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1
Maticzka, Daniel [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Modelling binding preferences of RNA-binding proteins." Freiburg : Universität, 2017. http://d-nb.info/1135134146/34.
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Khanal, Reecha. "Identification of RNA Binding Proteins and RNA Binding Residues Using Effective Machine Learning Techniques." ScholarWorks@UNO, 2019. https://scholarworks.uno.edu/honors_theses/128.
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Identification and annotation of RNA Binding Proteins (RBPs) and RNA Binding residues from sequence information alone is one of the most challenging problems in computational biology. RBPs play crucial roles in several fundamental biological functions including transcriptional regulation of RNAs and RNA metabolism splicing. Existing experimental techniques are time-consuming and costly. Thus, efficient computational identification of RBPs directly from the sequence can be useful to annotate RBP and assist the experimental design. Here, we introduce AIRBP, a computational sequence-based method, which utilizes features extracted from evolutionary information, physiochemical properties, and disordered properties to train a machine learning method designed using stacking, an advanced machine learning technique, for effective prediction of RBPs. Furthermore, it makes use of efficient machine learning algorithms like Support Vector Machine, Logistic Regression, K-Nearest Neighbor and XGBoost (Extreme Gradient Boosting Algorithm). In this research work, we also propose another predictor for efficient annotation of RBP residues. This RBP residue predictor also uses stacking and evolutionary algorithms for efficient annotation of RBPs and RNA Binding residue. The RNA-binding residue predictor also utilizes various evolutionary, physicochemical and disordered properties to train a robust model. This thesis presents a possible solution to the RBP and RNA binding residue prediction problem through two independent predictors, both of which outperform existing state-of-the-art approaches.
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Deumer, Claudia D. "RNA-binding proteins in yeast mitochondria." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2002. http://nbn-resolving.de/urn:nbn:de:swb:14-1035897639531-83407.
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This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.
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Loushin, Newman Carrie Lee. "Characterization of QKI RNA binding function /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004323.
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Crombie, Catriona Ann. "Histone hairpin binding protein, an RNA binding protein, essential for development." Thesis, University of Aberdeen, 2003. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602058.
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Histones are proteins found in the nuclei of eukaryotic cells where they are complexed to DNA in chromatin. Rephcation-dependent histones are expressed only during S-phase. Regulation of expression of replication-dependent histone genes requires a highly conserved hairpin RNA element in the 3' untranslated region of histone mRNAs. Replication-dependent histone mRNAs are not polyadenylated; their 3' end is formed by an endonucleolytic cleavage event, 3' of a hairpin element, which is recognised by the Hairpin Binding Protein, HBP (also known as Stem-Loop Binding Protein, SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage that generates the mature mRNA 3' end. The 3' hairpin, and presumably HBP, are also required for nucleocytoplasmic transport, translation and stability of histone mRNAs. It is therefore important to understand this interaction. The hairpin is highly conserved and I have demonstrated that residues in the hairpin loop are important for binding the HBP. This complimented structural studies that showed that the same residues are involved in stacking interactions in the RNA loop. In cell culture, expression of replication-dependent histone genes is S phase specific as is the expresion of HBP. Here I demonstrated that in Caenorhabditis elegans the HBP promoter is active in dividing cells during embryonic and postembryonic development. Depletion of HBP by RNAi leads to an embryonic lethal phenotype associated with defects in chromosome condensation. Postembryonic depletion of HBP results in defects in cell fate during late larval development, specifically in vulval development. A similar phenotype was obtained when histone H3 and H2A were depleted by RNAi suggesting that the phenotype of the hbp (RNAi) worms was due to a lack of histone proteins. I have confirmed this by showing that histone proteins are indeed reduced in hbp (RNAi) worms. I have also shown that depletion of HBP leads to a change in expression of a number of other proteins and specifically an up-regulation of a histone H3 like protein with an apparent molecular mass of 34 kDa. I have evidence that suggests that this protein is the centromer specific protein, CENP-A. As this protein was up-regulated when RNAi was used to deplete histones proteins, this suggests that there could be a compensatory mechanism that helps the animal to deal with the shortage of histone proteins.
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Whittington, Christi Leigh. "Molecular Dynamics of the RNA Binding Cavity of Influenza A Non-structural Protein 1 (NS1) RNA Binding Domain." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4256.
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Molecular dynamics simulations were performed on the influenza A non-structural protein 1 (NS1) RNA binding domain (RBD), a homodimer. Fourteen simulations were performed at 298K, nine ionized with 0.1M KCl and five with no ions. Several analysis techniques were employed to study RBD residue flexibility. The focus of the study was the RNA binding cavity formed by side chains of helix 2 (chain A) and helix 2’ (chain B) and cavity intermonomeric salt bridges. Opening of the salt bridges D29–R46’ and D29’–R46 was observed in several of the trajectories. The RNA binding cavity has large flexibility, where the dimension and shape change during the dynamics. One pair of residues surrounding the cavity and necessary for RNA binding, residues R38 and R38’, have motions during the simulations which cover the top of the cavity. There is correlation between the salt bridge breaking, flexibility of R38 and R38’, and the cavity size and shape changes. Possible RBD small molecule drug targets are these two salt bridges and the pair R38 and R38’. Disrupting the events that occur around these areas could possibly inactivate RNA binding function of the domain. These results could have implications in searching for potential molecules that effectively treat influenza A.
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Blancafort, Pilar. "Making conformation-specific RNA-binding zinc fingers." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0023/NQ47598.pdf.
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Waterman, David Geoffrey. "Structural studies on prokaryotic RNA-binding proteins." Thesis, University of York, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441058.
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Fesser, Stephanie Marion. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173105.
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Attig, Jan. "Impact of retrotransposon-derived RNA elements and their recognition by RNA binding proteins." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709161.
