Dissertations / Theses: 'Functional mRNA'

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Gape, Helen. "Microheterogeneity of porcine calpastatin and its functional implications." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267624.

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Wolf, Jana. "Structural and functional studies of mRNA PolyA tail processing complexes." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708435.

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Raher, Michael J. "Functional Analysis of Proteins Involved in Translational Regulation." Thesis, Boston College, 2003. http://hdl.handle.net/2345/433.

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Thesis advisor: Laura E. Hake
Cytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo
Thesis (BS) — Boston College, 2003
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program

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Fu, Xiaonan. "Functional study of miRNA-mRNA interactions in malaria mosquito An. gambiae." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/96216.

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Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown. The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration. To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction. Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection.
PHD

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Henscheid, Kristy L. "Functional conservation and RNA binding of the pre-mRNA splicing factor U2AF65 /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950821&sid=5&Fmt=2&clientId=11238&RQT=309&VName=PQD.

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Thesis (Ph. D.)--University of Oregon, 2007.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.

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Noerenberg, Marko. "Functional analysis of the KSHV ORF57 protein in mRNA nuclear export mechanisms." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574517.

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Kaposi's sarcoma-associated Herpesvirus (KSHV; HHV -8) is associated with multiple malignancies, including Kaposi's sarcoma. KSHV has two distinct life cycle phases, latent persistence and lytic replication. In contrast to other oncogenic Herpesviruses, lytic replication plays an important part in tumourigenicity and pathogenesis of KSHV. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. This in turn may lead to novel therapies, which could become an important strategy for the treatment of KSHV -associated diseases. Post-transcriptional regulation of RNA biogenesis is fundamental to KSHV lytic gene expression. The KSHV ORF57 protein plays an essential role in viral RNA processing, transcription, splicing, mRNA stability, nuclear mRNA export and translation. To date, it is unknown how ORF57 co-ordinates these many roles. Functional diversity of a protein can be achieved by post-translational modifications. We demonstrate that ORF57 is post-translationally methylated and inhibition of methylation has a dramatic effect on the ability of ORF57 to export intronless viral RN A out of the nucleus. This work shows that hypomethylation of ORF57 enhances its ability to bind RNA. Attempts were made to find ORF57 residues which are methylated and results identified PRMT5, a cellular protein methyltransferase as well as the putative demethylase MINA, which interact with ORF57. Furthermore, a proteomic-based approach was used to identify ce~lular proteins which are changed in their intracellular distribution or abundance upon ORF57 expression. This SILAC-based approach highlighted proteins and pathways affected by ORF57. Data showed changes in RNA processing pathways previously unknown to be affected by ORF57, e.g. mRNA polyadenylation and nucleoskeleton rearrangements. In addition, changes of putative, novel components of the human TREX complex were found. Data presented will provide a valuable resource not only to enhance our understanding of this viral pathogen but also to show new routes for drug intervention.

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Sampson, Natalie D. "Identification and functional characterisation of the novel pre-mRNA processing factor SCAF6." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272374.

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Muraru, Mariela I. "Functional analysis of Prp45p, a pre-mRNA splicing factor in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/15461.

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In this study we aimed to understand the role Prp45p executes within the spliceosome and the splicing mechanism. Part of this work was a detailed investigation into the relationships between the structure and the function of this protein by employing a screen for temperature-sensitive mutants. Prp45p is known to associate with the spliceosome throughout the splicing reactions but at what stage the protein is involved in spliceosome assembly i.e. pre-spliceosome, inactive or active spliceosome remained to be determined. Using a PRP45 conditionally regulated strain, it was found upon depletion of Prp45p from yeast cells and analysis of the in vivo splicing systems by native gel fractionation that the formation of the active spliceosome does not take place. This behaviour is compatible with Prp45p being a component of the Ntc-protein complex, which is known to be involved in this step of spliceosome assembly. Moreover, co-immunoprecipitation experiments with a tagged allele of the splicing factor Prp46p, confirmed the interaction between these two proteins suggested by two-hybrid screens. Using random PCR mutagenesis, there were identified two mutants with growth defects at 37°C. The mutants, named prp45-57 and prp45-113 contained mutations in two regions, designated A and B and located respectively upstream and, downstream of the absolutely conserved SNWKN motif. To understand which of the mutations were responsible for the temperature-sensitive phenotype, the substitutions in region A and/or region B were recreated by site-directed mutagenesis. It was proved that the phenotype requires mutations in both segments, which strongly suggests that regions A and B together play roles in protein function, perhaps through intra- or inter-protein interactions. However, co-immunoprecipitation experiments revealed that these substitutions in Prp45p do not alter the interaction with Prp46p in vitro. In order to investigate the role of these mutations within the cell, two strains were created that carry these mutations in a c-Myc tagged PRP45 ORF. When growing these strains at non-permissive temperature and employing a β-galactosidase assay, it was found that they had a mild effect on pre-mRNA splicing and do not affect the transcription/translation of the reporter genes.

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Wills, Quintin Frank. "The genetics of miRNA and mRNA expression in human lymphoblastoid cell lines." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572601.

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Human clinical genome-wide association studies (GWAS) have helped identify disease trait and pharmacogenomic loci without the need for biological under- standing. Molecular GWAS - associating genetic variation with traits such as gene expression - have been slow to fill the mechanistic gaps. While tissue specificity, lack of DNA resolution, and the need for better data integration are no dou bt important bottlenecks in molecular GWAS, there is also a very poor general understanding of which molecular phenotypes are important and how best to model them. Added to this is the clear need for a greater understanding of the strengths and weaknesses facing in vitro (and ex vivo) models as hypoth- esis generating and GWAS validation tools. The studies in this work focus on RNA expression in a popular human model: lymphoblastoid cell lines (LCLs). Chapters 2 and 3 examine microRNA (miRNA) and messenger RNA (mRNA) expression in a total of 300 genotyped human LCLs. The expression of only one miRNA could be associated with a nearby genetic variant. This result was observed in both the African and European samples studied, in a separate val- idation data set, and was technically validated with quantitative PCR. While limited genotype resolution and small sample sizes are likely to be important contributors to this low hit rate, the results strongly suggest experimental con- founders. Highly expressed miRNAs reflected the transformed nature of the cells, highly correlated miRNAs enriched for EBV and malaria associated tar- get mRNA genes, and several miRNAs that were differentially expressed be- tween the European and African samples suggested differential EBV transfer- mation. Chapter 4 presents a study on single cells from some of the same samples, to test the hypothesis that the lack of tissue spatial resolution is an important limiting factor in human genetic epidemiology. Experimental con- founders were also considered: sample growth was found to associate with the expression of several genes. Cell-to-cell gene correlations and distributions made it possible to propose how genes change their expression, functionally differ from each other, and are able to alter their behaviours without altered whole-tissue expression. The results suggest which type of genes are more likely to be susceptible to genetic effects, and propose promoter behaviours altered by genetic variants located near to 13 genes. From these whole-tissue and single cell results the broad conclusion is that, while LCLs are likely to be inappropriate for the study of miRNA genetics, their functional genomics at higher spatial resolution shows promise as a more mechanistic approach for the study of germline genetics.

