My work has centred around the identification of both novel HR proteins and novel, small molecule HR inhibitors. To further these aims, I have successfully employed high-throughput RNAi and small molecule screening strategies. RNAi screens are commonly used to identify genes involved in a given cellular process via genetic loss of function, whilst small molecule, cell based screens are a powerful tool in the drug discovery process.
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Laxman, Navya. "miRNA and Asymmetric siRNA : Small RNAs with Large Effects on Bone Metabolism." Doctoral thesis, Uppsala universitet, Endokrinologi och mineralmetabolism, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264451.
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RNA interference (RNAi) is a post-transcriptional gene silencing process elicited by double-stranded RNA, such as micro-RNA (miRNA) and small interfering RNA (siRNA). They are 18-25 nucleotide long, small non-coding RNAs acting as critical regulators in eukaryotic genome expression. They play an important role in regulating a wide range of biological processes such as cell cycle control, differentiation, aging and apoptosis. However, their role in supporting skeletal development and bone homeostasis is still poorly understood. Osteoporotic fractures constitute a tremendous and growing problem in our ageing populations, with an annual incidence of approximately 60000 osteoporotic fractures in Sweden. Osteoporosis is referred as the “Silent epidemic” because bone loss is gradual and a basically symptomless development until a fracture occurs. Results presented in this thesis provide a novel insight into crucial roles of miRNAs in regulating bone homeostasis. The initial aim for the thesis was to perform global miRNA expression profiling in human bone cells, and to correlate these levels to global mRNA levels. We identified and functionally characterized several miRNAs that were differentially expressed and acted in important bone signaling pathways such as the Wnt and BMP pathways. These miRNAs included hsa-miR-29b, hsa-miR-30c2 and hsa-miR-125b, which we found targeting genes highly relevant to bone metabolism e.g. COL1A1, SPARC, RUNX2, BGLAP and FRZB. Thereafter, the effect on the microRNAome upon external stimuli (e.g., Dexamethasone and Parathyroid hormone) was assessed by SOLiD sequencing. We observed a substantial difference in the expression of miRNAs between PTH and DEX treated cells. Understanding the changes in miRNAome in human bone cells under different conditions could provide new insight in bone remodeling, specifically differentiation and functional properties of osteoblasts. Based on these studies, we furthermore identified Dlx5 as potential common target of miR-203 and miR-320b and these miRNAs negatively regulate BMP-2-induced osteoblast differentiation. To activate the RNAi pathway, siRNA or miRNA molecules must be conveyed into the cytoplasm of target cells. Since challenges in cellular delivery of these small silencing RNA molecules so far have limited their clinical utility, we developed a new siRNA design that demonstrates a novel carrier-free cellular delivery. This development could potentially have a major impact in RNAi therapeutics. In conclusion, this thesis provides novel insight of miRNAs that play a major role in the regulation of bone remodeling and differentiation and functional properties of osteoblasts. Our findings may have diagnostic and/or therapeutic implications in disorders of bone metabolism.
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Zwirek, Monika. "Improving barley for biofuel production : investigating the role of 4CL and CCR in the lignin biosynthesis pathway." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/6785dbbb-f8a4-46f1-b7c4-0c3d0d4dcdd4.
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One of the challenges in the 21st Century is to overcome the recalcitrance of lignocellulose for the production of liquid biofuels. Lignin is one of the key factors in this recalcitrance. Grasses such as Miscanthus and switchgrass could become major sources of lignocellulose. Barley has potential as a genetically-tractable research model for such novel bioenergy crops and also as a bioenergy crop itself. This thesis concerns the 4CL and the CCR enzymes on the lignin pathway which were chosen as the targets to manipulate lignin in barley. They were selected because there is evidence that suppression of each of them in dicot species can lead to increased saccharification. The 4CL and CCR genes constitute multigene families where members have different expression patterns. RNAi was used to down-regulate 4CL1 and CCR1 using a constitutive promoter via Agrobacterium-mediated transformation of barley. From an extensive screen of the primary transformants for changes in protein level and lignin content, six CCR and four 4CL lines were taken forward for detailed analysis. Antibodies were also raised against barley 4CL and CCR recombinant proteins and these showed substantial reductions in the respective target protein levels in the RNAi lines. Both 4CL and CCR transgenic lines had significant reductions in lignin content, and CCR lines had changes in lignin structure due to changes in the proportions of acid soluble and acid insoluble lignin. No substantial consistent adverse effects on key agronomic traits were apparent in the 4CL and CCR transgenics. Selected 4CL and CCR transgenics had improved saccharification yield after using three different pretreatment methods, which is a desirable feature for biofuel production.
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Kaimoyo, Evans. "Study of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolites." Tucson, Arizona : University of Arizona, 2006. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1434%5F1%5Fm.pdf&type=application/pdf.
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Kaimoyo, Evans. "Study of the (+)-Pisatin Biosynthetic Pathway by RNAi and Development of a Novel Method to Elicit the Production of Plant Secondary Metabolites." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/193608.
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(+)-Pisatin, ([+]-[6aR,11aR]-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan) is the major phytoalexin of the garden pea (Pisum sativum L.). Despite being the first phytoalexin to be chemically characterized, its biosynthesis remains to be fully elucidated. RNA-mediated genetic interference (RNAi) was used to gain further insights into the (+)-pisatin biosynthetic pathway. The expression of three genes, isoflavone reductase (IFR) catalyzing the reduction of 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone to (-)-sophorol, sophorol reductase (SOR) involved in reducing (-)-sophorol to (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol and hydroxymaackiain-3-O methyltransferase (HMM) involved in methylation of (+)-6a-hydroxymaackiain to (+)-pisatin was silenced. The genes are transcriptionally co-regulated during (+)-pisatin biosynthesis, with the IFR and SOR proposed to function upstream of the HMM gene.Hairy roots expressing the HMM RNAi construct, deficient in (+)-pisatin biosynthesis were identified. However, these did not accumulate (+)-6a-hydroxymaackiain, precursor to (+)-pisatin. Instead they accumulated 2,7,4'-trihydroxyisoflavanone, daidzein, liquiritigenin and isoformononetin. The amino acid sequence of HMM is very similar to that of another methyltransferase, hydroxyisoflavanone-4'-O-methyltransferase (HI4MOT), found in most legumes. HI4?MOT catalyzes the methylation of 2,7,4'-trihydroxyisoflavanone (THI) to 2,7-dihydroxy-4'-methoxyisoflavanone, one of the earliest enzymatic steps in isoflavonoid biosynthesis. In pea, HI4OMT may be the same enzyme as "HMM" catalyzing the methylation of both THI and (+)-6a-hydroxymaackiain. Preventing the methylation of THI could divert pea intermediates to the production of daidzein and isoformononetin instead of (+)-pisatin.None of the transgenic hairy roots expressing the IFR RNAi construct were totally deficient in (+)-pisatin biosynthesis. However, all produced reduced amounts of (+)-pisatin, with one culture accumulating 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone, the substrate for IFR. Hairy roots expressing the SOR RNAi construct deficient in (+)-pisatin biosynthesis were identified. These accumulated (-)-sophorol, the substrate for SOR. These data provide evidence for the involvement of these genes and the intermediates with (-)-optical activity in (+)-pisatin biosynthesis.The elicitation of the biosynthesis of secondary metabolites in plant cell and tissue cultures by electric current was explored. Electric current was demonstrated to elicit the biosynthesis of secondary metabolites in pea hairy and intact roots, seedling, root and cell suspension cultures of various plant species. Electric current has potential for use as an elicitor of secondary metabolites in basic and commercial research ventures.
