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1
Warner, Katherine Deigan. "Structural studies of small molecule-RNA interactions in druggable RNA targets and fluorogenic RNAs." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708889.
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Hall, Adam. "Biogenesis of Y RNA-derived small RNAs." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/42404/.
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Small non-coding RNAs (sRNAs) constitute a significant portion of the transcriptome in eukaryotes. Many of these sRNAs regulate gene expression. Next-generation sequencing (NGS) has revealed a plethora of previously uncharacterised sRNAs with potential biological function, a number of which originate from longer RNAs. Here, the biogenesis of sRNAs derived from the non-coding Y RNAs (YsRNAs) was characterised as a model for understanding this emerging class of sRNA fragments. Y RNAs are highly conserved, 100 nt long molecules involved in DNA replication which bind to the autoimmune proteins Ro60 and La. YsRNAs are produced in cells undergoing apoptosis. Here, it was demonstrated that YsRNAs are generated from the 5’ and 3’ ends of all four Y RNAs in stressed and unstressed cells. Furthermore, production of these fragments was observed in both cancerous and non-cancerous cells. Although YsRNAs have been proposed to have gene silencing activity, experiments done here found that YsRNAs do not enter the microRNA pathway and are not generated by the gene silencing-related protein Dicer. Furthermore, experiments established that the enzyme which produces fragments from tRNAs, angiogenin, was also not responsible for YsRNA generation. Using mammalian cultured cells along with gene knockout and RNA interference (RNAi) technology, it was determined that RNase L contributed to YsRNA generation. Furthermore, the Y RNA binding protein Ro60 was shown to be essential for YsRNA production through a model of RNase protection. Analysis of deep sequencing data in Ro60 knockout cells revealed that many other sRNAs are also dependent on Ro60. Finally, a ‘high definition’ (HD) protocol to improve NGS detection of sRNAs was tested. The HD protocol was found to be better at detecting sRNAs than current methods. This will facilitate more efficient detection of novel sRNAs in the future.
3
Richter, Andreas S. [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Computational analysis and prediction of RNA-RNA interactions = Computergestützte Analyse und Vorhersage von RNA-RNA-Interaktionen." Freiburg : Universität, 2012. http://d-nb.info/1123475695/34.
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Garbutt, Jennifer S. "RNA interference in insects : persistence and uptake of double-stranded RNA and activation of RNAi genes." Thesis, University of Bath, 2011. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548101.
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RNA interference (RNAi) is a eukaryotic phenomenon where short double-stranded RNA molecules (dsRNAs) repress homologous sequences. In insects RNAi has been widely observed and has proved extremely useful as a reverse genetics tool to elucidate the function of newly identified genes, as well as showing potential as a novel insecticide. Unfortunately, however, not all insect species are equally susceptible to RNAi. This thesis explores whether persistence of dsRNA in insect hemolymph, uptake of dsRNA into insect tissue, or activation of RNAi genes could be limiting factors in RNAi experiments. Trials were conducted with the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. In M. sexta larvae dsRNA disappeared rapidly from the hemolymph in vivo. By comparison, exogenous dsRNA persisted longer in the hemolymph of B. germanica adults. These findings lead me to propose that the rate of persistence of dsRNA in insect hemolymph may be a key factor in determining the susceptibility of insect species to RNAi. Despite such rapid breakdown of dsRNA in M. sexta larvae uptake of exogenous dsRNA into hemocytes, fat body and midgut could be detected by quantitative RT-PCR in vivo and was experimentally investigated in hemocytes in vivo and in vitro using fluorescently labelled dsRNA. Furthermore, quantitative-RT-PCR revealed that the expression of two M. sexta RNAi genes dicer-2 and argonaute-2 (partial sequences of which were isolated during this study) was specifically upregulated in response to injection with dsRNA.
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Wilm, Andreas. "RNA-Alignments und RNA-Struktur in silico." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979837278.
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Salgado, Maria Paula Santos Cordeiro. "Structural studies of RNA-dependent RNA polymerases." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430559.
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Lai, Daniel. "Computational analysis of ribonucleic acid basepairs in RNA structure and RNA-RNA interactions." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57538.
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Ribonucleic acids (RNA), are an essential part of cellular function, transcribed from DNA and translated into protein. Rather than a passive informational medium, RNA can also be highly functional and regulatory. Certain RNAs fold into specific structures giving it enzymatic properties, while others bind to specific targets to guide regulatory processes. With the advent of next-generation sequencing, a large number of novel non-coding RNAs have been discovered through whole-transcriptome sequencing. Many efforts have been made to study the structure and binding partners of these novel RNAs, in order to determine their function and roles.
This work begins with a description of my R package R4RNA for manipulating RNA basepair data, the building blocks of RNA structure and RNA binding. The package deals with the input/output and manipulation of RNA basepair and sequence data, along with statistical and visualization methods for evaluation, interpretation and presentation. We also describe R-chie, a visualization tool and web server built on R4RNA that visualizes complex RNA basepairs in conjunction with sequence alignments. We then conduct the largest known evaluation of RNA-RNA interaction methods to date, running state-of-the-art tools on curated experimentally validated datasets. We end with a review of cotranscriptional RNA basepair formation, summarizing biological, theoretical and computational methods for the process, and future directions for improving classical methods in RNA structure prediction.
