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1
Smith, Nikki. "The role of cellular micro-RNAs in Epstein-Barr virus induced cellular transformation and oncogenesis". Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1344/.
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Micro-RNAs (miRNAs) are a class of non-coding RNA which post-transcriptionally regulate gene expression. Epstein-Barr Virus (EBV) transforms resting B-cells in vitro to establish continuously proliferating lymphoblastoid cell lines (LCLs) and is aetiologically linked to lymphomas. Little is known about the contribution of miRNAs to the transformation of B cells. We initially examined the regulation of the oncogenic miR-155, which is highly expressed in Hodgkin’s lymphoma but was reportedly absent in Burkitt’s lymphoma. We found that miR-155 was up-regulated by EBV-LMP1 expression, and that a reported defect of miR-155 processing in Burkitt’s lymphoma was a misinterpretation of data. Next, to identify cellular miRNAs and genes modulated during EBV-induced transformation, we compared the expression profiles of resting B cells and B cells either infected with EBV or stimulated to proliferate with CD40L and IL4. This revealed that a large proportion of miRNAs and genes differentially regulated by EBV and not by CD40L/IL4 were modulated by EBV interaction with its CD21 receptor complex, but these changes were maintained or amplified in LCLs; and included a set of tumour suppressor genes down-regulated by EBV. In addition, bioinformatics analysis indicated that EBV modulates the expression of multiple miRNAs predicted to target the same cellular genes.
2
Kheimar, Ahmed Mahmoud Osman [Verfasser]. "Tumor promoting functions of cellular telomerase RNA and viral RNAs in herpesvirus-induced cancer formation / Ahmed Mahmoud Osman Kheimar". Berlin : Freie Universität Berlin, 2017. http://d-nb.info/1147758247/34.
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3
Hewitt, Rachael Nicole. "Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence". Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7143/.
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Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.
4
Jeon, Young-Jun. "The Roles of Non-Coding RNAs in Solid Tumors". The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1426083854.
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Geisler, Sarah J. "Decapping of Long Noncoding RNAs Regulates Inducible Genes". Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1340141951.
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6
Wagner, Simone [Verfasser], i Bettina [Akademischer Betreuer] Kempkes. "Identification of cellular long non-coding RNAs regulated by the EBV nuclear antigen EBNA2 / Simone Wagner ; Betreuer: Bettina Kempkes". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1199265721/34.
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7
Rogowski, Karol Jerzy [Verfasser]. "Identification and characterization of non-coding RNAs and their associated proteins involved in cellular stress responses / Karol Jerzy Rogowski". Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1215099606/34.
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8
Balaratnam, Sumirtha. "BIOGENESIS AND FUNCTIONAL APPLICATIONS OF PIWI INTERACTING RNAs (piRNAs)". Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1531753741509242.
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9
Ostler, Jeffery Brent Jr. "Characterization of Pol IV and Pol V-Dependent Non-Coding RNAs Derived from aGeminivirus Genome". The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492698361649423.
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10
Navaroli, Deanna M. "Molecular Mechanisms of Endocytosis: Trafficking and Functional Requirements for the Transferrin Receptor, Small Interfering RNAs and Dopamine Transporter: A Dissertation". eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/592.
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Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping.
The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing.
Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.
11
Navaroli, Deanna M. "Molecular Mechanisms of Endocytosis: Trafficking and Functional Requirements for the Transferrin Receptor, Small Interfering RNAs and Dopamine Transporter: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/592.
Pełny tekst źródłaStreszczenie:
Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping.
The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing.
Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.
12
Bouckenheimer, Julien. "Rôle fonctionnel des longs ARN non codants dans l'adaptation et la pluripotence des cellules souches en culture". Thesis, Montpellier, 2016. http://www.theses.fr/2016MONT3505.
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Les applications des cellules souches pluripotentes humaines (CSP) dans le domaine biomédical sont particulièrement prometteuses, aussi bien au niveau expérimental qu’au niveau clinique. Leur utilisation comme source inépuisable de cellules permettant de tester et développer de nouvelles molécules thérapeutiques (notamment par modélisation de pathologies in vitro, criblage haut-débit et tests de cytotoxicité) s’ajoute à l’important potentiel qu’elles présentent en médecine régénérative et en thérapie cellulaire. Utilisables comme matériel biologique permettant de restaurer partiellement ou totalement un organe ou un tissu défaillant, il reste essentiel de vérifier l’intégrité génétique des lignées cellulaires utilisées afin de garantir une utilisation sécurisée pour le patient. Parmi les facteurs responsables de l’apparition d’anomalies génétiques chez les CSP, les conditions cultures jouent un rôle essentiel. Des techniques de culture inadaptées peuvent facilement provoquer l’émergence d’une instabilité génomique. Toute altération doit être détectée et documentée afin de pouvoir définir des critères d’acceptation préalable à leur utilisation clinique.Les CSP sont des cellules particulièrement sensibles au stress qui peut résulter de techniques de repiquage inappropriées. La dérive génétique qui découle de ce stress peut être précoce et apparaître dès les premiers passages des lignées cultivées. Notre équipe a pu tester de nombreuses méthodes de repiquage sur différentes lignées cellulaires pluripotentes. Nous avons notamment observé que des anomalies génétiques majeures caryotypiques (trisomies) et infra-caryotypiques (SNPs) ainsi que des changements phénotypiques (survie augmentée, acquisition de mobilité) apparaissaient rapidement suite à l’utilisation de techniques de repiquage basées sur l’utilisation d’enzyme de dissociation (TryPLE). Ces altérations apparaissent dans des lignées qui s’adaptent progressivement à la dissociation en cellules uniques (dissociation « single-cell ») provoquées par ces enzymes.Notre équipe étudie les conséquences cellulaires liées à