Dissertations / Theses: 'RNA Cleavage'

1

Forster, Anthony Carlyle. "Self-cleavage of plant pathogenic RNAs." Title page, contents and summary only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09phf7331.pdf.

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2

Mitchell, Michelle Hall. "Understanding structural mechanisms of endolytic RNA cleavage enzymes." Tallahassee, Florida : Florida State University, 2009. http://etd.lib.fsu.edu/theses/available/etd-07102009-145044.

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Thesis (Ph. D.)--Florida State University, 2009.
Advisor: Hong Li, Florida State University, College of Arts and Sciences, Institute of Molecular Biophysics. Title and description from dissertation home page (viewed on Oct. 26, 2009). Document formatted into pages; contains vi, 68 pages. Includes bibliographical references.

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3

Jin, Yan. "In vitro and in vivo studies of DNA cleavage and targeted cleavage of HIV REV response element RNA by metallopeptides." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1155606670.

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4

Marriott, Robert Edward. "Accelerated cleavage of phosphate esters." Thesis, University of Cambridge, 1994. https://www.repository.cam.ac.uk/handle/1810/272476.

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5

Hurst, Phillip C. "Lanthanum(III)-promoted cleavage of RNA and cyclic nucleotides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0015/NQ44460.pdf.

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6

Barman, Jharna. "Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8272.

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7

Ruan, Wenjie. "Evolution of two modes of intrinsic RNA polymerase transcript cleavage." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-136940.

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8

Jennebach, Stefan. "RNA polymerase I domain architecture and basis of rRNA cleavage." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-146779.

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9

Sheldon, Candice Claire. "Hammerhead mediated self-cleavage of plant pathogenic RNAs /." Title page, contents and summary only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phs544.pdf.

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10

Chambers, A. "RNA 3' cleavage and polyadenylation in oocytes, eggs and embryos of Xenopus laevis." Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380275.

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11

Ingle, Shakti Singh. "RNA structure investigation: a deuterium kinetic isotope effect/hydroxyl radical cleavage experiment." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12787.

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Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
The hydroxyl radical is widely used as a high-resolution footprinting agent for DNA and RNA. The hydroxyl radical abstracts a hydrogen atom from the sugar- phosphate backbone of a nucleic acid molecule, creating a sugar-based radical that eventually results in a strand break. It was shown previously that replacement of deoxyribose hydrogen atoms with deuterium results in a kinetic isotope effect (KIE) on hydroxyl radical cleavage of DNA. The KIE correlates well with the solvent accessible surface area of a deoxyribose hydrogen atom in DNA. We chose the structurally well-defmed sarcin-ricin loop (SRL) RNA molecule as a model system to extend the deuterium KIE/hydroxyl radical cleavage experiment to RNA. We observed a substantial KIE upon deuteration of the 5'-carbon of the ribose. Values ranged from 1.20 to 1.96, and depended on the position of the residue within the SRL. We found a smaller KIE upon 4'-deuteration. Values ranged from 1.05 to 1.23. Values of 5' and 4' KIEs correlate with the extent of cleavage and with the solvent accessible surface areas of ribose hydrogen atoms ofthe SRL. Gel electrophoresis of cleavage products reveals that the strand break is terminated at the 5' end by multiple chemical species. Upon 3'-radiolabeling a specifically 5'-deuterated SRL RNA molecule, we observed a KIE on the production of a cleavage product having a gel mobility different from that of a phosphate-terminated RNA strand. Reduction with sodium borohydride gave rise to an RNA fragment terminated by a 5'-hydroxyl group. These experiments are consistent with 5' hydrogen abstraction by the hydroxyl radical producing a 5'-aldehyde-terminated RNA strand that retains the nucleotide from which the hydrogen atom was abstracted. This is the first report of such a species. This chemistry has important implications for the interpretation of structural analysis experiments on RNA that rely on primer extension to synthesize eDNA copies of hydroxyl radical cleavage products. The different 5'-terminated products resulting from hydroxyl radical cleavage at a given nucleotide would yield cDNAs of two different lengths, thereby distributing the cleavage intensity over two nucleotides instead ofone and lowering the resolution ofthe experiment.

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12

Borda, Emily J. "Investigation of ribozyme structure and dynamics through photochemical crosslinking and metal ion cleavage /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/11616.

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13

Åström, Hans. "Studies on phosphate ester cleavage and development of oligonucleotide based artificial nucleases (OBAN's) /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-935-8/.

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14

Yeung, Man-lung, and 楊文龍. "Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31245055.

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15

Jourdan, Stefanie Simone. "The recognition and cleavage of RNA by members of the RNase E family." Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496130.

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16

Becaud, Jessica. "Towards RNase H mimics : artificial catalysts for the site specific cleavage of RNA /." [S.l.] : [s.n.], 2005. http://www.zb.unibe.ch/download/eldiss/05becaud_j.pdf.