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Guida, Claudia [Verfasser], and Martina [Akademischer Betreuer] Muckenthaler. "An RNAi screen identifies TLR2/6 as mediators of a novel inflammatory pathway for rapid hepcidin-independent hypoferremia / Claudia Guida ; Betreuer: Martina Muckenthaler." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180300475/34.
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Xu, Ning. "Adenoviral Control of RNAi/miRNA Pathways in Human Cells." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distribution], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9387.
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Dana, Foss. "Developing the P19 Protein as a Tool for Studying the RNA Silencing Pathway." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36122.
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RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes.
The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.
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Ayaz, Eyup Serdar. "Resonctructing Signaling Pathways From Rnai Data Using Genetic Algorithms." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613813/index.pdf.
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Cell signaling is a list of chemical reactions that are used for intercellular and intracellular communication. Signaling pathways denote these chemical reactions in a systematic manner. Today, many signaling pathways are constructed by several experimental methods. However there are still many communication skills of cells that are needed to be discovered. RNAi system allows us to see the phenotypes when some genes are removed from living cells. By observing these phenotypes, we can build signaling pathways. However it is costly in terms of time and space complexity. Furthermore, there are some interactions RNAi data cannot distinguish that results in many different signaling pathways all of which are consistent with the RNAi data. In this thesis, we combine genetic algorithms with some greedy approaches to find the topologies that fit the Boolean single knock-down RNAi experiments. Our algorithm finds nearly all of the results for small inputs in a few minutes. It can also find a significant number of results for larger inputs. Then we eliminate isomorphic topologies from the output set of this algorithm. This process fairly reduces the number of topologies. Afterwards we offer a simple scheme for suggesting new wet-lab RNAi experiments which is necessary to have a complete approach to find the actual network. Also we describe a new activation and deactivation model for pathways when the activation of the phenotype after RNAi knock-downs are given as weighted variables. We adapt the first genetic algorithm approach to this model for directly finding the most possible network.
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Hachoumi, Mounia el. "Die Funktion von Bx42/Skip im TGF-beta/Dpp Signal Transduktionsweg." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2007. http://dx.doi.org/10.18452/15635.
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Die Notwendigkeit von Bx42 für Drosophila Entwicklung und seine Beteiligung an unterschiedlichen zellulären Prozessen wurde mit Hilfe von RNA Interference (RNAi) demonstriert. Das ubiquitäre Ausschalten oder die Reduktion der Bx42 Expression mittels RNAi führte dabei zu embryonaler Letalität. Weiterhin führte eine gewebespezifische Induktion von Bx42 in Abhängigkeit der verwendeten Treiberlinien bei unterschiedlichen Temperaturen zu mehreren verschiedenen adulten Phänotypen. Diese Phänotypen waren die Grundlage für die Annahme, dass Bx42 eine Rolle in der Regulation mehrerer verschiedener Zellsignalwege spielt. In der Tat interagiert Bx42 mit den Proteinen des Notch-Signalweges Suppressor of hairless [Su(H)] und Notch intracellular domain (N-IC). Zusätzlich werden bei einer Verminderung von Bx42 die Notch Zielgene cut (ct) und enhancer of split m8 [e(Spl)m8] reprimiert (Negeri et al., 2002). In dieser Arbeit wurde die Beteiligung von Bx42 am TGF-ß/Dpp Signalweg untersucht. Es wurde gezeigt, dass Bx42 mit den TGF-ß/Dpp-Signalweg Proteinen Mad und Medea sowohl in vitro als auch in vivo interagiert. Die dabei verwendeten Methoden waren das Hefe-Zwei Hybrid-Sytem und Ni-NTA-Pulldown-Assays. Domänen der Smad Proteine (Mad und Medea), die für die Interaktion mit Bx42 notwendig sind, wurden mit Hilfe von Deletionskonstrukten untersucht. Es konnte gezeigt werden, dass die stark konservierte MH2-Domäne dieser Proteine für die Interaktion notwendig ist. Zudem belegten Versuche die genetische Interaktion zwischen Bx42 und Medea, in denen ein Bx42-RNAi-Phänotyp durch die gleichzeitige Überexpression von Medea gerettet werden konnte. Es ist bekannt, dass das humane Bx42-Homolog Skip sowohl mit den Proteinen Smad2 und 3 des TGF-ß/Activin Signalweg, als auch mit den Onkogenen Sno und Ski interagiert. Skip wirkt hier als Antagonist der Ski/Sno-Wirkung auf den TGF-ß/Activin-Signalweg und fungiert als Koaktivator (Leong et al., 2001). Die Interaktion zwischen Bx42 und der TGF-ß/Activin-Signalweg Komponente dSmad2, sowie mit dem Onkogen dSno konnte in dieser Arbeit auch für Drosophila bewiesen werden. Die Bedeutung dieser Wechselwirkung muss noch in weiteren Arbeiten analysiert werden. Der Einfluss der Bx42-RNAi-Induktion auf die TGF-ß/Dpp Zielgene distal-less (dll), optomotor blind (omb) und spalt (sal) wurde anhand von Reportergen Untersuchungen mit enhancer-trap-Linien und RNA in situ Hybridisierung untersucht. Es konnte gezeigt werden, dass das Ausschalten von Bx42 die Expression dieser Gene in ähnlicher Weise reprimiert, wie eine Elimination des TGF-ß/Dpp-Signals. Diese Ergebnisse unterstützen die Annahme, dass Bx42 in der Lage ist, TGF-ß/Dpp Zielgene durch eine Wechselwirkung mit Mad und Medea zu aktivieren.The importance of Bx42 in Drosophila development was demonstrated using Bx42-RNA interference. The ubiquitous downregulation of Bx42 generated embryonic lethality, indicating the importance of this protein in early development. The tissue specific induction of Bx42-RNAi resulted in several different phenotypes depending on the driver line and the temperature at which animals were raised. The phenotypes obtained were the key point for the assumption that Bx42 may play a role in the regulation of a number of different cellular signalling pathways. Indeed, within the Notch signalling pathway Bx42 interacts genetically with Suppressor of hairless [Su(H)] and Notch intracellular domain (N-IC). Additionally, the reduction of Bx42 negatively affected the expression of the Notch target Genes cut (ct) and enhancer of split m8 [e(Spl)m8] (Negeri et al., 2002). In this work, the involvement of Bx42 in the Dpp signalling pathway was investigated. It was shown that Bx42 interacts both in vitro and in vivo, as demonstrated by yeast two hybrid protein-protein studies and Ni-NTA pull-down assays, with the TGF-ß/ Dpp components Mad and Medea. Domains of Smads (Mad and Medea) required for Bx42 interaction were examined using deletion constructs of Smads and the importance of the well conserved MH2 domains of Mad and Medea for this interaction was revealed. Moreover, the rescue of the Bx42-RNAi phenotype by the simultaneous overexpression of Medea demonstrated the genetic interaction between Bx42 and Medea. Furthermore, evidences for the interaction of Bx42 with the TGF-ß/Activin pathway component dSmad2 and with the oncogene protein dSno were obtained from interaction assays. The human homologue of Bx42, Skip, also interacts with Smad2/3 or Sno. The meaning of this interaction in Drosophila has yet to be analysed. The influence of Bx42-RNAi induction on the expression of Dpp target genes distal less (dll), optomotor blind (omb) and spalt (sal) was also investigated using enhancer trap lines and RNA in situ hybridisation. In this way it was proven that these genes are suppressed as they are by elimination of Dpp signalling. These results suggest that Bx42 may be able to modulate positively TGF-ß/Dpp signalling through an interaction with the signalling transducer Mad and Medea.
