<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse : les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers : Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression. The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

No description available.

The repertoire of cellular functions mediated by Ribonucleic acid (RNA) molecules have expanded considerably during the last two decades. The role played by RNA in controlling and regulating gene expression in viruses, prokaryotes and eukaryotes has been a matter of continuous investigations. This interest has arisen primarily due to the discoveries of cisacting RNA structures like riboswitches, ribosensors and frameshift elements, which are found in either the 5’-, 3’-untranslated regions of mRNA or in the open reading frames. These structures control gene expression at the level of translation by either sequestering the Shine-Dalgarno (SD) sequence to regulate translation initiation or modulating ribosomal positions during an active translation process. Very often, these structures comprise of an RNA pseudoknot and it has been observed that these pseudoknots exist in a dynamic equilibrium with other intermediate structures. This equilibrium could be shifted by several factors including presence of ions, metabolites, temperature and external force. RNA pseudoknots represent the most versatile and ubiquitous class of RNA structures in the cell, whose unique folding topology could be exploited in a number of ways by the cellular machinery. In this thesis, a thorough study of programmed -1 ribosomal frameshifting (-1 PRF) process, which is a well known gene regulation event employed by many RNA viruses, was carried out. -1 PRF is a translation recoding process, necessary for viruses to main-tain a stoichiometric ratio of structural: enzymatic proteins. This ratio varies among different viral species. At the heart of this process, lies an RNA pseudoknot accompanied by a seven nucleotide long sequence motif, which pauses an actively translating ribosome on mRNA and causes it to shift its reading frame. The frameshift inducing efficiency of pseudoknot depends on multiple factors, for example the time scale of ribosomal pause and RNA unfolding, subsequent refolding of structure to native/intermediate states and/or environment conditions. With the aim of illustrating the fundamentals of the process, multiple factors involved in -1 PRF were studied. Chapters 2-4 represent distinct aspects of -1 PRF process, while Chapter 5 discusses a different work concerned with nucleocytoplasmic transport of tRNA carried out by nuclear export receptor Exporting. Chapter 1 gives an overview of the different regulatory activities with which RNA structures and sequences are found to be associated and the evolution of these stud-ies. It discusses the different types of structural motifs found to constitute tertiary RNA structure and secondary structure prediction and determination techniques. A brief description of ab initio RNA structure modeling and other relevant tools and methodologies used in this work has been presented. Details of techniques used in each study have been provided in relevant chapters. Chapter 2 describes how local factors like ionic conditions, hydration patterns, presence of protonated residues and single residue mutations affect the structural dynamics of an RNA pseudoknot involved in -1 PRF from a plant luteovirus. Single residue mutations in the loop regions or certain base-pair inversions in the stem regions of pseudoknot increase the frameshift inducing ability of the pseudoknot structure, while some others decrease this efficiency. However, it was not clear how the changes made to the wild-type (WT) RNA pseudoknot from Beet Western Yellow Mosaic virus were affecting the global structure in terms of its dynamics and other parameters. To study this, multiple all-atom molecular dynamics simulations (MD) were performed on WT and mutant structures created in silico. The effect of presence and absence of magnesium ions on the structural geometry was also studied. The analysis was done to identify the increase/decrease in the number of hydrogen bonds formed by Watson-Crick base-pairs in stem region or non Watson-Crick pairs between stem