Rozprawy doktorskie: „Specific protein/RNA recognition”

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Miranda, Rafael. "Sequence Specific RNA Recognition by Pentatricopeptide Repeat Proteins: Beyond the PPR Code". Thesis, University of Oregon, 2018. http://hdl.handle.net/1794/23135.

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Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that bind RNAs through a simple 1-repeat:1-nucleotide manner. Nucleotide specificity is determined by an amino acid code, the PPR code. This modular interaction mode, predictable code for nucleotide specificity, and simple repeating architecture make them a promising scaffold for engineering proteins to bind custom RNA sequences and binding site prediction of native PPR proteins. Despite these features, the alignments of the binding sites of well-characterized PPR proteins to the predicted binding sites often have mismatches and discontinuities, suggesting a tolerance for mismatches. In order to maximize the ability to predict the binding sites of native PPR proteins and effectively generate designer PPR proteins with predictable specificity, it will be important to address how affinity and specificity is distributed across a PPR tract. I developed a high- throughput bind-n-seq technique to rapidly and thoroughly address these questions. The affinity and specificity of the native PPR protein, PPR10 was determined using bind-n- seq. The results demonstrate that not all of PPR10’s repeats contribute equally to binding affinity, and there were sequence specific interactions that could not be explained by the PPR code, suggesting alternate modes of nucleotide recognition. A similar analysis of four different designer PPR proteins showed that they recognize RNA according to the code and lacked any alternate modes of nucleotide recognition, implying that the non- canonical sequence specific interactions represent idiosyncratic features of PPR10. This analysis also showed that N-terminal and purine specifying repeats have greater contributions to binding affinity, and that longer scaffolds have a greater tolerance for mismatches. Together, these findings highlight the challenges for binding site prediction and present implications for the design of PPR proteins with minimum off-target binding. This dissertation contains previously published and unpublished co-authored material.
10000-01-01

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2

Zanier, Katia. "Regulation of histone gene expression : solution structure determination by NMR of the 3' histone mRNA hairpin and implications for specific protein-RNA recognition". Thesis, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269984.

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Ramos, Andres. "NMR studies of specificity in RNA-protein recognition". Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625038.

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Chan, Yin-tung Crystal, i 陳燕彤. "Demonstration of specific physical interaction between CHOP mRNA and intracellular proteins". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47169369.

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The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response. Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3. Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays. This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE.
published_or_final_version
Biochemistry
Master
Master of Philosophy

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Davies, Holly Gibs. "MSY4, a sequence-specific RNA binding protein expressed during mouse spermatogenesis /". Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/10307.

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Giorgini, Flaviano. "Functional analysis of the murine sequence-specific RNA binding protein MSY4 /". Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10293.

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Young, David James, i n/a. "The recognition of stop codons by the decoding release factors". University of Otago. Department of Biochemistry, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090603.104834.

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Termination of protein synthesis involves the recognition of one of three stop codons (UAG, UAA or UGA) and hydrolysis of the nascent polypeptide chain from the peptidyl-tRNA on the ribosome. Unlike sense codons, which are decoded by aminoacyl-tRNAs, stop codons are decoded by proteins known as release factors. The decoding release factors occupy the same site as aminoacyl-tRNA, interacting directly with the stop codon at the decoding centre and inducing peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Eubacteria have two codon-specific decoding release factors - RF1, which recognizes UAG and UAA, and RF2, which recognizes UGA and UAA. Biochemical studies identified two tripeptide 'anticodon' motifs, PXT in RF1 and SPF in RF2, which structural studies have shown occur in exposed loops (anticodon loops) on the surface of the proteins. Structures of isolated release factors show a compact 'closed' conformation whereas structures of release factors bound to the ribosome show them to be in a highly extended 'open' conformation. This suggests that a large conformational change in the release factor must take place upon or before binding to the ribosome. This transition has been invoked as a mechanism for how translational fidelity is maintained (Rawat et al, 2003), however, small angle X-ray scattering data from E. coli RF1 suggest the decoding release factors are also in the open conformation in solution challenging this mechanism. Mora et al. (2003a) presented evidence that swapping the anticodon loop of RF2 with that of RF1 switched the stop codon specificity of the release factor. Recent structures of the decoding release factors bound to the ribosome showed that there was a second structural element of the release factor, the tip of helix α5, involved in recognition of the first base of the stop codon. The objectives of this thesis were to investigate both the anticodon loop and the helix α5 region for their roles in stop codon recognition, and to investigate whether there is a conformational change in the release factors on binding to the ribosome. The anticodon loop was investigated using chimeras of E. coli RF1/RF2 and E. coli RF1/C. elegans mitochondrial RF1 (MRF1) within the anticodon loop. An RF1 variant containing the RF2-specific SPF tripeptide motif did not switch stop codon specificity showing that the tripeptide motifs are not sufficient determinants for the codon specificity of RF1 and RF2 as was originally proposed. Surprisingly repeating the complete swap of the RF1 anticodon loop to that of RF2 did not switch the stop codon specificity as claimed in Mora et al. (2003a). The studies in this thesis identified additional regions of the anticodon loop of the release factor that are important for stop codon recognition. Two of the RF1/RF2 anticodon loop variants produced showed altered codon specificity recognizing all three standard stop codons and the sense codon UGG. These variants provided unexpected insights into the mechanism of stop codon recognition and can explain why there are two release factors in eubacteria. The C. elegans MRF1 contains a novel anticodon loop that is shorter and lacks the classical PXT motif. E. coli RF1/C. elegans MRF1 chimeras showed that this anticodon loop could function in E. coli RF1 and maintain the same codon specificity. While size and sequence within the loop together are important for recognition clearly there is more than one way RF1-type release factors can recognize the UAG and UAA stop codons. Vertebrate mitochondria use four stop codons, two of the standard stop codons, UAA and UAG, and the reassigned arginine codons AGA and AGG. Two vertebrate mitochondrial release factors have been identified, mtRF1a and mtRF1 (renamed here mRF1[Canonical] and mRF1[Noncanonical]). Bioinformatic studies showed mRF1[C] had similar helix α5 and anticodon loop regions to classical RF1s. mRF1[NC] had different helix α5 and anticodon loop regions and was hypothesized to recognize the non-standard stop codons AGA and AGG. E. coli RF1/Human mRF1[NC] chimeras were constructed that showed that the helix α5 and anticodon loop regions are important for stop codon recognition. Nevertheless the chimeras showed poor activity at the AGA and AGG stop codons on E. coli 70S ribosomes suggesting that mRF1[NC] has evolved to function exclusively on 55S mitoribosomes. A release factor variant of RF2 was designed that had the potential to trap this E. coli factor in the closed conformation in solution by disulphide bond formation. The RF2 double cysteine variant was successfully expressed and purified. The disulphide bond between the two cysteines was detected directly by mass spectrometry in a high proportion of molecules, showing the closed form of RF2 exists in solution. The RF2 closed form variant was shown to have release activity concomitant with the proportion of the open form in the RF preparation showing that the conformational change is required for normal release factor function. Preliminary binding studies have suggested that the RF2 closed form variant can bind to the ribosome. The ability of the closed form of RF2 to bind to the ribosome allowed a mechanism of translational fidelity to be proposed from the studies in this thesis; the release factor would recognize the stop codon in the decoding centre and, if cognate, the conformational change would occur allowing peptidyl-tRNA hydrolysis.