All content chapters of this thesis has been peer-reviewed and published. The work on R4RNA has led to two publications, with the package used to great visual effect by various publications and also adopted by the RNA structure database Rfam. My assessment of RNA-RNA interaction is at present the only published evaluation of its kind, and will hopefully become a benchmark for future tool development and a guide to selecting appropriate tools and algorithms. Our published review on RNA cotranscriptional folding is well-received, being the first review specifically on its topic.Science, Faculty of
Graduate
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Poolsap, Unyanee. "Computational methods for predictions of RNA pseudoknotted secondary structures and RNA-RNA interactions." 京都大学 (Kyoto University), 2011. http://hdl.handle.net/2433/147348.
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Fritz, Sarah E. "Molecular basis of the DExH-box RNA helicase RNA helicase A (RHA/DHX9) in eukaryotic protein synthesis." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437413252.
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Pereira, Tiago Campos. "Estudo de possiveis aplicações médicas da interferencia por RNA." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316861.
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Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy MaiaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Lackey, Jeremy. "New methods for the synthesis of RNA, novel RNA pro-drugs and RNA microarrays." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92290.
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The demand for synthetic oligonucleotides has grown exponentially over the past decades as genome sequencing, functional genomics, polymerase chain reaction (PCR)-based detection methods, and gene silencing via RNA interference (RNAi) consume enormous numbers of DNA and RNA oligonucleotides. Although various RNA synthesis chemistries now allow oligoribonucleotides to be produced routinely, the higher complexity and cost of RNA (over DNA) has somewhat limited its availability.A major goal of this thesis work was aimed at finding ribonucleoside synthons that potentially benefit two critical aspects of RNA manufacturing: yield and ease of post-synthesis processing. Towards these goals, we developed methods for the synthesis of RNA using 2'-O-Lv and 2'-O-acetal Lv (ALE) ribonucleoside derivatives. Deprotection of the RNA chains consisted of a three-step deprotection scheme, which eliminated the need for any harsh basic hydrolytic steps, generally composed of: (1) treatment with anhydrous NEt3 (r.t., 1 h) to deblock the phosphate's cyanoethyl groups; (2) hydrazinolysis (r.t., 30 min 4 h) to simultaneously deprotect the nucleobases and 2'-OH positions, and (3) fluoride treatment (r.t., 30 min) to effect cleavage from the controlled pore glass solid support. Significantly, the rather mild conditions to remove 2'-O-Lv or 2'-O-ALE protecting groups did not lead to RNA strand scission. Furthermore, in the case of 2'-O-ALE protection, higher step-wise monomer coupling yields (~98.7%) was possible, since the ALE protection is less bulky than conventional silyl protection, i.e. TBDMS. Furthermore, both 2'-O-Lv or 2'O-ALE chemistries are completely compatible with the synthesis cycles used by all automated gene synthesizers.
With adjustments in protecting group strategies for the 5'-OH, exocyclic amino nucleobase groups and the development of a light-labile solid support, two other major goals were achieved: (1) the first in situ synthesis of RNA on microarrays, and (2) synthesis of chemically modified RNA strands with 2'-O-acetal ester and 2'-O-acetal ester pyrrolidines in order to increase lipophilicity and cellular permeability over native RNA. When RNA synthesis was carried out with 5'-O-NPPOC 2'-O-ALE monomers on a microarray ("chip"), deprotection typically involved (1) cleavage of the photolabile 5'-protecting group; (2) treatment with anhydrous NEt3 (r.t., 1 h) to deblock the phosphate's cyanoethyl groups; (3) hydrazinolysis (r.t., 30 min 4 h) to simultaneously deprotect bases and 2'-OH positions. The latter step could also be accomplished with ethylenediamine at room temperature. An RNase A assay was performed as "proof-of-principle" to demonstrate the value of a DNA-RNA microarray for studying enzyme kinetics and specificity on oligonucleotide based libraries. We showed that RNase A acts effectively on a DNA-RNA substrate with measurable kinetics analogous to those of the reference substrates.
The novel 2'-O-modified RNA were tested as short interfering RNA pro-drugs ("pro-siRNA") that would cross the cell membrane and be hydrolyzed (at the 2'-O-ester groups) by ubiquitous esterases to release the active (siRNA) molecules. Indeed, both siRNA and pro-siRNA prepared via 2'-O-ALE chemistry were shown to be active in an RNAi luciferase gene knockdown assay, confirming the integrity of the synthesized RNA strands and the promise of the pro-siRNA approach.
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Jensen, Morten Bernt. "Characterisation of an Arabidopsis RNA-dependent RNA polymerase." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437828.
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Shechner, David M. "The structural basis of RNA-catalyzed RNA polymerization." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/58463.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Cataloged from PDF version of thesis.