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Brackett, David Michael. "Ligand binding and catalysis in an RNA aptamer /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Hill, G. R. "NMR studies of DNA and RNA binding proteins." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604060.
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HMG-D is a 112-residue, non-histone chromosomal protein from <i>Drosophila melanogaster </i>and is a member of the class of non-sequence specific HMGB proteins. The present project was based on the observation that other HMGB complexes that had been solved by NMR had a phenylalanine residue at a key interfacial location (corresponding to position 12 in HMG-D), whereas those like HMG-D that gave few intermolecular NOE cross peaks generally had a tyrosine at this location. This tyrosine was known to be involved in hydrogen-bonding to the DNA in a related complex that had been solved crystallographically. The Y12F mutant of full-length HMG-D was expressed and purified in isotope-labelled form suitable for NMR spectroscopy, and a set of multidimensional triple resonance experiments used to derive assignments for the backbond resonances of the protein both free and in complex with the dA<sub>2 </sub>bulge DNA. Sidechain assignments for the protein were obtained by a combination of “CCH”-transfer-based experiments and NOE spectra, while nearly complete assignments for the DNA in the complex were obtained from a combination of homonuclear 2D NOESY and TOCSY experiments together with filtered NOESY experiments where just cross peaks between protons both of which were not coupled to heteronuclei were selected. Filtered NOESY-based experiments were used to observe intermolecular NOE cross peaks in isolation, and, in contrast to the case of the wild-type complex, these experiments yielded around 50 intermolecular interactions. Together with an extensive set of assigned intramolecular NOE constraints, these formed the basis for a calculation of the structure of the complex starting from random conformations of both protein and DNA chains, which resulted in an NMR structure for the complex that had good precision over the structured region (residues 3-70 of the protein and stem 1 of the DNA).
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Sang, Andrea Elizabeth. "Molecular and physiological studies of RNA-binding proteins." Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244469.
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Robertson, Alexander De Jong. "Understanding regulation of mRNA by RNA binding proteins." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/87474.
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Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 167-187).<br>Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA and protein are controlled by the proteins' inherent affinities for different RNA sequences as well as other features such as translation and RNA structure which affect the accessibility of mRNA. The stabilities of mRNA transcripts are regulated by nonsense-mediated mRNA decay (NMD), a quality control degradation pathway. In this thesis, I present a novel method for high throughput characterization of the binding affinities of proteins for mRNA sequences and an integrative analysis of NMD using deep sequencing data. This thesis describes RNA Bind-n-Seq (RBNS), which comprehensively characterizes the sequence and structural specificity of RNA binding proteins (RBPs), and application to the developmentally-regulated splicing factors RBFOX2, MBNL1 and CELF1/CUGBP1. For each factor, the canonical motifs are recovered as well as additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in UGC-containing motifs. In a project investigating how NMD shapes the embryonic transcriptome, this thesis presents integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). Over 200 direct UPF1 binding targets are identified using crosslinking/immunoprecipitation-sequencing (CLIP-seq). Results from ribosome foot printing show that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression.<br>by Alexander De Jong Robertson.<br>Ph. D.
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Wolf, Joshua Jaeger. "Post-transcriptional coordination by an RNA-binding protein." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57893.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>RNA-binding proteins can regulate the stability, localization, and translation of their target mRNAs. Post-transcriptional regulation can orchestrate dynamic changes in gene expression, and can coordinate multiple cellular processes in response to various stimuli. Filamentous growth in Saccharomyces cerevisiae is a morphogenetic switch that occurs in response to nitrogen starvation and requires alterations in cell growth, cell cycle, and cell wall functions. Tyl element retrotransposition is also induced under conditions of nitrogen starvation. I describe a role for the RNA-binding protein Khdl in regulating these two responses to environmental stress through its mRNA targets. I identified the RNA targets of Khdl using in vivo crosslinking and immunoprecipitation (CLIP), combined with deep sequencing. This produced a high-resolution map of Khdl binding sites across the transcriptome, and provided unprecedented insight into its biological functions. Khdl regulates multiple post-transcriptional regulatory loops to coordinate the components of filamentous growth and Tyl retrotransposition. Although similar mechanisms were known to transcriptionally regulate these processes, the posttranscriptional coordination is a novel discovery. The feed-forward regulation that Khdl confers on FLO11, which encodes a protein required for filamentous growth, enables asymmetric expression between mother and daughter cells to switch between filamentous and yeast form growth. In this thesis, I describe regulation of gene expression by RNA-binding proteins, methods to identify their target transcripts and recognition sequences, the KH domain, known functions of Khdl, and the phenotypes it coordinates. My work represents the first application of CLIP to budding yeast, and the growing understanding of RNA-binding proteins in this organism facilitated the placement of Khdl into its posttranscriptional regulatory network. While many questions remain regarding the role Khdl plays in regulating cellular activities, this thesis addresses its direct role in key processes.<br>by Joshua Jaeger Wolf.<br>Ph.D.
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Brugiolo, Mattia. "The dynamic RNA-binding behavior of SR proteins." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-191373.
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In the cell, the genetic information encoded in the DNA is transcribed to RNA. All RNAs that are transcribed in the cell are initially produced as precursor RNAs, which have to undergo various steps of processing to obtain their mature form. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family.
SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs.
The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments.
To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general.
By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm.
I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences.
Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.
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Zinnall, Ulrike. "Functional characterization of the RNA-binding protein HDLBP." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/23301.
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Der Sekretionsweg ist essenziell für die Funktion von Zellen und beginnt, wenn mRNAs, die für Membran- und Sekretionsproteine codieren, an das endoplasmatische Retikulum (ER) gebracht werden. Allerdings ist wenig darüber bekannt, inwiefern RNA-bindende Proteine zur Erkennung und Translation von ER lokalisierten mRNAs beitragen.