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17

Usher, Louise. "RACE-Seq identifies the Argonaute-2 cleavage products of RNA interference-based oligonucleotides." Thesis, University of Westminster, 2018. https://westminsterresearch.westminster.ac.uk/item/qq4w2/race-seq-identifies-the-argonaute-2-cleavage-products-of-rna-interference-based-oligonucleotides.

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The recent announcement of the first successful Phase III clinical trial of a RNA interference (RNAi)-based therapeutic is a major achievement in the field. Synthetic RNAi therapeutic oligonucleotides are either first cleaved by Dicer or incorporate directly into the Argonaute-2 RNA-induced silencing complex (AGO2-RISC) and directs the protein complex to homologous RNA. Cleavage of target RNA occurs opposite bases 10-11 when counting from the 5' end of the hybridized siRNA guide strand. The capture and identification of these cleaved products by 5' Rapid Amplification of cDNA Ends and Sanger sequencing remains the gold standard for confirming Argonaute-2 mediated RNAi cleavage. Next Generation Sequencing of 5' RACE has brought new insights into the biological activity of RNAi-based oligonucleotides. This work currently represents the largest undertaking using RACE-Seq to investigate AGO2-RISC-mediated activity. RACESeq reported the expected RISC-cleaved product for each of the oligonucleotides investigated. Additionally, RACE-Seq analysis revealed that some of the oligonucleotides could be processed into multiple active siRNA molecules. Analysis of the activity of a Dicer substrate siRNA targeting transthyretin revealed that this molecule by-passed Dicer processing but still induced RNAi activity. In examining RACE-Seq peak profiles, an on-target mechanism of action (MOA) for up to four active siRNA derived from siRNA19 is proposed. The shRNA19 RACE-Seq assay predicted that this hairpin molecule probably exists as two distinct forms, one with a 7-nucleotide loop and the other with a 5-nucleotide loop. The project also focused on optimising the library preparation, data filtering and data presentation for RACE-Seq. A simplified, low computation data analysis pipeline was designed and used to align the filtered dataset to a reference sequence and to count the 5' ends. RACE-Seq is presented as a suitable solution for investigating, discriminating and quantifying specific RNA cleavage events and visualizing evidence for an on-target MOA of RNAi based oligonucleotide therapeutics.

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18

Joyner, Jeff C. "Synthesis and Evaluation of Catalytic Metallodrugs and Analysis of RNA Cleavage by Mass Spectrometry." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343764674.

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19

Hardy, Jessica. "Human cleavage factor I (CFIm) and its role in alternative polyadenylation of pre-mRNA." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:a3ba5d10-b3fa-4ab7-9709-a0d642e21543.

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For many human protein-coding genes, alternative cleavage and polyadenylation (APA) of pre-mRNA generates distinct 3' untranslated regions (3'UTRs) with differing regulatory potential. Widespread 3'UTR shortening via APA occurs in proliferative cell states, including cancer, where it can lead to oncogene overexpression. There has therefore been significant interest in identifying factors which influence poly(A) site choice in different physiological states. The multi-subunit human cleavage factor I complex (CFIm), a core component of the mammalian pre-mRNA cleavage machinery, has been identified as a potential master regulator of APA, as its depletion leads to widespread 3'UTR shortening. However, mechanistic understanding of how CFIm influences poly(A) site selection, and how its activity is regulated, is lacking. In this work, gene editing was used to generate cell lines with substantial, permanent depletion of the 25 kDa or 68 kDa subunits of CFIm (CFIm25 and CFIm68), which exhibited the expected 3'UTR shortening for representative transcripts. Reversal of this 3'UTR shortening by CFIm25 or CFIm68 re-expression provided the basis for a complementation assay, which allowed various aspects of CFIm25 and CFIm68 function to be investigated in vivo. The capacity of CFIm25 to recognise UGUA RNA sequences was shown to make an important contribution to poly(A) site selection transcriptome-wide, and a novel function for the C-terminal arginine/serine-rich (RS domain) of CFIm68 in poly(A) site selection was identified. The potential contribution of CFIm post-translational modification (PTM) to APA regulation was also explored. Novel acetylation sites on CFIm25 and CFIm68 were identified, as well as extensive serine phosphorylation in the CFIm68 RS domain. Complementation analysis revealed that phosphomimetic mutations in this RS domain inhibited distal poly(A) site selection, suggesting a potential role for CFIm68 phosphorylation in APA regulation. Taken together, the findings presented here provide insights into several important determinants of CFIm function, and the complementation assay developed provides a useful tool for future investigations.

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20

Zhao, Hongwei. "A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System." Oxford, Ohio : Miami University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1218547019.