Includes bibliographical references.
The Class I ligase is an artificial ribozyme that catalyzes a reaction chemically identical to a single turnover of RNA-dependent RNA polymerization. Such an activity would have been requisite for the emergence of a self-replicase ribozyme, an enzyme that, according to the RNA World hypothesis, would be fundamental for the emergence of life. Demonstrating the plausibility of RNA-catalyzed self-replication, the Class I ligase catalytic machinery was previously harnessed to produce general RNA polymerase ribozymes. Hence, this ligase represents a robust model system for studying both the potential role RNA may have played in the origins of life and RNA catalysis in general. Through a combination of crystallographic and biochemical experiments, we have sought to elucidate the structure and mechanism of this ribozyme. As a starting point for our experiments, the crystal structure of the self-ligated product was solved to 3.0 Angstrom resolution, revealing a tripodal architecture in which three helical domains converge in the vicinity of the ligation junction. A handful of tertiary interactions decorate this tripod scaffold; among them were two instances of a novel motif, the A-minor triad. The structure elucidated interactions that recognize and bind the primer-template duplex and those that position the reaction electrophile. It furthermore revealed functional groups that compose the active site. Biochemical evidence and the position of these groups lead us to propose a reaction mechanism similar to that used by proteinaceous polymerases. Using a slowly reacting mutant, 3.05-3.15 Angstrom crystal structures were solved of unreacted, kinetically trapped ligase-substrate complexes bound to different metal ions. Comparison of the Ca2+- and Mg2+-bound structures explains the preference of the ligase for Mg 2+. Moreover, these structures revealed features missing in the product structure: interactions to the 5'-triphosphate and an active site catalytic metal ion. While this metal is positioned in a manner similar to the canonical "Metal A" of proteinaceous polymerases, the role of "Metal B" might have been supplanted by functional groups on the RNA. Kinetic isotope experiments and atomic mutagenesis of two active site functional groups imply that they may act in concert to electrostatically aid transition-state stabilization.
by David M. Shechner.
Ph.D.
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Tomescu, Alexandra Iulia. "Fluorescence studies of influenza RNA and RNA polymerase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:77f33f82-76f7-4154-912e-5c92bd4bf9c6.
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The influenza A virus genome consists of eight single-stranded segments of negativesense viral RNA (vRNA) with highly conserved, partially complementary termini. These termini associate in a double-stranded RNA structure, known as a panhandle, which is bound by the viral RNA-dependent RNA polymerase and can serve as a promoter in both viral transcription and replication. In part A of this thesis, I use a combination of classical biochemistry techniques and fluorescence techniques (both at the ensemble and single-molecule level) for a quantitative investigation of the interaction between purified influenza A RNA polymerase and the individual 5' and 3' conserved termini of the vRNA segments, as well as the double-stranded vRNA promoter. Furthermore, I report the first direct, real-time observation of the promoter changing its structure when bound by the polymerase and show that the structure assumed agrees best with the corkscrew model. In part B of this thesis, I use fluorescence to detect RNA: I design and test a singlemolecule biosensor aimed at probing the presence of influenza A RNA in a sample, on the one hand, and I use click-chemistry to fluorescently label very shorty RNAs (3-25nt) that have been generated in an in vitro transcription reaction, on the other. The biosensing assay I propose can be further developed for diagnostic purposed, while click-chemistry labelling of short RNAs can be optimised and extended such that it becomes a reliable alternative to the use of radiolabels.
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Brown, James W. "RNA polymerase binding sites and polyadenylated RNAs in archaebacteria /." The Ohio State University, 1988. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487591658174843.
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Wright, Sam Mathew. "Structural and biophysical studies of RNA-dependent RNA polymerases." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:d5c2a16d-e1e2-4c22-aca5-70f72aa96853.
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RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.
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Hahn, Daniela. "Brr2 RNA helicase and its protein and RNA interactions." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5775.
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The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.
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Kimura, Richard Henry. "The SINE RNA stress response and Alu RNA activity /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2003. http://uclibs.org/PID/11984.
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D'Abramo, Claudia M. "Biochemical characterization of the BVDV RNA-dependent RNA polymerase during initiation and elongation of RNA synthesis." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111870.
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The RNA-dependent RNA polymerase (RdRp) of viruses belonging to the Flaviviridae family, including the hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) is critical for viral replication. The major goal of this PhD study was to biochemically characterize the role of the polymerase during initiation and elongation of RNA synthesis, utilizing the BVDV RdRp as a model system. We showed that the BVDV polymerase efficiently incorporates chain-terminating nucleoside analogues, which ultimately arrest RNA synthesis. The incorporated chain-terminators, however, can be removed from the primer terminus in the presence of pyrophosphate (PPi). These results suggest that the phosphorolytic excision of incorporated chain-terminators is a possible mechanism that can diminish the efficiency of this class of compounds against viral RdRps. The chain-terminators then served as valuable tools in subsequent experiments to analyze the functional role(s) of the RdRp-associated GTP-specific binding site (G-site) and the consequences of GTP binding during the initiation of RNA synthesis. The results provide biochemical evidence for the existence of a G-site in the BVDV enzyme, and suggest that GTP binding controls template positioning during de novo initiation. Finally, through the development of a novel ribonuclease-based footprinting assay, it was determined that catalytically active complexes contact the newly synthesized RNA during elongation of RNA synthesis with approximately 6-7 base pairs. The polymerase moves along the template according to the position where RNA synthesis is arrested. Taken together, this study provides novel insight into mechanisms involved during initiation and elongation of RNA replication of viruses belonging to the Flaviviridae family. The ability of RdRps to excise incorporated chain-terminators points to possible shortcomings of nucleoside analogue inhibitors that are under development as antiviral agents for the treatment of infection with HCV.