In dieser Arbeit haben wir das humane RNA-bindende Protein HDLBP charakterisiert. Wir haben durch PAR-CLIP-, Zellfraktionierungs- und RNA-Seqeuenzierexperimente festgestellt, dass HDLBP an mehr als 80% aller ER lokalisierten mRNAs bindet. Analysen zu HDLBPs Bindungsmotiv haben gezeigt, dass HDLBP vorwiegend an ein CU-haltiges Motiv in der codierenden Sequenz (CDS) hauptsächlich von ER lokalisierten mRNAs bindet. Im Gegensatz dazu enthalten zytosolische HDLBP gebundene mRNAs weniger Bindungsstellen und diese treten sowohl in der CDS als auch in 3‘ untranslatierten Regionen auf. Dies zeigt, dass sich ER lokalisierte mRNAs von Zytosol lokalisierten mRNAs in ihrer Sequenzzusammensetzung hinsichtlich der HDLBP Bindungsstellen unterscheiden.
Weitere Analysen des PAR-CLIP-Experiments ergaben, dass HDLBP mit RNA-Komponenten des Signalerkennungspartikels (SRP) und der 40S ribosomalen Untereinheit interagiert. Durch BioID-Experimente haben wir Proteine in unmittelbarer Nähe zu HDLBP bestimmt und konnten damit die Assoziation von HDLBP mit Komponenten des Translationsapparates und des SRPs bestätigen.
Funktionelle Studien, bei denen wir CRISPR-Cas9 erzeugte HDLBP Knockout (KO) Zelllinien in Kombination mit Ribosomen-Profiling verwendet haben, haben gezeigt, dass HDLBP die Translation von mRNAs fördert, die an HDLBP gebunden und am ER lokalisiert sind.
Letztlich haben in vivo Experimente mit Nacktmäusen ergeben, dass HDLBP KO eine Abnahme der Lungentumorbildung verursacht, was die Relevanz von HDLBP für die Tumorprogression hervorhebt. Insgesamt zeigt unsere Arbeit eine generelle Funktion von HDLBP bei der Translation von ER lokalisierten mRNAs.<br>The secretory pathway is essential for proper cell functioning and starts when mRNAs encoding membrane and secretory proteins are targeted to the endoplasmic reticulum (ER). However, little is known about the contribution of RNA-binding proteins to the recognition, localization and translation of ER-localized mRNAs.
In this work, we characterized the human RNA-binding protein HDLBP. We identified that HDLBP binds to more than 80% of all ER-localized mRNAs by PAR-CLIP, cell fractionation and RNA-sequencing experiments. Analysis of the HDLBP binding motif showed that it predominantly binds to a CU-containing motif and forms high affinity multivalent interactions primarily in the coding sequence (CDS) of ER-localized mRNAs. In contrast, we identified that cytosolic HDLBP mRNA targets show less HDLBP binding sites randomly distributed between the CDS or 3’ untranslated regions. This indicates that ER-localized mRNAs per se differ from cytosol-localized mRNAs in their sequence composition with regard to HDLBP binding sites.
Further PAR-CLIP analysis revealed that HDLBP interacts with RNA components of the signal recognition particle (SRP) and the 40S ribosomal subunit. We identified by BioID experiments proteins in close proximity to HDLBP and confirmed the association of HDLBP with components of the translational apparatus and the SRP.
Functional studies using CRISPR-Cas9 HDLBP knockout (KO) cell lines in combination with ribosome profiling demonstrated that HDLBP promotes the translation of its ER-localized target mRNAs. We validated this finding by pSILAC experiments and detected the corresponding decrease in protein synthesis of proteins encoded by mRNAs that are bound by HDLBP and ER-localized.
Lastly, in vivo experiments with nude mice showed that HDLBP KO resulted in a decrease of lung tumor formation highlighting the relevance of HDLBP for tumor progression. Overall, these results demonstrate a general function for HDLBP in the translation of ER-localized mRNAs.
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Morris, Katherine Louise. "Investigation of RNA binding proteins regulated by mTOR." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39742.
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The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which plays a key role in the transduction of cellular energy signals, in order to coordinate and regulate a wide number of processes including cell growth and proliferation via control of protein synthesis and protein degradation. For a number of human diseases where mTOR signalling is dysregulated, including cancer, the clinical relevance of mTOR inhibitors is clear. However, understanding of the mechanisms by which mTOR controls gene expression is incomplete, with implications for adverse toxicological effects of mTOR inhibitors on clinical outcomes. mTOR has been shown to regulate 5’ TOP mRNA expression, though the exact mechanism remains unclear. It has been postulated that this may involve an intermediary factor such as an RNA binding protein, which acts downstream of mTOR signalling to bind and regulate translation or stability of specific messages. This thesis aimed to address this question through the use of whole cell RNA binding protein capture using oligo‐d(T) affinity isolation and subsequent proteomic analysis, and identify RNA binding proteins with differential binding activity following mTOR inhibition. Following validation of 4 identified mTOR‐dependent RNA binding proteins, characterisation of their specific functions with respect to growth and survival was conducted through depletion studies, identifying a promising candidate for further work; LARP1. Having selected LARP1 from depletion screens, overexpression co‐IP experiments conducted alongside known binding partner PABP and subsequent arrays allowed for preliminary identification of mRNAs to which LARP1 binds. Finally, we showed evidence for differential binding of mRNA subsets between LARP1 and PABP, opening a new caveat for the role of the effector protein LARP1 in mTOR dependent gene expression regulation.
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Dwyer, Holly. "Characterization of putative Porphyromonas gingivalis RNA-binding proteins." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3550.