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Raza, Sobia. "Modelling and analysis of macrophage activation pathways." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5898.
Full textAbstract:
Macrophages are present in virtually all tissues and account for approximately 10% of all body mass. Although classically credited as the scavenger cells of innate immune system, ridding a host of pathogenic material and cellular debris though their phagocytic function, macrophages also play a crucial role in embryogenesis, homeostasis, and inflammation. De-regulation of macrophage function is therefore implicated in the progression of many disease states including cancer, arthritis, and atherosclerosis to name just a few. The diverse range of activities of this cell can be attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its physiology depending on its environment; for instance in response to different types of pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by the macrophages capacity to adjust rapidly its transcriptional profile in response to a given stimulus. This includes interferons which are a group of cytokines capable of activating the macrophage by interacting with their cognate receptors on the cell. The different classes of interferons activate downstream signalling cascades, eventually leading to the expression (as well as repression) of hundreds of genes. To begin to fully understand the properties of a dynamic cell such as the macrophage arguably requires a holistic appreciation of its constituents and their interactions. Systems biology investigations aim to escape from a gene-centric view of biological systems. As such this necessitates the development of better ways to order, display, mine and analyse biological information, from our knowledge of protein interactions and the systems they form, to the output of high throughput technologies. The primary objectives of this research were to further characterise the signalling mechanisms driving macrophages activation, especially in response to type-I and type- II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to data analysis and modelling. In order to achieve this end I have explored and developed methods for the executing a ‘systems-level’ analysis. Specifically the questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the similarities and differences between the macrophage response to different types of interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the macrophage transcriptome affected by siRNA targeting of key regulators of the interferon pathway? (d) To what extent does a model of macrophage signalling aid interpretation of the data generated from functional genomics screens? There is general agreement amongst biologists about the need for high-quality pathway diagrams and a method to formalize the way biological pathways are depicted. In an effort to better understand the molecular networks that underpin macrophage activation an in-silico model or ‘map’ of relevant pathways was constructed by extracting information from published literature describing the interactions of individual constituents of this cell and the processes they modulate (Chapter-2). During its construction process many challenges of converting pathway knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be addressed. The final model comprised 2,170 components connected by 2,553 edges, and is to date the most comprehensive formalised model of macrophage signalling. Nevertheless this still represents just a modest body of knowledge on the cell. Related to the pathway modelling efforts was the need for standardising the graphical depiction of biology in order to achieve these ends. The methods for implementing this and agreeing a ‘standard’ has been the subject of some debate. Described herein (in Chapter-3) is the development of one graphical notation system for biology the modified Edinburgh Pathway Notation (mEPN). By constructing the model of macrophage signalling it has been possible to test and extensively refine the original notation into an intuitive, yet flexible scheme capable of describing a range of biological concepts. The hope is that the mEPN development work will contribute to the on-going community effort to develop and agree a standard for depicting pathways and the published version will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse macrophages to interferon stimulation, genome wide expression profiling was performed and an explorative-network based method applied for analysing the data generated (Chapter-4). Although transcriptomics data pertaining to interferon stimulation of macrophages is not entirely novel, the network based analysis of it provided an alternative approach to visualise, mine and interpret the output. The analysis revealed overlap in the transcriptional targets of the two classes of interferon, as well as processes preferentially induced by either cytokine; for example MHC-Class II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by IFN-β. To further investigate the contribution of individual proteins towards generating the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress the expression of selected target genes. However in macrophages and other cells equipped with pathogen detection systems the act of siRNA trasfection can itself induce a type-I interferon response. It was therefore necessary to contend with this autocrine production of IFN-β and optimise an in vitro assay for studying the contribution of siRNA induced gene-knock downs to the interferon response (described in Chapter-5). The final assay design incorporated LPS stimulation of the macrophages, as a means of inducing IFN-β autonomously of the transfection induced type-I response. However genome-wide expression analysis indicated the targeted gene knock-downs did not perturb the LPS response in macrophages on this occasion. The optimisation process underscored the complexities of performing siRNA gene knockdown studies in primary macrophages. Furthermore a more thorough understanding of the transcriptional response of macrophages to stimulation by interferon or by LPS was required. Therefore the final investigations of this thesis (Chapter-6) explore the transcriptional changes over a 24 hour time-course of macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage pathway model in interpreting the response to the three stimuli. Taken together the work described in this thesis highlight the advances to be made from a systems-based approach to visualisation, modelling and analysis of macrophage signalling.
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Jagannath, Aarti. "Studies on the RNA interference pathway in mammalian cells." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711608.
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Au, Lauren. "Evolutionary Patterns of small RNA Pathway Genes in Hymenopteran Insects." Scholarship @ Claremont, 2019. https://scholarship.claremont.edu/scripps_theses/1342.