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Samsonova, Anastasiia. "Structural and functional insights into YB-1 and Lin28 interplay in mRNA regulation." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL037.

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La régulation de l'ARNm dans les cellules humaines est l'un des mécanismes essentiels permettant aux cellules de s'adapter à un nouvel environnement et de répondre aux signaux entrants. En effet, il est bien plus efficace pour une cellule de réguler l'homéostasie protéique au niveau de la traduction des ARNm plutôt qu'en jouant sur la transcription ou les mécanismes de dégradation des protéines. Dans les mécanismes de régulation de la traduction de l'ARNm, les protéines de liaison à l'ARN (RBP) jouent un rôle clé.Au cours de ce travail de recherche, nous avons démontré que Lin28, une RBP humaine avec un domaine de choc froid peut interagir avec l'ARNm en coopération avec YB1, une protéine très abondante contenant aussi un domaine de choc froid, essentielle dans la constitution des mRNPs. L'interaction entre ces deux protéines est basée sur leur haute similitude de structure, et permet potentiellement à Lin28a de réguler la traduction nombreux mRNPs en utilisant YB-1 comme «badge d'entrée» au mRNP.Pour étudier l'interaction entre Lin28 et YB-1, différentes méthodes de biologie structurale et cellulaire ont été utilisées. D'abord, l'oligomérisation des domaines de choc froid de Lin28 et YB-1 en présence d'ARNm a été démontrée in vitro, et les résidus d'acides aminés impliqués dans ce processus ont été mis en évidence par spectroscopie RMN. Ensuite, la colocalisation dans le cytoplasme de Lin28 et YB-1 a été démontrée dans un contexte cellulaire. Finalement, nous avons révélé les conséquences fonctionnelles de cette interaction, par exemple dans le cadre de la prolifération et de la différenciation cellulaires. Ces résultats éclairent un nouveau rôle de Lin28 dans le développement et l'adaptation des cellules cancéreuses à leur environnement
The mRNA regulation in human cells is one of the key mechanisms allowing the cells to adapt to a new environment and to respond to incoming signals. In terms of protein synthesis, the regulation of mRNA translation is a preferable process for cells compared to a more rigid mechanism of transcription or degradation. The RNA-binding proteins (RBPs) play a key role in the mRNA translation regulation.In the present work, we made an effort to demonstrate that a human RBP containing a cold shock domain, Lin28a, can act in cooperation with another cold shock protein YB-1, a core protein of mRNPs. The interplay between two cold shock proteins is based on their high structure similarity, that potentially gives Lin28 an opportunity to regulate the mRNA target translation in a general way using YB 1 as an “entry badge” to the mRNP.To demonstrate the interplay between Lin28 and YB 1, several methods of structural and cellular biology were used in the present study. The oligomerization of Lin28-CSD and YB-1-CSD upon RNA binding was shown in vitro, and the amino acid residues responsible for that were highlighted by NMR spectroscopy. Then, the colocalization of Lin28 and YB-1 was demonstrated in cell cytoplasm. Also, the protein interplay was shown to have functional consequences, e.g. for cell proliferation and differentiation

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Aibara, Shintaro. "Structural and functional characterization of the Mex67:Mtr2 complex in mRNA nuclear export." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708892.

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Thomas, Anne L. "Structural and functional characteristics of novel mRNA isoforms of the death-promoting gene bax." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285747.

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Serdar, Lucas D. "The Functional Relationship between the Nonsense-Mediated mRNA Decay Pathway and the Prematurely Terminating Ribosome." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1554304118763865.

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Zielke, Barbara [Verfasser], and Thomas [Akademischer Betreuer] Stamminger. "The HCMV encoded mRNA-export factor pUL69 : functional conservation within the Betaherpesvirinae and identification of mRNA-targets during infection / Barbara Zielke. Betreuer: Thomas Stamminger." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2012. http://d-nb.info/1022737279/34.

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Worrell, Harrison. "CSPG4 in osteosarcoma : functional roles and therapeutic potential." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33146.

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Osteosarcoma is the most common primary malignancy of bone. 5-year survival has remained stable at around 60-70% for 40 years. However, a number of patients will suffer from recurrent and/or metastatic disease representing a large unmet clinical need. CSPG4 is a transmembrane protein which is expressed on a number of progenitor cells and tumour types. Preliminary work had found CSPG4 present in osteosarcoma tumour samples. In this study, CSPG4 mRNA and protein expression was demonstrated in clinical samples and model cell lines. CSPG4 mRNA is overexpressed in osteosarcoma samples compared to mature osteoblast cells, the putative cell of origin for osteosarcoma. In a cohort of patients, CSPG4 protein expression was found on 86% of samples. Furthermore, CSPG4 expression was demonstrated in U2OS, MG63, HOS, HOS-MNNG and 143B osteosarcoma cell lines. CSPG4 protein expression was successfully deleted in 143B cells using CRISPR/Cas9 technology. Two stable CSPG4-negative cell lines were produced. CSPG4 expression was then reintroduced into negative cell lines, as well as the parental 143B cell line. This created a panel of 6 cell lines with differing CSPG4 expression. Furthermore, siRNA treatment of U2OS, MG63, 143B and U87MG cell lines reduced CSPG4 expression. These cells provided another panel with varying CSPG4 expression for in vitro investigation. In vitro experiments failed to demonstrate a role for CSPG4 in osteosarcoma tumorigenesis. The CRISPR/Cas9 cell panel found that CSPG4 expression did not influence cell proliferation, adhesion and spreading on fibronectin or collagen-I, cell migration, chemosensitivity or anchorage-independent growth. Similarly, the siRNA cell panel found that CSPG4 expression did not influence cell proliferation or anchorage-independent growth. In vivo experimentation did not demonstrate a role for CSPG4 in mediating osteosarcoma tumour growth or metastatic spread. Treatment with a sc-Fv antibody fragment failed to demonstrate specific toxicity of CSPG4-positive cell lines. These results indicate that CSPG4 plays no role in osteosarcoma tumour cell behaviour. However, due to its wide expression pattern it represents a viable therapeutic option for drug targeting.