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21

Ruan, Wenjie [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "Evolution of two modes of intrinsic RNA polymerase transcript cleavage / Wenjie Ruan. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1017688303/34.

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22

Jennebach, Stefan [Verfasser], and Patrick [Akademischer Betreuer] Cramer. "RNA polymerase I domain architecture and basis of rRNA cleavage / Stefan Jennebach. Betreuer: Patrick Cramer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1025047257/34.

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23

Wang, Yu. "Investigations of functionalized cyclodextrins as artificial enzymes on the cleavage of RNA and DNA analogues." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0004/NQ42987.pdf.

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24

Wallace, Andrew J. "Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374489993.

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25

Lindell, Magnus. "Lead(II) as a Tool for Probing RNA Structure in vivo." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis: Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5780.

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26

Han, Bo W. "Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/782.

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Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

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27

Han, Bo W. "Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/782.

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Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

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28

Kang, Ting-Wei Patrick. "Studies Of Molecular Structure-Function Relationships For A Pyrrolysine-Containing Methyltransferase And Novel Rna-Cleaving Protein Nucleic Acids." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1230879583.

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29

Johnstone, Nicholas. "Aluminium and lead complexes of Nitrogen/Oxygen Donor ligands. Cyclic ester polymerisation and studies towards the understanding of lead induced RNA cleavage." Thesis, University of Sussex, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504336.

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30

Bradford, Seth Stephen. "The Design and Evaluation of Catalytic MetalloDrugs Targeting HCV IRES RNA: Demonstration of a New Therapeutic Approach." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1345132549.

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31

Luo, Weifei. "The coupling of transcription termination by RNA polymerase II to MRNA 3' end processing in Saccharomyces cerevisiae /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.

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Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006.
Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

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32

Oruganty, Aparna. "Role of the Cytoplasmic Polyadenylation Element Binding Proteins in Neuron: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/648.

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Genome regulation is an extremely complex phenomenon. There are various mechanisms in place to ensure smooth performance of the organism. Post-transcriptional regulation of gene expression is one such mechanism. Many proteins bind to mRNAs and regulate their translation. In this thesis, I have focused on the Cytoplasmic Polyadenylation Element Binding family of proteins (CPEB1-4); a group of sequence specific RNA binding proteins important for cell cycle progression, senescence, neuronal function and plasticity. CPEB protein binds mRNAs containing a short Cytoplasmic Polyadenylation Element (CPE) in 3’ untranslated Region (UTR) and regulates the polyadenylation of these mRNAs and thereby controls translation. In Chapter II, I have presented my work on the regulation of mitochondrial function by CPEB. CPEB knockout mice have brain specific defects in mitochondrial function owing to a reduction in Electron transport chain complex I component protein NDUFV2. CPEB controls the translation of this NDUFV2 mRNA and thus affects mitochondrial function. A consequence of this reduced bioenergetics is reduced growth and branching of neurons, again emphasizing the importance of this pathway. Chapter III focuses on the role of CPEB4 in neuronal survival and protection against apoptosis. CPEB4 shuttles between nucleus and cytoplasm and becomes nuclear in response to stimulation with ionotropic glutamate receptors, focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose; nuclear CPEB4 affords protection against apoptosis in ischemia model. The underlying cause for nuclear translocation is reduction in Endoplasmic Reticulum calcium levels. These studies give an insight into the function and dynamics of these two RNA binding proteins and provide a better understanding of cellular biology.

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33

Anta, Rodríguez Héctor. "Characterization of the role of the CPEB family of RNA-binding proteins in neurodegeneration." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/664116.