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Porphyromonas gingivalis (P. gingivalis) is a gram-negative, anaerobic bacterium recognized as a major player in progression of periodontal disease. P. gingivalis survives in the oral cavity while being exposed to dynamic environmental conditions such as pH, temperature, nutrient availability and host immune responses such as oxygen tension and nitrosative stress. Survival and pathogenesis of P. gingivalis in the oral cavity require mechanisms to regulate gene expression in response to the extracellular signals. Little is known about the regulatory mechanisms of P. gingivalis in the oral cavity, so it is important to investigate and characterize these regulatory mechanisms. Adaptation to environmental cues using riboregulation is a significant mechanism for post-transcriptional regulation in bacteria. Using bioinformatics, we have identified a putative RNA-binding protein in P. gingivalis: RBP. Bioinformatic studies have led to the selection of HUβ and HUα nucleoid associated proteins as controls for RNA binding. I hypothesize that the candidate proteins RBP, HUβ and HUα bind RNA in P. gingivalis. The first aim is to show that RBP, HUβ and HUα bind RNA. Using electrophoretic mobility shift assays with IRE RNA and synthesized RNA motifs, I have confirmed that the proteins do bind RNA. The second aim is to isolate and sequence the P. gingivalis RNA that bind to RBP, HUβ and HUα. I have isolated the RNAs that bound the proteins and determined identity of the RNA using high throughput sequencing. Finally, I have identified an antibody that specifically binds RBP to use for in vivo immunoprecipitation of RNA-protein complexes from P. gingivalis. In conclusion RBP, HUβ and HUα are novel RNA binding proteins in P. gingivalis, and further investigation of these proteins is necessary to understand the mechanisms of gene regulation in P. gingivalis.
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Collins, K. M. "Target recognition by multi-domain RNA-binding proteins." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1460867/.
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Multi-functional RNA binding proteins regulate and coordinate the many steps of RNA metabolism. Accurate functioning of these processes is vital in cells and misregulation has been linked to many human diseases. RNA binding proteins contain multiple RNA binding domains. The ability to perform multiple functions depends on the recognition of a diverse range of targets and domains are used combinatorially to achieve this. In this thesis I ask how the sequence specificity of low affinity RNA-binding domains and the interplay between said domains plays a role in RNA target selectivity. Within this question I focus on three proteins; TUT4, a uridyl transferase involved in the regulation of both non-coding RNAs and histone mRNA; FMRP, a translational repressor whose loss in cells is the cause of Fragile X Syndrome; and RBM10 a regulator of alternative splicing and miRNA biogenesis. I found that through the use of separate RNA binding domains both TUT4 and RBM10 are able to exert flexibility in target recognition; TUT4 by using two CCHC-type zinc fingers, working independently to recognise short RNA stretches; and RBM10 by using different subsets of domains to recognise either specific high affinity splice site sequences or pre-miRNAs. In FMRP the determination of the sequence specificity of KH1 allowed us to isolate its contribution to target selection. In a secondary objective, looking at methodologies used in RNA-protein interaction, SIA was improved to make it both less laborious and to reduce the sample requirements, and with FMRP a novel mutational strategy was used in combination with SIA to determine the sequence specificity of this low affinity domain. In summary these data extend our understanding of the RNA binding mechanisms of the three systems studied and introduces improved or novel methodologies to the future study of protein-RNA interactions.
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Wang, Hai. "Design, synthesis and RNA binding of aminoglycoside antibiotics /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9820848.
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Moro, Alberto Maria. "Functional characterization of the RNA binding protein RALY." Doctoral thesis, University of Trento, 2013. http://eprints-phd.biblio.unitn.it/1086/1/Albertomaria_Moro_thesis_Final_version.pdf.
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Of 25000 genes encoded from genome, more than 90% are subject to alternative splicing or other post-transcriptional modifications. All these events produce a high number of different proteins that form the basis for the high variety of cells. The RNAbinding proteins (RBPs) play crucial roles in this variability by regulating many steps of biological processes regarding RNA metabolism. The heterogeneous nuclear ribonucleoproteins (hnRNPs) belong to big family of RBPs involved in many aspects of RNA metabolism including RNA stability, intracellular transport and translation. More recently, RALY, a RNA-binding protein associated with the lethal yellow mutation in mouse, has been identified as new member of the hnRNP family even if, its biological function remains still elusive.
My PhD project aimed to characterize human RALY and to assess its function in mammalian cells. Initially I dentified the expression pattern of this protein into the cell and I characterized the functional nuclear localization sequence that localizes RALY protein into the nuclear compartment. In order to better understand the role of RALY in the cells, I identified the proteins component of RALY-containing complexes using a new assay named iBioPQ (in vivo-Biotinylation-Pulldown-Quant assay). I also performed polyribosome profiling assay to check the resence of RALY in translating mRNAs. Moreover, a microarray assay was performed in order to identify potential mRNAs whose metabolism appears dependent on RALY expression. Taken together, the results that I obtained suggest that RALY is involved in mRNA metabolism. Unfortunately more studies remain to do before shedding some light on the biological
role of RALY in mammals
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Malaurie, Emilie. "Structural and ligand binding studies of EDEN-BP and CPEB1 RNA binding proteins." Thesis, University of Nottingham, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546689.
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Stark, Jeremy M. "SR proteins can function during alternative splicing to mediate exon/exon associations /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5020.
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Zhong, Jun. "A double-stranded RNA binding protein that is important for murine spermatogenesis and growth /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/10301.
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Kok, Kin-hang. "Roles of human double-stranded RNA binding proteins TRBP and PACT in RNA interference." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B38523218.
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Kok, Kin-hang, and 郭健恆. "Roles of human double-stranded RNA binding proteins TRBP and PACT in RNA interference." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B38523218.
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Best, Andrew Jonathan. "Identification and characterisation of RNA targets of the RNA binding proteins TRa2a and TRa2B." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2775.