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Li, Kaiyong [Verfasser], and Bruno [Akademischer Betreuer] Kyewski. "Identification of a functional mRNA-miRNA network in mTEC development / Kaiyong Li ; Betreuer: Bruno Kyewski." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177149249/34.

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Cheah, Sern Yih. "Understanding the risk and functional importance of schizophrenia genetic factors." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/101340/1/Sern%20Yih_Cheah_Thesis.pdf.

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This thesis investigated the role of genetics in schizophrenia. Particular schizophrenia risk genes were examined to determine their role in the development of specific schizophrenia symptoms. These genes were further studied to determine how they interact to cause schizophrenia symptoms. One important schizophrenia risk gene (BDNF) was studied in detail to determine its effect on gene expression and epigenetic changes in brains of schizophrenia individuals. This thesis has contributed to a better understanding of the role of genetics in the development of particular symptoms of schizophrenia. The results presented in this thesis may ultimately lead to more accurate diagnosis and targeted treatment for schizophrenia leading to more effective treatments with less side-effects.

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Yue, Bai-Gong. "Regulation of adenovirus alternative pre-mRNA splicing : Functional characterization of exonic and intronic splicing enhancer elements." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-454.

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Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.

Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro.

Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.

Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.

Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

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Lau, Agatha. "RNA-binding of LC3 to the AU-rich element of fibronectin mRNA, a structural and functional study." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0003/MQ46043.pdf.

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Jeridi, Aicha [Verfasser], and Vigo [Akademischer Betreuer] Heissmeyer. "Functional analysis of the mRNA decay factor Lsm1 in the immune system / Aicha Jeridi ; Betreuer: Vigo Heissmeyer." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1202011322/34.

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Kiesler, Eva. "Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans." Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-218.

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Jin, Shao-Bo. "Molecular Cloning and Functional Characterization of Factors Involved in Post-transcriptional Gene Expression." Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-24.

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Yu, Yang. "REGULATION OF PRE-MRNA SPLICING IN MAMMALIAN CELLS: IDENTIFICATION AND CHARACTERIZATION OF INTRONIC AND EXONIC SILENCERS." Connect to text online, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1184182785.

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Younis, Shady. "Functional characterization of the biological significance of the ZBED6/ZC3H11A locus in placental mammals." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-329190.

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The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

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Bach, Frauke [Verfasser], and Iris [Akademischer Betreuer] Bruchhaus. "Exploring the vacuolin gene family, mRNA production, localization and functional characterization in the Mycobacterium marinum - Dictyostelium discoideum model system / Frauke Bach. Betreuer: Iris Bruchhaus." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2014. http://d-nb.info/1053811187/34.

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Serrat, Farran Xènia 1993. "Unraveling the functional roles of sftb-1/SF3B1 and prpf-4/PRPF4B in Caenorhabditis elegans splicing and human disease." Doctoral thesis, Universitat Pompeu Fabra, 2019. http://hdl.handle.net/10803/668152.

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Caenorhabditis elegans provides a powerful experimental system for understanding fundamental questions in biomedical research, including the consequences of pre-mRNA splicing alterations. Here, we have applied CRISPR and RNA-sequencing technologies to investigate the functions of two evolutionary conserved splicing factors that have been linked to disease in different ways. We have established that cancer-related mutations in sftb-1 cause splicing alterations in C. elegans. The use of distinct alleles has uncovered novel synthetic lethal interactions between sftb-1 mutations and further perturbations in the U2 snRNP, while holding the potential to be expanded to other biological processes. Moreover, we have humanized an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Finally, we have initiated the functional characterization of the splicing regulatory kinase prpf-4 in this nematode by studying its dynamic subcellular location pattern and the requirement of its kinase activity during C. elegans development.
El nematode Caenorhabditis elegans és un sistema experimental que pot ajudar a resoldre qüestions fonamentals en recerca biomèdica, incloses les conseqüències d’alteracions en el processament de pre-ARN missatgers o splicing. En aquest treball, hem aplicat les tècniques de CRISPR i seqüenciació d’ARN per a investigar les funcions de dues proteïnes involucrades en splicing, les quals s’han conservat al llarg de l’evolució i s’han associat a diverses malalties de formes diferents. Hem observat que mutacions en el gen sftb-1 relacionades amb càncer alteren el procés de splicing en C. elegans. Mitjançant l’ús de diferents al·lels, hem descobert noves interaccions de letalitat sintètica entre mutacions en el gen sftb-1 i alteracions addicionals en el complex U2 snRNP. El sistema té el potencial de ser utilitzat per a identificar altres interaccions amb diferents processos biològics. A més, hem humanitzat un domini de la proteïna SFTB-1 per sensibilitzar els cucs als fàrmacs pladienolide B i herboxidiene, que actuen com a moduladors de l’splicing. Finalment, hem iniciat la caracterització funcional de la quinasa reguladora del procés de splicing prpf-4 en aquest nematode mitjançant l’estudi de la seva localització subcel·lular dinàmica i del requeriment de la seva activitat quinasa durant el desenvolupament de C. elegans.

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Galloway, Summer E. "Functional characterization of conserved domains within the L protein component of the vesicular stomatitis virus RNA-dependent RNA polymerase implications for transcription and MRNA processing /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2010r/galloway.pdf.

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Jansson, Anna M. "Targeting Infectious Disease : Structural and functional studies of proteins from two RNA viruses and Mycobacterium tuberculosis." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-196623.

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The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections. Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera. The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

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Ruppert, Ann-Kathrin [Verfasser], Peter [Akademischer Betreuer] Nürnberg, Peter [Akademischer Betreuer] Schneider, and Peter [Akademischer Betreuer] Kloppenburg. "Integrative functional genomic search for regulatory DNA sequence polymorphisms influencing DNA methylation and mRNA expression in hippocampal brain tissue / Ann-Kathrin Ruppert. Gutachter: Peter Nürnberg ; Peter Schneider ; Peter Kloppenburg." Köln : Universitäts- und Stadtbibliothek Köln, 2015. http://d-nb.info/1084872617/34.