Full textAbstract:
Small RNAs are short noncoding RNA sequences of 20-30 nucleotides. There are three classes of small RNAs: small interfering RNA (siRNA), microRNA (miRNA), and Piwi- interacting RNA (piRNA). There is a comprehensive understanding of the small RNA pathways in D. melanogaster and how these pathways function, due to D. melanogaster being a widely studied model organism. From studies of the small RNA pathways in D. melanogaster, it is known that the small RNA pathways are extremely important in defending the host genome against viruses and retrotransposons and regulating target genes. However, very little is known about small RNA pathways outside of D. melanogaster and whether or not small RNA pathways are highly conserved in distantly related insects. The objective of this study was to identify the small RNA pathway genes in representative hymenopteran insects and determine the level of conservation across this diverse group. By BLAST searching small RNA pathway proteins against the genomes of five bees, two wasps, three ants, and two sawflies, I found there are more small RNA pathway genes present in hymenopteran insects than in D. melanogaster. By constructing Bayesian gene trees using BEAST v1.10.4 to determine how the identified homologs are related, I found that the piRNA pathway genes Piwi and Aubergine have undergone dramatic duplication in most hymenopteran lineages, whereas AGO1, AGO2, and especially AGO3 are less dynamic in expansion. Finally, alignment of small RNA protein sequences revealed large-scale length differences between certain genes, suggesting functional diversification of these genes.
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Lee, Mark N. "Genomic Approaches to Dissect Innate Immune Pathways." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10692.
Full textAbstract:
The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.
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Rogozina, Anastasia. "The pathway to transcriptionally active Escherichia coli RNAP-T7A1 promoter complex formation." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-110852.
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Hale, Jared J. "Transfer RNA nuclear export pathways in Saccharomyces cerevisiae." Connect to resource, 2009. http://hdl.handle.net/1811/37270.
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North, Melanie. "The pathway taken by 5S RNA in oocytes of Xenopus laevis." Thesis, University of Canterbury. Microbiology, 1996. http://hdl.handle.net/10092/6148.
Full textAbstract:
In Xenopus oocytes, 5S ribosomal RNA (5S RNA) is synthesised prior to other ribosomal components, and stored in the cytoplasm in ribonucleoprotein particles (RNPs). At vitellogenesis, when ribosome assembly begins, 5S RNA is imported into the nucleus and targeted to the amplified nucleoli for assembly into the 60S ribosomal subunit. In this thesis, I have investigated some of the steps of this pathway taken by 5S RNA.
In vivo assays using a series of mutant 5S RNAs revealed that only one mutant, with base substitutions in loop A, was defective for binding the 7S RNP storage protein, TFIIIA. All mutants were capable of binding to ribosomal protein L5, a precursor to ribosome assembly. Four of the mutants tested were defective for incorporation into 60S subunits, possibly due to a loss of recognition sites for
interactions with other ribosomal proteins. Nucleolar localisation studies showed that the defective ribosome incorporation of these mutants was not due to defective nucleolar targeting. Taken together, these results reveal that different structural features of 5S RNA and different oocyte factors are required for different steps in the pathway taken by 5S RNA.
Nucleolar localisation studies also revealed that a large proportion of oocyte-type 5S RNA and L5 in the nucleus are not associated with nucleoli. In contrast, somatic-type 5S RNA was predominantly asssociated with nucleoli, suggesting that nuclear factors directly recognise the sequence differences between the two types of 5S RNA. These could be nucleolar components, which have a higher affinity for somatic-type, or nucleoplasmic factors which have a higher affinity for oocyte-type.
Finally, the mechanism by which 7S RNPs are sequestered in the cytoplasm of previtellogenic oocytes was investigated. The results show that neither cytoskeletal or membrane structures are responsible for cytoplasmic retention. Other possibilities for cytoplasmic retention are discussed.
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Haac, Mary Etna Richter. "Genetic factors affecting the RNA interference pathway of Aedes aegypti mosquitoes." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/53506.
Full textAbstract:
Aedes aegypti mosquitoes are the vectors of many significant arboviruses that cause
tremendous social and economic impact. RNA interference (RNAi) plays a crucial role in the
vector competence of mosquitoes and is often targeted in studies involving mosquito innate
immunity, genetics-based vector control strategies, and the development of viral-resistant
transgenic mosquitoes. In general, RNA interference is induced by double stranded RNA
(dsRNA) and results in the inhibition of cognate gene expression. There are several different
RNA interference pathways, with distinct functions and mechanisms. The micro RNA pathway
is important for endogenous gene regulation and development. The endogenous small
interfering RNA (endo-siRNA) pathway functions in gene regulation and protection of the
genome from the deleterious effects of transposable elements. The exogenous siRNA (exosiRNA)
pathway is a major contributor to mosquito innate immunity and vector competence by
limiting viral replication during infection. Lastly, the piwi RNA (piRNA) pathway primarily
functions in protecting the genome from the deleterious effects of transposable elements.
While the structure and function of many genes involved in Drosophila RNAi have been
characterized, the corresponding mosquito orthologs have only been peripherally described or
remain unknown. Thus, the overall purpose of this study is to improve the understanding of
mosquito RNAi mechanisms by identifying and analyzing genetic factors involved in the various
pathways. This research especially focuses on characterizing and analyzing putative doubleiii
stranded RNA binding proteins (dsRBPs) important to the function of the RNAi initiator and
effector complexes. Two genes, r2d2 and r3d1 are orthologs of Drosophila genes known to
have important roles in the RNAi initiator complex. A third member of the same family, which
we refer to as extra loquacious (exloqs), appears to have no known orthologs outside of the
Aedes genus. Structural characterization of these genes included identification of single
nucleotide polymorphisms (SNPs), novel exons and alternative splice variants. RT-PCR assays
were utilized to examine differential expression of all three genes in specific tissues and
developmental stages. Sub-cellular fractionation assays enabled intracellular localization of the
RNAi proteins within Ae. aegypti cells. Co-immunoprecipitation of tagged dsRBPs revealed
protein-protein interactions between specific dsRBPs and known RNAi factors. In addition, an
exo-siRNA sensor was designed and tested in-vivo and in-vitro with the purpose of facilitating
the identification of novel genetic factors involved in this anti-viral pathway. Lastly, TALENbased
gene disruption was successfully employed to knockout the exloqs gene in Ae. aegypti
mosquitoes, enabling further analysis into the function of this gene. The research described in
this document provides further insight into mosquito innate immunity and gene regulation, which
is important to the advancement of genetics-based vector control strategies.Ph. D.
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Mankin, Danielle N. "MC3R and MC4R Knockdown via RNA Interference." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/37.
Full textAbstract:
Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.
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Liu, Ming-lai, and 廖明麗. "Roles of microRNAs in hepatocellular carcinoma: biomarkers, matabolisms and pathway regulators." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46918929.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
Full textAbstract:
The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Weick, Eva-Maria. "Genetic and functional characterisation of piRNA pathway factors in Caenorhabditis elegans." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708028.
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Freidhoff, Paul. "Using RNA Mimicry of Viroids to Uncover New Noncoding RNA Structural Motifs and Pathways." Thesis, University of the Sciences in Philadelphia, 2020. http://pqdtopen.proquest.com/#viewpdf?dispub=27665966.