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Cattelin, Céline. "Exploration de la diversité des protéines à solénoïdes alpha, régulatrices de l'expression des gènes des organites dans les lignées eucaryotes photosynthétiques et étude de la dynamique conformationnelle des protéines à "PentatricoPeptide Repeats"". Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS158.

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Au sein des Archaeplastida (eucaryotes photosynthétiques ayant acquis un chloroplaste suite à une endosymbiose avec une cyanobactérie ancestrale) les génomes chloroplastiques et mitochondriaux des algues vertes et des plantes terrestres sont régulés de manière post-transcriptionnelle, principalement par des protéines à solénoïde alpha codées dans le noyau. Ces facteurs nucléaires sont composés de motifs répétés dégénérés (protéines PPR et OPR, respectivement pentatricopeptide repeat et octatricopeptide repeats) interagissant de façon spécifique avec une partie de la séquence de leur ARN cible et forment de grandes familles de paralogues. Les protéines PPR sont très abondantes chez les plantes terrestres tandis que les OPR le sont chez les algues vertes. Ces expansions différentielles, en parallèle de l'évolution du métabolisme des ARN dans les organites pourraient refléter des adaptations génétiques préservant la phototrophie dans diverses conditions et niches écologiques. Chez les autres Archaeplastida (algues rouges et Glaucophytes) et chez les eucaryotes issus d'une endosymbiose avec une microalgue ancestrale comme les Diatomées, la régulation des génomes des organites reste peu explorée. Un premier objectif de ma thèse a été de décrire la diversité et la dynamique évolutive des protéines à solénoïde alpha connues ou candidates pour la régulation de l'expression du génome des organites et ce, dans l'ensemble des eucaryotes photosynthétiques. Pour les identifier, j'ai développé une approche combinant détection d'homologie lointaine de séquence et classification indépendante de la similarité entre séquences. J'ai validé cette approche en retrouvant et complétant les familles OPR et PPR connues chez les espèces modèles Chlamydomonas reinhardtii et Arabidopsis thaliana. J'ai montré que les expansions d'OPR étaient restreintes au sein des Chlorophytes et qu'en dehors des algues vertes et des plantes terrestres, les protéines à PPR et à OPR étaient peu nombreuses, suggérant que d'autres acteurs de la régulation de l'expression des génomes des organites restent à découvrir. J'ai également identifié plusieurs dizaines d'autres familles de protéines à solénoïde alpha adressées aux organites dans tous les protéomes étudiés, certaines aux fonctions encore inconnues et dont la caractérisation expérimentale dans des organismes modèles serait pertinente. Dans un second temps, j'ai utilisé des approches de dynamique moléculaire pour mieux comprendre l'affinité et la spécificité des liaisons entre les PPR et leurs ARN cibles. J'ai notamment étudié la dynamique des motifs répétés et la géométrie des sites de liaison des nucléotides en fonction de leur position dans la séquence des motifs PPR, y compris les effets du nombre de répétitions et de la présence ou non des domaines N- et C-terminaux, en plus de l'évolution de la conformation globale de la protéine. Nos résultats suggèrent le rôle de la flexibilité des protéines PPR, tant au niveau de la protéine que du motif dans la liaison à sa cible ARN et sa pertinence pour l'affinité et la spécificité de la reconnaissance des nucléotides
In Archaeplastida (photosynthetic eukaryotes that acquired a chloroplast following endosymbiosis with an ancestral cyanobacterium) the chloroplast and mitochondrial genomes of green algae and land plants are regulated post-transcriptionally, mainly by alpha-solenoid proteins encoded in the nucleus. These nuclear factors are composed of degenerate repeat motifs (PPR and OPR proteins, respectively pentatricopeptide repeat and octatricopeptide repeats) that interact specifically with part of their target RNA sequence and form large families of paralogs. PPR proteins are very abundant in terrestrial plants while OPRs are abundant in green algae. These differential expansions, in parallel with the evolution of RNA metabolism in organelles, may reflect genetic adaptations that preserve phototrophy under different conditions and ecological niches. In other Archaeplastids (red algae and Glaucophytes) and in eukaryotes that originate from endosymbiosis with an ancestral microalga such as the Diatoms, the regulation of organelle genomes remains poorly explored. A first objective of my thesis was to describe the diversity and evolutionary dynamics of known or candidate alpha-solenoid proteins for the regulation of organelle genome expression in all photosynthetic eukaryotes. To identify them, I developed an approach that combines distant sequence homology detection and sequence similarity independent classification. I validated this approach by finding and completing the known OPR and PPR families in the model species Chlamydomonas reinhardtii and Arabidopsis thaliana. I showed that OPR expansions were restricted within Chlorophytes and that outside of green algae and land plants, PPR and OPR proteins were few in number, suggesting that other players in the regulation of organelle genome expression remain to be discovered. I also identified several dozen other families of organelle-addressed alpha-solenoid proteins in all the proteomes studied, some of which have as yet unknown functions and whose experimental characterisation in model organisms would be relevant. In a second step, I used molecular dynamics approaches to better understand the affinity and specificity of binding between PPRs and their target RNAs. In particular, I studied the dynamics of the repeat motifs and the geometry of the nucleotide binding sites as a function of their position in the PPR motif sequence, including the effects of the number of repeats and the presence or absence of N- and C-terminal domains, in addition to the evolution of the overall conformation of the protein. Our results suggest the role of PPR protein flexibility, both at the protein and motif level, in binding to its RNA target and its relevance to the affinity and specificity of nucleotide recognition

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Bronicki, Lucas M. "Characterization of Multiple Exon 1 Variants and Neuron-specific Transcriptional Control of Mammalian HuD". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23682.

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The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.

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Oberstrass, Florian Christophe. "Novel modes of protein-RNA recognition in post-transcriptional gene regulation studied by NMR spectroscopy". kostenfrei kostenfrei, 2007. http://e-collection.ethbib.ethz.ch/view/eth:30123.

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Topping, Katherine P. "Structural studies on serotype-specific opsonic antibody recognition of protective streptococcal M protein epitopes". Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294877.

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Lyons, James Geoffrey. "Enhanced Feature Extraction from Evolutionary Profiles for Protein Fold Recognition". Thesis, Griffith University, 2016. http://hdl.handle.net/10072/365732.