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Tejedor, Vaquero Juan Ramón 1984. "Systematic functional analyses of spliceosomal components reveal novel mechanisme of alternative splicing regulation." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/385718.

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Alternative splicing is an essential regulatory layer of gene expression that expands the coding potential of the genome in multicellular organisms. The spliceosome -the sophisticated machinery involved in intron removal- allows versatile regulation of gene expression programs. The splicing process relies on the dynamic interplay between hundreds of components of the spliceosome, and the steps at which the complex process of the splicing reaction can be regulated remain largely unknown. The main objective of this thesis has been to develop high- throughput approaches to systematically identify novel regulators of alternative splicing, as well as to study the mechanisms by which they modulate splice site choice. We have identified a variety of regulators of Fas/CD95 alternative splicing within and outside of the splicing machinery and provide novel insights into connections between iron homeostasis and alternative splicing regulation. Using computational networks, we carried out a systematic functional analysis of the spliceosome components and their regulatory potential. Our results reveal the extensive regulatory plasticity of core spliceosome components throughout its assembly process. They also identified links between alternative splicing and iron homeostasis, providing a mechanism by which iron modulates alternative splicing through regulation of the RNA binding properties of a Zinc knuckle domain in the SR regulatory protein SRSF7. The results of this thesis highlight the value of high throughput technologies and network analyses to study complex molecular mechanisms, and unveils novel functional connections between the splicing machinery and other cellular processes.
El procesamiento alternativo del pre-ARNm constituye uno de los pilares esenciales en la regulación de la expresión génica y expande la capacidad codificadora del genoma en organismos multicelulares. El Espliceosoma – la maquinaria encargada de la eliminación alternativa de los intrones- permite una regulación multifacética de los programas genéticos en el interior de la célula. El proceso de corte y empalme se sustenta en la interacción dinámica de cientos de componentes del Espliceosoma, y los distintos niveles de regulación de la compleja reacción de splicing permanecen aun sin descubrir. El objetivo principal de esta tesis se ha centrado en el desarrollo de tecnologías sistematicas de alto cribado para identificar reguladores potenciales del procesamiento alternativo del pre- ARNm, así como los mecanismos implicados en su regulación. Hemos identificado una gran variedad de reguladores del procesamiento alternativo de Fas/CD95, tanto componentes esenciales del espliceosoma como factores implicados en otros procesos biologicos, y hemos observado una conexión inédita entre la regulación del splicing alternativo y el proceso de homeostasis modulado por hierro. Mediante el uso de redes computacionales, hemos llevado a cabo un análisis sistemático y funcional de los componentes del Espliceosoma y hemos identificado el potencial regulador de los mismos en la reacción de corte y empalme. Nuestros resultados reflejan una inmensa plasticidad de los factores esenciales del Espliceosoma a lo largo de toda a reacción de ensamblaje. Además, hemos conseguido identificar el mecanismo potencial por el cual la homeostasis del hierro ejerce su función en splicing alternativo a través de la modulación de la actividad de unión a RNA -mediada por un dominio de unión a cinc- en la proteína reguladora de splicing SRSF7. Los resultados de esta tesis enfatizan la relevancia de las tecnologías emergentes de alto cribado y el análisis de redes computacionales en el estudio de complejos mecanismos moleculares, y desvelan nuevas conexiones funcionales entre la maquinaria de splicing y otros procesos celulares.

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Hofmann, Katharina Bettina [Verfasser], Patrick [Akademischer Betreuer] Cramer, Patrick [Gutachter] Cramer, and Reinhard [Gutachter] Lührmann. "Transcriptome maps of general eukaryotic RNA degradation factors and identification and functional characterization of the novel mRNA modification N3-methylcytidine / Katharina Bettina Hofmann ; Gutachter: Patrick Cramer, Reinhard Lührmann ; Betreuer: Patrick Cramer." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1209738457/34.

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Diebold, Marie-Laure. "Etude biochimique, structurale et fonctionnelle du complexe chaperonne d'histone/facteur d'élongation Spt6/Iws1." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ008/document.

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Les ARN messagers (ARNm) fonctionnels sont produits au cours d'un mécanisme complexe qui allie la transcription, qui permet la synthèse d'un pré-ARNm, la maturation de ce transcrit et son export. De plus, ces différentes machineries vont devoir faire face à la structure compacte de la chromatine, nécessitant une activité de décondensation/recondensation de la chromatine qui est notamment régulée par les mécanismes épigénétiques. Un très grand nombre de facteurs sont donc requis pour la production des ARNm fonctionnels . Parmi ces facteurs, les protéines Spt6 et Iws1 sont impliquées dans le mécanisme général de la transcription, dans la modulation de la structure de la chromatine et la maturation et l'export des ARNm. Ces travaux de thèse ont permis de caractériser biochimiquement, structuralement et fonctionnellement ces deux protéines, leur complexe et leur interaction avec d'autres effecteurs de la transcription. Ces travaux ont notamment permis de comprendre en termes moléculaires et fonctionnels (i) comment Spt6 est recrutée par l'ARN polyméraseII au cours de la transcription et (ii) comment le complexe Spt6/Iws 1 est formé. Ils ont également permis d'identifier de nouveaux interactants potentiels de Spt6, et notamment le facteur d'élongation de la transcription TFIIS. Ces travaux ont ainsi permis de révéler le rôle essentiel et extrêmement complexe joué par Spt6 et Iws1 lors de la production d'un ARNm, mais également de permettre l'étude future de leur interaction avec d'autres facteurs transcriptionnels
Production of functional messenger RNA (mRNA) requires a complex mechanism that couples transcription with maturation and export of the mRNA. In addition to this mechanism, chromatin needs to be unwound to allow the transcription machinery access the DNA, this unwinding being also highly regulated. Thus, production of a functional mRNA requires a huge number of factors implicated in these different processes. Among these proteins Spt6 and Iws1 are participating in the mechanism of transcription, chromatin unwinding, and maturation and export of the mRNA. The work carried out during this thesis has enabled the biochemical, structural and functional characterization of these proteins, their complex and their interaction with other effectors of transcription. This work has specifically enabled the molecular and functional characterization (i) of the recruitment of Spt6 by RNA polymerase II and (ii) of the formation of the Spt6/Iws1 complex. Moreover, this work has identified putative new partners of Spt6, not ably the elongation factor TFIIS. Thus, our work has highlighted the essential and complex role of Spt6 and Iws1 during the production of functional mRNA, and has also enabled future studies of the complexes formed by these two proteins with other transcriptional factors

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Chang, Chung-Te. "SRAG functions as a New mRNA export co-adaptor." Thesis, University of Sheffield, 2012. http://etheses.whiterose.ac.uk/2119/.