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Alzheimer’s disease (AD) is the most common type of dementia in the elderly. It is associated to a progressive loss of memory, problems in learning and behaviour changes. This disease is characterized by the accumulation of extracellular amyloid β (Aβ) aggregates and intracellular deposits of hyperphosphorylated Tau protein. Both aggregates trigger neuronal apoptosis and glial inflammation, leading to the cognitive decline found in AD patients. Interestingly, the serine protease tissue plasminogen activator (tPA), which is induced by Aβ, has been found to play a dual, dose-dependent role in the disease. Physiological levels of tPA confers neuroprotection through plasmin generation and Aβ degradation. In contrast, high doses of tPA activate intracellular signalling pathways in neurons and glial cells, inducing neuronal apoptosis and inflammation. However, the molecular mechanisms that govern the regulation of tPA expression in AD have still not been fully elucidated. In this work, we demonstrate that Aβ-induced tPA expression is regulated by translational control. In particular, our results show that CPEB1 and CPEB4, two members of the cytoplasmic polyadenylation element binding protein (CPEB) family of RNA-binding proteins, control local tPA synthesis in response to Aβ. Specifically, Aβ promotes tPA mRNA translation in the dendritic spines through synaptic polyadenylation and synaptic cleavage and polyadenylation, a mechanism that is impaired in the absence of CPEB1 or CPEB4. Our results also demonstrate that the pre-mRNA 3'-end processing machinery required for the efficient cleavage and polyadenylation of mRNAs is also present in the synaptic terminals. Finally, we have found that, similarly to tPA, CPEB4 is upregulated in the synaptic terminals in response Aβ and in vivo in the brain of AD patients.
La enfermedad de Alzheimer (EA) es la demencia más común en la tercera edad. Está asociada a una pérdida progresiva de memoria, problemas de aprendizaje y cambios de comportamiento. Esta enfermedad se caracteriza por la acumulación de agregados extracelulares de proteína β-amiloide (Aβ) y depósitos intracelulares de proteína Tau hiperfosforilada. Ambos agregados inducen apoptosis neuronal e inflamación mediada por las células de la glia, lo cual desencadena el declive cognitivo característico de los enfermos de EA. En este sentido, se ha demostrado que una serina proteasa, el activador del plasminógeno tisular (del inglés "tissue plasminogen activator", tPA), cuya expresión se induce por Aβ, juega un doble papel clave en la enfermedad en función de sus niveles. Por un lado, unos niveles fisiológicos de tPA pueden ser neuroprotectores a través de la generación de plasmina, con la consiguiente degradación del Aβ. Por otro lado, unos niveles altos de tPA activan cascadas de señalización intracelular en neuronas y células de la glia, lo que induce apoptosis neuronal e inflamación. Los mecanismos moleculares que rigen la regulación de la expresión de tPA en la EA no se conocen con claridad. En este trabajo, demostramos que la expresión de tPA inducida por Aβ está regulada por control traducional. En concreto, nuestros resultados muestran que CPEB1 y CPEB4, dos miembros de la familia CPEB de proteínas de unión a RNA (del inglés "cytoplasmic polyadenylation element binding, CPEB), controlan la síntesis de tPA en respuesta a Aβ. Concretamente, el Aβ promueve la traducción del ARNm de tPA en las espinas sinápticas a través de poliadenilación sináptica, y procesamiento y poliadenilación alternativos sinápticos, un mecanismo que se ve interrumpido en ausencia de CPEB1 o CPEB4. Nuestros resultados también demuestran que la maquinaria de procesamiento de los extremos 3' del pre-ARNm necesaria para llevar a cabo este proceso está presente en los terminales sinápticos. Por último, hemos encontrado que, al igual que tPA, CPEB4 se sobreexpresa en los terminales sinápticos en respuesta a Aβ, así como en el cerebro de pacientes con EA.

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Burns, David M. "Post-Transcriptional Control of Human Cellular Senescence: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/491.

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The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.

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35

Kan, Ming-Chung. "Analysis of CPEB Family Protein Member CPEB4 Function in Mammalian Neurons: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/362.

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Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.

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36

Raoelijaona, Raivoniaina. "Compréhension des rôles des complexes Nob1/Pno1 et RPS14/Cinap dans la maturation cytoplasmique de la petite sous-unité ribosomique (pré-40S) chez les eucaryotes." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0221/document.