ce phénomène d’adaptation des CSP provoquée par la dissociation « single-cell ». Grâce à des techniques de séquençage dernière génération (RNA-Seq), nous avons comparé les profils transcriptomiques de CSP repiquées par des techniques standard (comme le passage mécanique) et par des techniques basées sur la dissociation « single-cell » (comme le passage enzymatique par TryPLE). Cette comparaison a montré au niveau transcriptionnel une surexpression spectaculaire d’ARNs non codants appartenant à une classe récemment décrite : les longs ARNs non codants (lncRNAs).L’objectif principal de ce travail de thèse a été d’évaluer le niveau d’implication de ces lncRNAs dans le processus d’adaptation des CSP en culture, et leur rôle fonctionnel potentiel. Nous avons ainsi dans un premier temps déterminé in silico quels lncRNAs étaient différentiellement exprimés dans les CSP adaptées, et après validation expérimentale par biologie moléculaire des candidats les plus prometteurs, nous avons utilisé des tests fonctionnels (notamment par RNA interférence (siRNA)) afin de déterminer le rôle de ces lncRNAs dans la machinerie cellulaire et la pluripotence des CSP. Autour de ce projet principal, nous avons essayé de comprendre les mécanismes régissant les changements phénotypiques et comportementaux provoquées par la dissociation « single-cell ». Nous avons notamment pu suggérer la mise en place d’un phénomène de transition épithélio-mésenchymateuse (EMT) chez des CSP dissociées. Enfin, l’attractivité que représente un sujet d’étude récent comme les lncRNAs et la disponibilité croissante de publications les concernant nous ont poussé à publier une revue approfondie ainsi qu’une méta-analyse sur l’implication des longs ARN non codants dans le développement précoce de l’embryon et dans les cellules souches pluripotentesThe actual and future applications of human pluripotent stem cells (PSC) in the biomedical field are highly promising. Their use for the discovery of new therapeutic drugs through the development of high-throughput screening tests, cytotoxicity tests and in vitro disease modeling has been added to their tremendous interests in regenerative medicine and cellular therapy. As a source of biological material that can be used to restore partially or totally the lost functions of a damaged organ or tissue, or as a source of normal cells to study human development or test putative new drugs, their genomic integrity has to be thoroughly assessed. Therefore, an effective optimization of their culture conditions has to be considered, in order to control the absence of genomic instability and prevent their potential emergence. Any genetic or epigenetic alteration resulting from cell culturing must be detected in order to define and characterize acceptance criteria for scientific and medical purposes.PSC are particularly sensitive to stress resulting from unappropriated passaging techniques, which cause rapid genetic drift. Indeed, our team observed that many genomic abnormalities arise from aggressive single cell, enzymatic based, passaging methods, and that substantial phenotypical changes such as increased survival after cell dissociation and variation in cell shape can then occur.In order to understand the mechanisms governing the emergence of those adverse alterations, the team focused on the consequences resulting from the adaptation of PSC to single-cell dissociation. By using new generation sequencing techniques as RNA-Seq, we compared transcriptomics of PSC passaged by standard techniques (such as mechanical passaging) versus single-cell enzymatic dissociation (such as TRyPLE-based single-cell passaging). This comparison showed that the most striking difference in the gene expression pattern between adapted and non adapted cells concerned the dramatic overexpression of RNAs from a recently discovered class: long non-coding RNAs (lncRNAs).The aim of this thesis work was to determine to which extent some of these lncRNAs were functionally linked to adaptation of PSC. In order to address this matter, we first investigated in silico which lncRNAs were upregulated by single-cell dissociation, and after experimental validation of lncRNA candidates by molecular biology, we performed functional in vitro analysis (notably by siRNA-mediated loss of function) and sought their cellular localization in order to decipher their role in the cellular machinery and their level of implication. Beside this main project, other auxiliary projects were grafted. The observation of major changes in cell phenotype and behavior led to the investigation of the global mechanisms governing these modifications, underlining the potential role of epithelial-to-mesenchymal transition provoked by single-cell dissociation. Finally, the global attractiveness of lncRNAs and the emergence of exponential documentation concerning non-coding RNAs prompted the writing of an extensive review and meta-analysis concerning the implications of lncRNAs during embryo development and in pluripotent stem cells
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Conine, Colin C. "Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation". eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/724.
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During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.
14
Conine, Colin C. "Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation". eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/724.
Pełny tekst źródłaStreszczenie:
During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.
15
Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation". eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/825.
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XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
16
Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation". eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/825.
Pełny tekst źródłaStreszczenie:
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
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Palsule, Geeta. "Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656.
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Gore, Panter Shamone Robinette. "Genetic and Functional Studies of LociAssociated with Atrial Fibrillation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1396521127.
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Hunt, Sarah Louise. "Cellular proteins required for rhinovirus RNA translation". Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313880.
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Bailey, Daniel John. "Cellular proteins in picornavirus replication". Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298484.
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Kandan-Kulangara, Febitha. "Poly(ADP-ribose) polymerase-1 (PARP-1) and RNA interference (RNAI) during cell death". Doctoral thesis, Université Laval, 2013. http://hdl.handle.net/20.500.11794/25972.