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Proteins are important biological macromolecules that play important roles in al- most all biological reactions. The function of a protein is dependent on the shape it folds in to, which is in turn dependent on the protein’s amino acid sequence. Ex- perimental approaches for determining a protein’s 3D structure are expensive and time consuming, so computational methods for determining the structure from the amino acid sequence are desired. Methods for directly computing the 3D structure of a protein exist, however they are impractical for large proteins and high resolution models due to the large search space. Instead of trying to directly find the 3D struc- ture from first principles, the primary structure can be compared to proteins with known 3D structure. A ‘fold’ is a way of classifying proteins with the same major secondary structures in the same arrangement and with the same topological con- nections. Protein Fold Recognition (PFR) is an important step towards determining a protein’s structure, simplifying the protein structure prediction problem. This is a multi-class classification problem solvable using machine learning techniques. The PFR problem has been widely studied in the past, with feature extraction approaches including using counts of amino acids and pairs of amino acids, physic- ochemical information, evolutionary information from the Position Specific Scoring Matrix (PSSM), and structural information from its predicted secondary structure. These approaches do work, but with limited success. Current state of the art features use information from the PSSM as well as the predicted secondary structure.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith School of Engineering
Science, Environment, Engineering and Technology
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Ceribelli, A. "PROTEIN AND RNA IMMUNOPRECIPITATION FOR THE IDENTIFICATION OF SPECIFIC SERUM AUTOANTIBODIES IN SYSTEMIC AUTOIMMUNE RHEUMATIC DISEASES". Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/365538.

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Serum autoantibodies play a key role in systemic autoimmune rheumatic diseases for diagnostic, classification and prognostic purposes. Research of new autoantibodies has been very active in the last decade in rare connective tissue diseases such as systemic sclerosis and poly/dermatomyositis, with new biomarkers entering the clinical practice. Immunoprecipitation of protein and/or RNA components of the target autoantigens constitutes the gold standard method for the discovery of new autoantibodies in a screening setting but is considered time- and labor-intensive and, accordingly, is performed only in a few laboratories worldwide. As a result, alternative techniques such as ELISA and immunoblotting are often preferred for large-scale testing, despite the lack of standardization. The aims of the present project are (1) to set up protein- and RNA- immunoprecipitation in our laboratory and (2) to describe serum autoantibodies identified in our series of patients affected by systemic autoimmune rheumatic diseases. During the PhD program we were able to perform correctly protein-and RNA-immunoprecipitation in our laboratory as demonstrated by positive reference sera, and then by the identification of known but also new and rare autoantibodies, as represented by two new patterns immunoprecipitated in systemic sclerosis, corresponding to serum anti-hnRNP-L and anti-mitochondrial antibodies. In psoriatic arthritis we also analyzed the concentration of circulating levels of LL37, a recently established target of autoimmune response at the skin level, and we identified an increased production in a subset of patients. In conclusion, performing protein- and RNA-immunoprecipitation as a screening method in our laboratory allows a more complete and specific autoantibody analysis that cannot be performed by the commercial techniques available nowadays, and further analysis of the role of LL37 in psoriatic arthritis patients may help in the identification of a new biomarker in this condition.

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Pryor, Anne M. "Growth-regulated expression and G0-specific turnover of the mRNA that encodes AH49, a mammalian protein highly related to the mRNA export protein UAP56". Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1069259604.

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Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xii, 187 p.; also includes graphics (some col.) Includes bibliographical references (p. 175-187). Available online via OhioLINK's ETD Center

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Beebe, Kirk. "Substrate recognition through modular domains : protein tyrosine phosphatase SHP-1 and tail-specific protease (TSP) /". The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488203158827833.

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Roy, Poorna. "Deconstructing the ribosome: specific interactions of a ribosomal RNA fragment with intact and fragmented L23 ribosomal protein". Thesis, Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/47579.

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The complexity of translation is a classical dilemma in the evolution of biological systems. Efficient translation requires coordination of complex, highly evolved RNAs and proteins; however, complex, highly evolved RNAs and proteins could not evolve without efficient translation system. At the heart of this complexity is the ribosome, itself a remarkably complex molecular machine. Our work illustrates the ribosome as deconstructed units of modification. Here we have deconstructed a segment of the ribosome to interacting RNA-protein units. L23 interacts in vivo with both Domain III (DIII) and Domain IIIcore (DIIIcore) independently of the fully assembled ribosome. This suggests that DIIIcore represents the functional rRNA unit in DIII-L23 interaction. Furthermore, L23peptide sustains binding function in vitro with both DIII and DIIIcore independently of any stabilizing effects from the globular domain of L23. The ability of L23peptide to form a 1:1 complex with both DIII and DIIIcore suggests that L23peptide is the functional rProtein unit in DIII-L23 interaction. We believe that our results will stimulate interest and discussions in the significance of 3D architecture and units of evolution in the ribosome. The ubiquity of the ribosome in cellular life prognosticates that our results impact and appeal to biologists, chemists, bioinformaticists, as well as the general scientific community.

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SUN, LEI. "SUBSTRATE SPECIFIC CONTRIBUTIONS OF THE PROTEIN SUBUNIT OF E.COLI RNASE P TO SUBSTRATE RECOGNITION AND CATALYSIS". Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1188406954.

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Sun, Meng. "Retrovirus-Specific Differences in Matrix (MA) and Nucleocapsid (NC) Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343844761.

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Dhondge, Hrishikesh. "Structural characterization of RNA binding to RNA recognition motif (RRM) domains using data integration, 3D modeling and molecular dynamic simulation". Electronic Thesis or Diss., Université de Lorraine, 2023. http://www.theses.fr/2023LORR0103.