Full textAbstract:
There is broad interest in ncRNAs within technology. The functions of ncRNAs are linked to RNA structural elements, but most of the structure-function relationships of ncRNAs are unknown, which limits the use RNA in technology. Viroids are the first ncRNAs observed. They are unique pathogens because they exclusively hijack ncRNA pathways by mimicking structures found in cellular RNAs. This makes viroids excellent models to study structural-functional relationships of cellular ncRNAs. In this research, we studied a structural element, the sarcin/ricin domain motif, found in PSTVd. This structural element is important in replication and processing, but the same structural element was not found in all viroids belonging in the Pospiviroidae family (a family of biochemically related viroids) using traditional methods including secondary structure prediction. Viroids in the Pospiviroidae utilize similar biochemical pathways including replication and RNA processing pathways. Furthermore, they utilize similar cellular factors in the processing and replication pathways so we have hypothesized that every Pospiviroidae forms a similar structural element like an SRD. We used computational methods including alignment, molecular dynamics, and RNA footprinting to show that every Pospiviroidae could form an SRD-like motif. The work also suggests that there could be undiscovered subsets of SRD mimics. The knowledge of conserved structural elements in RNA processing could be applied in RNA technology. In addition to structural analysis, our work resulted in the development of new analysis methods. These included an enrichment method to remove large RNAs from an RNA mixture without the removal of small to moderate sized RNAs. We have also progressed in developing a new cell-based RNA assay which will analyze the effects of cellular biochemical factors on viroid processing.
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Du, Tingting. "Dissecting Small RNA Loading Pathway in Drosophila melanogaster: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/356.
Full textAbstract:
In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type.
Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways.
Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.
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Durovic, Peter Vincent. "Characterisation of a novel pathway for ribosomal RNA maturation in Sulfolobus acidocaldarius." Thesis, University of British Columbia, 1993. http://hdl.handle.net/2429/41498.
Full textAbstract:
Since the initial proposition that the archaebacteria form a primary kingdom as
distinct as that of the eubacteria or the eukaryotes, sequence data generated from the
ribosomal RNA genes have flooded the databases and periodicals. Phylogenetic trees based on
these sequences have been constructed to map the finest details of topology and branching
order within the archaebacteria. Yet, despite the plethora of sequence data, relatively little was
discovered regarding rRNAgene regulation, transcript processing and requirements for mature
ribosome function. The aim of this study is to analyze possible novel regulatory mechanisms
in the rRNA genes of the extremely thermoacidophilic archaebacterium Sulfolobus
acidoccddccrius.
The three ribosomal RNA genes were cloned and sequenced. The gene organization was
confirmed to differ from that of the halophilic archaebacteria and the eubacteria: the 5S gene
was not linked to the 16S and 23S operon, and the operon lacked recognizable tRNA
sequences. Southern hybridization unveiled, and sequence data confirmed a long-standing
confusion regarding species identity. The previously published Sulfolobus acidocaldarius 5S
sequence was shown to have been attributed to the wrong species.
Mapping experiments showed that both transcripts initiated downstream of a
previously defined archaebacteria! promoter sequence. While sequence data showed the 5S
transcript start site and end site to be coincidental with the mature 5S termini, the 16S-23S
transcript was shown to contain a 143 nucleotide transcribed leader sequence, a 138
nucleotide intergenic sequence, and a trailer sequence of at least 105 nucleotides. Inverted
repeat sequences within these transcribed non-coding regions allow for the formation of
numerous stem-loops conforming to a semi-conserved archaebacterial structure. While no
processing took place within the 5S transcript, extensive processing of the 16S-23S transcript
was observed. Of the 12 processing sites mapped, only 6 could be accounted for in the context
of precursor processing and maturation events known directly or inferred by analogy from the
halophilic archaebacteria and the eubacteria. Alignment of the remaining sites revealed a
non-trivial sequence and structural similarity. If the novel processing indeed took place in the postulated context, it would mark a
radical departure from the expected maturation mechanism thought to predate the speciation
of archaebacteria and eubacteria. To examine this possibility, in vitro transcripts from
judiciously selected DNA fragments were subjected to cell-free extract. Analysis of the resultant
cleavage products confirmed the presence not only of a novel processing activity mediated by a
ribonucleoprotein complex but also of a novel processing pathway. Based on the locations of
the novel processing sites within the primary 16S-23S transcript, a model for transcriptional
regulation independent of polycistronic linkage is presented.Medicine, Faculty of
Medical Genetics, Department of
Graduate
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Phillips, Jack O. "RNA interaction studies : a pathway to the development of a surface-based technology." Thesis, University of Portsmouth, 2016. https://researchportal.port.ac.uk/portal/en/theses/rna-interaction-studies(0618a5e7-95d6-4d8e-9850-b9b6de8c5a70).html.
Full textAbstract:
Ribonucleic acid (RNA) has long been thought of as the bearer and the machinery of translating the genetic code. It is now known that RNA is capable of diverse functions both in a cellular and in a synthetic context which have highlighted RNA as an important biological molecule to be studied and as a potential toolkit for the researcher. Research into RNA has uncovered a wealth of subgroups of RNA with different functions including the bacterial small non-coding RNAs (sRNAs). These sRNAs and other RNA subgroups are now known to be key cellular regulators of important activities such as virulence and stress response and thus emphasise their importance in antibiotic research. Many RNAs regulate their function and the function of other biomolecules by specific interactions with other RNAs, proteins, small molecules and even ions. However, identifying specific meaningful RNA interactions and their consequences is troublesome and methods to study these interactions are slow and laborious. This work has focused on developing protocols for applying existing and novel techniques to studying important RNA interactions which has led to the invention of a new technology for creating functional multi RNA arrays. Initial work employed structural and binding analyses to investigate the details of three importants RNA interactions with divalent ions or the chaperone protein Hfq, which provided clarity on previous data. Subsequently, efforts were made to develop methods to studying RNA interactions in detail but with medium throughput with surface plasmon resonance imaging, not previously used for studying RNA interactions. Finally, protocols were developed to create stable and functional, multi RNA arrays that were subsequently validated for use in screening RNA interactions in high-throughput with the demonstration of specific RNA-RNA, RNA-protein and RNA-small molecule interactions. This work sets the precedent for the utilisation of higher throughput methods in studying RNA interactions towards the greater understanding and exploitation of RNA functions and capabilities.
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Ovrén, Caroline. "Knockdown of the ERK pathway using siRNA in cultured chicken cardiomyocytes." Thesis, Linköpings universitet, Biologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-104567.