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L’activation de la poly(ADP-ribose) polymérase-1 (PARP-1) en réponse aux dommages à l’ADN est impliquée dans diverses réponses cellulaires, de la réparation de l’ADN à la mort cellulaire. Dans l'annexe I, nous avons décrit différentes techniques indispensables pour détecter le métabolisme de PARP-1 en réponse aux dommages à l’ADN in vitro et in vivo. Les travaux de cette thèse se concentrent sur le rôle de PARP-1 dans la mort cellulaire. PARP-1 est clivée et inactivée par des caspases pendant l’apoptose ; j’ai donc utilisé une PARP-1 non-clivable pour étudier le rôle de l’activation et de la fragmentation de PARP-1 dans la mort cellulaire induite par les UVB. Nous avons observé que, contrairement aux fibroblastes de peau humaine exprimant la PARP-1, les fibroblastes avec un "knockdown" de PARP-1 sont résistants à l’apoptose induite par les UVB, phénotype pouvant être totalement inversé par ré-expression de PARP-1 sauvage mais pas de PARP-1 non-clivable par les caspases, suggérant un rôle significatif du clivage de PARP-1 en réponse à la mort cellulaire induite par les UVB (chapitre 2). Dans ce contexte, nous avons récemment passé en revue comment les substrats non clivables par des caspases peuvent être utilisés comme outil important pour démystifier le rôle de ce clivage pour la mort comme pour la vie, avec l’exemple spécifique de PARP-1 non-clivable par les caspases (chapitre 3). Curieusement, en utilisant l’ARNi comme outil d’étude du rôle de PARP-1 dans la mort cellulaire, nous avons observé que l’ARNi stable (shRNA) de nombreux gènes, incluant PARP-1, échoue lors de l’apoptose, en raison de l’inactivation catalytique par clivage par une caspase de l’endoribonucléase Dicer-1, indispensable pour la régulation de l’ARNi et des miARN (chapitre 4). Cependant, nous avons découvert que l’ARNi transitoire persiste plusieurs jours même après induction de l’apoptose, soulignant des différences entre les ARNi stable et transitoire dans la dynamique de "knockdown" génétique et dans la dépendance de la fonction de Dicer-1 (chapitre 5). En résumé, mon travail a permis la découverte des avantages et des limites de l’ARNi durant l’apoptose et le rôle de PARP-1 dans la mort cellulaire induite par les UVB.
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Osborn, Maire. "Cellular RNA Targeting by Platinum (II) Anticancer Therapeutics". Thesis, University of Oregon, 2014. http://hdl.handle.net/1794/17920.
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Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis.
Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets.
This dissertation includes previously published and unpublished co-authored material.
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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II". Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.
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Brown, E. C. "Cellular proteins involved in translation of human rhinovirus RNA". Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596963.
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Translation of picornavirus RNA takes place by internal initiation, determined by the presence of an internal ribosome entry site (IRES) in the 5'-untranslated region of the genomic RNA. Efficient translation from the human rhinovirus-2 (HRV-2) IRES is dependent on host cell trans-acting factors. These include unr, p38 and polypyrimidine tract binding protein (PTB). This thesis details the investigation into how these factors act to promote translation from the HRV-2 IRES. Unr, an RNA-binding protein with five cold-shock domains (CSDs), binds to the HRV-2 IRES and this interaction was studied by crude and then fine mapping of the binding sites of unr on the IRES. The functions of the CSDs of unr were investigated by point mutation of each of the CSDs and testing the ability of these mutants to bind the IRES and stimulate translation from it. p38, a WD-motif protein with no RNA-binding activity, was expressed using recombinant baculovirus-infected insect cells. An in vivo interaction between unr and p38 was demonstrated, and the effect of p38 on unr's binding to the HRV-2 IRES was tested in vivo. After gaining insight into the complexes of unr and p38 that form on the IRES, the function of p38 in translation from the HRV-2 IRES was demonstrated. Unr and PTB were also used as tools to compare the factor requirements of the HRV-2 and poliovirus IRESs for efficient translation. Finally, an investigation was made into the cellular role of unr, in terms of the cellular mRNAs that unr binds, and those whose translation it stimulates.
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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.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
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.
by Tanya Todorova.
Ph. D.
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Mullani, Nowsheen. "An RNA Signature Links Oxidative Stress To Cellular Senescence". Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS560.pdf.
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Le stress oxydatif est l’une des voies menant à la sénescence cellulaire. Bien que les dommages causés par les espèces réactives de l'oxygène aux protéines et à l'ADN soient bien décrits, notre compréhension de la manière dont la transcription peut participer à l'apparition de la sénescence est encore limitée. Au niveau de la transcription, le stress oxydatif entraîne l’accumulation d’ARN promoteurs (ARNAu) et d’ARN amplificateur (ARNs), conséquence de la libération défectueuse du RNAPII de la chromatine, un phénomène connu sous le nom de RNAPII crawling. Nous avons observé que l'exploration de RNAPII était également détectée en aval d'une petite série de gènes connus pour être régulés par HP1Υ au niveau de leur terminaison. L'exploration de ce phénomène a donné un résultat inattendu, en ce sens qu'il a révélé un effet inhibiteur du peroxyde d'hydrogène sur le complexe exosome d'ARN impliqué dans la dégradation des ARN polyadénylés. Le RNAPII rampant a pour résultat la transcription de séquences d’ALU situées au voisinage des promoteurs et amplificateurs et en aval de gènes sans intron et de petites séries de gènes contenant un intron. Comme les séquences ALU contiennent des séquences A codées par le génome, elles doivent normalement être dégradées par l’exosome de l’ARN. Cependant, comme le stress oxydatif inhibe également cette activité d'ARNase, les ARNm contenant des séquences d'ALU transcrites par hasard se stabilisent et sont détectés dans le cytoplasme et même dans les fractions de polysomes. Ce phénomène peut participer à l'apparition de la réponse à l'interféron associée au stress oxydatifOxidative Stress is one of the routes leading to cellular senescence. While the damages that reactive oxygen species inflict on proteins and DNA are well described, our insight on how transcription may participate in the onset of senescence is still limited. At a transcriptional level, oxidative stress results in accumulation of promoter RNAs (uaRNAs) and enhancer RNAs (eRNAs) as a consequence of defective release of the RNAPII from the chromatin a phenomenon known as RNAPII crawling. We observed that RNAPII crawling was also detected downstream of a small series of genes known to be regulated by HP1Υ at the level of their termination. Exploring this phenomenon yielded an unexpected result in the sense that it revealed an inhibiting effect of hydrogen peroxide on the RNA exosome complex involved in degradation of polyadenylated RNAs. The crawling RNAPII results in the transcription of ALU sequences located in the neighborhood of promoters and enhancers and downstream of intron-less genes and of small series of intron-containing genes. As ALU sequences contain genome encoded A tracts, they should normally be degraded by the RNA exosome. Yet, as oxidative stress also inhibits this RNAse activity, mRNAs containing serendipitously transcribed ALU sequences get stabilized and are detected in the cytoplasm and even polysome fractions. This phenomenon may participate in the onset of the interferon response associated with oxidative stress
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation". eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
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The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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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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Stassinopoulos, Ioannis A. "Interactions of picornavirus internal ribosome entry sites with cellular proteins". Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322940.