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Cette thèse a été réalisée dans le cadre d'un projet Européen plus vaste (ITN RNAct) dans lequel des approches informatiques et biologiques étaient combinées pour progresser vers la synthèse de nouveaux domaines protéiques capables de se fixer sur des séquences spécifiques d'ARN. L'objectif spécifique de cette thèse était de concevoir et développer des outils informatiques pour mieux exploiter les connaissances existantes sur les domaines à Motif de Reconnaissance de l'ARN (RRM) lors de la modélisation 3D des complexes RRM-ARN. Les domaines RRMs représentent 50% de toutes les protéines fixant l'ARN et sont trouvées dans environ 2% de toutes les régions codantes du génome humain. Cependant, du fait de la grande diversité des domaines RRMs, il n'y a eu jusqu'à présent que très peu de succès rapportés dans la conception de nouveaux domaines RRMs. La contribution centrale de cette thèse est la construction d'une base de données relationnelle appelée (InteR3M) qui intègre des informations de séquence, de structure et de fonction sur les domaines RRMs. La base de données InteR3M (href{https://inter3mdb.loria.fr/}{https://inter3mdb.loria.fr/}) contient 400,892 instances de domaines RRM (dérivées d'entrées UniProt) et 1,456 structures 3D déterminées expérimentalement (dérivées d'entrées PDB), qui correspondent à seulement 303 instances distinctes de domaines RRM. De plus, InteR3M contient 459,859 interactions atomiques entre RRM et acides nucléiques, dérivées de 656 structures 3D dans lesquelles le domaine RRM forme un complexe avec un ARN ou un ADN. Au cours du processus de collecte de données, des incohérences ont été détectées dans la classification de plusieurs instances de domaines RRMs dans les bases de données de domaines protéiques populaires CATH et Pfam. Ceci m'a conduit à proposer une approche originale (CroMaSt) pour résoudre ce problème, à partir de la mise en correspondance des instances structurales de domaines RRMs entre ces deux bases de données et de l'alignement structural des domaines sans correspondance avec une structure prototype du domaine RRM. Le workflow CroMast est disponible sur le Workflow Hub Européen (href{https://workflowhub.eu/workflows/390}{https://workflowhub.eu/workflows/390}). Les informations de séquence et de structure intégrées dans la base de données InteR3M ont ensuite été utilisées pour aligner entre eux tous les domaines RRM et cartographier toutes les interactions RRM-ARN sur cet alignement en vue d'identifier les différents modes de liaison de l'ARN aux domaines RRM. Ceci a conduit au développement, avec nos partenaires RNAct de VUB (Vrije Universiteit Brussel), de l'outil `RRMScorer'. Cet outil contribue au déchiffrage du code de reconnaissance RRM-ARN en calculant les probabilités de liaison entre les nucléotides de l'ARN et les acides aminés des domaines RRM à certaines positions de l'alignement. Les contacts atomiques entre RRMs et ARN ont aussi été utilisés pour identifier des motifs d'ancrage, c'est-à-dire des prototypes des positions 3D atomiques (relatives au squelette protéique) d'un nucléotide interagissant par empilement (`stacking') avec un acide aminé aromatique conservé. Ces ancres peuvent être utilisées comme des contraintes dans un protocole d'amarrage ancré (`anchored docking'). Le pipeline `RRM-RNA dock' est présenté ici et il intègre à la fois les motifs d'ancrage extraits de la base de données InteR3M et les scores de liaison de RRMScorer. Finalement, la simulation en dynamique moléculaire (MD) est un autre outil informatique testé dans cette thèse pour contribuer à la modélisation 3D des complexes RRM-ARN. Des protocoles MD préliminaires mais prometteurs sont décrits au titre d'essais visant à distinguer entre les complexes RRM-ARN à liaison forte ou faible
This thesis was carried out in the frame of a larger European project (ITN RNAct) in which computer science and biology approaches were combined to make progress towards the synthesis of new protein domains able to bind to specific RNA sequences. The specific goal of this thesis was to design and develop computational tools to better exploit existing knowledge on RNA Recognition Motif (RRM) domains using 3D modeling of RRM-RNA complexes. RRMs account for 50% of all RNA binding proteins and are present in about 2% of the protein-coding regions of the human genome. However, due to the large diversity of RRMs, there have been very few successful examples of new RRM design so far. A central achievement of this thesis is the construction of a relational database called `InteR3M' that integrates sequence, structural and functional information about RRM domains. InteR3M database (href{https://inter3mdb.loria.fr/}{https://inter3mdb.loria.fr/}) contains 400,892 RRM domain instances (derived from UniProt entries) and 1,456 experimentally solved 3D structure (derived from PDB entries) corresponding to only 303 distinct RRM instances. In addition, InteR3M stores 459,859 atom-atom interactions between RRM and nucleic acids, retrieved from 656 3D structures in which the RRM domain is complexed with RNA or DNA. During the data collection procedure, inconsistencies were detected in the classification of several RRM instances in the popular domain databases CATH and Pfam. This led me to propose an original approach (CroMaSt) to solve this issue, based on cross-mapping of structural instances of RRMs between these two domain databases and on the structural alignment of unmapped instances with an RRM structural prototype. The CroMaSt CWL workflow is available on the European Workflow hub at href{https://workflowhub.eu/workflows/390}{https://workflowhub.eu/workflows/390}. Sequence and structural information stored in InteR3M database was then used to align RRM domains and map all RRM-RNA interactions onto this alignment to identify the different binding modes of RNA to RRM domains. This led to the development, with RNAct partners at VUB (Vrije Universiteit Brussel), of the `RRMScorer' tool. This tool contributes to decipher the RRM-RNA code by computing binding probabilities between RNA nucleotides and RRM amino acids at certain positions of the alignment. Atomic contacts between RRMs and RNA were also used to identify anchoring patterns, i.e. prototypes of 3D atomic positions (relative to the protein backbone) of a nucleotide stacked on a conserved aromatic amino acid. These anchors can be used as constraints in anchored docking protocols. The `RRM-RNA dock' docking pipeline is presented here and integrates both anchoring patterns extracted from InteR3M and binding scores from RRMScorer. Finally, molecular dynamic (MD) simulation is another computational tool tested in this thesis to contribute to the 3D modeling of RRM-RNA complexes. Promising preliminary MD protocols are described as attempts to distinguish between strongly and weakly binding RRM-RNA complexes

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20

Dong, Shuyun. "Transcript-Specific Cytoplasmic Degradation of YRA1 Pre-mRNA Mediated by the Yeast EDC3 Protein: A Dissertation". eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/352.

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mRNA degradation is a fundamental process that controls both the level and the fidelity of gene expression. Using a combination of bioinformatic, genomic, genetic, and molecular biology approaches, we have shown that Edc3p, a yeast mRNA decay factor, controls the stability of the intron-containing YRA1 pre-mRNA. We found that Edc3p-mediated degradation of YRA1 pre-mRNA: 1) is a component of a negative feedback loop involved in the autoregulation of YRA1, 2) takes place in the cytoplasm, 3) is independent of translation, 4) occurs through a deadenylation-independent decapping and 5΄ to 3΄ exonucleotic decay mechanism, and 5) is controlled by specific cis-acting elements and trans-regulatory factors. Cis-regulation of YRA1 pre-mRNA degradation is complicated and precise. Sequences in exon1 inhibit YRA1 pre-mRNA splicing and/or promote pre-mRNA export in a size-dependent but sequence-independent manner. Sequences in the intron dictate the substrate specificity for Edc3p-mediated decay. Five structurally different but functionally interdependent modules were identified in the YRA1 intron. Two modules, designated Edc3p-responsive elements (EREs), are required for triggering an Edc3p-response. Three other modules, designated translational repression elements (TREs), are required for repressing translation of YRA1 pre-mRNA. TREs enhance the efficiency of the response of the EREs to Edc3p by inhibiting translation-dependent nonsense-mediated mRNA decay (NMD). Trans-regulation of YRA1 pre-mRNA is governed by Yra1p, which inhibits YRA1 pre-mRNA splicing and commits the pre-mRNA to nuclear export, and the RNP export factors, Mex67p and Crm1p, which jointly promote YRA1 pre-mRNA export. Mex67p also appears to interact with sequences in the YRA1 intron to promote translational repression and to enhance the Edc3p response of YRA1 pre-mRNA. These results illustrate how common steps in the nuclear processing, export, and degradation of a transcript can be uniquely combined to control the expression of a specific gene and suggest that Edc3p-mediated decay may have additional regulatory functions in eukaryotic cells.