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DEAD box RNA helicases play important roles in many cellular processes including splicing, mRNA export and translation. The two domains of many DEAD box RNA helicases adopt radically different conformations according to whether they are nucleotide free, ADP or ATP bound. This change in conformation is often harnessed to drive subunit rearrangements in multiprotein complexes. Assembly of the TREX complex which plays a role in mRNA export requires a DEAD box helicase, UAP56 to bind ATP. Here we show that a novel mRNA export co-adaptor, SRAG, binds UAP56 in a mutually exclusive manner with REF, yet both REF and SRAG are found in a fully assembled TREX complex. Interestingly, REF and SRAG stimulate ATP hydrolysis and RNA helicase activity. This implies that UAP56 goes through at least two rounds of ATP hydrolysis to assemble TREX. Within assembled TREX, SRAG functions as an mRNA export co-adaptor and binds synergistically with REF to the TAP mRNA export factor, whose recruitment to TREX triggers UAP56 loss. Depletion of REF or SRAG alone in vivo has a modest effect on mRNA export, but their combined knockdown causes a drastic mRNA export block. Interestingly, the TAP:SRAG interaction is dependent on methylation of SRAG. SRAG binds to TAP in a manner which is mutually exclusive with the TREX component THOC5 and yet TAP, SRAG and THOC5 are found in a single complex in vivo. These data indicate that TREX undergoes substantial rearrangements during its assembly and interaction with TAP, and these rearrangements are driven by UAP56 dependent ATP hydrolysis.

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Chapman, Tajekesa Kudzaishe Pamacheche. "Regulation of PABP1 function by differential post-translational modification." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25875.

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Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.

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Cordiner, Ross Andrew Alex. "The cellular functions of the microprocessor complex." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25877.

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DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.

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He, Weihai. "SM-like proteins function in mRNA degradation of Saccharomyces cerevisiae." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/279964.

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The degradation of mRNA is a critical step in determining the regulation and levels of gene expression. A family of Sm-Like (Lsm) proteins containing the "Sm" sequence motif has been identified, which forms a complex with U6 snRNA and functions in pre-mRNA splicing. Moreover, Lsm proteins interact with mRNA decay factors in yeast two-hybrid analysis. One of the Lsm proteins, Lsm1p, is required for the efficient decapping of mRNA during degradation. Lsm proteins might exist as a complex since they coimmunoprecipitate with each other. Based on these observations we hypothesized that the family of Lsm proteins might be involved in mRNA degradation. We showed that mutations in seven Lsm proteins (Lsm1-Lsm7) caused inhibition of decapping. These Lsm proteins co-immunoprecipitated with Dcp1p (the decapping enzyme), Pat1p (a decapping activator) and with mRNA, indicating that the Lsm proteins (Lsm1--Lsm7) play a direct role in promoting the decapping step of mRNA degradation. In addition, the Lsm proteins seem to form distinct complexes that affect different aspects of mRNA metabolism. A nuclear Lsm complex consisting of Lsm2p through Lsm8p, interacts with U6 snRNA and promotes pre-mRNA splicing. A cytoplasmic Lsm complex consisting of Lsm1p through Lsm7p binds mRNA and functions in mRNA degradation. The cytoplasmic Lsm complex plays a second role in protecting the 3 ' UTR of mRNA from trimming. Mutations in the cytoplasmic Lsm complex caused trimming of mRNAs. The ski mutations, which cause defects in 3' to 5' mRNA degradation suppressed the temperature-sensitive growth of lsm mutants by preventing the further degradation of trimmed mRNAs. These results argue that Lsm complex might bind to the 3' UTR of mRNAs and protect it from trimming. In addition, the 3' to 5 ' degradation of the trimmed mRNAs might at least in part account for the temperature-sensitive growth of the lsm and the pat1 mutants.

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Hill, Christopher G. "Studies in microrna function and gene dysregulation in ovarian cancer." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53086.

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Ovarian cancer results from the dysregulation, in normal ovarian epithelial cells, of genes responsible for the control of critical biological processes. Since their discovery 20 years ago, microRNAs have increasingly been implicated in that dysregulation due to their role mediating gene expression; changes in microRNA expression levels in cancer have been linked with tumor growth, proliferation and metastasis. Their imputed involvement in cancer has led to the possibility of their use as biomarkers and to their potential clinical use. Using mRNA and microRNA microarray analysis to compare human gene expression in normal ovarian surface epithelial (OSE) cells and epithelial ovarian cancer (EOC) cells, we explored the interactions between microRNAs and genes. First, we validated in silico predictions of microRNA targets by comparing them with in vitro evidence after exogenous microRNA transfection. We found that pairs of microRNAs with identical 7-nt (nucleotide) seed regions shared 88% of their predicted targets and 55% of their in vitro targets, confirming the importance of the seed as a targeting mechanism. But more importantly, we found that even a single nucleotide change in the seed region can result in a significant shift in the set of targeted genes, implying strong functional conservation of the seeds and their corresponding binding sites. Next, we discovered a 3-element network motif which explains the upregulation of nearly 800 genes in ovarian cancer which, as predicted microRNA targets, might be expected to be down- regulated. This model shows that, under certain circumstances, repressor genes which are down- regulated in cancer can apparently override the repressive effects of microRNAs, resulting in the upregulation of predicted microRNA targets. Finally, we developed a phenomenological network model, based on the Pearson correlation of microarray gene expression data, to identify subnetworks dysregulated in cell cycle and apoptosis. While our methodology reported many genes previously associated with ovarian cancer, it significantly suggested potentially oncogenic genes for further investigation. This network model can easily be extended to identify dysregulated genes in other cancers.