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Böhm, Stefanie. "Non-protein-coding RNA : Transcription and regulation of ribosomal RNA." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-102718.
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Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript
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Marr, Edward John. "RNA interference (RNAi) for selective gene silencing in Astigmatid mites." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25722.
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Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.
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Jady, Béata Erika. "RNA-guided post-transcriptional modification of spliceosomal small nuclear RNAs." Toulouse 3, 2001. http://www.theses.fr/2001TOU30156.
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Kable, Moffett Lee. "Kinetoplastid RNA editing : analysis of the mechanism of guide RNA directed uridylate insertion into precursor messenger RNA /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/9295.
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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.
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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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Croci, R. "RNA DEPENDENT RNA POLYMERASE: A VALUABLE TARGET TO BLOCK VIRAL REPLICATION IN SINGLE-STRANDED (+)SENSE RNA VIRUSES." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/243352.
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The (+)strand RNA viruses include a very large group of viruses that cause epidemic diseases in humans, including dengue fever and gastroenteritis. The human (+)RNA viruses include Flaviviruses (FV) and Norovirus (NV). Both encode for proteins essential for viral replication, such as the RNA dependent RNA polymerase (RdRp). Since human cells lack RdRp, it appears as one of the most promising targets for antivirals development. I worked on the identification of new non-nucleotide inhibitors against FV and NV, using RdRp as the main target. In this context, suramin and NF023 have been identified in my lab as NV RdRp inhibitors that, however both are hampered in their application by pharmacokinetics problems. To overcome such problems, I analyzed the potential inhibitory role of Naf2, a fragment derived from these two molecules. Although Naf2 showed a low inhibitory activity, the crystal structures of NV RdRp/Naf2 complex revealed a new binding site. To further map this new site, I tested a Naf2 related molecule, PPNDS. The crystal structures of the RdRp/PPNDS complex revealed interesting features about the new binding site. I also focused on structurally related molecules synthesized following structure-driven information. NV RdRp crystal structures in complex with one of these compounds (Cpd6) were analyzed, providing new knowledge on the interactions between a small fragment and NV RdRps, establishing a platform for structure-guided drug optimization. In parallel to the NV work, I screened in silico a library of compounds against FV RdRp. One of the best compounds identified (HeE1-2Tyr) was able to inhibit the RdRp activity and several FVs in cell-based assays. Although the crystallographic analyses don't reveal clear enough electron density for the inhibitor, indirect evidence suggests that HeE1-2Tyr interferes with the RdRp priming loop that appears disordered.
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Nip, Ka Ming. "RNA-Bloom : de novo RNA-seq assembly with Bloom filters." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62590.
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High-throughput RNA sequencing (RNA-seq) is primarily used in measuring gene expression, quantifying transcript abundance, and building reference transcriptomes. Without bias from a reference sequence, de novo RNA-seq assembly is particularly useful for building new reference transcriptomes, detecting fusion genes, and discovering novel spliced transcripts. This is a challenging problem, and to address it at least eight approaches, including Trans-ABySS and Trinity, were developed within the past decade. For instance, using Trinity and 12 CPUs, it takes approximately one and a half day to assemble a human RNA-seq sample of over 100 million read pairs and requires up to 80 GB of memory. While the high memory usage typical of de novo RNA-seq assemblers may be alleviated by distributed computing, access to a high-performance computing environment is a requirement that may be limiting for smaller labs. In my thesis, I present a novel de novo RNA-seq assembler, “RNA-Bloom,” which utilizes compact data structures based on Bloom filters for the storage of k-mer counts and the de Bruijn graph in memory. Compared to Trans-ABySS and Trinity, RNA-Bloom can assemble a human transcriptome with comparable accuracy using nearly half as much memory and half the wall-clock time with 12 threads.Science, Faculty of
Alumni
Graduate
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DeBlasio, Daniel. "NEW COMPUTATIONAL APPROACHES FOR MULTIPLE RNA ALIGNMENT AND RNA SEARCH." Master's thesis, University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4070.