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The ancient South American birds called tinamous (Tinamidae) have the smallest hearts known among birds and their cardiomyocytes have previously been shown to express significantly lower levels of the mitogen-activated protein kinase ERK compared to the more modern chicken (Gallus gallus). ERK is a well-known mediator of growth signalling in the heart, especially in hypertrophy. The aim of this project was to assess the effect of ERK knockdown on proliferation in cultured chicken cardiomyocytes. By transfecting these cells with a lipoplexed siRNA, ERK mRNA levels were knocked down to approximately half (45%, SD: 27%) compared to cells transfected with a negative control siRNA. The knockdown was coupled with a decreased proliferative response to insulin-like growth factor 1 (IGF-1) and foetal bovine serum (FBS). In conclusion, the ERK pathway was confirmed to be instrumental also in proliferative signalling. The results also support the notion that ERK itself is the rate-limiting step of this MAPK cascade. The low native expression of ERK in tinamou cardiomyocytes is expected to impose a strict limit on proliferative growth in response to various stimuli in these hearts. The genetic changes leading to higher expression levels, and with it the potential for larger hearts, in modern birds would have led to greatly increased evolutionary fitness by way of an increased aerobic scope and the ability to sustain flight.
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Parhad, Swapnil S. "Adaptive Evolution of piRNA pathway in Drosophila." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/981.
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Major fraction of eukaryotic genomes is composed of transposons. Mobilization of these transposons leads to mutations and genomic instability. In animals, these selfish genetic elements are regulated by a class of small RNAs called PIWI interacting RNAs (piRNAs). Thus host piRNA pathway acts as a defense against pathogenic transposons. Many piRNA pathway genes are rapidly evolving indicating that they are involved in a host-pathogen arms race. In my thesis, I investigated the nature of this arms race by checking functional consequences of the sequence diversity in piRNA pathway genes.
In order to study the functional consequences of the divergence in piRNA pathway genes, we swapped piRNA pathway genes between two sibling Drosophila species, Drosophila melanogaster and Drosophila simulans. We focused on RDC complex, composed of Rhino, Deadlock and Cutoff, which specifies piRNA clusters and regulates transcription from clusters. None of the D. simulans RDC complex proteins function in D. melanogaster. Rhino and Deadlock interact and colocalize in D. simulans and D. melanogaster, but D. simulans Rhino does not bind D. melanogaster Deadlock, due to substitutions in the rapidly evolving Shadow domain. Cutoff from D. simulans stably binds and traps D. melanogaster Deadlock. Adaptive evolution has thus generated cross-species incompatibilities in the piRNA pathway which may contribute in reproductive isolation.
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Almeida, Miguel Duarte Dias de Vasconcelos [Verfasser]. "Insights into the function and evolution of nematode RNAi pathways / Miguel Duarte Dias de Vasconcelos Almeida." Mainz : Universitätsbibliothek Mainz, 2019. http://d-nb.info/118828309X/34.
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Shi, Zhen. "Genetic and genomic analysis of small RNA pathways in nematodes." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11093.
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Small noncoding RNAs, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs), regulate developmental and defense pathways in animals. While many small RNA silencing protein cofactors have been identified, much more is to be learned from a dynamic and quantitative perspective to reveal the underlying mechanisms and designing principles of each pathway. In this dissertation, I present studies that examine the temporal dynamics of small RNA pathways - one from an evolutionary time scale among the nematode species, and one from finely staged Caenorabditis elegans during the first larval stage. I also describe works identifying new cofactors functions in the miRNA pathway, potentially through regulating the spatial dynamics of the miRNA silencing complex.
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Jan, Calvin H. "Diverse RNA processing pathways important for post-transcriptional gene regulation." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/65169.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis. Vita.
Includes bibliographical references.
Cis-acting elements in 3' untranslated regions (UTRs) of mRNAs are crucial to the regulation of gene expression. Animal microRNAs (miRNAs) each target hundreds of mRNAs, which are recognized by pairing to nucleotides 2-7 of the miRNA. MicroRNAs mature through sequential RNase III cleavage of characteristic stem-loop precursors. Cleavage by Drosha defines the premiRNA hairpin, which is then cleaved by Dicer to generate a mature miRNA. This biogenesis pathway ensures high fidelity definition of miRNA 5' ends, which determine target specificity. Small RNAs from Caenorhabditis elegans and Drosophila melanogaster are extensively surveyed here using high-throughput sequencing. Analysis of these libraries led to the discovery of a novel miRNA biogenesis pathway, the mirtron pathway. Unlike canonical miRNAs, mirtrons are defined by intron splicing. The excised intron lariat is debranched and folds into a pre-miRNA hairpin that is cleaved by Dicer. Because of the accuracy of the spliceosome, the mirtron pathway also allows for high fidelity miRNA maturation. The trans-acting siRNAs (tasiRNAs) found in plants also reproducibly generate discrete small RNA species. TasiRNAs align to their parent locus (a TAS gene) in a distinctive 21-nt phase. This phasing is crucial; only siRNAs in the appropriate phase have sufficient complementarity to recognize their targets. The register of this phase is established by miRNA cleavage of the TAS transcript. Analysis of siRNAs sequenced from Physcomitrella patens reveals a conserved pathway in which P. patens TAS genes all possess two cleavage sites for miR390, the miRNA that cleaves TAS3 in Arabidopsis. A second miR390 site was found in Arabidopsis TAS3 that is bound by the miRNA but not cleaved. This interaction is important in triggering tasiRNA production from TAS3 transcripts. A novel approach to mRNA 3' end identification is applied here to determine 3' UTRs in C. elegans. C. elegans UTRs are typically 150 nt long and have a higher density of miRNA seed sites than mammals. Ten percent of genes are alternatively polyadenylated. Approximately 1000 convergent gene pairs were found to use bidirectional poly(A) sites. This architecture maximizes gene density and demonstrates the influence of 3' end formation on the evolution of gene topology.
by Calvin H. Jan.
Ph.D.
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Spinelli, Pietro. "Functional studies of the RNA helicases Vasa and Tdrd9 in the piRNA pathway." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV047/document.