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Schmier, Brad J. "The Molecular Machinery Critical to the Degradation of Cellular RNA". Scholarly Repository, 2012. http://scholarlyrepository.miami.edu/oa_dissertations/714.
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Exoribonucleases are indispensable for cellular RNA metabolism. RNA processing, end-turnover, and degradation all require the concerted action of exoribonucleases. In this thesis, two families of exoribonucleases that act in the final steps of RNA decay pathways are explored. The first of these is the RNR superfamily of processive 3’→5’ RNases with major roles in both mRNA and stable RNA degradation. The initial focus of this work is the structural and enzymatic characterization of an unusual RNR family enzyme from the radiation-resistant bacterium Deinococcus radiodurans. This enzyme is demonstrated biochemically to be an RNase II-type enzyme (DrII), based on its sensitivity to secondary structure. Analysis of the DrII X-ray structure reveals that a novel, winged-HTH domain has replaced the canonical RNA binding clamp typical of RNR family proteins. The exposed architecture of DrII’s RNA binding surface offers an explanation for the nuclease’s ability to approach within 3-5 nt of a duplex, an important mechanistic difference from the well-studied E. coli RNase II. The open, clamp architecture of DrII may have broader relevance to mechanisms of duplex RNA recognition in the RNR superfamily. RNA decay by processive exonucleases such as RNR family proteins leaves 2-5 nt nanoRNA limit products that are further degraded to mononucleotides by nanoRNases. In E. coli, the DEDD family enzyme Oligoribonuclease (ORN) executes nanoRNA decay and represents the first major family of nanoRNases, with homologs widely conserved in eubacteria and eukaryotes. The B. subtilis NanoRNase A (NrnA), a DHH family phosphoesterase, represents a second major class of nanoRNases, with broad phylogenetic distribution in organisms that lack orn homologs. The second major focus of this thesis is a structural and mechanistic study of this nanoRNase machinery. The atomic structure of the B. subtillis nanoRNase NrnA is described, and unveils a bi-lobal architecture similar to the 5’→3’ DNase RecJ, where the catalytic DHH domain is linked via a partially helical connector to the C-terminal RNA binding domain. NrnA is a highly dynamic molecule, adopting both open and closed conformations. Co-crystallization with several substrates shows that NrnA has a nanoRNA specific substrate-binding patch that offers a structural explanation for its 3’→5’ nanoRNase activity. This RNA binding site feeds substrate to the DHH active site in an orientation opposite to the 5’→3’ path proposed for RecJ. Surprisingly, NrnA also maintains a weak 5’→3’ activity on certain substrates, and thus possesses both 5’→3’ and 3’→5’ exonuclease activities. In conclusion, an overall model is presented for how DHH family exonucleaess can degrade nucleic acids from both the 5’→3’ and 3’→5’ directions. Thus, the studies described in this thesis offer both an atomic and a biochemical view of the macromolecular machinery critical to the degradation of RNA.
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Tedeschi, Frank A. Tedeschi. "IDENTIFICATION OF CELLULAR RNA BINDING SITES OF DEAD-BOX HELICASES". Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1531217057171378.
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Gerst, Catherine. "Epissage in vivo et in vitro de rnas premessagers d'adenovirus-2 : etude des interactions rna - facteurs nucleaires". Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13159.
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McNally, Beth Anne. "A role for cytoplasmic PML in the cellular antiviral response". Columbus, Ohio : Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1133377007.
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Wong, Tsz-lo, i 黃子璐. "Cellular role of miR-143 in cervical cancer". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48274045.
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Cervical cancer is a largely preventable malignancy due to the availability of cytology screening and vaccination against the essential initiation factor of cervical carcinogenesis, human papillomavirus (HPV). However, cervical cancer remains a significant medical burden worldwide, particularly in developing countries where large scale screening or vaccination programs are not financially feasible. Molecular tests such as HPV DNA tests have the potential to improve the speed and sensitivity of cervical cancer screening but suffer from limited specificity. Additional adjunct molecular markers are therefore desirable for enhancing molecular tests. Our previous research has revealed miR-143, a microRNA downregulated in a number of cancers, could be detected in liquid based cytology samples and is significantly reduced in cervical cancer samples and cell lines. Cellular role of miR-143 and mechanism behind its downregulation remain an unknown in cervical carcinogenesis. To explore the cellular roles of miR-143 in cervical cancer, a construct expressing miR-143 was transfected into cervical cancer cell lines HeLa, SiHa and C33A. miR- 143 overexpression was verified by qPCR. The miR-143 overexpressing cell lines were used to conduct a number of cellular function assays. It has been reported that miR-143 is able to suppress cell growth in HPV-positive HeLa. We followed up the findings and revealed miR-143 overexpression in HPV-negative C33A did not suppress cell growth in an MTT cell proliferation assay. ERK5 and KRAS, two targets of miR-143, are downregulated in colon cancer and bladder cancer to suppress cell grwoth. However, mRNA level of ERK5 and KRAS were not altered in all three miR-143 overexpressed cervical cancer cell lines, suggesting that miR-143 may not target ERK5 and KRAS transcriptionally in cervical cancer. Ability of miR-143 in regulating cell differentiation was evaluated by the expression of K10, an early keratinocyte differentiation marker. K10 was upregulated only in miR-143 overexpressed HeLa and SiHa as revealed by qPCR. A parallel increase in hSkn-1a mRNA, a transcription factor of K10, was also observed specifically in the two miR-overexpressed HPV-positive cell lines. miR-143 level is inversely correlated with cytology grading and progression of cervical disease, hinting its role in mediating cell migration and invasion during cancer progression and metastasis. A reduction of cell migration as demonstrated in wound healing assay and in vitro transwell migration assay was observed exclusively in miR-143 overexpressed HeLa and SiHa. miR-143 overexpression in C33A did not introduce any effect in cell migration. A reduction of cell invasion was also observed merely in miR-143 overexpressed HeLa and SiHa as revealed in a transwell invasion assay. Apart from studying the cellular roles of miR-143 in cervical cancer, this study has also explored mechanisms behind miR-143 downregulation in cervical cancer owing to the fact that certain miR-143 mediated cellular functions were observed only in HPV-positive cervical cancer cell lines. We hypothesized that HPV E6 and E7 oncoprotein may downregulate miR-143 in cervical cancer. The hypothesis was supported by our findings where normal cervical epithelial cell line immortalized by E6 and E7 had an undetectable level of endogenous miR-143 level. The same primary cells immortalized by shp16-hTERT expressed residual amounts of miR-143 as revealed by qPCR. Owing to the low miR-143 expression in shp16-hTERTimmortalized normal cervical epithelial cell line, downregulation of miR-143 in cervical cancer cell lines may also be contributed to hTERT overexpression and p16 silencing. Overall, miR-143 plays an important role in suppressing cell proliferation, enhancing keratinocyte differentiation marker expression, reducing migration and invasion in HPV-positive cervical cancer. Downregulation of miR-143 level may be an effect as manifested by E6 and E7 in HPV-positive cervical cancer. Differential cellular effects in miR-143 overexpressed HPV-positive and HPV-negative cervical cancer cell lines suggest that HPV oncoprotein mediates miR-143 cellular functions.published_or_final_version
Pathology
Master
Master of Medical Sciences
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Babendure, Jeremy R. "Utilizing RNA structure as a tool in molecular and cellular biology". Diss., Connected to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3190000.