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In this thesis we explore the the theory and history behind RNA alignment. Normal sequence alignments as studied by computer scientists can be completed in $O(n^2)$ time in the naive case. The process involves taking two input sequences and finding the list of edits that can transform one sequence into the other. This process is applied to biology in many forms, such as the creation of multiple alignments and the search of genomic sequences. When you take into account the RNA sequence structure the problem becomes even harder. Multiple RNA structure alignment is particularly challenging because covarying mutations make sequence information alone insufficient. Existing tools for multiple RNA alignments first generate pair-wise RNA structure alignments and then build the multiple alignment using only the sequence information. Here we present PMFastR, an algorithm which iteratively uses a sequence-structure alignment procedure to build a multiple RNA structure alignment. PMFastR also has low memory consumption allowing for the alignment of large sequences such as 16S and 23S rRNA. Specifically, we reduce the memory consumption to $\sim O(band^2*m)$ where $band$ is the banding size. Other solutions are $\sim O(n^2*m)$ where $n$ and $m$ are the lengths of the target and query respectively. The algorithm also provides a method to utilize a multi-core environment. We present results on benchmark data sets from BRAliBase, which shows PMFastR outperforms other state-of-the-art programs. Furthermore, we regenerate 607 Rfam seed alignments and show that our automated process creates similar multiple alignments to the manually-curated Rfam seed alignments. While these methods can also be applied directly to genome sequence search, the abundance of new multiple species genome alignments presents a new area for exploration. Many multiple alignments of whole genomes are available and these alignments keep growing in size. These alignments can provide more information to the searcher than just a single sequence. Using the methodology from sequence-structure alignment we developed AlnAlign, which searches an entire genome alignment using RNA sequence structure. While programs have been readily available to align alignments, this is the first to our knowledge that is specifically designed for RNA sequences. This algorithm is presented only in theory and is yet to be tested.M.S.
School of Electrical Engineering and Computer Science
Engineering and Computer Science
Computer Science MS
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Digard, Paul Eugene. "Analysis of the influenza virus RNA-dependent RNA poymerase complex." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304251.
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Xu, Guorong. "RNA CoMPASS: RNA Comprehensive Multi-Processor Analysis System for Sequencing." ScholarWorks@UNO, 2012. http://scholarworks.uno.edu/td/1531.
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The main theme of this dissertation is to develop a distributed computational pipeline for processing next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate hundreds of millions of short reads for each DNA/RNA sample. There are many existing bioinformatics tools developed for the analysis and visualization of this data, but very large studies present computational and organizational challenges that are difficult to overcome manually. We designed a comprehensive pipeline for the analysis of RNA sequencing which leverages many existing tools and parallel computing technology to facilitate the analysis of extremely large studies. RNA CoMPASS provides a web-based graphical user interface and distributed computational pipeline including endogenous transcriptome quantification and additionally the investigation of exogenous sequences.
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Sarver, Michael. "STRUCTURE-BASED MULTIPLE RNA SEQUENCE ALIGNMENT AND FINDING RNA MOTIFS." Bowling Green State University / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1151076710.
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Phosiwa, Maanda Noaxe. "Molecular characterization of a porcine picobirnavirus RNA-dependent RNA polymerase." Diss., Pretoria : [s.n.], 2009. http://upetd.up.ac.za/thesis/available/etd-07152009-175205/.
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Bakker, Saskia. "RNA packaging and uncoating in simple single-stranded RNA viruses." Thesis, University of Leeds, 2012. http://etheses.whiterose.ac.uk/2801/.
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Simple (non-enveloped) small, positive-sense single-stranded RNA viruses infect hosts from all kingdoms of life. However, their assembly and uncoating processes remain poorly understood. For turnip crinkle virus (TCV), 3D reconstructions by cryoelectron microscopy (cryo-EM) are shown for the native and the expanded form. The expanded form is a putative disassembly intermediate and exhibits pores that are large enough to allow exit of single-stranded RNA. Biochemical experiments revealed the expanded form is protease-sensitive, although the RNA genome remains protected from ribonuclease. Virus particles complexed with ribosomes are shown by negative stain EM. Proteolysis causes release of some coat protein from the capsid, while the capsid remains largely intact. Proteolysed particles have lost their icosahedral symmetry and show a protuberance in negative stain EM. Taken together, these results suggest expansion and subsequent proteolysis are essential steps in the uncoating process of TCV, and that the capsid plays multiple roles consistent with ribosome-mediated genome uncoating to avoid host anti-viral activity. Similarly, 3D cryo-EM reconstructions are presented for native equine rhinitis A virus (ERAV) an expanded particle containing no RNA. The native virus fits well with the ERAV crystal structure. The empty particle is a putative disassembly intermediate representing a stage after the release of the RNA genome. A mechanism is suggested that is consistent with the RNA release from the endosome without exposure to the endosomal contents. A crystal structure is presented of satellite tobacco necrosis virus (STNV) virus-like particles containing a small RNA fragment. The coat protein structure is identical to that of native STNV. Although density internal to the coat protein shell has been observed in the experiment that corresponds to earlier experiments, no unambiguous RNA structure can be built into the density. Together, the results presented here shed some light on the life cycle of three of these viruses.