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Les ribosomes sont des complexes nucléoproétiques responsables de la traduction. Chez les eucaryotes, la biogenèse du ribosome est un processus complexe très régulé qui fait intervenir un nombre important de facteurs d’assemblages (~200). La construction d’un ribosome est initiée dans le nucléole puis continue dans le nucléoplasme et se termine dans le cytoplasme. La maturation cytoplasmique de la petite sous-unité ribosomale implique la dissociation séquentielle des facteurs d’assemblage tardifs et la maturation finale de l’ARNr 18S. Ce processus est catalysé par l’endonucléase Nob1 qui assure la coupure de l’extrémité 3’ du précurseur de l’ARNr 18S (pré-18S) aboutissant à sa forme mature. Ce mécanisme est coordonné par la protéine Pno1 qui est le partenaire de Nob1. Des informations détaillées sur l’architecture des particules pré-ribosomiques nous ont permis de mieux comprendre les différents intermédiaires de la biogenèse. Cependant, certains aspects fonctionnels comme la conformation adoptée par Nob1 pour assurer la coupure du site D du pre-18S reste encore flou. L’objectif de mon travail a été de mieux comprendre les aspects très tardifs de la maturation cytoplasmique du ribosome. Pour ce faire, nous avons redéfini l’organisation modulaire de l’endonucléase Nob1 chez les eucaryotes pour ensuite étudier son mode d’interaction avec son partenaire Pno1. Des tests fonctionnels in vitro ont été effectués pour étudier le rôle de Pno1 dans la régulation de la coupure par Nob1.Nos résultats nous ont permis de montrer que le domaine catalytique de Nob1 adopte une conformation atypique. En effet le domaine PIN est composé de deux fragments (res 1-104 and 230-255) séparé par une boucle interne qui est importante pour la reconnaissance avec son partenaire Pno1. Nos études nous ont également montré que Pno1 inhibe l’activité de Nob1 probablement en reconnaissant directement l’ARNr substrat, masquant ainsi le site de coupure de l’endonucléase. Ces résultats sont complémentaires et cohérents avec les données structurales de cryo-EM de la particule pré-40S humaine récemment publiées. En effet, Nob1 est dans une conformation incapable de couper le pré-ARNr puisque son domaine catalytique se retrouve à une distance d’environ 30Å de son ARN substrat. Ce phénomène implique donc des changements de conformations ou encore la nécessité de protéine accessoire pour déplacer certains facteurs. La protéine Cinap est impliqué dans la maturation de l’ARNr 18S. Nos études d’interaction avec les protéines localisées au niveau de la plateforme (à savoir RPS14, RPS26, le complexe Nob1/Pno1) ont permis de montrer que Cinap pouvait former un complexe tripartite avec l’endonucléase Nob1 et son partenaire Pno1. De plus, Cinap est capable de reconnaitre RPS26 dans un complexe RPS14-dépendant. Il est important de noter que RPS26 est un composant de la petite sous-unité qui remplace Pno1 dans le ribosome mature. De ce fait le recrutement de RPS26 au sein du pré-ribosome nécessite la dissociation de Pno1 et cet échange serait assurée par Cinap. Sur la base des travaux effectués, nous pouvons proposer un modèle de maturation où la formation du complexe Cinap/Pno1 induirait un changement de conformation permettant à Nob1 de reconnaitre son substrat et donc de catalyser la coupure du site D qui aboutit à la maturation de l’ARNr 18S et donc à la production de la sous-unité 40S mature
Ribosomes are translational machineries universally responsible of protein synthesis. In eukaryote, ribosome assembly is a complex and highly regulated process that requires coordinated action of more than 200 biogenesis factors. Ribosome assembly is initiated in the nucleolus, continues in the nucleoplasm and terminates in the cytoplasm. The cytoplasmic maturation events of the small ribosomal subunit are associated with sequential release of the late assembly factors and concomitant maturation of the pre-rRNA. During final maturation of the small subunit, the pre-18S rRNA is cleaved off by the endonuclease Nob1, which activity is coordinated by its binding partner Pno1. Detailed information on pre-ribosomal particle architectures have been provided by structural snapshots of maturation events. However, key functional aspects such as the architecture required for pre-rRNA cleavage have remained elusive. In order to better understand these late steps of cytoplasmic pre-40S maturation, we first redefine the domain organization of Nob1, then study its binding mode with Pno1 using different tools such as sequence analysis, structure prediction and biochemical experiments and, we then performed functional assay to elucidate the role played by Pno1 during the pre-18S rRNA maturation.Our results have shown that eukaryotic Nob1 adopts an atypical PIN domain conformation: two fragments (res 1-104 and 230-255) separated by an internal loop, which is essential for Pno1 recognition. We also found out that Pno1 inhibits Nob1 activity likely by masking the cleavage site. Our findings further support the recently published cryo-EM structure of the pre-40S, where Nob1 displays an inactive conformation. Moreover, 18S rRNA 3’-end cleavage has to happen and this implies structural rearrangement or requirement of some accessory proteins such as Cinap, an atypical kinase involved in pre-18S processing. Studying the interplay between proteins localized in the pre-40S platform (RPS14, RPS26, Nob1/Pno1 complex) has shown that Cinap is able to form a trimeric complex with Nob1 and its binding partner Pno1. Furthermore, Cinap can recognize RPS26 in a RPS14-dependent manner, which had already been studied with its yeast counterpart. It is important to note that RPS26 is the ribosomal protein replacing Pno1 in the mature ribosome. Our finding clearly suggests a mechanism where RPS26 recruitment to the ribosome requires Pno1 dissociation. This exchange would be carried out by Cinap. Therefore, we can suggest a simplified model as follow: upon binding with Pno1, the newly formed complex (Cinap/Pno1) will trigger a conformational change, which will allow the endonuclease Nob1 to reach its substrate (D-site) and perform its cleavage resulting in mature 18 rRNA generation

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37

Azad, Robert Navid. "Hydroxyl radical cleavage of nucleic acids: understanding RNA cleavage profiles and identifying DNA structural motifs." Thesis, 2014. https://hdl.handle.net/2144/15123.