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Lombardi, Olivia. "Investigating the role of mRNA capping enzyme in C-MYC function." Thesis, University of Dundee, 2017. https://discovery.dundee.ac.uk/en/studentTheses/4816aeec-c481-4494-9a07-56e74a83c08e.

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C-MYC is a transcription factor and a potent driver of many human cancers. In addition to regulating transcription, C-MYC promotes formation of the mRNA cap which is important for transcript maturation and translation. However, the mechanistic details of C-MYC-dependent mRNA capping are not fully understood. Since anti-cancer strategies to directly target the C-MYC protein have had limited success, enzymatic co-factors or effectors of C-MYC present attractive alternatives for therapeutic intervention of C-MYC-driven cancers. mRNA capping enzyme (CE) initiates mRNA cap formation by catalysing the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide. The involvement of CE in C-MYC-dependent mRNA capping and C-MYC function has not yet been explored. Therefore, I sought to determine whether C-MYC regulates CE, and whether CE is required for C-MYC function. I found that C-MYC promotes CE recruitment to RNA polymerase II (RNA pol II) transcription complexes and to regions proximal to transcription start sites on chromatin. Consistently, C-MYC increases RNA pol II-associated CE activity. Interestingly, cells driven by C-MYC are highly dependent on CE for C-MYC-induced target gene expression and cell transformation, but only when C-MYC is overexpressed; C-MYC-independent cells or cells retaining normal control of C-MYC expression are insensitive to CE inhibition. C-MYC expression is also dependent on CE. Taken together, I present a bidirectional regulatory relationship between C-MYC and CE which is potentially therapeutically relevant. Studies here strongly suggest that inhibiting CE is an attractive strategy to selectively target cancer cells which have acquired deregulated C-MYC.

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Heimiller, Joseph Karl. "Genome-wide analysis of splicing requirements and function through mRNA profiling." Thesis, University of Colorado at Boulder, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3607314.

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The RNA-binding proteins U2AF and PTB play important roles in gene expression in many eukaryotic species. Although U2AF and PTB have been well-studied, their functional requirements have not been investigated on a genome-wide scale. In this thesis, I analyze RNA expression data to determine the requirement of the general splicing factor U2AF in S. pombe and to identify genes misregulated in Drosophila PTB mutants. I find that many introns are insensitive to U2AF inactivation in a Schizosaccharomyces pombe U2AF59 mutant, prp2.1. Bioinformatics analysis indicates that U2AF-insensitive introns have stronger 5' splice sites and higher A/U composition. The importance of intronic nucleotide composition was further investigated using wild type RNA expression data sets. I show that nucleotide composition is a relatively important factor for regulated intron retention in a variety of species. I also analyzed the RNA-binding protein PTB using RNA Seq data to reveal genes misregulated in PTB mutants in D. melanogaster. I identify misregulation of alternative splicing in PTB mutants and putative PTB binding sites. In the PTB embryonic lethal mutant, which shows dorsoventral patterning defects, I show that dorsal fate genes are significantly up-regulated. I present a model to link PTB to dorsal closure defects. This thesis provides the first genome-wide analysis of U2AF in S. pombe and PTB in Drosophila melanogaster.

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Hannigan, Molly M. "The Functions and Regulation of mRNA Processing During Male Germ Cell Development." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1554291304563947.

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Grasso, Laura. "The regulation and function of mRNA cap methylation in pluripotency and differentiation." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/ea974122-f82d-4cca-afb2-9ea43c1587e4.

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The synthesis of the N7-methylguanosine cap at the 5’ end of pre-mRNA occurs co-transcriptionally and is catalysed by a series of enzymes including the N7 RNA methyltransferase (RNMT), which along with its recently discovered activating subunit RAM, methylates the cap. RAM, which contains an RNA binding domain, is required to promote RNMT activity both in vitro and in vivo. Although the biochemical function of RAM has been characterized, its biological relevance remains elusive to date. The addition of the cap moiety is a crucial event in gene expression as it affects several processes within the mRNAs life cycle including mRNA processing, stability and translation. In stem cells, every step of mRNA metabolism is tightly regulated to maintain the undifferentiated state, allowing the expression of pluripotency genes and the concomitant repression of the lineage-specific ones. Here, I describe a critical role for the mRNA cap methylation in the maintenance of pluripotency. RNMT and RAM are highly expressed in mESCs compared to differentiated cells. The reprogramming of MEFs to iPS totally restores the elevated expression levels of RNMT and RAM suggesting that high levels of the two proteins are a feature of pluripotent cells. Even more exciting, the same expression is conserved amongst species as also hESCs and hiPSCs exhibit high levels of RNMT and RAM compared to fibroblasts. So far, RNMT and RAM were described as a complex in all cells lines examined and it was assumed that are similarly regulated, instead surprisingly, during in vitro neural differentiation a specific reduction in RAM protein levels is observed. Gain- and loss-of-function studies have been employed to demonstrate that specifically high RAM levels are required for the maintenance of pluripotency. In fact, RAM depletion causes a major reduction in the methyl cap levels of important pluripotency factors, ultimately resulting in a decreased of the protein levels. Therefore, RAM is found to function as modulator of RNMT activity, whereby it promotes cap methylation of fundamental transcripts required for the maintenance of ESCs pluripotency. I have also found that during differentiation RAM is down regulated post-transcriptionally, and therefore current studies are focused on investigating the role of RAM phosphorylation at Serine-36, which correlates with proteosomal degradation. Together the data corroborate previous findings about the methyl cap formation being a critical and regulated process within gene expression and propose a novel implication of this modification in the maintenance of pluripotency.

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Hammarlöf, Disa L. "EF-Tu and RNase E : Essential and Functionally Connected Proteins." Doctoral thesis, Uppsala universitet, Mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-159682.

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The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme. We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode. RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

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Kiri, Arpna. "The isolation and function of the 3'untranslated region of the myosin heavy chain genes of skeletal muscle." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325611.

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Tubeuf, Helene. "Développement de stratégies de criblage de mutations d'épissage dans des gènes de prédisposition au cancer. Demystifying the splicing code: new bioinformatics insights for the interpretation of genetic variants A staggering number of genetic variations affect the splicing pattern of BRCA2 exon 7: validation of the predictive power of splicing-dedicated silico analyses MLH1 exon 7, an emblematic exon sensitive to intronic mutations but not to alterations of exonic splicing regulators, sheds light into the performance of SRE-dedicated bioinformatics approaches Calibration of pathogenicity of partial splicing defects: The model of BRCA2 Exon 3." Thesis, Normandie, 2019. http://www.theses.fr/2019NORMR009.