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Thesis (Ph. D.)--University of California, San Diego, 2005.Title from first page of PDF file (viewed Mar. 6, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 122-131).
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Nurmohamed, Salima. "Communication between the Escherichia coli RNA degradative machineries and cellular metabolism". Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611286.
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Deb, Maharshi Krishna. "Generation of antisense RNAs at convergent gene loci in cells undergoing senescence". Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30274.
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La sénescence, qui est un mécanisme antitumoral majeur, est définie comme un état d'arrêt irréversible de la prolifération cellulaire en réponse à un stress comme l'activation illégitime d'oncogènes. Les cellules qui entrent en sénescence subissent de profonds changements de leur épigénome. Les ARNs antisens sont suspectés de joue des rôles importants dans le contrôle du destin cellulaire et dans des processus cellulaires variés. Dans la levure, le variant d'histone H2A.Z co-opère avec les machineries du RNAi et de l'hétérochromatine pour réprimer sur les loci de gènes convergents l'apparition d'antisens dus à des défauts de terminaison de transcription de un des deux gènes. Chez les mammifères, l'existence et la régulation de tels transcrits antisens restent inconnues. De façon intéressante, la déplétion du variant d'histones H2A.Z est connue pour induire la sénescence. Nous nous sommes donc demandés si la sénescence est accompagnée de la régulation de tels transcrits antisens sur les gènes convergents, si la régulation par H2A.Z est conservée et si ces transcrits pouvaient avoir un rôle fonctionnel. Dans un modèle de sénescence induite par les oncogènes in vitro, nous avons identifiés par RNA-Seq brin spécifiques plusieurs loci de gènes convergents où des ARN antisens pourraient être générés par des défauts de terminaison de transcription sur le gène convergent. Des analyses en profondeurs sur deux loci ont confirmé que les transcrits antisens sont effectivement générés par un tel mécanisme (appelé "read-through transcriptionnel"). Nous avons appelé ces antisens START RNAs (pour " Senescence Triggered Antisense Read-through Transcripts). Nous avons par la suite montré que ces STARTs répriment l'expression du gène pour lequel ils sont antisens. Finalement, nous avons montré qu'ils sont réprimés par H2A.Z dans les cellules en prolifération. Nous proposons donc un modèle où la progression en sénescence s'accompagne d'une diminution de H2A.Z, ce qui se traduit par l'induction de transcrits antisens régulateurs sur une famille de loci de gènes convergents dus à des défauts de contrôle de la terminaison de la transcriptionCellular senescence represents one of the major fail-safe mechanisms that counteracts tumour development is defined as a state of irreversible cell cycle arrest as a consequence of stress response such as oncogenic challenge. Such cells undergoing Oncogene-induced Senescence (OIS) display profound alternation in their epigenome as their chromatin are largely decorated with prominent drivers of constitutive heterochromatin.Antisense RNA-mediated gene regulation has been attributed to play diverse roles in mediating various cellular processes and cell fates per-se. In yeast, histone variant H2A.Z cooperates with RNAi and heterochromatin machinery to regulate antisense transcription at convergent gene loci which can otherwise generate pervasive read-through transcripts owing to improper transcription termination. In mammals, whether such antisense transcripts (occurring by read-through transcription at convergent gene pairs) exist and how they are regulated remains unknown. Interestingly, the depletion of the human H2A.Z histone variant has been reported to induce cellular senescence. We thus wondered if the regulation of particular antisense transcripts at convergent gene pairs occurs in senescence, if their regulation by H2A.Z is conserved in mammals and, if so, if a functional significance can be attributed to these transcripts. To this end we took advantage of a well-established in-vitro OIS model. Briefly, we analysed genome wide strand specific RNA-seq analysis of cells undergoing Oncogene Induced Senescence. This led us to identify numerous convergent gene loci associated with accumulation of transcripts downstream of the designated transcription termination site in senescent cells, and extending to generate an antisense to the next gene located in the opposite strand of the convergent gene pair. We confirmed the RNA-seq data at two of such convergent loci. An RNAi based approach revealed that at least two of these transcripts are generated by transcriptional read-throughs. Hence we designated such pervasive transcripts as Senescence Triggered Antisense Read-through Transcripts (START). Importantly, we further found that the two STARTs for which we performed in depth studies repress the expression of the gene for which they are antisense. Finally, we demonstrate that the histone variant H2A.Z suppresses the accumulation of STARTs in proliferative cells. Since it also prevents senescence induction, this suggests that expression of START is important for cellular senescence. This has lead us to propose a model that human cells undergoing OIS are associated with loss of H2A.Z that leads to the wide spread accumulation of read-through transcripts owing to impaired termination control
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Ozes, Ali Rayet. "Targeting the long non coding RNA HOTAIR in cancer". Thesis, Indiana University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10154781.