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Jamieson, Lauren Vilda. "The RNA-dependent RNA polymerase of the hepatitis A virus." Thesis, University of Bath, 1998. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268194.
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Zhou, Yu. "Application of RNA Bioinformatics in decoding RNA structure and regulation." Paris 11, 2008. http://www.theses.fr/2008PA112234.
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Ma thèse porte sur le développement de méthodologies informatiques et bioinformatiques pour résoudre des problèmes provenant de questions biologiques liées à l’ARN, telles que la prédiction de structures, l’identification de structures communes, la découverte de cibles des micro-ARN, la prédiction de la régulation de l’épissage, et le design (ou repliement inverse) d'ARN. Le premier chapitre concerne la mise en place d’une méthode itérative pour la prédiction des structures secondaires des introns de groupes 1, incluant les pseudo-nœuds, et la développement d’une base de données complète sur les introns de groupe 1. Dans le deuxième chapitre, je décris mon travail sur l’analyse bioinformatique de la structure des sites d’incorporation de la Pyrrolysine, le 22ème acide aminé, dans des gènes d’archae. Les troisième et quatrième chapitres sont consacrés au développement et à la mise en œuvre de deux méthodes d’analyse de données expérimentales pour la recherche, dans les séquences d’ARN, de cibles de micro-ARN, et de sites de fixation de protéines impliquées dans le processus d’épissage des introns. Enfin, le cinquième chapitre présente un algorithme de design de structures d’ARN avec des contraintes de motifs, faisant appel à des manipulations d’automates et de grammaires non contextuellesMy thesis focuses on the application of RNA bioinformatics analysis to solve the problems originated from biological requirements, ranging from structure prediction, common structure identification, microRNA target discovery, splicing regulation prediction, and RNA design (inverse folding). The first chapter concerns the establishment of an iterative method for the secondary structure prediction of group I introns including pseudo-knots, and the development of a comprehensive group I intron sequence and structure database. In the second chapter, I describe my work on bioinformatics analysis of the Pyrrolysine (Pyl, 22nd amino acid) insertion structure in Pyl-associated genes in archaea. The third and fourth chapters are devoted to develop two methods of experimental data analysis for identification of micro-RNA target sites, and for determination of binding sites of a RNA binding protein implicated in pre-mRNA splicing, independently. Finally, the fifth chapter presents an algorithm for RNA design under motif constraints, involving manipulation of automata and context-free grammars
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Stoppel, Rhea. "Chloroplast RNA metabolism." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-152718.
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Lawrence, Michael S. (Michael Scott) 1975. "[RNA polymerase ribozymes]." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/31193.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Title supplied by cataloger from abstract.
Includes bibliographical references.
The RNA World is a hypothetical ancient evolutionary era during which RNA was both genome and catalyst. During that time, RNA was the only kind of enzyme yet in existence, and one of its chief duties was the replication of RNA. This scenario presupposes that among all possible RNA sequences, there exist RNA replicase ribozymes, capable of synthesizing RNA using the information in an RNA template. The goal of the present work is to provide experimental evidence in support of this conjecture, by isolating such ribozymes in the laboratory. We created a large pool of RNA molecules each containing a previously isolated RNA ligase ribozyme and a large stretch of random RNA. Applying in vitro evolution to select for molecules that could extend a tethered RNA primer using nucleoside triphosphates, we isolated nine distinct classes of polymerase ribozymes. Two of these rudimentary polymerases were further evolved to the point that they each could add 14 nucleotides to an untethered primer-template. One of them was subjected to a detailed further characterization. The polymerization it catalyzes was shown to be accurate, with an average fidelity of nearly 97%. It was shown to be general, with primer-templates of all sequences and lengths being accepted as substrates. Finally, it was shown to be partially processive, with the polymerase achieving processivity as high as 90% in a few instances. The polymerase is currently limited by its low affinity for the primer-template. Future work will focus on improving primer- template binding, in order to produce a polymerase that can synthesize longer RNA.
Michael S. Lawrence.
Ph.D.
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Zhang, Tongchuan. "RNA Structural Modelling." Thesis, Griffith University, 2021. http://hdl.handle.net/10072/404155.
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This thesis outlines the development of several methods for improving RNA secondary and tertiary structure prediction. Novel non-coding RNAs (ncRNAs) are discovered to play various roles in living organisms. A profound understanding of their structures is crucial for learning their functional mechanisms. In Chapter 2, a statistical RNA energy scoring function was developed based on a distance-scaled finite ideal-gas reference state (DFIRE_RNA). The energy score is tested by discriminating native and near-native structures from other decoy structures. We showed that this energy function has a higher success rate in detecting native and near-native structures than other energy functions. In Chapter 3, RNAcmap was established to perform RNA contact prediction based on homology search and evolutional covariance analysis. RNAcmap extended the applicability of evolution-based contact prediction from RNAs within manually curated RNA families to all RNAs. This method has the performance in contact prediction comparable to those generated from the default Rfam alignment. In Chapter 4, RNAmrf was developed to construct multiple sequence alignment based on the probabilistic graph theory. This method for the first time is able to account for pseudoknot base-pairing in sequence alignment that was ignored by existing covariance models. The new method was demonstrated to improve over existing methods in aligning sequences with pseudoknots as well as prediction of pseudoknot base pairs. These tools are available at https://sparks-lab.org/ and should facilitate the tasks of predicting RNA secondary and tertiary structures.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
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Swiatecka-Hagenbruch, Monika. "Phagenähnliche RNA-Polymerasen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15924.