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Dominguez, Palao Francisco. "Interferon induction by paramyxoviruses : investigations into specific RNA:protein interactions". Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/10750.

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RNA:protein interactions are central in many cellular processes, including activation of innate immune responses against microbial infection. Their study is essential to better understand the diverse biological events that occur within cells. However, isolation of RNA:protein complexes is often laborious and requires specialized techniques. This thesis is concerned with attempts to develop an improved purification protocol to isolate specific RNA:protein complexes. Taking advantage of the specific interaction of the Pseudomonas aeruginosa PP7 protein with its cognate RNA binding site, termed the PP7 recognition sequence (PRS), the aim was to identify cellular proteins involved in activating cell-signalling pathways, including the interferon-induction cascade, following viral infection with stocks of parainfluenza virus 5 (PIV5) rich in copyback defective interfering (DI) particles. Copyback DI genomes are powerful inducers of IFN and, here, I show they also activate the induction of IL-6, IL-8 and TNFα; cytokines that also have antiviral properties. Following the successful cloning of the PRS into a copyback DI genome, we investigated conditions for optimal in vitro capture of DI-PRS:protein complexes by PP7 on Dynabeads. When tested, the protocol led to the successful capture of ILF3 and PKR, two dsRNA binding proteins induced by IFN. We further developed a tap-tagging system to minimize the presence of non-specifically bound proteins to Dynabeads that may interfere with future mass spectrometry analysis. To isolate DI-PRS RNA:protein complexes from infected cells, attempts were made to rescue replicating DI-PRS genomes in the context of wild type PIV5. Similarly, efforts were made to isolate influenza A virus RNPs that contained the PRS in the neuraminidase (NA) gene from infected cells using the PP7-based protocol developed. However, for reasons discussed, unfortunately RNA:proteins complexes were not successfully purified from infected cells in either case.

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Anderson, Ross Calley. "Expression and characterisation of a novel poly(A)-binding protein, PABP5". Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/5942.

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The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein.

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23

Nilsson, Mikael. "Protein-DNA recognition : in vitro evolution and characterization of DNA-binding proteins /". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4269.

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Patschull, Lafitte-Laplace Anathe Olivia Maria. "In silico ligand fitting/docking, computational analysis and biochemical/biophysical validation for protein-RNA recognition and for rational drug design in diseases". Thesis, Birkbeck (University of London), 2014. http://bbktheses.da.ulcc.ac.uk/84/.

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Kaposi’s sarcoma-associated herpesvirus, is a double-stranded DNA γ - herpesvirus and the main causative agent of Kaposi’s sarcoma (KS). γ - herpesviruses undergo both lytic and latent replication cycles; and encode proteins that modulate host transcription at the RNA level, by inducing decay of certain mRNAs. Here we describe a mechanism that allows the viral endo-/exonuclease SOX to recognise mRNA targets on the basis of an RNA motif and fold. To induce rapid RNA degradation by subverting the main host mRNA degradation pathway SOX was shown to directly bind Xrn1. This may shed light as to how some viruses evade the host antiviral response and how mRNA degradation processes in the eukaryotic cell are involves in this.

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25

Soler, Calvo Nuria. "Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus". Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/31631.

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El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana.
Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime.
Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631
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VIVARELLI, SILVIA. "New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2010. http://hdl.handle.net/10281/10747.

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The aim of the first work (presented in the Chapter 2) was to investigate the role performed by SRPK2 kinase in the regulation of alternative splicing in SH-SY5Y neuroblastoma cells after paraquat treatment (a complex I mitochondrial respiratory chain inhibitor). Alternative splicing is a versatile form of genetic control whereby a common precursor messenger RNA (pre-mRNA) is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. This process is particularly important in the nervous system and its essential nature is underscored by the finding that its misregulation is a common feature of human diseases, including neurodegenerative pathologies. Our approach to gain insight the regulation of neuron specific pre-mRNA splicing brought us to the characterization of SRPK2 kinase because this protein phosphorylates Serine/Arginine-rich domain (RS-domain)-containing proteins and it is expressed almost exclusively in the nervous system. In order to understand how SRPK2 intracellular localization and activity are regulated, in first instance we performed a mutational analysis. These mutants have been characterized by transient transfection in SH-SY5Y neuroblastoma cells. We analysed SRPK2 intracellular localization both under physiological condition and after generating stress through mitochondrial damage, since mitochondrial damage and oxidative stress are found in many neurodegenerative diseases. We also determined the effect of this stress treatment on SRPK2 phosphorylation and its nuclear translocation. We did this first by using the minigene E1A, an alternative splicing reporter system, and also by analysing both SR proteins intracellular localization and their phosphorylation status. This work showed that not only paraquat treatment increased the phosphorylated SRPK2 fraction, but also that a specific phosphorylation at its 581 residue could be connected with the nuclear translocation of SRPK2. Consequently, this nuclear translocation brought to a splicing change in the isoform ratio of the minigene reporter system. After the drug treatment we also observed a specific speckled enlarged pattern coupled with an increase in the phosphorylation level for the SR classical proteins, known targets of SR protein kinase 2. These findings supported a functional link between the nuclear translocation and the activity of this neuronal specific kinase. In the second line of our research (presented in the Chapter 3) we performed experiments assessing the function of the mammalian 3’ end processing factor CFIm68 in the mRNA export, thus confirming its action as an adaptor for TAP/NXF1 mRNA export receptor. In particular I helped to demonstrate that the tethering of CFIm68 promoted mRNA export by designing and performing an RNA FISH assay. I used a RNA-biotinylated probe that detected the intracellular localisation of an mRNA reporter construct co-transfected with the CFIm68 protein or control proteins. Therefore we observed an increase of the probe fluorescent cytoplasmic signal only in the presence of the overexpressed CFIm68 but not with other control proteins, observation confirmed by further Real Time PCR data. In the third line of our research (presented in the Chapter 4) we reported that CFIm68 was also involved in the 3’ end cleavage of mammalian histone transcripts (not polyadenylated) by interacting with the LSM11 U7 snRNP component both in vitro and in vivo thus increasing the efficiency of the 3’ end processing in vivo. In this context I performed the Bimolecular Fluorescence Complementation (BiFC) analysis. I co-transfected the CFIm68 and the LSM11 proteins (or its MPL loss-of-function mutant) fused respectively with the C-terminus and the N-terminus of a Venus-Yellow Fluorescent Protein. Thus I detected a nuclear fluorescent complementation in more than 90% of the cells (or not complementation with the MPL mutant counterpart). This data supported our whole characterized observation concerning the involvement of this mammalian cleavage factor in 3’ histone mRNAs processing.