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Ovarian cancer (OC) takes the lives of nearly 14,000 US women every year. Although platinum is one of the most effective drugs in treating ovarian cancer, the development of platinum resistance is one of the biggest challenges facing patients. I have shown that the long non-coding RNA HOTAIR contributes to platinum-resistant OC and determined the regulators and targets of HOTAIR during the platinum-induced DNA damage response. My published data supports the role of HOTAIR in contributing to DNA damage induced cellular senescence and secretion of pro-inflammatory cytokines leading to cisplatin resistance. My unpublished work (under review) analyzed the interaction of HOTAIR with the PRC2, its known interacting partner. In this study, I developed a novel strategy blocking HOTAIR-PRC2 interaction and resensitized ovarian tumors to platinum in mouse studies. The results offer a pre-clinical proof of concept for targeting long non-coding RNAs as a therapeutic approach and may represent a strategy to overcome chemotherapy resistance in tumors exhibiting high expression of HOTAIR, a frequent observation in high grade serous OC.
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Singh, Harinder. "CAVEOLAE AS SPATIO-TEMPORAL COMPARTMENTS FOR ROS/RNS GENERATION AND NITROXIDATIVE STRESS SIGNALING". Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/275561.
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Cell BiologyPh.D.
During inflammatory conditions excessive production of reactive oxygen (ROS) and nitrogen species (RNS), peroxynitrite, is implicated in the development of vascular pathologies. Our previous studies showed that both NADPH oxidase enzyme complexes and eNOS localize to endothelial caveolae microdomains. Additionally, caveolae internalization has been shown as an activating mechanism for enzyme eNOS. However, roles of caveolae in ROS/RNS generation and downstream signaling roles in activating endothelial cells are not well known. Hypothesis: Caveolae act as, a) micro-environments in providing spatio-temporal reaction compartments for ROS/RNS generation, tyrosine nitration of proteins, b) platforms to propagate localized nitroxidative signaling in inducing endothelial cell activation and dysfunction (ICAM-1, VCAM-1 expression), and c) intracellular redox signaling endosomes to regulate adhesion molecule expression. Objectives: The aim of the study was to investigate whether, a) caveolae compartmentalize ROS, regulate localized tyrosine nitration of proteins, b) nitroxidative-signaling in the endothelium is compartmentalized in caveolae, c) dynamin-2-dependent internalization of caveoale is important for activating redox signaling, and d) caveolae compartments can be targeted to reduce endothelial ROS Methods and results: Cultured primary bovine aortic endothelial cells were stimulated with TNFα to generate ROS/RNS. Blockade of NADPH oxidase (gp91ds-tat) or scavenging of peroxynitrite (Uric acid) inhibited TNFα-induced protein tyrosine nitration, activation of the NFkB, and upregulation of ICAM-1/VCAM-1 expression. To test the role of caveolae in this process, cultured cells were depleted of caveolin-1 (siRNA). Similar to inhibitors, TNFα failed to induce protein-tyrosine nitration, activate NFkB or enhance adhesion molecule expression in cells lacking caveolin-1. These findings were corroborated in vivo using Cav1KO animals. Our results show that several caveolar residing proteins were nitrated on tyrosine in response to TNFα. Here, immunoprecipitation of cell lysates with an anti-nitrotyrosine antibody revealed Src-family kinases (SFK) in the precipitated fraction. Moreover, SFK nitration was lost in cells depleted of caveolin-1. Given that SFK nitration is associated with enzyme activation, cells were pretreated with PP2 to inhibit SFK activity. We found that PP2 attenuated the NFkB and adhesion molecule pathway activated by TNFα. Depletion of dynamin-2 (Dyn2siRNA) or inhibiting GTPase activity (Dynasore) also showed reductions in ROS generation, NFkB redox signaling and ICAM-1/VCAM-1 expression. Development of caveolae targeting peptide tagged with gp91ds-tat showed inhibitions in compartmentalized ROS production. Conclusions: Caveolae act as sites for ROS/RNS production where resident redox-sensitive second messengers are activated and propagate signals that regulate endothelial inflammatory phenotype. Targeting NADPH oxidase enzyme specifically in caveolae can be used a therapeutic strategy to limit vascular oxidative stress.
Temple University--Theses
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Le, Roy Florence. "Clonage et caractérisation de protéines associées à la RNase L. Identification d'ARNm cellulaires régulés par la RNase L". Montpellier 2, 2000. http://www.theses.fr/2000MON20054.
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Les interferons (ifns) induisent de nombreux genes impliques dans le controle de la proliferation et de la differenciation cellulaires, ainsi que dans la modulation des reponses immunitaires et dans la defense contre l'infection virale. Le systeme 2-5a/rnase l est un des systemes induit par les ifns. L'ifn induit l'expression des genes 2-5a synthetases qui, activees par des arns double-brin (viraux ou cellulaires), polymerisent l'atp en une serie d'oligoadenylates relies par des liaisons phosphodiesters 2'-5' (2-5a). Le 2-5a active une endoribonuclease latente, la rnase l, qui degrade les arns simple brin, entrainant une inhibition de la synthese proteique. L'activite de la rnase l est regulee par le 2-5a et par son inhibiteur proteique, rli (rnase l inhibitor). Afin d'elucider le mecanisme d'action de la rnase l, nous avons clone des partenaires de la rnase l. Nous avons identifie, par purification biochimique suivie d'un microsequencage, une proteine associee a la rnase l impliquee dans la terminaison de la traduction. Parallelement, par la technique du double-hybride, nous avons clone un autre partenaire de la rnase l, implique dans l'initiation de la traduction mitochondriale : if2-mt. L'etude des partenaires de la rnase l nous a amene a etudier le role de la rnase l dans la metabolisme des arnm mitochondriaux. Notre etude montre que la rnase l et son inhibiteur sont presents dans les mitochondries et que la rnase l est impliquee dans l'effet antiproliferatif de l'ifn via la degradation des arnm mitochondriaux.