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Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben.Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes.
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Forrest, Sophie. "Use of RNA aptamers to further elucidate the role of FMDV RNA-dependent RNA polymerase (3Dpol) in replication." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574540.
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Replication of poliovirus involves complexes between the viral RNA-dependent-RNA polymerase 3Dpol, its precursor 3CD and the primer Vpg, although the details of replication initiation and elongation are not fully understood. Much less is known about replication complexes of other picornaviruses, especially FMDV. To gain insight into these mechanisms, we carried out in vitro selection to produce RNA aptarners to FMDV 3Dpol. Aptamers are small nucleic acid molecules that can bind targets with affinities and specificities to rival those of antibodies, but offer significant advantages as molecular tools. They are generated by an in vitro selection process termed SELEX and can be modified chemically for use both in vitro and in live cells and to facilitate their detection.
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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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Kajitani, Takuiya. "Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi." Kyoto University, 2018. http://hdl.handle.net/2433/232146.
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Attwater, James. "Ice as a medium for RNA-catalysed RNA synthesis and evolution." Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/246525.
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A critical event in the origin of life is thought to have been the emergence of a molecule capable of self-replication and evolution. According to the RNA World hypothesis, this could have been an RNA polymerase ribozyme capable of generating copies of itself from simple nucleotide precursors. In vitro evolution experiments have provided modern examples of such ribozymes, such as the R18 RNA polymerase ribozyme, exhibiting basic levels of this crucial catalytic activity; R18’s activity, however, falls far short of that required of an RNA replicase, leaving unanswered the question of whether RNA can catalyse its self-replication. This thesis describes the development and use of a novel in vitro selection system, Compartmentalised Bead-Tagging (CBT), to isolate variants of the R18 ribozyme with improved sequence generality and extension capabilities. CBT evolution and engineering of polymerase ribozymes, together with RNA template evolution, allowed the synthesis of RNA molecules over 100 nucleotides long, as well as the RNA-catalysed transcription of a catalytic hammerhead ribozyme. This demonstrates the catalytic capabilities of ribozyme polymerases. The R18 ribozyme was also exploited as an analogue of a primordial replicase, to determine replicase behaviour in different reaction environments. Substantial ribozyme polymerisation occurred at −7˚C in the liquid eutectic phase of water-ice; increased ribozyme stability at these low temperatures allowed longer extension products to be generated than at ambient temperatures. The concentration effect of eutectic phase formation could also yield RNA synthesis from dilute solutions of substrates, and provide quasicellular compartmentalisation of ribozymes. These beneficial physicochemical features of ice make it a potential protocellular medium for the emergence of primordial replicases. Ice also could serve as a medium for CBT, allowing the isolation of a polymerase ribozyme adapted to the low temperatures in the ice phase, demonstrating the primordial potential and modern feasibility of ribozyme evolution in ice.
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Yin, Peng. "Structural and functional studies of reovirus RNA-dependent RNA polymerase complex." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ32040.pdf.
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Cheng, Chi-Ping. "VIRAL RNA ELEMENTS AND HOST GENES AFFECTING RNA RECOMBINATION IN TOMBUSVIRUSES." UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/436.
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RNA recombination is a major factor driving viral evolution and contributing to new disease outbreaks. Therefore, understanding the mechanism of RNA recombination can help scientists to develop longer lasting antiviral strategies. Tombusviruses are one of the best model RNA viruses to study RNA virus recombination. My goals were to dissect the mechanism of tombusviral RNA recombination. To do so, in my thesis, I describe my results on the roles of (i) the viral replicase and the viral RNA templates; and (ii) the effect of host factors on tombusvirus recombination events. To study the mechanism of RNA recombination without the influence of selection pressure on the emerging recombinants, we developed an in vitro RNA recombination assay based on viral RNA templates and purified viral replicase preparations. Using this in vitro assay, we demonstrated that replicase driven template switching is the mechanism of recombination, whereas RNA ligation seems less likely to be a major mechanism. In addition, we also studied the role of RNA substrates, in more detail. Our results showed that viral replicase preferred to use functional RNA domains in the acceptor RNAs over random switching events. Host factors may also play important roles in RNA recombination. Using yeast as a model system for studying replication and recombination of a tombusvirus replicon, we identified 9 host genes affecting tombusvirus RNA recombination. Separate deletion of five of these genes enhanced generation of novel viral RNA recombinants. Further studies on one of these genes, XRN1, a 5-3 exoribonuclease, indicated that it might be involved in degradation of tombusvirus RNAs. Lack of Xrn1p resulted in accumulation of truncated (partially degraded) replicon RNAs, which became good templates for RNA recombination. To further study Xrn1p, we overexpressed Xrn4p of Arabidopsis thaliana, a functional analogue of the yeast Xrn1p, in Nicotiana benthamiana plants. After superinfecting the Xrn4p-overexpressing N. benthamiana with tombusvirus, truncated tombusvirus genomic and subgenomic RNA1 were observed. Some of the identified tombusvirus variants were infectious in protoplasts and could systemically infected N. benthamiana plants. Overall, this is the first report that a single host gene can affect rapid viral evolution and RNA recombination.