and loop. Ionic and water densities were analyzed and the role of potential ribosome-pseudoknot interaction was elaborated. With the aim of mimicking ribosome induced unfolding of an RNA pseudoknot, steered molecular dynamics pulling experiments were performed. This work was done primarily to understand the unfolding pathway of Hairpin(H)-type pseudoknots in general and the intermediate structures formed. Chapter 3 describes the thermodynamics and mechanics associated with the mechanical pulling of -1 PRF inducing RNA pseudoknot and its mutants described in previous chapter. Analysis of the trajectories reveal relative unfolding patterns in terms of disruption of various hydrogen bonds. This study allowed us to pinpoint the kind of intermediate structures being formed during pulling and whether these intermediate structures correspond to any known secondary structures, such as simple stem-loops. This information could be used for gaining insights into the folding pathways of these structures. An RNA pseudoknot stimulates -1 PRF in conjunction with a heptanucleotide “slippery site” and an intervening spacer sequence. A comprehensive study of analyzing the sequence signatures and composition of all overlapping gene segments harboring these frameshift elements from four different RNA virus families was carried out. Chapter 4 describes the sequence composition of all overlapping gene segments in Astroviridae, Coronaviridae, Retroviridae and Luteoviridae viral families which are known to employ -1 PRF process for maintaining their protein products. Sequence analysis revealed preference for GC bases in the structure forming sequence regions. A comparative study between multiple sequence alignment and secondary structure prediction revealed that while pseudoknots have a clear preference for specific base-pairs in their stem regions, viral families that employ a hairpin loop as -1 PRF structure, doesn’t show this preference. Information derived from secondary structure prediction was then used for RNA ab initio modeling to generate tertiary structures. Furthermore, the structural parameters were calculated for the helices of the frameshift inducing pseudoknots and were compared with the values calculated for a set of non -1 PRF inducing H-type pseudo-knots. This study highlighted the differences between -1 PRF pseudoknots and other H-type pseudoknot structures as well as specific sequence and structural preferences of the former. Chapter 5 discusses the dynamics of a tRNA transport factor Exportint (Xpot), which transports mature tRNA molecules from nucleus to cytoplasm and belongs to Importitβ family of proteins. The global conformational dynamics of other transport receptors has been reported earlier, using coarse-grained modeling and Elastic Network Models (ENMs), but a detailed description of the dynamics at an all-atomic resolution was lacking. This transport requires association of Xpot with RanGTP, a G-protein, in the nucleus and hydrolysis of RanGTP in the cytoplasm. The chain of events leading to tRNA release from Xpot after RanGTP hydrolysis was not studied previously. With these objectives, several molecular complexes containing Xpot bound to Ran or tRNA or both in the GTP and GDP ligand states as well as free Xpot structures in nuclear and cytosolic forms were studied. A combination of conventional and accelerated molecular dynamics simulations was used to study these molecular complexes. The study highlighted various aspects associated with tRNA release and conformational change which occurs in Xpot in cytosolic form. The nuclear to cytosolic state transition in Xpot could be attributed to large fluctuations in C-terminal region and dynamic hinge-points located between specific HEAT repeats. A secondary role of Xpot in controlling the quality of tRNA transport has been proposed based on multiple sequence and structure alignment with Importin-β protein. The loss of critical contacts like hydrogen bonds and salt bridges between Xpot/Ran and Xpot/tRNA interface was evaluated in order to study the initial effects of RanGTP hydrolysis and how it influences receptor-cargo binding. This study revealed various aspects of tRNA transport process by Xpot, not understood previously. The results