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Uchikawa, Emiko. "A structural approach of RNA-protein recognition and kinetics of binding in two examples : tRNA aminoacylation by arginyl-tRNA synthetase and 7SK stabilization by LaRP7". Strasbourg, 2011. http://www.theses.fr/2011STRA6052.

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Dans la cellule, les interactions ARN-protéines jouent un rôle fondamental dans divers processus impliqués dans la régulation de l'expression du message génétique. Si l'épissage de l'ARN pré-messager, la polyadénylation, le transport et l'adressage, la stabilité et la traduction sont des régulations de type post-transcriptionnel, les interactions ARN-protéines ont également un rôle-clé dans le domaine de la transcription. Effectivement, en addition aux capacités de codage, qui font de l'ADN et de l'ARN des supports de l'information génétique, la grande variabilité de structures et la flexibilité de l'ARN créent de nombreux sites uniques et le potentiel pour des régulations complexes. Les protéines se liant à l'ARN utilisent une bibliothèque de motifs structuraux pour reconnaître la séquence et la structure de leurs ARN cibles, ce qui conduit à un large éventail d'interactions, de labiles à stables. Ce manuscrit décrit nos travaux consistant à révéler les détails d'interactions RNA-protéines au niveau moléculaires, dans différents exemples concernant deux champs de la biologie cellulaire, la traduction et la transcription du code génétique. Nos cibles ont été choisies afin d'apporter des informations sur des interactions critiques pour la survie cellulaire et représentent différents modes de liaison de protéines à des ARN. Nous avions pour but d'utiliser la cristallographie aux rayons X, qui est une méthode fiable et reconnue pour la finesse des informations à l'échelle atomique que l'on peut en obtenir, et nous avons développé pour chaque cible un protocole de purification conduisant à une préparation homogène et cristallisable. Nous décrivons également les divers tests biophysiques et biochimiques ayant été utilisés pour caractériser nos échantillons
In the cell, RNA-protein interactions are fundamental to many processes involved in the regulation of gene expression, including pre-mRNA splicing, polyadenylation, editing, transport, cytoplasmic targeting, mRNA turnove and translation. In addition to these post-transcriptional processes, RNA-prote in interactions may also play a key rôle in transcription. Indeed, in addition to its coding capacity, which makes both DNA and RNA recipients of the genetic message, the high variability and conformationnal flexibility of RNA structure creates a number of unique binding sites and the potential for complex regulation by RNA binding proteins. These use a large Iibrary of structural modules in order to recognize RNAs in a combination of sequence- or structure-dependent ways, leading to a wide range of transient to more stable interactions. This manuscript describes our endeavour to reveal the details of RNAprotein interactions at the molecular level in several examples taken in two different fields of cell biology, transcription and translation. Our targets were chosen to better understand the molecular foundation of interactions critical for the cell survival, and represent different binding modes ofproteins to RNA. Aiming to use X-ray crystallography, a well-accepted and reliable mean to analyze recognition details at atomic resolution, we developed for each target a purification protocolleading to homogeneous preparations that were used for crystallization and subjected to various anai}'ses, including functional assays and biophysical characterization

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Zhang, Da Jiang. "Involvement of the Polypyrimidine Tract-Binding Protein-Associated Splicing Factor (PSF) in the Hepatitis Delta Virus (HDV) RNA-Templated Transcription". Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31095.

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Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

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29

Ranjit, Srijana. "Role and Regulation of Fat Specific Protein (FSP27) in Lipolysis in 3T3-L1 Adipocytes: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/484.

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The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in levels of circulating FFAs by sequestration and storage of FFAs in adipocytes may prevent deleterious effects of FFAs on insulin sensitivity. Recently our lab and others have shown that the storage of TG in adipocytes is promoted by a novel protein, Fat Specific Protein 27 (FSP27). Although, these studies also revealed FSP27 to be a lipid droplet associated protein that suppresses lipolysis to enhance TG accumulation in adipocytes, the role of FSP27 in lipolysis remains largely undetermined. Therefore, this study investigates the role and regulation of FSP27 in adipocytes in both the basal state, as well as during lipolysis. The studies presented here show FSP27 to be a remarkably short-lived protein (half-life=15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, I tested the hypothesis that lipolytic agents like the cytokine, TNF-α and the catecholamine isoproterenol modulate FSP27 protein levels to regulate FFA release. Consistent with this concept, TNF-α markedly decreased FSP27 mRNA and protein along with lipid droplet size as it increased lipolysis in cultured adipocytes. Similarly, FSP27 depletion using siRNA mimicked the effect of TNF-α to enhance lipolysis, while maintaining stable FSP27 protein levels by expression of HA epitope-tagged FSP27 blocked TNF-α mediated lipolysis. In contrast, the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 protein and a delayed degradation rate that corresponds to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism to restrain excessive lipolysis by catecholamines, is mimicked by forskolin or 8-Bromo-cAMP treatment, and prevented by Protein Kinase A (PKA) inhibitor KT5720 or PKA depletion using siRNA. These results show that isoproterenol stabililizes FSP27 via the canonical PKA pathway and increased cAMP levels. However, the work presented here also suggests that FSP27 does not get phosphorylated in response to isoproterenol treatment, and the stabilization of FSP27 is independent of isoproterenol mediated lipolysis. The data presented in this thesis not only identifies the regulation of FSP27 as an important intermediate in mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol, but also suggests that FSP27 may be a possible therapeutic target to modulate lipolysis in adipocytes.

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30

Subramaniam, Srisunder. "Studies of conformational changes and dynamics accompanying substrate recognition, allostery and catalysis in bacteriophage lambda integrase". The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1111655332.

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31

Ramsay, Milele. "Effects of a putative Reb1 protein binding site on IME4 sense and antisense transcription and sporulation in Saccharomyces cerevisiae". ScholarWorks@UNO, 2009. http://scholarworks.uno.edu/td/1012.

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Genome transcription is much more widespread than has been traditionally thought because our view of a "gene" or "transcription unit" has changed dramatically over the past 4 to 5 years with the identification of many different non-coding ribonucleic acids. In the yeast, Saccharomyces cerevisiae, meiosis and sporulation are an important part of the life cycle and IME4 gene expression is required for these processes. IME4 sense transcript levels of expression are influenced by the level of its complementary non-coding antisense strand by mechanisms that are currently unknown. The a1-alpha2 heterodimer binding in the downstream 3' region of IME4 is one component required for repression of IME4 antisense transcription. However, this thesis shows that the general regulatory protein Reb1 is also required in this system. Reb1 involvement is most likely to create a nucleosome-free zone in the promoter region of the IME4 antisense strand therefore contributing to transcription.