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Alternative splicing – the production of multiple messenger RNA isoforms from a single gene – is regulated in part by RNA binding proteins. The overall aim of this study was to identify and characterise novel targets of the RNA binding proteins Tra2α and Tra2β, in order to further understand their biological functions. Tra2β is implicated in male germ cell development and in the initial stages of this project, I utilised data from a previous Tra2β HITS-CLIP experiment to validate and characterise novel RNA targets from the mouse testis using minigenes. These included a large testis-enriched exon from Nasp and a posion exon from Tra2a. The identification of a Tra2β-responsive poison exon within the Tra2a gene suggested that Tra2β may directly regulate Tra2α protein expression. Subsequent experiments in a human breast cancer cell line revealed that following depletion of Tra2β, Tra2α is up-regulated, and could functionally compensate in splicing regulation. Tra2β is also up-regulated in several human cancers and we hypothesised that Tra2β may regulate alternative splicing programmes of functional importance in cancer. Therefore for the majority of this project, I investigated RNA targets of Tra2α and Tra2β in the human invasive breast cancer cell line MDA-MB-231. Two transcriptome-wide approaches were used to identify RNA targets in this study. Firstly, I used iCLIP to map the transcriptome-wide binding sites of Tra2β in MDA-MB-231 cells. Secondly, I used RNA-seq to investigate the functional effect of joint Tra2 protein depletion on the transcriptome. Combining the iCLIP and RNA-seq data facilitated the identification of target exons which were both directly bound by Tra2β and functionally responsive to Tra2 protein depletion. Unexpectedly, Tra2 protein dependent exons included both alternative and constitutively spliced exons. A Gene Ontology enrichment analysis of the experimentally validated exons revealed that Tra2 protein dependent exons were functionally enriched in genes associated with chromosome biology. These included a functionally important exon from CHEK1, which encodes a key DNA damage response protein. Joint depletion of Tra2α and Tra2β led to reduced expression of the full-length CHK1 protein, accumulation of the DNA damage marker γH2AX and decreased cell viability. Together, this data suggests that human Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation which are important for cell viability.
29
Kylberg, Karin. "Transcription and transport of a messenger RNP particle : novel regulatory mechanisms /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-318-4/.
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Silva, Ana. "Structural and functional studies of several RNA-binding proteins." Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516585.
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Todorova, Tanya (Tanya Todorova). "Function and regulation of PARP13 binding to cellular RNA." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97789.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.<br>Cataloged from PDF version of thesis. Vita.<br>Includes bibliographical references.<br>Poly(ADP-ribose) polymerase-13 (PARP13) is a member of the PARP family of proteins - enzymes that use NAD+ to synthesize a posttranslational protein modification called poly(ADP-ribose) (PAR). PARPs function in multiple cellular pathways, and recently several members of the family have been implicated in regulating various steps in RNA metabolism, from splicing to translation and decay. PARP1 3 is the best-understood RNA-regulatory PARP. Initially discovered as a host immune factor, PARP13 functions by binding viral transcripts via its four CCCH-type zinc fingers and targeting them for degradation. In the context of the immune response PARP1 3 can also inhibit the translation of its viral targets and enhance the activity of other RNA-binding viral receptors, such as RIG-1. More recently PARP13 was shown to also indirectly regulate the cellular transcriptome by inhibiting the activity of Argonaute 2 (Ago2), a member of the miRNA silencing pathway. While itself catalytically inactive, PARP13 is modified by PAR and can target Ago2 for modification by a yet unknown PARP. However, it remains unclear if RNA binding is required for this function of PARP1 3. Indeed, even though multiple viruses are known to be restricted by PARP13, cellular mRNA targets of PARP13 binding and regulation have not yet been identified. Here we show that PARP1 3 binds endogenous RNA and regulates the cellular transcriptome. We identify TRAILR4 mRNA as the first cellular target of PARP13 regulation and demonstrate that PARP13 represses TRAILR4 expression posttranscriptionally by binding to a specific region in the 3' untranslated region of the transcript and targeting it for degradation in a primarily 3'-5' decay mechanism. By inhibiting the expression of TRAILR4 - a decoy pro-survival receptor of the apoptotic ligand TRAIL, PARP1 3 regulates the cellular response to TRAIL and acts as a pro-apoptotic factor. We also examine possible mechanisms of regulation of PARP1 3 function. We identify the RNA-helicase DHX30 as a constitutive PARP1 3-interacting protein and show that the two proteins co-regulate a subset of cellular transcripts. We further demonstrate that the PAR-binding domain of PARP1 3 inhibits RNA binding, while PARP1 3 interaction with PARP5a and covalent modification with PAR appear to be mutually exclusive with RNA binding.<br>by Tanya Todorova.<br>Ph. D.
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Freese, Peter (Peter Dale). "Biochemical and functional characterization of human RNA binding proteins." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/115601.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2018.<br>This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references (pages 205-226).<br>RNA not only shuttles information between DNA and proteins but also carries out many other essential cellular functions. Nearly all steps of an RNA's life cycle are controlled by approximately one thousand RNA binding proteins (RBPs) that direct RNA splicing, cleavage and polyadenylation, localization, translation, and degradation. Despite the central role of RBPs in RNA processing and gene expression, they have been less well studied than DNA binding proteins, in part due to the historical dearth of technologies to probe RBP binding and activity in a high-throughput, comprehensive manner. In this thesis, I describe the affinity landscapes of the largest set of human RBPs to date elucidated through a highthroughput version of RNA Bind-N-Seq (RBNS), an unbiased in vitro assay that determines the primary sequence, secondary structure, and contextual preferences of an RBP. The 78 RBPs bound an unexpectedly low diversity of RNA motifs, implying convergence of binding specificity toward a small set of RNA motifs characterized by low compositional complexity. Offsetting the low diversity of sequence motifs, extensive preferences for contextual features beyond short linear motifs were observed, including bipartite motifs, flanking nucleotide content, and preference for or against RNA structure. These features likely refine which motif occurrences are selected in cells, enabling RBPs that bind the same linear motif to act on distinct subsets of transcripts. Additionally, RBNS data is integrated with complementary in vivo binding sites from enhanced crosslinking and immunoprecipitation (eCLIP) and functional (RNAi/RNA-seq) data produced through collaborative efforts with the ENCODE consortium. These data enable creation of "RNA maps" of RBP activity in pre-mRNA splicing and gene expression levels, either with (eCLIP) or without (RBNS) crosslinking-based assays. The mapping and characterization of RNA elements recognized by over 200 human RBPs is also presented in two human cell lines, K562 and HepG2 cells. Together, these novel data augment the catalog of functional elements encoded in the human genome to include those that act at the RNA level and provide a basis for how RBPs select their RNA targets, a fundamental requirement in more fully understanding RNA processing mechanisms and outcomes.<br>by Peter Freese.<br>Ph. D.