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Les protéines Piwi sont exprimées dans les gonades (testicules et ovaires) des animaux où elles s'associent à des petits ARN nommés piRNAs (Piwi-interaction RNAs) et répriment les éléments transposables, défendant ainsi de l'intégrité du génome. En effet, les animaux knock-out pour les protéines Piwi montrent une perte de piRNAs et une activation des éléments transposables avec des conséquences catastrophiques: l'arrêt du développement des cellules germinales, due potentiellement à des dommages du génome qui entraîne l'infertilité. Le « Silencing » est réalisé soit par l'activité endonucléase guidée par les piRNAs de la protéine Piwi cytosolique ou par le recrutement de la machinerie de répression transcriptionelle sur les loci génomiques des cibles par Piwi nucléaire. La biogenèse des piRNAs peut être divisée en deux voies, une primaire et une secondaire. De longs ARN précurseurs simple-brin sont transformés en piRNAs matures de 25-30 nt qui sont chargés dans les protéines Piwi. Une fois chargée avec le piRNA, Piwi se lie aux transcrits de transposons ayant la séquence complémentaire et les clive en générant deux ARN, dont l'un peut être chargé dans une nouvelle protéine Piwi, produisant un piRNA secondaire. Des études génétiques ont identifié plusieurs facteurs protéiques essentiels à ce processus. Certain de ces facteurs sont des hélicases à ARN dont le rôle spécifique reste inconnu, principalement parce que ce sont des enzymes dynamiques et l'identification de leurs cibles et leurs partenaires protéiques avec des approches biochimiques standards est difficile.Dans la première partie de cette thèse nous décrivons comment l'introduction d'une mutation ponctuelle dans la boîte DEAD de l'hélicase à ARN Vasa (DEAD à DQAD) peut bloquer son activité in vivo et figer le complexe transitoire de biogenèse qui contient VASA et les deux protéines Piwi responsables de la biogenèse secondaire.La résolution de la structure de VASADQ en complexe avec l'ATP ou l'AMPPNP a révélé les détails moléculaires de cette inhibition et a expliqué le phénotype observé in vivo. VASADQ a un taux d'hydrolyse de l'ATP réduit, car après hydrolyse, le phosphate libre est bloqué à l'intérieur du site actif en raison d'une liaison hydrogène supplémentaire formée avec la Gln mutée. La réduction de l'hydrolyse de l'ATP se traduit par une faible liaison à l'ARN mesurée par des expériences biophysiques. L'introduction de la même mutation chez l'homologue murin de VASA (MVH) produit un phénotype de dominant négatif où MVHDQ est agglutinée sur le complexe RNP contenant les protéines Piwi et les piRNAs.Dans la deuxième partie de cette thèse, nous avons introduit la même mutation dans TDRD9, une autre hélicase à ARN impliquée dans la voie des piRNAs mais dont la fonction est inconnue. Nous avons d'abord exprimé, purifié TDRD9 et montré que la mutation dans son domaine hélicase DEVH à DQVH abolit complètement son activité ATPase sans impacter sur sa stabilité. Par la suite, nous avons généré une souris Knock-in et analysé son phénotype. Les souris Knock-in mâles sont stériles et présentent un blocage au début de la spermatogenèse qui est probablement une conséquence des dommages de l'ADN générés par l'activation des éléments transposables. Ces éléments, comme Line-1, présentent un défaut de méthylation à leur loci génomiques, mais qui ne semble pas être contrôlé par la voie piRNA dans le mutant, étant donné que les protéines Piwis sont correctement chargées avec les piRNAs dérivés de Line-1.Dans l'ensemble, nous avons étudié le rôle moléculaire de deux hélicases à ARN dans la voie des piRNAs, nous avons élucidé le rôle de VASA et nous montrons que l'activité ATPase de TDRD9 est essentielle pour la régulation des transposons au cours de la spermatogenèse de la sourisPIWI proteins are expressed in the gonads (testis and ovary) of animals where they associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements, defending the integrity of the genome. Indeed, animal knock-outs of Piwi proteins display a loss of piRNAs and activation of transposon sequences with catastrophic consequences: block in germ cell development potentially due to genome damage, resulting in infertility. Silencing is achieved either by piRNA-guided endonuclease activity of cytosolic Piwi protein or by recruitment of transcriptional repression machinery on target genomic loci by nuclear Piwi. Biogenesis of piRNAs can be divided in primary and secondary pathway. Primary pathway describes how long single-stranded RNA precursors are processed into mature 25-30 nt piRNAs and loaded into Piwi proteins. Piwi-loaded piRNAs bind and cleave complementary transposon transcripts generating two RNA products, one of which can be loaded into a new Piwi protein, generating a secondary piRNA. Different protein factors are essential in this process as identified by genetic studies. Few of these factors are putative RNA helicases but their specific role is unknown, mainly because RNA helicase are dynamic enzymes and identification of their targets and protein partners with standard biochemical approaches is challenging.In the first part of this thesis I describe how the introduction of a point mutation in the DEAD box of the RNA helicase Vasa (DEAD to DQAD) can block its activity in vivo and freeze a transient biogenesis complex that contains Vasa and the two Piwi proteins responsible for secondary biogenesis.Crystal structure of VASADQ in complex with ATP or AMPPNP revealed the molecular details of this inhibition and explained the phenotype observed in vivo. VasaDQ has a reduced ATP hydrolysis rate because after hydrolysis the free phosphate is blocked inside the active site due to an additional hydrogen bond formed with the mutated Gln. The reduction in ATP hydrolysis is mirrored by an impaired RNA binding activity as measured with biophysical experiments. Introduction of the same mutation in the mouse homologue of Vasa (MVH) has a dominant-negative phenotype where MVHDQ is clump on an ribonucleoprotein (RNP) complex containing piRNAs and mouse Piwi proteins.In the second part of this thesis I introduce the same mutation in TDRD9, another RNA helicase involved in piRNA pathway with an unknown function. First I expressed and purified TDRD9 and showed that DEVH to DQVH mutation in its helicase domain completely abolishes its ATPase activity but do not affects its stability. Next I created a knock-in mutation in the mouse genomic locus for Tdrd9 and analysed the resulting phenotype in the mutant. Knock-in mice are male sterile with an early block in spermatogenesis that is probably a consequence of uncontrolled DNA damages generated by de-repressed transposon elements. These elements, like Line-1, fail to be correctly methylated at their genomic loci in the Tdrd9 mutant. Although Tdrd9 is important for Line-1 transposon silencing, it is likely not via a role in piRNA biogenesis since Piwi proteins are correctly loaded with Line-1 derived piRNAs. Interestingly a drop in piRNAs that derives from SINE elements is observed in the mutant, probably reflecting a role for Tdrd9 in sorting primary transcripts into MILI and MIWI2 during DNA de novo methylation.Overall I investigated the molecular role of two RNA helicases in the piRNA pathway, elucidating the role of Vasa and show that the ATPase activity of Tdrd9 is essential for transposon regulation in mouse spermatogenesis
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Stadler, Bradford Michael. "Interaction of a Mammalian Virus with Host RNA Silencing Pathways: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/322.
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In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of Dicer, a key component of RNA silencing mechanisms.
Our work demonstrates that adenovirus encodes two miRNAs within the loci of the virus-associated RNA I (VA RNA I). We find that one of these miRNAs, miR-VA “g”, enters into a functional, Argonaute-2 (Ago-2)-containing silencing complex during infection. Currently, the cellular or viral target genes for these miRNAs remain unidentified. Inhibition of the function of the miRNAs during infection did not affect viral growth in a highly cytopathic cell culture model. However, studies from other viruses implicate viral miRNAs in the establishment of latent or chronic infections.
Additionally, we find that adenovirus infection leads to the reduced expression of Dicer. This downregulation does not appear to be dependent on the presence of VA RNA or its associated miRNAs. Rather, Dicer levels appear to inversely correlate with the level of viral replication, indicating that another viral gene product is responsible for this activity. Misregulation of Dicer expression does not appear to influence viral growth in a cell culture model of infection, and also does not lead to gross changes in the pool of cellular miRNAs. Taken together, our results demonstrate that RNA silencing pathways are active participants in the process of infection with human adenovirus. The production of viral miRNAs and the regulation of cellular Dicer levels during infection implicate RNA silencing mechanisms in both viral fitness as well as potential host defense strategies.