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High-resolution techniques to characterize the three-dimensional structure of nucleic acids are critical for understanding the mechanisms of action of biologically important RNA and DNA molecules. Methods based on chemical probing have been particularly useful in gaining insight into the structures of nucleic acids in solution. The hydroxyl radical has been widely adopted as a chemical probe for DNA and RNA structure since its first application to protein-DNA footprinting. This dissertation describes efforts to improve upon the current model of how the hydroxyl radical cleaves the RNA backbone, through the use of specifically deuterated ribonucleoside triphosphates (NTPs). The synthesis and purification of deuterated NTPs are described in detail, as well as their application to the study of two RNAs: the sarcin-ricin loop (SRL) RNA - a biologically active region of ribosomal RNA - and a short RNA designed to lack secondary structure. Measurement of deuterium kinetic isotope effects (KIEs) on the cleavage of these RNAs suggests that it is possible to use this experiment to identify the GUA base triple structural motif that is commonly found in RNA. Abstraction of a 5' ribose hydrogen atom in RNA yields a fragment containing a 5'-aldehyde terminus with the sugar and base intact. Comparison of primer extension products of cleaved SRL RNA with or without deuterium substituted at the C5' ribose position of uracil residues demonstrated that the 5' aldehyde-terminated fragment can serve as a template for reverse transcription. Implications of the presence of a 5'-aldehyde terminus on hydroxyl radical cleavage analysis are discussed in the context of reverse transcriptase-mediated primer extension, a commonly used method. Structural features of naked DNA molecules with known protein binding sequences were explored using hydroxyl radical cleavage analyzed by capillary gel electrophoresis. An application was written in MATLAB to deconvolute and integrate cleavage intensities of hundreds of peaks in an electropherogram. In many cases, comparison of the cleavage profile to the minor groove width found in an X-ray co-crystal structure of the DNA-protein complex revealed a high degree of correlation.

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Brown, Abigail Leigh. "Competing RNA Structures and Their Effects on HDV Antigenomic RNA Self-cleavage and mRNA Processing." Diss., 2010. http://hdl.handle.net/10161/3131.

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HDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle.

A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product.

The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.

Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.

Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.

Dissertation

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39

Lin, Hsuan-Yung, and 林瑄詠. "Cleavage of coronavirus RNA is associated with cellular endoribonuclease RNase L." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/xmz73c.

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碩士
國立中興大學
獸醫病理生物學研究所
106
Among the cellular endoribonucleases, RNase L is a well-studied endoribonuclease associated with antiviral defense induced by innate immunity. RNase L cleaves viral and host RNA including 28S and 18S rRNA predominantly after single-stranded UA and UU dinucleotides. In coronaviruses, the cleavage of coronaviral RNA and the features for the cleavage preference by RNase L has not been previously probed. In an attempt to test whether coronavirus defective interfering (DI) RNA with transcription regulating sequence (TRS) is able to synthesize subgenomic DI RNA 12.7 (sgmRNA 12.7), it was unexpectedly found by Northern blot assay that, in addition to predicted sgmRNA 12.7, an RNA fragment designed ST RNA with a size less than sgmRNA 12.7 was also found. Subsequent study demonstrated that the cleaved site for the ST RNA is located downstream of 12.7 sgmRNA TRS, in the loop region of stem-loop II and after UU dinucleotides. The cleaved DI RNA 12.7 fragment ST RNA was identified with Northern blot assay in both uninfected and bovine coronavirus (BCoV)-infected HRT18-cells, indicating that the cellular factors are responsible for the cleavage. The similar cleavage was also found in HEK-293T and A549 cells. To characterize the features of the cleavage, mutagenesis followed by Northern blot assay was performed. It was found that the cellular factor preferentially cleaved after UU or UA dinucleotides, and the sequences upstream and downstream of UU dinucleotides has influence on the efficiency of the cleavage, consisting with the general criteria of cleavage by cellular RNase L. Since the cleavage of 28S and 18S rRNA indicates the activation of RNase L, the cleavage of rRNA as well as DI RNA 12.7 found in A549 cells suggested that the cleavage of DI RNA 12.7 is correlated to RNase L. In conclusion, we for the first time demonstrated that the cleavage of coronavirus genome is correlated to RNase L. Accordingly, since similar cleavage was also found in cells (HRT-18 and HEK-293T cells) from which RNase L are not inducible, it is proposed that these cells may have basal levels of activated RNase L, which is independent of innate immunity and may serve as a non-specific role for the fast cleavage of foreign single-stranded RNA.

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Nwe, Kido. "Lanthanide(III)-macrocyclic complexes as catalysts for RNA cleavage and para-cest agents." 2008. http://proquest.umi.com/pqdweb?did=1546798211&sid=6&Fmt=2&clientId=39334&RQT=309&VName=PQD.

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Thesis (Ph.D.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Nov. 25, 2008) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Morrow, Janet R. Includes bibliographical references.