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Idris, Jalilah. "Investigating novel roles of RNA binding proteins SAFB1 and SAFB2 in RNA processing and in cellular stress". Thesis, University of Bristol, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738206.
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Jeyaraj, Selvi Chrysolyte. "A role for the mRNA-stabilizing protein HuR in protection from cellular ATP depletion". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1186773861.
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Öhrmalm, Christina. "Functional characterization of the cellular protein p32 : a protein regulating adenovirus transcription and splicing through targeting of phosphorylation /". Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6794.
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Schonauer, Melissa. "Intersection of RNA Processing and Fatty Acid Synthesis and Attachment in Yeast Mitochondria". Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194674.
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Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient
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Mina, Ibarra Leonardo Bruno. "Cellular mRNA decay factors involved in the hepatitis C virus life cycle". Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7215.
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The group of positive strand RNA ((+)RNA) viruses includes numerous plant, animal and human pathogens such as the hepatitis C virus (HCV). Their viral genomes mimic cellular mRNAs, however, besides acting as messengers for translation of viral proteins, they also act as templates for viral replication. Since these two functions are mutually exclusive, a key step in the replication of all (+) RNA viruses is the regulated exit of the genomic RNAs from the cellular translation machinery to the viral replication complexes. By using a model system that allows the replication of the plant (+) RNA Brome Mosaic Virus in yeast, it was shown by our group that the cellular decapping activators Dhh1, LSm1 and PatL1 play a key role in such transition. The LSm1 protein is a subunit of the LSm1-7 complexes. We have recently shown that these complexes interact directly with sequences in the BMV genome and that these interactions regulate the translation and replication of BMV. Interestingly, the LSm1 homolog in bacteria, Hfq, is required for the replication of the (+) RNA bacteriophage Qβ in E. coli.Since all (+)RNA viruses need to regulate the exit of the viral genome from the cellular translation machinery to replication and these proteins are conserved from yeast to humans, We hypothesized that the human homologues of Lsm1-7/Dhh1/Pat1 are required for the replication of human (+) RNA viruses. In this work we proved this hypothesis by showing that HCV translation and replication depend on these cellular proteins. Moreover, the requirement of these factors for efficient HCV RNA translation was linked exclusively to the 5´ and 3´ nontranslated regions (NTRs) of the viral genome. Furthermore, the LSm1-7 complex specifically interacts in vitro with essential cis-acting HCV RNA elements located in the NTRs.
El grupo de virus de RNA de cadena de polaridad positiva ((+)RNA) incluye numerosos patógenos de plantas, animales y humanos, tales como el virus de la Hepatitis C (VHC). Sus genomas virales imitan a los RNAm celulares, sin embargo, además de actuar como mensajeros para la traducción de proteínas virales, también actúan como moldes para la replicación viral. Debido a que estas dos funciones son mutuamente excluyentes, un paso clave en la replicación de todos los virus (+) RNA es la salida regulada del RNA genómico desde la maquinaria de traducción celular hacia las complejos de replicación virales. Utilizando un sistema modelo que permite la replicación del virus del mosaico del bromo (VMB), un virus (+) RNA de plantas, en levaduras, nuestro grupo ha demostrado que los activadores de decapping celulares Dhh1, LSm1 y Pat1 juegan un papel clave en este paso. La proteína LSm1 es una subunidad del complejo celular LSm1-7. Hemos demostrado recientemente que dichos complejos interactúan directamente con secuencias específicas en el genoma del VMB y que dichas interacciones regulan la traducción y replicación del VMB. De manera interesante, la proteína homologa de LSm1 en bacterias, Hfq, es necesaria para la replicación del bacteriófago (+) RNA Qβ en E. coli.
Debido a que todos los virus (+) RNA necesitan regular la salida del genoma viral desde la maquinaria traduccional de la célula hacia los complejos de replicación virales, y que las proteínas Dhh1, LSm1-7 y Pat1 están conservadas de levaduras a humanos, nuestra hipótesis es que lo homólogos humanos de Lsm1-7/Dhh1/Pat1 son requeridos para la replicación de virus (+)RNA de humanos. En este trabajo, hemos demostrado esta hipótesis mostrando que la traducción y la replicación del virus de la hepatitis C (VHC) dependen de estas proteínas celulares. Más aún, el requerimiento de estos factores para una eficiente traducción del genoma del VHC está vinculado de manera exclusiva a las regiones no traducidas (RNT) 5´ y 3´ del genome viral. Además, el complejo cellular LSm1-7 interactúa in vitro de manera específica con elementos esenciales del RNA del VHC que actúan en cis localizados en las RNTs.
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York, Ashley D. "A study of viral and cellular factors in the regulation of the influenza virus RNA-dependent RNA polymerase". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5958fafd-4c91-4434-910e-29e2dd0539b9.
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The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.
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Mahmoudi, Massoud. "Induction of Interferon Messenger RNA and Expression of Cellular Oncogenes in Human Lymphoblastoid Cells". Thesis, North Texas State University, 1986. https://digital.library.unt.edu/ark:/67531/metadc798199/.
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The purposes of this study was to demonstrate the induction of alpha interferon mRNA in Sendai virus-induced Namalava cells, to follow the level of alpha interferon mRNA synthesis at the transcriptional level, and to determine whether the Namalava cell line expresses the c-myc oncogene and to what degree. The amount of c-myc message deteted in Namalva cell RNA was about one-tenth that of Daudi cell RNA, whereas no difference in the amount of the c-Ha-ras message was observed between the two cell lines.