presented in this thesis illustrate the role of RNA sequence elements and pseudoknots present in RNA viruses in modulating -1 PRF process and how multiple environmental factors affect -1 PRF inducing ability of the structure. From the studies of Xpot and its complexes, the effects of GTP hydrolysis leading to tRNA dissociation have been presented and the progression of conformational transition in Xpot after tRNA dissociation has been highlighted. Chapter 6 summarizes major conclusions of this thesis work. The refolding of single stranded RNA chains, subjected to a previous unfolding simulation is studied. Appendix A describes this work and initial results. Appendix B describes the effect of improved molecular dynamics force fields, containing corrections for χ torsion angle for RNA, on the conformation of tertiary RNA structures. Part of the work presented in this thesis has been reported in the following publications. 1.Asmita Gupta and Manju Bansal. Local Structural and Environmental Factors De-fine the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process. J. Phys. Chem. B. 118 (41), pp 11905-11920. 2014 2.Asmita Gupta, Senthilkumar Kailasam and Manju Bansal. Insights Into Nucleo-cytoplasmic Transport of tRNA by Exportin-t. Manuscript under review. List of manuscripts that are being prepared from the work reported in Chapter 3 in this thesis. 1 Asmita Gupta and Manju Bansal. The role of sequence effects on altering the un-folding pathway of an RNA pseudoknot: a steered molecular dynamics study. Manuscript in preparation. 2 Asmita Gupta and Manju Bansal. Molecular basis for nucleocytoplasmic transport of tRNA by Exportin-t. Journal of Biomolecular Structure and Dynamics, May;33 Suppl 1:59-60, 2015

Les adénovirus (AdV) sont des virus à ADN se répliquant dans le noyau de la cellule hôte. Pour pouvoir se répliquer, ils détournent la machinerie cellulaire à leur profit. Au cours de l’entrée dans la cellule, les particules virales utilisent la machinerie de transport des microtubules pour rejoindre le noyau. Les AdV interagissent avec la dynéine, moteur moléculaire associé aux microtubules, pour être transportés vers le compartiment nucléaire. Ils se lient alors aux pores nucléaires, structures ancrées dans l’enveloppe nucléaire (EN). Une fois aux pores nucléaires, les capsides virales se désassemblent pour libérer et importer leur génome. Les mécanismes de détachement des microtubules, de translocation nucléaire et d’import du génome des AdV impliquent des facteurs de la machinerie de transport nucléocytoplasmique. Cependant, le mécanisme exact utilisé par les virus pour atteindre les pores nucléaires n’est pas clairement défini. Le transport nucléocytoplasmique est composé de différents facteurs et est hautement régulé dans les cellules. Le transport actif de cargos est dû à des facteurs d’import et d’export interagissant avec RanGTP. Le principal facteur d’export est CRM1 et il est connu pour être essentiel dans la translocation des AdV vers l’EN. L’inhibition de CRM1 par la Leptomycine B conduit à l’accumulation des AdV au centrosome, le principal Centre Organisateur des Microtubules (COMT) des cellules de mammifères. Nous avons donc étudié le rôle de CRM1 dans la libération du génome adénoviral. Nous avons analysé l’interaction des AdVs avec le COMT et nous avons observé que l’absence de facteurs cytoplasmiques ainsi que la perte d’intégrité des microtubules n’affectaient pas leur accumulation au COMT. En revanche, nous avons identifié et caractérisé un mutant de CRM1, qui reste fonctionnel pour l’export physiologique de cargo mais qui induit un retard important dans la translocation des AdV vers l’EN. Nous avons utilisé l’imagerie sur cellules vivantes pour analyser l’infection de l’AdV dans des cellules mitotiques et ceci a permis de révéler le rôle de CRM1 dans la libération du génome de ce virus. Nous avons également identifié un partenaire viral potentiel pour CRM1 parmi les protéines associées au génome viral, la Terminal Protein (TP). Cette protéine possède un signal d’export nucléaire et est un substrat de CRM1. Nos données soulignent le rôle de CRM1 comme un médiateur essentiel au désassemblage total de la capside adénovirale, qui favorise la libération du génome et son import.