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Zalma, Carre Alison. "Monitoring folding pathways for large RNAs using site-directed spin-labeling techniques." Thesis, Texas A&M University, 2005. http://hdl.handle.net/1969.1/4904.
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The function of biomolecules is very sensitive to structure. Folding in proteins and nucleic acids is a hierarchical process progressing from primary to secondary, then tertiary, and finally, quaternary structures. RNA in its folded form performs a variety of biological activities. Obtaining intramolecular distance measurements makes it possible to generate structural models along the folding pathway that may be related to the overall function of the molecule. Distances can be measured by Site-Directed Spin-Labeling (SDSL), in which nitroxyl spin-label probes are attached and observed by EPR spectroscopy. Spin-labels can provide information concerning structure and conformational changes because they are particularly sensitive to molecular motion and interspin distances. Continuous-wave EPR spectroscopy has been commonly applied to detect and monitor nitroxide spin-label probes within biological systems. A previous published SDSL study from this laboratory investigated a 10-mer RNA duplex model system with spin-label probe succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate; however, an increased spin-labeling efficiency was observed with an isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO). In this thesis, a 4-isocyano TEMPO spin-label probe replaced the previously used succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate in 10-mer SDSL studies. The influence of labeling with the 4-iscocyano TEMPO spin-label in a 10-mer RNA model system was investigated with thermal denaturation, Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS), Electron Paramagnetic Resonance (EPR) spectroscopy, and reverse phase high performance liquid chromatography (RP-HPLC). In the 10-mer RNA duplex model system a 4-isocyano TEMPO spin-label is individually attached to one strand and two strands are annealed to measure distances. This methodology is limited to systems in which two oligonucleotides are annealed together. To circumvent this limitation and also to explore single-strand dynamics a new methodology was implemented, double spin-labeling. Double spin-labeled single-stranded RNA was investigated as a single-strand and within a duplex via MALDI-TOF-MS, EPR spectroscopy and RP-HPLC. A double spin-labeling strategy in this work will be applicable to large complex RNAs like Group I intron of Tetrahymena thermophilia.
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Caulfield, Thomas R. "Structural basis for the fidelity of translation modeling the accommodation pathway /." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/22553.
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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008.Committee Chair: Harvey, Stephen C; Committee Member: Hud, Nicholas V; Committee Member: Oyelere, Adegboyega; Committee Member: Wartell, Roger.
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Akhtar, Y. "Studies on the maturation pathway of ribosomal precursor RNA : Analysis of Xenopus ribosomal RNA synthesised by transcription in vitro." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382054.
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Haley, Benjamin. "A Biochemical Dissection of the RNA Interference Pathway in Drosophila melanogaster: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/9.
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In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster.
To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway.
Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target.
In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.
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Vu, Ngoc T. "Regulation and Mechanistic Functions of Caspase-9 RNA Splicing." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3578.
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Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.
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Chang, Ya-Wen. "The Ras/PKA pathway controls transcription of genes involved in stationary phase entry in Saccharomyces cerevisiae." Columbus, Ohio : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061214472.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xiii, 108 p.; also includes graphics. Includes abstract and vita. Advisor: Paul K. Herman, Dept.of Molecular, Cellular, and Developmental Biology. Includes bibliographical references (p. 96-108).
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Richardson, Megan Leigh. "Identifying Novel Transcriptional Effectors of the Juvenile Hormone Pathway in Aedes aegypti." Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/98538.
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Aedes aegypti is the primary vector for dengue, zika, chikungunya, and yellow fever viruses. Disease transmission through this mosquito places over 40% of the world's population at risk of contracting one or more of these pathogens. Current control strategies such as insecticide application have failed or carry additional burdens, such as off-target toxicity to mammals and birds. Our lab proposes utilizing a conserved arthropod hormone pathway, juvenile hormone (JH), related to growth and reproduction to curb these vector populations and reduce disease transmission. Additionally, JH is nontoxic to birds and mammals; it requires incredibly high doses to have lethal effects. We hypothesize that JH-responsive genes expressed early in the adult are responsible for her reproductive capacity and by manipulating the signaling downstream of the receptor, we will be able to decrease the female's fecundity and limit vector populations.
Via bioinformatics screening of RNA-sequencing data using the New Tuxedo pipeline, we identified 47 potential transcription factor candidates. With the use of in vitro culturing of the mosquito's reproductive tissues in the presence of a translation inhibitor, we identified two early JH responsive gene candidates, FoxA and zinc finger 519, p-value <0.05. The functional characterization of these two remains to be seen, however, in Drosophila melanogaster, they both have roles in chromatin remodeling and require protein partners to carry out long range interactions.Master of Science in Life Sciences
The mosquito, Aedes aegypti, is responsible for the spread of a myriad of viruses such as dengue, zika, and chikungunya. Currently, these infections have no vaccine or treatment available and transmission rates continue to steeply rise in response to the spread of breeding grounds. Popular insecticides carry detriments such as off-species toxicity and continuous application to treatment areas. Our lab proposes an alternative to these chemical insecticides by manipulating a developmental pathway in the mosquito. The Juvenile Hormone pathway is conserved in arthropods, responsible for growth and reproduction, and the hormone is nontoxic to mammals. Through the combination of bioinformatics and genomics studies, we have identified two JH-responsive gene candidates that are potential regulators of this pathway.
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Wang, Wei. "Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/805.
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PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance.
By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila.
How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.
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Wang, Wei. "Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/805.
Full textAbstract:
PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance.
By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila.
How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.
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Gera, Joseph F. "An analysis of mRNA decay pathways in Chlamydomonas reinhardtii /." abstract and full text PDF (UNR users only), 1998. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9907755.
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Kumar, Aseem. "Identification and characterization of interferon and double-stranded RNA signal transduction pathways." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0019/NQ27678.pdf.
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Lévesque, Kathy. "hnRNP A2 is a protein involved in the trafficking pathway of HIV-1 genomic RNA." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98748.
Full textAbstract:
HIV-1 genomic RNA trafficking is an important event which remain unclear. Different cellular proteins are involved in this process and the heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) is suggested to play a role by its potential implication in ribonucleoprotein complex which is related with the microtubule network. We identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) is involved in this process. To investigate the potential function of hnRNP A2 in the trafficking, protein expression was decreased using small interfering RNA and the impact on RNA localization, protein expression patterns and levels of genomic RNA in new virions was looked at. The results obtained suggested that hnRNP A2 knockdown impedes transport of the genomic RNA and causing its accumulation at the MTOC (microtubule organizing center). These data show the importance of the hnRNP A2 protein in genomic RNA trafficking and its role in HIV-1 RNA localization.