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32

Laugsch, Magdalena, Jochen Seebach, Hans Schnittler i Rolf Jessberger. "Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-127228.

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SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

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33

Lee, Soojin. "Structure and dynamics in proteins Part I. Structural origins of specific DNA recognition by GFI-1 ; Part II. Structural and dynamic studies of [gamma]S-crystallin and OPJ, implications for cataract formation /". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1189025356.

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34

Laugsch, Magdalena, Jochen Seebach, Hans Schnittler i Rolf Jessberger. "Imbalance of SMC1 and SMC3 Cohesins Causes Specific and Distinct Effects". Public Library of Science, 2013. https://tud.qucosa.de/id/qucosa%3A27288.

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SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

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35

Giblin, Sean. "Investigating cell lineage specific biosynthesis of tenascin-C during inflammation". Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:8c7306d8-53cf-4131-a134-f74885e37cc9.

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The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.

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36

Agostini, Federico 1985. "Predictions of RNA-binding ability and aggregation propensity of proteins". Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/318159.

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RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interactions with RNA protect unstructured protein domains from aberrant associations or aggregation. Nevertheless, the mechanisms that prevent protein aggregation and the role of RNA in such processes are not well understood. In this work, I will describe algorithms that I have developed to predict protein solubility and to estimate the ability of proteins and transcripts to interact. I will illustrate applications of computational methods and show how they can be integrated with high throughput approaches. The overarching goal of my work is to provide experimentalists with tools that facilitate the investigation of regulatory mechanisms controlling protein homeostasis.
Las proteínas de unión de ARN son responsables de controlar el destino de una multitud de transcriptos codificantes y no codificantes. De hecho, la formación de complejos de ribonucleoproteínas (RNP) afina la regulación de una serie de eventos post-transcripcionales e influye en la expresión génica. Recientemente, se ha observado que las proteínas con capacidad no canónica de unión al ARN se enriquecen en las regiones estructuralmente desordenadas y de baja complejidad, que son las que participan generalmente en asociaciones funcionales y disfuncionales. Por lo tanto, es posible que interactuar con el ARN pudiera ser una manera de proteger las proteínas no estructuradas de asociaciones aberrantes o de agregación. Sin embargo, los mecanismos que impiden la agregación de proteínas y la función del ARN en tales procesos no están bien descritas. En este trabajo, se describen los me ́todos que he desarrollado para predecir la solubilidad de proteínas y para estimar la capacidad de transcriptos y proteínas de interactuar. De otra parte, voy a ilustrar sus aplicaciones y explicar como los métodos de bajo rendimiento han evolucionado a un mayor rendimiento. El objetivo final es proporcionar instrumentos a los investigadores experimentales que se pueden utilizar para facilitar la investigación de los mecanismos reguladores que controlan la homeostasis molecular.

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37

Millichap, Peter. "Immune responses of the insect Manduca sexta towards the bacterium Photorhabdus luminescens". Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501624.

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The Gram-negative bacterium Photorhabdus luminescens is a pathogen of insects. It is able to secrete a variety of toxins and effectors against its host in order to escape its immune defences. The model insect Manduca sexta is able to mount a variety of humoral and cellular responses against pathogen attack. Ultimately these prove ineffective against P. luminescens. The pre-treatment of M. sexta with Escherichia coli provides protection against the pathogenesis of P. luminescens. Here, I use RNA interference and Fluorescence-assisted cell sorting techniques to investigate interactions between pathogen and host to further elucidate the roles of various host factors in mounting the immune response. I also investigate the nutrient requirements of the bacteria for pathogenesis. I show data that peptidoglycan recognition protein (PGRP) is essential for the up-regulation of antimicrobial peptides, an important immune defence. I also show that P. luminescens has a requirement for two types of iron during pathogenesis of M. sexta. And lastly I show that P. luminescens is able to avoid phagocytosis, another important immune defence.

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38

Flügel, Veronika M. Verfasser], Sabine [Akademischer Betreuer] Schneider i Aymelt [Akademischer Betreuer] [Itzen. "Expressed Cyclopeptide Libraries as Selective Inhibitors for RNA-Protein Interactions. Structural Basis for the Site-Specific Incorporation of Non-Natural Amino Acids into Peptides and Proteins / Veronika M. Flügel. Gutachter: Aymelt Itzen ; Sabine Schneider. Betreuer: Sabine Schneider". München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1064075541/34.

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39

Erramouspe, Pasilio Pablo Joaquin. "The Facilitates Chromatin Transcription (FACT) complex as a novel target for the treatment of Neuroendocrine Prostate Cancer". Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/229973/1/Pablo%20Joaquin_Erramouspe%20Pasilio_Thesis.pdf.

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This thesis is about the role of The Facilitates Chromatin Transcription (FACT) complex as a potential target for Neuroendocrine Prostate Cancer (NEPC). I studied FACT complex in different models representing the main stages of prostate cancer progression and found that FACT complex expression correlated to the lethal NEPC phenotype. The FACT-inhibiting drug, CBL1037, was more effective at reducing cell viability as compared to the standard-of-care chemotherapy drugs, cisplatin and carboplatin. Significantly lower dose of CBL0137 was required to achieve 50% cell death. These results are promising and support further investigation of CBL0137 as anti-cancer therapy in late-stage prostate cancer.

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40

Viljanen, Johan. "A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases". Doctoral thesis, Linköpings universitet, Organisk Kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11612.

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This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification. A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized. A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems. Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.

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41

Rivas, Cruz Manuel A. "Medical relevance and functional consequences of protein truncating variants". Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:a042ca18-7b35-4a62-aef0-e3ba2e8795f7.

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Genome-wide association studies have greatly improved our understanding of the contribution of common variants to the genetic architecture of complex traits. However, two major limitations have been highlighted. First, common variant associations typically do not identify the causal variant and/or the gene that it is exerting its effect on to influence a trait. Second, common variant associations usually consist of variants with small effects. As a consequence, it is more challenging to harness their translational impact. Association studies of rare variants and complex traits may be able to help address these limitations. Empirical population genetic data shows that deleterious variants are rare. More specifically, there is a very strong depletion of common protein truncating variants (PTVs, commonly referred to as loss-of-function variants) in the genome, a group of variants that have been shown to have large effect on gene function, are enriched for severe disease-causing mutations, but in other instances may actually be protective against disease. This thesis is divided into three parts dedicated to the study of protein truncating variants, their medical relevance, and their functional consequences. First, I present statistical, bioinformatic, and computational methods developed for the study of protein truncating variants and their association to complex traits, and their functional consequences. Second, I present application of the methods to a number of case-control and quantitative trait studies discovering new variants and genes associated to breast and ovarian cancer, type 1 diabetes, lipids, and metabolic traits measured with NMR spectroscopy. Third, I present work on improving annotation of protein truncating variants by studying their functional consequences. Taken together, these results highlight the utility of interrogating protein truncating variants in medical and functional genomic studies.