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Le développement du séquençage de l’ADN à haut débit a grandement facilité le criblage de variations génétiques dans le génome des patients. Désormais, l’un des principaux défis de la génétique médicale n’est donc plus la détection des variations, mais leur interprétation fonctionnelle et clinique. Récemment, nous avons montré, à l’aide de tests fonctionnels basés sur l’utilisation de minigènes, que bien que le nombre de mutations d’épissage, et en particulier celles qui affectent sa régulation, est actuellement sous-estimé, l’effet de ces variations pourrait être dorénavant prédit à l’aide d’outils bioinformatiques spécifiques. Nous avons ainsi étendu l’évaluation du caractère prédictif de ces quatre nouvelles approches bioinformatiques par une étude comparative des scores générés par ces approches avec des données expérimentales obtenues pour un total d’environ 1200 variations exoniques. Nos travaux ont ainsi démontré la fiabilité de ces approches, utilisées seules ou en combinaison, et ont permis de proposer des recommandations quant à leur utilisation en tant qu’outils de filtration pour prioriser les variations à analyser dans des tests fonctionnels axés sur l’épissage. Néanmoins, une analyse mutationnelle exhaustive ciblée sur l’exon 7 de MLH1, a mis en évidence l’échec apparent de ces approches, pourtant validées par des études menées sur l’exon 7 de BRCA2, l’exon 10 de MAPT et l’exon 5 de MSH2, laissant suggérer que ces méthodes pourraient ne pas s’appliquer de manière équivalente à tous les exons et/ou à tous les gènes. En effet, nous avons montré que cet exon était doté de caractéristiques particulières, i.e. de sites d’épissage remarquablement forts, lui conférant une résistance totale aux mutations de régulation d’épissage et mettant en échec les outils de prédictions. Ces données contribuent à mieux déterminer les limitations de ces outils bioinformatiques tout en contribuant à leur amélioration. En dépit de ces avancées, l'évaluation de la pathogénicité des mutations d'épissage reste complexe, en particulier celles conduisant à des anomalies d'épissage en phase et/ou partielles. En utilisant, comme modèle d’étude, des variations à l’origine du saut partiel de l’exon 3 de BRCA2, nos résultats ont révélé que l’activité tumeur-suppressive de BRCA2 tolère une réduction substantielle du niveau d’expression, étant donné qu’un allèle produisant jusqu’à 70% de transcrit codant une protéine déficiente n’est pas nécessairement associé à un risque élevé de développer un cancer. L’ensemble de ces données a d’importantes implications dans le diagnostic moléculaire et la prise en charge des patients et de leurs apparentés, avec un bénéfice direct pour les familles évocatrices d’une prédisposition héréditaire et devrait contribuer à l’interprétation de VSI identifiées par séquençage à haut débit dans toute autre pathologie d’origine génétique
The development of high-throughput DNA sequencing has greatly facilitated the screening of genetic variations within patient genome. Henceforth, one of the main challenges in medical genetics is no longer the detection of variations, but their functional and clinical interpretation. Recently, we showed by using splicing reporter minigene assays, that although splicing mutations, and in particular those affecting its regulation, are more prevalent than initially estimated, they could now be predicted by using dedicated bioinformatics tools. We thus extended the evaluation of the predictive power of these four newly developed computational approaches by a comparative study of the scores obtained by these approaches with experimental data for a total of about 1200 exonic variations. Our findings have demonstrated the reliability of these approaches, used alone or in combination, and allow to offer recommendations for their use as a filtration tool to prioritize the variations to be analysed as a priority in splicing-dedicated functional assays. Nevertheless, an exhaustive mutational analysis targeting MLH1 exon 7, has highlighted the apparent failure of these approaches, yet validated by studies focused on BRCA2 exon 7, MAPT exon 10 and MSH2 exon 5, suggesting that these methods might not be equivalently applicable to all exons and/or genes. Indeed, we have shown that this exon has particular characteristics, i.e. remarkably strong splice sites, conferring it a total resistance to splicing regulation mutations and defeating prediction tools. These findings help to better determine the limitations of these bioinformatics tools while contributing to their improvement. In spite of these advances, the pathogenicity assessment of splicing mutations remains complicated, especially of those leading to in-frame and/or partial splicing anomalies. By using variant-induced partial BRCA2 exon 3 skipping as a model system, we showed that BRCA2 tumor suppressor function tolerates a substantial reduction in expression level, as BRCA2 allele producing as much as 70% of transcript encoding deficient protein may not necessarily confer high-risk of developing cancer. Altogether, these data have important implications in the molecular diagnosis and clinical management of patients and their relatives, with a direct benefit for hereditary cancer-suspected families and should contribute to the interpretation of VSI identified by high throughput sequencing in any other genetic disease

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Bhattacharyya, Debmalya. "Translation Modulation of Cellular mRNA by G-Quadruplex Structures." Kent State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1469636863.

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Blasco, Moreno Bernat 1986. "Uncovering a novel function of the exonuclease Xrn1 in viral and cellular mRNA translation." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/664207.