33
Kheirollahi, Kouhestani Mahsa. "Analysis of RNA binding proteins using stable cell lines." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2504.
Full textAbstract:
Over 94% of human genes are alternatively spliced. Alternative splicing regulation is necessary for some developmental pathways. Many proteins including RNA binding proteins such as STAR family, hnRNPs, SR and SR-like proteins are involved in alternative splicing regulation. Some members of these proteins also have important roles in gene expression, transcription, signal transduction, RNA metabolism, cell cycle regulation and cancer. Although these proteins are known to be involved in alternative splicing control of specific targets, mechanisms of many of their actions and protein interaction partners are unknown. The aim of present study was the identification and characterization of protein interaction partners of some of these proteins including: STAR family (T-STAR, Sam68), hnRNP proteins (hnRNP G, hnRNP G-T) and SR-like proteins (Tra2β). The cDNAs of T-STAR, wild type Sam68 and P439R mutant Sam68 were cloned into a FLAG epitope encoding vector. The next step was generation of stable cell lines expressing these FLAG tagged proteins. To induce protein expression, stable cell lines were treated with tetracycline. The wild type Sam68-FLAG tagged protein was nuclear, while the P439R mutant Sam68-FLAG had cytoplasmic localization. Using immunoprecipitation and mass spectroscopy, both FLAG tagged proteins and co-purified proteins were purified and identified. RHA (RNA helicase A) was detected as an interacting partner for T-STAR, wild type and P439R mutant Sam68. Also, CCAR1 (cell division cycle and apoptosis regulator 1) was detected as a further partner for wild type and mutant Sam68. In addition, stable cell lines expressing FLAG tagged hnRNP G, hnRNP G-T and Tra2β were generated. These proteins and their candidate protein partners were pulled down and detected using immunoprecipitation and mass spectroscopy. Some of these detected proteins such as hnRNP C, hnRNP CL1 and RNA binding motif protein, X-Linked-Like 1 were common interacting candidates for both hnRNP G and hnRNP G-T. In addition to confirmation the roles of hnRNP G and hnRNP G-T in reduction of cell growth, the over-expression of hnRNP G-T led to remarkable cell morphological changes and alternative splicing of some target genes. In conclusion, in this project new interacting protein partners for T-STAR, Sam68, hnRNP G, hnRNP G-T were detected which their roles need to be tested within the cell.
34
Brown, James W. "RNA polymerase binding sites and polyadenylated RNAs in archaebacteria /." The Ohio State University, 1988. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487591658174843.
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SPATARO, CLARISSA. "IDENTIFICATION OF NEW MYC DEPENDENCIES AMONG RNA-BINDING PROTEINS." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/909490.
Full textAbstract:
Increased expression and activity of the MYC protein is a widespread cancer
hallmark and renders tumor cells addicted to sustained activation of a variety of
other gene products. Identification of those dependencies can offer new
therapeutic approaches against MYC-driven tumors. Previous studies showed that
RNA processing events have a critical role in MYC-induced tumorigenesis and
survival. Moreover, we and others observed that multiple genes encoding RNABinding
Proteins (RBPs) were positively regulated upon MYC activation in various
cell types. Hence, we hypothesized that the activity of specific RBPs could become
rate-limiting for the growth and/or survival of MYC-overexpressing cells. Toward
the identification of those RBPs, we set up high-throughput genetic dropout
screens, involving both RNAi and CRISPR/Cas9 technologies. We designed
lentiviral shRNA and sgRNA libraries targeting 730 RBPs, which we transduced in
cell lines allowing controlled super-activation of MYC, in order to identify genes
whose expression was specifically required in this condition. A series of candidates
emerged from our screens, including UPF1 and XRN1, two RBPs involved in mRNA
turnover and in particular in nonsense-mediated mRNA decay (NMD). Biological
validation in different systems confirmed our screening results and allowed us to
extend our observations to other NMD factors, thus identifying NMD as a critical
pathway in MYC-overexpressing cells. Addressing the mechanisms underlying the
synthetic lethality between MYC and NMD shall not only allow us to unravel this
unexpected crosstalk, but shall also pave the way toward the development of new
therapeutic opportunities against MYC-dependent tumors.
36
DeLaney, Elizabeth Erin. "RNA Recognition by the Pattern Recognition Receptor RIG-I: Roles of RNA Binding, Multimerization, and RNA-dependent ATPase Activity." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1405015903.
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Cass, Danielle Marie. "Role of two RNA binding properties in pre-mRNA splicing /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950811&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
Full textAbstract:
Thesis (Ph. D.)--University of Oregon, 2007.<br>Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-80). Also available for download via the World Wide Web; free to University of Oregon users.
38
Long, Jennifer Connie. "Identification of in vivo RNA tragets of the RNA-binding proteins Acinus and hnRNP A1." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4227.