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Cicmil, Nenad. "Biochemical and structural studies of the enzymes involved in RNA cleavage and modification /." 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3314749.

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Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2008.
Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2972. Advisers: Colin Wraight; Raven H. Huang. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

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Chen, Jyh-Yeu, and 陳治宇. "Cleavage of Birnaviral RNA by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/61904720949675627314.

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Wang, Wei-Lun, and 汪惟倫. "Hammerhead ribozymes on the cleavage of VP5 RNA in infectious pancreatic necrosis virus (IPNV) at low temperature." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/65367701652998611726.

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碩士
國立臺灣大學
漁業科學研究所
88
Infectious pancreatic necrosis virus (IPNV) is one of the contagious and widespread fish diseases. IPNV belongs to the Birnaviridae family of virus, which has bisegmented ds-RNA genomes and often causes seriously damages to the aquaculture in Taiwan. VP5 is encoded from the small ORF located at 5' end of A segment. VP5 protein had been demonstrated to play a dual role on the regulation of virus replication and up-regulation of the survival factor Mcl-1 during IPNV infection. In this study, we designed highly specific hammerhead ribozymes to test the cleavages of VP5 RNA. Sixteen predicted cleavage sites (14 in loop and 2 in stem) of hammerhead ribozyme were selected according to the secondary structure of VP5 cDNA sequence by GCG Mfold program. The ribozyme RNAs and VP5 RNA (partial sense RNA of IPNV) were both synthesized by in vitro transcription. Five (4 in loop and 1 in stem) out of 16 hammerhead ribozymes (predicted cleavage site) possess the trans-cleavage activity at 37C in vitro. The trans-cleavage activities of RZ-5 and RZ-8 are concentration-dependent of divalent-cation (Mg++) fashion at 25C in vitro. Divalent-cations including Ca++, Co++, Mn++, Cd++, and Cu++ as Mg++ act as cofactors in trans-cleavage reaction of hammerhead ribozymes. The addition of chemical reagents (PEG-6000, urea, and formamide) can improve the cleavage of VP5 RNA by hammerhead ribozymes (RZ-5 & RZ-8). The trans-cleavage capabilities of RZ-5 and RZ-8 at low temperature (17C and 25C), revealing that these two hammerhead ribozymes have strong potential for ex vivo or in vivo trans-cleavage in fish cells to prevent IPNV infection.

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Rao, Ping. "Influenza virus polymerases determination of the cap binding site and the crucial role of CA endonuclease cleavage site in the cap snatching mechanism for the initiation of viral messenger RNA synthesis /." Thesis, 2003. http://wwwlib.umi.com/cr/utexas/fullcit?p3116140.

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Melnychuk, Stephanie. "A Mechanistic Study in Methanol: Cleavage of RNA Models and Highly Stable Phosphodiesters with Dinuclear Zn(II) Complexes." Thesis, 2008. http://hdl.handle.net/1974/1432.

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Phosphoryl transfer reactions are vital to life. In response to the slow intrinsic rates of phosphoryl transfer, Nature has evolved a series of enzymes designed to accelerate these reactions and allow them to occur at biologically relevant rates. These metallo-enzymes are largely characterized by bi- or tri-nuclear active sites with effective dielectric constants that more closely resemble those of organic solvents than water. This project was designed to better understand the mechanisms by which metalloenzymes cleave phosphodiesters with poor leaving groups. The stability of the phosphodiester is central to the storage of genetic information in DNA and RNA. The cleavage of a series of more reactive RNA models, 2-hydroxylpropyl aryl phosphates 1a-g, catalyzed by a dinuclear Zn(II)2 complex of 53 in methanol was explored. A solution of 53:Zn(II)2:(-OCH3) was observed to accelerate the decomposition of 1a-g with rates that were 10^11-10^12-fold greater than the methoxidepromoted reaction at ss pH 9.47, approaching rate accelerations achieved by natural enzymes. The remarkable activity of 53:Zn(II)2:(-OCH3) and 36:Zn(II)2:(-OCH3) towards the cleavage of 1a-g probed the study of the decomposition of diribonucleotides(3'-> 5')UpU and (3'-> 5')ApC in methanol. The 53:Zn(II)2:(-OCH3)- and 36:Zn(II)2:(-OCH3)-catalyzed decomposition of UpU achieved k2 values of 1.21 ± 0.17 and (7.04 ± 0.99) x 10^-2 M^-1s^-1. The reactivity of ApC in the presence of these systems was unimpressive, however Zn(II) ions in ethanol resulted in the isomerization of 3'-> 5')ApC to (2'-> 5')ApC providing support for the existence of a pentacoordinate phosphorane intermediate. The pentacoordinate phosphorane was further explored through the reaction of 36:Zn(II)2:(-OCH3) with the cyclic phosphate 58 and 2-hydroxylpropyl methyl phosphate (59). In the presence of 36:Zn(II)2:(-OCH3) the rate of isomerization of 59/59a (kobs = (4.7 ± 0.5) x 10^-3 s^-1) exceeded that of expulsion of the methoxy group (kobs = 1.62 x 10^-3 s^-1), thus confirming the existence of a pentacoordinate phosphorane intermediate (60)and providing support for a two-step phosphodiester cleavage reaction. The catalytic efficiency of 36:Zn(II)2:(-OCH3) towards the cleavage of stable phosphodiesters probed its application towards the decomposition of dimethyl phosphate (2) in methanol-d4. The exchange of OCH3 for OCD3 occurred with kcatmax = (2.27 ± 0.03) x 10^-6 s^-1.
Thesis (Master, Chemistry) -- Queen's University, 2008-09-12 13:09:42.427