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42

Tran, Anh-Nhi. "A Genetic Survey of the Pathogenic Parasite Trypanosoma cruzi". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3425.

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43

Bosch, Gutiérrez Almudena. "Role of Ring1B in ephitelial to mesenchimal transition, invasion and migration of mammary epithelial cells". Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7230.

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The Polycomb group (PcG) family of proteins form chromatin-modifying complexes essential for embryonic development, and stem cell renewal and are commonly deregulated in cancer. There are several reports that address the possible implication of PcG proteins in tumor progression and metastasis, but very little is known about the specific role of these proteins in tumor progression and invasion. On the other hand, the molecular processes of the worst cancer prognosis, metastasis, which leads to an incurable disease, are yet incompletely elucidated. Here we show a role for Ring1B, a PcG protein, in three processes related to metastasis: in the Epithelial-mesenchymal transition (EMT), a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors, an in the migration and the invasion of mammary epithelial cells.
Las proteínas del grupo Polycomb (PcG) forman complejos modificadores de la cromatina esenciales en el desarrollo embrionario y en la renovación de las células madre, y su desregulación ha sido asociada al cáncer. Varios estudios muestran la posible implicación de las proteínas de PcG en la progresión tumoral y en la metástasis, pero a pesar de ello se sabe muy poco de los procesos moleculares en los que estas proteínas están participando. Por otro lado, los procesos moleculares responsables del peor pronóstico en cáncer, la metástasis, que continua siendo una enfermedad incurable, siguen sin estar completamente elucidados. En esta disertación mostramos el papel de Ring1B, una proteína del PcG, en tres procesos implicados en la metástasis: en la transición epitelio-mesénquima (EMT), un proceso morfogénico crítico en el desarrollo embrionario y durante la progresión de varios cánceres epiteliales, y en la migración y la invasión de las células epiteliales mamarias.

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44

Mills, Nicholas L. "Chemical and structural components of specific RNA recognition". Diss., 2007. 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:3286658.

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45

CHEN, ZHENG-XIAN, i 陳正憲. "Identification of a cotton specific pathogen recognition protein". Thesis, 1993. http://ndltd.ncl.edu.tw/handle/83091695993171682719.

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46

Hsu, Wen-Yuan, i 許文苑. "Characterization for the Function of RNA Recognition Motif 3 Mutant of RNA Binding Protein Rbp1p". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/gfjva2.

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碩士
國立臺灣大學
分子醫學研究所
106
Rbp1p, as a RNA binding protein, was first identified as a negative growth regulator in Saccharomyces cerevisiae. Protein composition of Rbp1p contains three RNA recognition motifs (RRMs), two glutamine-rich regions, and one asparagine-methionine-proline-rich (NMP) region in the C terminus. Our previous studies have shown that deletion of RBP1 resulting in a hyper ager-invasive growth in ∑1278b strain. Recently, we had found that over-expressing Rbp1p-RRM mutants, especially Rbp1p-rrm3, into yeast induced a hyper-invasion growth phenotype. This hyper-invasion growth phenotype had not only been found in ∑1278b strain but also in BY4741 strain, which had no invasive ability because of its flo8-mutation. We had previously predicted eight putative phosphorylation sites of Rbp1p, which mainly are located at C-terminus. According to the mass-spectrometry-based results, phosphorylation at threonine 637 (T637) would change in response to glucose deprivation. We generated an antibody specifically recognizing T637 phosphorylation, and observed a high phosphorylation level at T637 when over-expressing Rbp1p-rrm3 into yeast. However, the invasion phenotype of Rbp1p was unchanged regardless of T637 phosphorylation state. It indicated that T637 phosphorylation is not sufficient to regulate the Rbp1p-dependant invasive ability. Here we showed that eight putative phosphorylation sites of Rbp1p partially participated in regulating the Rbp1p-dependant invasive ability. Furthermore, the deletion of SNF1 and BCY1 decreased the Rbp1p-rrm3-induced hyper-invasion. According to previous microarray results, we found that the mRNA levels of FLO genes family increased when over-expressing Rbp1p-rrm3 as compared to Rbp1p. The increased FLO genes were FLO1, FLO9, FLO10 and FLO11. These FLO genes were involved in filamentous growth in Saccharomyces cerevisiae, such as flocculation, adhesion and invasion. Here we showed that most increasing mRNA levels of these FLO genes were consistent to the filamentous growth relative phenotypes; however, transcription was not sufficient to reflect these phenotypes. The translation of these FLO genes are needed to be further investigated.

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47

Ganguly, Abantika. "Probing Macromolecular Reactions At Reduced Dimensionality : Mapping Of Sequence Specific And Non-Specific Protein-Ligand lnteractions". Thesis, 2012. https://etd.iisc.ac.in/handle/2005/2478.

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During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

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48

Ganguly, Abantika. "Probing Macromolecular Reactions At Reduced Dimensionality : Mapping Of Sequence Specific And Non-Specific Protein-Ligand lnteractions". Thesis, 2012. http://etd.iisc.ernet.in/handle/2005/2478.

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During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

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49

Lyon, Angeline Marie. "Biophysical studies of an expanded RNA recognition motif from the Bruno protein". Thesis, 2009. http://hdl.handle.net/2152/ETD-UT-2009-08-229.

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RNA recognition motifs (RRMs) are a ubiquitous class of proteins which bind RNA in a sequence-specific fashion, often with high affinity. The mechanisms through which this single protein domain recognizes diverse RNA sequences is not fully understood. High-resolution three-dimensional structures are particularly important in understanding the structural features required for RNA recognition and binding. This work presents the structure of an expanded RRM domain from the Drosophila melanogaster Bruno protein. The Bruno protein is involved in establishing proper body patterning during development. This is accomplished through the translational repression of several mRNAs, in particular, the oskar mRNA. Previous work has identified an expanded RRM domain within the Bruno protein. This RRM requires an additional forty amino acids prior to the start of the canonical RRM domain for high affinity RNA binding. The protein was found to contain a canonical RRM domain comprised of four anti-parallel [beta] strands and two [alpha] helices. The RRM is preceded by a ten amino acid loop that interacts with [alpha]₁ and [beta]₂, while the remaining amino acids are flexible in solution. Interestingly, the deletion of these residues does not alter the fold or stability of the RRM domain. Thus, these additional residues must be involved in RNA binding, as they are not required for structure. From these studies, the Bruno RRM represents a new example of protein features required for recognition and high affinity binding of RNA.
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50

Dar, Arvin Christopher. "Catalytic switching and substrate recognition mechanisms of the RNA dependent protein kinase PKR". 2006. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=449911&T=F.

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