Full textAbstract:
RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression; however, little is known about the endogenous transcripts to which they bind. Here, I have used the ultra-violet cross-linking and immuno-precipitation (CLIP) technique to identify RNA targets directly bound to two RNA-binding proteins: Acinus and hnRNP A1. Acinus (apoptotic chromatin condensation inducer in the nucleus) contains a region that is homologous to the RNA binding domain of the Drosophila splicing regulator sex-lethal, and a serine and arginine rich region similar to that seen in the SR family of proteins, which function extensively in splicing. Furthermore it is a component of the multi-protein spliceosome complex, and I have demonstrated it can directly bind polyadenylated RNA. I have shown that Acinus displays a diffuse nuclear localisation pattern, however, overexpression of an epitope-tagged protein results in its accumulation in enlarged nuclear speckles. Together these results suggest a role in pre-mRNA splicing. Acinus is cleaved during apoptosis by caspase-3, resulting in a truncated protein with chromatin condensation inducing activity (Sahara et al., 1999). Accordingly, I have demonstrated that overexpression of epitope-tagged Acinus results in an increased number of cells exhibiting an apoptotic phenotype. The proteolytic fragment contains the RNA binding region, and to determine if the role of Acinus in apoptosis is mediated by RNA interactions I utilised CLIP to identify in vivo RNA targets. I have identified several mRNA targets of Acinus and found that the binding sites in those mRNA targets predominantly map to constitutively expressed exons. This is in agreement with the exon junction complex, of which Acinus is a component, being deposited on mRNAs after splicing. These results may indicate that Acinus is a core RNA binding factor of the exon junction complex. To complement this approach, I also performed CLIP with a known alternative splicing regulator, hnRNP A1. In this manner, the binding site preferences could be compared between the two proteins. As expected, the majority of hnRNP A1 binding sites are located in introns, corresponding with their identified role of antagonizing pre-mRNA splicing by binding intronic splicing elements. Interestingly, a number of the CLIP tags are located in, or adjacent to, alternatively spliced events suggesting a role for hnRNP A1 in the regulation of alternative splicing of these specific pre-mRNAs. In addition to pre-mRNA splicing hnRNP A1 also functions in the cellular stress response. Upon environmental stresses it relocates to the cytoplasm and accumulates in cytoplasmic foci known as stress granules (Guil et al., 2006). Here I show some of the targets identified by CLIP are regulated by hnRNP A1 in times of cellular stress. In summary, I have identified two novel subsets of RNAs, bound by Acinus or hnRNP A1 in vivo. I have shown these proteins exhibit distinct binding preferences, which correspond to their biological function. This work is consistent with hnRNP A1 acting as an alternative splicing regulator, and provides evidence for a dual role of Acinus in mRNA splicing and apoptosis. This study also demonstrates the power of the CLIP technique, as identification of in vivo RNA targets allows greater understanding of the mechanisms by which RNA-binding proteins exert their regulatory control.
39
Howe, Peter W. A. "NMR studies of the RNA binding domain of U1A protein and its complexes with RNA." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313879.
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Nashchekin, Dmitri. "A Y-box protein/RNA helicase complex links mRNP assembly on the gene to mRNA translation /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-811-8/.
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Nguyen, Loc Tien. "BIV TAR RNA binding glycine mutant Tat peptides| An integrated modeling and binding assay approach." Thesis, San Jose State University, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=1602943.
Full textAbstract:
<p> Interactions between viral encoded regulatory proteins and RNA target sequences control gene expression of Lentiviruses, including human immunodeficiency virus (HIV). Bovine immunodeficiency virus (BIV) provides a simpler model of interaction between the viral trans-activating protein (Tat) and trans-activation response RNA element (TAR), using Tat peptides binding to TAR RNA fragments. The resulting characterization of the hinge region of native BIV TAR-Tat complex was confirmed by more comprehensive calculations, involving an exhaustive generation of lattice chains. This modeled 2-residues per move of the native 11-mer Tat peptide and a 28-nucleotides TAR fragment. But these sorts of coarse-grained calculations, upon substitution of Gly at key hinge region positions, are not fully sensitive to the local flexibility of amino acid side chains optimized for packing and possible interaction with relevant all-atom RNA structure. An overall binding destabilization effect is indicated for the single substitution at 78 and double substitution of Gly at positions 75 and 78. Destabilization effects were further examined, and model data showed that it included both potential and flexibility effects. Future studies require 1-residue per move approach and building all-atom models to fully examine molecular interactions of TAR-Tat complexes.</p>
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Prichard, Lisa. "The role of the IQ motif, a protein kinase C and calmodulin regulatory domain, in neuroplasticity, RNA processing, and RNA metabolism /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/6302.
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Sidiqi, Mahjooba. "The structure and RNA-binding of poly (C) protein 1." University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0077.