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Dall'Ara, Mattia. "RNA/RNA interactions involved in the regulation of Benyviridae viral cicle." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ019/document.
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Pour préserver l’intégrité de leur génome, les virus multipartite à ARN nécessitent une forte multiplicité d’infection qui représente un coût biologique inapproprié en terme de réplication virale. Dans cette étude, un réseau d’interaction entre ARN génomiques (ARNg), constitué d’au moins un type de chaque ARNg est proposé. Un tel réseau permet de réduire les coûts biologiques liés à la réplication en assurant une reconnaissance intermoléculaire et une mobilisation d’un complexe RNP modulaire maintenant l’intégrité du génome. Un tel complexe est considéré comme l’unité infectieuse mobile assurant la dissémination du virus dans la plante entière. Le but de cette thèse a été de démontrer l’existence d’interactions entre les ARNg du beet necrotic yellow vein virus (BNYVV) et de déterminer l’incidence de ces interactions sur le cycle viral. Une formule génomique a été déterminée pour différentes plantes et tissus. Les ARNg ont tous été co-détectés dans des cellules isolées issues de tissus infectés. Un domaine d’interaction entre l’ARN1 et 2 a été identifié in vitro et in silico puis évaluée in vivo par des approches de mutagenèse et de complémentationMultipartite RNA virus condition to provide a complete set of genomic segments in each infected cell implies a high level of MOI that results in an unsustainable biological cost in terms of viral replication. In the proposed model, to minimize the cost of the genome integrity preservation, a network of RNA/RNA interactions determines the recognition and the mobilization of at least one of each genomic RNAs in a modular RNP complex. Such complex must be considered as the mobile infectious unit of the segmented genome during viral spread in the plant. The Aim of this thesis was to experimentally determine the existence of RNA/RNA interactions between BNYVV RNAs and their implication in the viral cycle. BNYVV genomic segments have been co-detected within isolated single cells from systemic tissues where they accumulate to reach set point genome formulas. In the model where vRNAs interact each other to form the minimal mobile infective unit, RNA1 and RNA2 interaction domain has been identified in silico and in vitro. The rationale of such an interaction has been provided in vivo using BNYVV and Beet soil-borne mosaic virus chimeras
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Hunter, Lydia Jane. "The role of RNA-dependent RNA-polymerase 1 in antiviral defence." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648332.
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Turnbull, Carly. "Sequence and structure requirements of Y RNA-derived small RNA biogenesis." Thesis, University of East Anglia, 2014. https://ueaeprints.uea.ac.uk/49478/.
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Numerous small non-coding RNAs have been identified in mammalian cells, including microRNAs and piwi-interacting RNAs. The patterns of gene expression within cells can be altered in response to cellular stress. To examine the effects of cellular stress on all small RNA types, a number of human cell lines were treated with Poly(I:C), a mimic of viral infection, and the levels of small RNAs were examined by next generation sequencing. Surprisingly, we did not find many differentially expressed microRNAs, but we discovered a new class of small RNAs that were 30-35 nucleotides and showed up-regulation following Poly(I:C) treatment. These slightly longer small RNAs were derived from many different types of non-coding RNA and only very few small RNAs were derived directly from messenger RNAs. Small RNAs derived from various types of RNA were validated by northern blot. Further sequencing libraries were prepared for Poly(I:C)-treated and untreated MCF7 cells, as well as Poly(I:C)-treated and untreated SW1353 cells. The human Y5 RNA gene was chosen as an example of a Poly(I:C)-induced small RNA-producing gene. This gene was cloned into an expression construct and systematically mutated to alter the sequence or secondary structure of the resulting Y RNA. These mutant plasmids were expressed in mouse cells and the effect on small RNA production determined. These individual mutants together helped to determine a region vital for cleavage, and that it is the structure rather than the sequence within this region that is important. A high-throughput method was also implemented, involving the generation of large pools of plasmids containing all possible sequences within a particular region of the RNA gene. These pools were expressed in mouse cells and the mutants that were expressed and processed into small RNAs were sequenced. These experiments showed that the formation of the large internal loop determines the internal cleavage site.
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Hengrung, Narin. "Structure of the RNA-dependent RNA polymerase from influenza C virus." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:694e16a6-f94e-4375-a1f9-7e250aea7343.
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The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.
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Grierson, Patrick Michael. "The BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337865492.
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Burger, Kaspar. "CDK9 links RNA polymerase II transcription to processing of ribosomal RNA." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-167037.
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