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46

Kubíková, Jana. "Štěpení substrátů isoformami savčího Diceru." Master's thesis, 2016. http://www.nusl.cz/ntk/nusl-345029.

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Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...

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47

Kersebohm, Tim [Verfasser]. "PNA-ligand bioconjugates as potential building blocks for sequence-specific, metal-mediated DNA-RNA-cleavage / presented by Tim Kersebohm." 2005. http://d-nb.info/974180912/34.

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48

Rose, Scott Daniel. "Classes of polyadenylation sites revealed by native gel electrophoresis of in vitro assembled complexes and sensitivity to U RNA cleavage." Thesis, 1988. http://hdl.handle.net/1911/16288.

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The sequences that comprise a polyadenylation signal are varied. With the exception of the conserved hexanucleotide AAUAAA, other required sequence elements vary from site to site. This variability in sequence content may be indicative of different types or classes of polyadenylation signals. We have used an in vitro polyadenylation system to investigate the possibility that classes of poly(A) sites exist. Precursor RNAs from the SV40 late and adenovirus 2 L3 polyadenylation sites were examined for differences in: assembly into RNA-protein complexes; sequence requirements for complex assembly; and interactions with small nuclear ribonucleoproteins (snRNPs). Both SV40 late and L3 precursor RNA required an intact hexanucleotide and downstream sequence elements for complex formation. The stability of the complexes assembled using the two precursor RNAs was different. The L3 RNA complex was unstable in the presence of the anion poly(ACU); whereas the SV40 late complex or a chimeric L3/SV40 late complex were not. SV40 late and L3 precursor RNAs associated with the Sm protein determinant (common to U1, U2, U4/U6, U5 and U7 snRNPs) and a U1 snRNP-specific protein determinant early in the polyadenylation reaction. This association was reduced as the polyadenylation reaction progressed. Formation of the polyadenylation specific complexes was shown to require the small nuclear RNAs (snRNAs) U1, U2 and U4. The two polyadenylation precursor RNAs showed a different sensitivity to oligonucleotide directed RNase H cleavage of U RNAs. The SV40 late site was sensitive to cleavages of U1, U2 and U4 RNA. The L3 site showed sensitivity to only U4 cleavage. When the two polyadenylation signals were preceded by a functional intron with 5$\sp\prime$ and 3$\sp\prime$ splice sites, sensitivity to U RNA cleavages was altered. However even as chimeric polyadenylation splicing templates, the two sites exhibited different sensitivities to U4 cleavage in the region of U4 in which it hybridizes to U6 RNA. The observed differences in complex stability, and sensitivity to U RNA cleavage suggest that different classes of polyadenylation sites exist.

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Mathews, Ryan. "Cleavage of an RNA analog by mononuclear zinc(II) macrocyclic complexes and metal ion and metallodrug interactions with deoxyribonucleic acids." 2008. http://proquest.umi.com/pqdweb?did=1594480911&sid=1&Fmt=2&clientId=39334&RQT=309&VName=PQD.

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Thesis (Ph.D.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Jan. 22, 2009) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Morrow, Janet R. Includes bibliographical references.

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50

Skružný, Petr. "Analýza vybraných sekundárních struktur nukleových kyselin." Master's thesis, 2010. http://www.nusl.cz/ntk/nusl-296541.

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This work introduces a database of experimentally verified structures of nucleic acids which were collected from published scientific literature. The database is annotated and the structures are analysed from the perspective of quality and it was found that the experimentally obtained data are not always sufficient - their supporting evidence is often limited and their quality is not convincing. This work also discusses some of the problems, that can be encountered when the structures are experimentally probed. Contents of the database were compared to the RFAM database and despite of its small range it contains 80 new structures. The complete database of 166 structures can be possibly used to optimise software used to predict derived structures of nucleic acids. Furthermore, the work presents several possible ways of improvement of the quality of contained structures.

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Dissertations / Theses on the topic 'RNA Cleavage'

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