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Chambers, A. "RNA 3' cleavage and polyadenylation in oocytes, eggs and embryos of Xenopus laevis". Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380275.
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Plakos, Kory. "Platinum-seq: High-throughput mapping of small-molecule platinum adducts on cellular RNA". Thesis, University of Oregon, 2017. http://hdl.handle.net/1794/22269.
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Methods to map small-molecule interactions with cellular RNAs are important for understanding endogenous activation, such as in riboswitches, as well as the potential for exogenous compounds to target RNA. Cisplatin is one of the most widely used of the platinum anticancer drugs that are prescribed in approximately 40-50% of all chemotherapy treatments (Dyson and Sava, 2006; Harper et al., 2010). Despite nearly 40 years of experience with this class of drugs, we still lack a comprehensive understanding of the targets of Pt compounds and their effects on cells. Pt(II) compounds are well-known DNA and RNA crosslinking agents, but the latter area is under-studied. In order to better understand the impacts of cisplatin and other platinum(II)-derived small molecules on cellular RNA, we have developed a technique we call “Platinum-seq,” which couples reverse transcription mapping of platinated RNAs to high-throughput sequencing. Chapter 1 is a study of cisplatin and a novel click-functionalized platinum compound (2-ADAP Pt) binding to the HDV ribozyme, a small catalytic RNA. Chapter 2 moves our platinum mapping approaches from low-throughput, sequencing gel based methods into next-generation sequencing for high-throughput analysis of all platinum sites in cellular RNA, a method we have named “Platinum-seq.” Chapter 3 is a study of differential gene expression of Saccharomyces cerevisiae treated with cisplatin and a second novel platinum(II) compound (azaplatin), using data acquired from the work in Chapter 2. Chapter 4 describes recent efforts to implement pre-enrichment of sequencing targets using click chemistry followed by DNA hybridization, in order to enrich for platinated fragments before sequencing library construction. Together, this work represents a significant step forward in advancing analysis of Pt(II) binding to cellular RNA, a potentially important target for this widely used class of anticancer compounds. Methods developed here are broadly applicable to genome-wide identification of platinum accumulation on DNA as well, which has not been pursued despite the extensive use of these compounds.
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Rubilar, Guzman Paulina. "Cellular host factors involved in the translation of the HIV-1 genomic RNA". Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1009/document.
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Le virus de l’immunodéficience humaine de type 1 (VIH-1) est un virus à simple brin positif qui appartient au genre Lentivirus dans la famille retroviridae et qui constitue l’agent étiologique du SIDA pandémique.Pendant le cycle réplicatif du VIH-1, la traduction de protéines virales dépend exclusivement de la machinerie traductionnelle cellulaire. Pour cette raison, nous avons cherché à comprendre le rôle de quelques facteurs cellulaires qui pourraient contrôler la traduction du VIH-1 à différents nivaux. Nous avons centré nos recherches sur la traduction de l’ARN génomique (ARNg) du virus qui sert en même temps de génome pour être encapsidé et comme ARN messager pour la traduction des protéines virales Gag et Gag-Pol. 1) Le rôle de l’hélicase d’ARN DDX3 dans la traduction du VIH-1. L’ARNg du VIH-1 possède une région 5’ non traduite très structurée, raison pour laquelle nous avons spéculé sur un possible rôle de DDX3 dans la traduction du VIH-1. Nous avons utilisé une combinaison de techniques in vitro et ex vivo afin de pouvoir démontrer que DDX3 était capable de lier et faire des complexes avec l’ARN de la région 5’ non traduite pour promouvoir l’initiation de la traduction. Nous avons aussi pu démontrer que DDX3 formait des complexes avec les facteurs d’initiation de la traduction PABP, eIF4G et eIF4E. 2) Le changement programmé du cadre de lecture (PRF) dans l’ARN génomique du VIH-1. La traduction de la polyprotéine Gag-Pol du VIH-1 nécessite un décalage de phase de 1 nucléotide en arrière. Ce mécanisme permet la synthèse des protéines Gag et Gag-Pol avec des ratios de 95 et 5% respectivement à partir du même ARN. Cette proportion doit être conservée pour assurer la réplication du virus. Nous avons utilisé un système de double gène rapporteurs et un système de réplication complète du provirus pour montrer que la protéine associé aux granules de stress TIAR pouvait contrôler la réplication viral en régulant la proportion de ribosome qui assurentHuman Immunodeficiency virus type 1 (HIV-1) is a positive strand RNA virus belonging to the lentivirus genus of the retroviridae family and it is the etiological agent of the pandemic AIDS, which is a major health concern worldwide. Throughout HIV-1 replication cycle, the production of viral proteins depends exclusively on the cellular translational machinery. This is the reason why we have explored the role of some cellular factors that could control HIV-1 translation at different stages. We have focused our studies on the translation of the full length genomic RNA (gRNA), which serves both as genome for viral encapsidation and as a messenger for translation of Gag and Gag-Pol viral polyproteins.1) The role of the RNA helicase DDX3 in HIV-1 translation Initiation The fact that HIV-1 possesses a highly structured 5’ untranslated region (5’UTR) prompted us to speculate that DDX3 may be involved in HIV-1 translation. We used a combination of in vitro and ex-vivo approaches to show that DDX3 was able to bind and form complexes with the 5’-UTR of HIV-1 to assist translation initiation. We also demonstrated that DDX3 can form a complex with initiation factors such as PABP, eIF4G and eIF4E. 2) Programmed Ribosomal Frameshift (PRF) in the genomic RNA of HIV-1Translation of HIV-1 Gag-Pol polyprotein requires a -1 PRF. This mechanism allows the synthesis of Gag and Gag-Pol polyproteins, using the same mRNA template, at ratios of 95 and 5% respectively. Keeping the -1PRF ratio is important as any change leads to reduction in virus infectivity.By means of a dual reporter construct and full provirus replication system we were able to demonstrate that the stress granules associated protein TIAR, controls HIV-1 infectious progeny by regulating the ratio of the HIV-1 PRF