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The exonuclease Xrn1 degrades messenger RNAs (mRNAs) and controls transcription. By using a model system that replicates brome mosaic virus (BMV) in yeast, here we show that Xrn1 promotes translation initiation of BMV RNA. This function is linked to the highly structured 5’UTR and the coding sequence. The exonuclease activity of Xrn1 is required for its role in viral RNA translation. In contrast, the role of Xrn1 in translation is independent from its function in transcription. Importantly, ribosome profiling analyses reveal that Xrn1 acts as a translational activator of a specific group of cellular mRNAs enriched for glycosylation functions. As for BMV RNA, these cellular mRNAs depend on the exonuclease activity of Xrn1 for translational activation and contain long and structured 5’UTR. Our results reveal a new cross-talk between mRNA degradation and translation, and uncover an unexpected function of the exonuclease Xrn1 in viral and cellular translational control. Xrn1 és una exonucleasa que degrada l’ARN missatger (ARNm) i en regula la transcripció. Emprant un sistema model basat en el virus del mosaic del brom (BMV) i el llevat S. cerevisiae, en aquest treball es demostra que Xrn1 promou l’inici de la traducció de l’ARN viral. Aquesta funció en traducció està lligada a la regió no traduïda a l’extrem 5’ i a la seqüència codificant. L’activitat exonucleasa pròpia de Xrn1 és necessària per tal que actuï en traducció. En canvi, la funció de Xrn1 en transcripció és independent de la funció en traducció. Mitjançant estudis amb perfil de ribosomes, es va constatar que Xrn1 també actua com a activador de la traducció d’un subgrup d’ARNm cel·lulars. Aquests, codifiquen per funcions relacionades amb la glucosilació. Tal i com s’observa per l’ARN de BMV, aquests ARNm cel·lulars depenen de l’activitat exonucleasa de Xrn1 per a la seva traducció i tenen una elevada estructura secundària a la regió no traduïda de l’extrem 5’. En conjunt, els nostres resultats descriuen un nou exemple de la comunicació existent entre la degradació i la traducció de l’ARNm i revelen que Xrn1 té una funció inesperada en el control de la traducció de l’ARNm viral i cel·lular.
Xrn1 és una exonucleasa molt conservada entre espècies i que té un paper cabdal en la degradació de l’ARN missatger (ARNm) i la regulació de la transcripció. Emprant un sistema model basat en el virus del mosaic del brom (BMV) i el llevat Saccharomyces cerevisiae, en aquest treball es demostra que Xrn1 promou la traducció de l’ARN viral. Aquesta funció en traducció està lligada a la regió no traduïda a l’extrem 5’ i a la seqüència codiﬁcant. A més, mitjançant l’anàlisi amb perﬁls de polisomes s’observa que Xrn1 assisteix la iniciació de la traducció de l’ARN viral i que interacciona directament amb la maquinària de traducció. Tenint en compte que Xrn1 par􀀁cipa en la degradació i la transcripció d’ARNm, es van crear diferents mutants per tal d’analitzar la relació entre aquestes funcions i el rol de Xrn1 en traducció. En primer lloc, es va observar que l’ac􀀁vitat exonucleasa pròpia de Xrn1 és necessària per tal que Xrn1 actuï en traducció. Expressant la exonucleasa nuclear (Rat1) al citoplasma de cèl·lules xrn1∆, s’aconsegueix compensar els defectes en degradació de l’ARN i en la taxa de creixement. En canvi, no es rescaten els defectes en la traducció de l’ARN viral. En segon lloc, u􀀁litzant un mutant de Xrn1 que no pot ser importat al nucli (Xrn1∆NLS) es va concloure que la funció de Xrn1 en transcripció és independent de la funció en traducció. Tot seguit, mitjançant estudis amb perﬁl de ribosomes, es va constatar que Xrn1 també actua com a ac􀀁vador de la traducció d’un subgrup d’ARNm cel·lulars. Aquests, codiﬁquen per funcions relacionades amb la glucosilació i estan enriquits en proteïnes del re􀀁cle endoplasmà􀀁c. Tal i com s’observa per l’ARN de BMV, aquests ARNm cel·lulars depenen de l’ac􀀁vitat exonucleasa de Xrn1 per a la seva traducció i tenen una elevada estructura secundària a la regió no traduïda de l’extrem 5’. En conjunt, els nostres resultats descriuen un nou exemple de la comunicació existent entre la degradació i la traducció de l’ARNm i revelen que Xrn1 té una funció inesperada en el control de la traducció de l’ARNm viral i cel·lular.

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Swift, Robert. "Structure-function relationships in enzymes of the nucleotidyltransferase superfamily RNA ligation and mRNA capping /." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3354742.

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Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed June 16, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 137-147).

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Welch, Kasey C. "Expression Of Nicotinic Acetylcholine Receptor mRNA As a Function Of Age In Whole Hippocampus Preparations From Wistar Rats." Diss., CLICK HERE for online access, 2008. http://contentdm.lib.byu.edu/ETD/image/etd2371.pdf.

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Cenik, Can. "Studies on the Evolution and Function of Introns in 5' Untranslated Regions." Thesis, Harvard University, 2011. http://dissertations.umi.com/gsas.harvard:10020.

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The function and evolution of introns have been topics of great interest since introns were discovered in the 1970s. Introns that interrupt protein-coding regions have the most obvious potential to affect coding sequences; therefore, their evolution have been studied most intensively. Splicing of introns within untranslated regions does not contribute directly to the diversity of proteins, yet ~35% of human transcripts contain introns within the 5' untranslated region (UTR). The evolution and possible functions of 5'UTR introns (5UIs) remain largely unexplored. Here we undertook a genome-wide functional analysis of 5UIs. Our main results are as follows: First, the distribution of these introns in the human genome is nonrandom. While genes with regulatory roles are enriched in having 5UIs, genes encoding proteins that are targeted to the endoplasmic reticulum and mitochondria are surprisingly depleted of these introns. Second, we offered and supported a model whereby gene encoding secretory and nuclear-encoded mitochondrial proteins share a common regulatory mechanism at the level of mRNA export, which is dependent on the absence of 5'UTR introns. Specifically, the upstream element in a given transcript, be it an intron or RNA elements near the 5' end of coding sequences (CDS), dictates the mRNA export pathway used. Finally, we discovered a strong correlation between existence of 5'UTR introns and sequence features near the 5' end of CDS. We developed an integrated machine-learning framework that can predict absence of 5UIs using solely the sequence near the 5' end of CDS. Our model achieved >80% accuracy when validated against nuclear-encoded mitochondrial transcripts. Specific RNA elements predictive of 5UI absence are found in ~40% of human transcripts spanning a wide spectrum of functions. By analyzing hundreds of large-scale datasets, we functionally characterized the transcripts with these RNA elements; revealing their association with translational regulation. These RNA elements were bound by proteins interacting with the Exon Junction Complex in vivo suggesting a molecular mechanism that links these elements to their downstream effects in mRNA export and translational regulation. While some 5'UTR introns might be evolving neutrally, our results, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns.

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Neu, Ancilla [Verfasser], and Remco [Akademischer Betreuer] Sprangers. "Structure, dynamics and function of proteins in the mRNA decay pathway / Ancilla Neu ; Betreuer: Remco Sprangers." Tübingen : Universitätsbibliothek Tübingen, 2016. http://d-nb.info/1165235986/34.

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Dissertations / Theses on the topic 'Functional mRNA'

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