Full textAbstract:
[Truncated abstract] Regulation of mRNA stability is an important posttranscriptional mechanism involved in the control of gene expression. The rate of mRNA decay can differ greatly from one mRNA to another and may be regulated by RNA-protein interactions. A key determinant of mRNA decay are sequence instability (cis) elements often located in the 3' untranslated region (UTR) of many mRNAs. For example, the AU rich elements (AREs), are such well characterized elements, and most commonly involved in promoting mRNA degradation, and specific binding of proteins to these elements leading to the stabilization of some mRNAs. Other cis-elements have been described for mRNA in which mRNA stability is a critical component of gene regulation. This includes the androgen receptor (AR) UC-rich cis element in its 3'UTR. The AR is a key target for therapeutics in human prostate cancer and thus understanding the mechanism involved in regulating its expression is an important goal. The [alpha]CP1 protein, a KH-domain containing RNA-binding protein has been found to bind this UC-rich region of the AR and is thought to play an important role in regulating AR mRNA expression. [alpha]CP1 protein is a triple KH (hnRNP K homology) domain protein with specificity for Crich tracts of RNA and ssDNA (single stranded DNA). Relatively little is known about the structural interaction of [alpha]CP1 with target RNA cis elements, thus the present study aimed to better understand the nature of interaction between 30 nt 3'UTR UC-rich AR mRNA and [alpha]CP1 protein using various biophysical techniques, in an attempt to determine which [alpha]CP1 domain or combination of domains is involved in RNA-binding. These studies could ultimately provide novel targets for drugs aimed to regulate AR mRNA expression in prostate cancer cells. At the commencement of this study little was known about the structure of the [alpha]CP1- KH domains and their basis for poly (C) binding specificity. ... Additional studies addressed the significance of the four core recognition nucleotides (TCCC) using a series of cytosine to thymine mutants. The findings verified some of the results predicted from structural studies, especially the need for maximum KH binding to a core tetranucleotide recognition sequence. Our mutational studies of the four core bases confirmed the importance of cytosine in positions two and three as no binding was observed, while some binding was observed when the fourth base was mutated. In summary, the work presented in this thesis provides new detailed insight into the molecular interactions between the [alpha]CP1-KH domain and AR mRNA. Furthermore, these studies shed light on the nature of protein/mRNA interactions in general, as well as the specific complex that forms on AR mRNA. These studies have provided new understanding into the mode of [alpha]CP1 binding at a target oligonucleotide binding site and, provide a foundation for future studies to define structure of multiprotein/oligonucleotide complexes involved in AR mRNA gene regulation. Understanding the detailed interaction between the AR mRNA and [alpha]CP1 could provide possible targets for drug development at reducing AR expression in prostate cancer cells by interfering with the interaction of [alpha]CP1 and AR-mRNA.
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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.
Full textAbstract:
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.<br>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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Zhang, Tong. "Characterization of the shuttling properties of RNA-binding TIA proteins." Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210999.
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Durie, Danielle. "RNA Binding Protein HuR Regulates the Expression of Bcl-xL." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23206.
Full textAbstract:
The RNA-binding protein HuR controls key cellular processes by binding target
mRNAs and regulating them at
various
post-transcriptional levels. HuR
can function as
an
Internal
Ribosome
Entry
Site (IRES)
trans-acting factor
that
regulates the IRES-mediated
translation of XIAP.
Since
XIAP and Bcl-xL
expression was reported to be co-regulated, we
investigated whether
HuR
is also a
regulat
or of Bcl-xL expression. We found that HuR binds
the 3’end of the Bcl-xL 5’UTR
in-vitro. In U2OS cells, we showed that loss of HuR by
siRNA significantly increased Bcl-xL protein expression
while
Bcl-2 and Mcl-1 levels
remained unchanged. We found that
the HuR-dependent
Bcl-xL
increase was
through
translation,
shown by polysome
profiling.
Possible transcriptional, stability
and splicing
changes were eliminated.
At the physiological level HuR levels did not impact cell survival
but altered mitochondrial morphology,
partially through Bcl-xL.
Thus, HuR may be involved
in maintaining proper mitochondrial
function
by controlling Bcl-xL expression.
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Swale, Christopher. "RNA binding and assembly of human influenza A virus polymerases." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV053/document.
Full textAbstract:
Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-β<br>Influenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family
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Valeiras, Brenda. "Probing RNA binding specificities of AID/APOBEC proteins by iCLIP." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288562.
Full textAbstract:
The AID/APOBEC protein family comprises a group of cytosine deaminases found in vertebrates that are capable of modifying cytosine to uracil in the context of RNA or singlestranded DNA. They exert diverse valuable physiological functions including antibody diversification and restriction of viral infection. However, off-target mutations have also been shown to contribute to cancer development, making it crucial to better understand the interactions and mechanisms that regulate AID/APOBEC activity and editing site fidelity. In this regard, a new focus on RNA as a putative regulator of AID/APOBECs has recently emerged. Regardless of whether it is used or not as a substrate for deamination, most members of the family have been shown to retain the ability to bind RNA, emphasizing a potential regulatory role for this interaction. However, little is known about AID/APOBECs RNA binding specificity. A promiscuous binding has been suggested in some cases while in vitro evidence for other members of the family indicate a certain level of specificity. Therefore, to thoroughly unravel the AID/APOBECs RNA binding specificity, in my doctoral research I applied cross-linking and immunoprecipitation (iCLIP), an unbiased technique that allows identification of protein-bound RNAs with nucleotide resolution in living cells. As a first approach, I adapted the technique for its use in yeast and probed the RNA binding of AID and APOBEC3G, revealing different degrees of preference for small structured RNAs and recognition of particular sites within them. I then expanded the analysis to mammalian cells (HEK293T) and evaluated an extended set of APOBECs finding that, even in the presence of a broader and more complex pool of RNAs, small RNAs were still significantly bound by some members of the family. Furthermore, the comparative analysis of AID, APOBEC1, APOBEC3G, APOBEC3A and APOBEC3B iCLIP data obtained in my research, revealed shared and individual preferences for certain RNAs, suggesting a degree of binding specificity among APOBECs. In summary, my thesis outlines for the first time a comprehensive analysis of the RNA binding specificity of different AID/APOBECs in vivo, including the description of novel interactions with nucleotide resolution. The results obtained are of great value and open the field for further investigation of the specific meaning and validation of each preferential binding, providing new insights into understanding the role of AID/APOBEC interaction with RNA.
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Spellman, Rachel Claudine Helen. "Investigation of polypyrimidine tract binding protein function by RNA interference." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614210.
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Zinnall, Ulrike [Verfasser]. "Functional characterization of the RNA-binding protein HDLBP / Ulrike Zinnall." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1241116946/34.
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