L’ARN polymérase II (ARNPII), l’enzyme responsable de la transcription des ARN messagers, procède au décodage du génome des organismes vivants. Cette fonction requiert l’action concertée de plusieurs protéines, les facteurs généraux de la transcription, par exemple, formant un réseau d’interactions protéine-protéine, plusieurs étant impliquées dans la régulation de l’ARNPII à différents niveaux. La régulation de la transcription a été largement étudiée durant les quatre dernières décennies. Néanmoins, nous en connaissons peu sur les mécanismes qui régulent l’ARNPII avant ou après la transcription. Dans la première partie de cette thèse, nous poursuivons la caractérisation du réseau d’interactions de l’ARNPII dans la fraction soluble de la cellule humaine, travail qui a débuté précédemment dans notre laboratoire. Ce réseau, développé à partir de la méthode de la purification d’affinité en tandem couplée à la spectrométrie de masse (AP-MS) et à des méthodes d’analyses bioinformatiques, nous amène une foule d’informations concernant la régulation de l’ARNPII avant et après son interaction avec la chromatine. Nous y identifions des protéines qui pourraient participer à l’assemblage de l’ARNPII telles des chaperonnes et les protéines du complexe R2TP/prefoldin-like ainsi que des protéines impliquées dans le transport nucléocytoplasmique. Au centre de ce réseau se trouvent RPAP4, une GTPase qui semble se positionner à l’interface entre ces protéines régulatrices et l’ARNPII. Nous avons donc entamé l’étude la fonction de RPAP4, ce qui nous a menés à la conclusion que RPAP4 est essentielle à l’import nucléaire de l’ARNPII au noyau, où elle exerce sa fonction. Nous avons également montré que les motifs G et GPN sont essentiels à la fonction de RPAP4. Le traitement des cellules avec le bénomyl nous montre aussi que la fonction de RPAP4 et l’import nucléaire de l’ARNPII requièrent l’action des microtubules. La deuxième partie de la thèse s’intéresse à une autre protéine positionnée au centre du réseau, RPAP2. Cette dernière partage plusieurs interactions avec RPAP4. Elle est aussi essentielle à la localisation nucléaire de l’ARNPII et interagit directement avec celle-ci. RPAP4 et RPAP2 étant toutes deux des protéines cytoplasmiques qui font la navette entre le noyau et le cytoplasme, nous présentons des évidences que RPAP4 est impliquée dans l’export nucléaire de RPAP2 pour permettre à celle-ci d’être disponible dans le cytoplasme pour l’import de l’ARNPII dans le noyau. Dans la troisième partie de la thèse, nous étudions plus en profondeur les modifications post-traductionnelles de RPAP4, ce qui nous aide à mieux comprendre sa propre régulation et sa fonction auprès de l’ARNPII. RPAP4 est phosphorylée en mitose par la MAP kinase ERK5. Cette phosphorylation favorise l’interaction entre RPAP4 et RPAP2, ce qui empêche RPAP2 d’interagir avec l’ARNPII pendant la mitose, prévenant du même coup, son interaction avec la chromatine pendant cette phase du cycle cellulaire où la transcription est presque inexistante. / RNA polymerase II, the enzyme responsible for transcription of messenger RNA, decodes the genome of living organisms. This function requires the concerted action of several proteins, including transcription factors, which form a protein-protein interaction network. Many of them are implicated in the regulation of RNAPII transcription. Although regulation of transcription has been largely studied during the last four decades, little is known about mechanisms that regulate RNAPII prior and after the transcription reaction. In the first part of this thesis, we continue the characterization of the RNAPII interaction network of RNAPII in the soluble fraction of the human cell. This network, developed using tandem affinity purification method coupled with mass spectrometry (AP-MS) and bioinformatic analysis, provides a wealth of information about RNAPII regulation prior and after its interaction with chromatin for transcription. We identified proteins that can be involved in RNAPII assembly, including chaperones and the cochaperone complex R2TP prefoldin-like, and proteins involved in nucleocytoplasmic shuttling. RPAP4 is a GTPase that occupies a central position in this network being at the interface between these regulatory proteins and RNAPII. We therefore started to study the function of RPAP4, which lead us to conclude that RPAP4 is essential for RNAPII nuclear import. We also report that G domains and the GPN motif are essential for RPAP4 function. Treatment of the cells with benomyl suggests that microtubules are required for RPAP4 function and RNAPII nuclear import. The second part concerns another protein found in the network that is also centrally positioned in the network, called RPAP2. RPAP2 shares many interactions with RPAP4. This protein is also essential for the nuclear import of RNAPII as it interacts directly with it. RPAP4 and RPAP2 being cytoplasmic proteins that shuttle between the cytoplasm and the nucleus, we show evidences that RPAP4 is implicated in RPAP2 nuclear export to make it available for RNAPII nuclear import. In the third part, we study RPAP4 post-translational modifications, which help us to understand its own regulation and its function with RNAPII. RPAP4 is phosphorylated in mitosis by the MAP kinase ERK5. This phosphorylation promotes the interaction between RPAP4 and RPAP2. It prevents RPAP2 and RNAPII interaction and RNAPII chromatin localization in mitosis where transcription is mostly nonexistent.

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