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Dans la levure S. cerevisiae, la mitose nécessite le positionnement du fuseau mitotique le long de l’axe cellule mère-bourgeon (future cellule fille) afin d‘assurer une bonne ségrégation des chromosomes. Ce phénomène requiert le fonctionnement de deux mécanismes impliquant les protéines Kar9 et Dyn1. Durant la métaphase, Kar9 se positionne de manière asymétrique le long du fuseau mitotique, avec une accumulation notable sur les microtubules qui émanent de l’ancien « spindle pole body » (SPB; l’équivalent du centrosome dans les vertébrés), qui est normalement dirigé vers le bourgeon. Dans le cas d’un défaut d’alignement du fuseau mitotique, un mécanisme appelé « Spindle Position Checkpoint » (SPOC) inhibe la sortie de mitose et la cytokinèse, afin de permettre un réalignement correct du fuseau mitotique. La principale cible de ce checkpoint est une GTPase Tem1. Dans le cas d’alignement correct du fuseau mitotique, Tem1 active une voie de signalisation appelée le « Mitotic Exit Network » (MEN) qui permet de mener à la sortie de mitose et à la cytokinèse. Lors de la transition métaphase/anaphase Tem1 se positionne asymétriquement sur les SPBs jusqu’à se concentrer majoritairement sur l’ancien SPB. Des données récentes ont montré que des composants du MEN, Tem1 inclus, sont également impliqués dans la régulation de la localisation de la protéine Kar9 à l’SPB, et dans l’établissement d’une polarité correcte des SPBs durant la métaphase. En effet, Kar9 se positionne plus symétriquement dans le cas des mutants du MEN que dans le type sauvage, ce qui engendre des problèmes d’orientation du fuseau et de ségrégation des SPBs. Nous cherchons à élucider comment l’activité du MEN régule la localisation de Kar9 et l’orientation du fuseau mitotique en métaphase alors que les fonctions du MEN liées à la sortie de mitose restent bloquées jusqu’à la télophase. Nous avons émis l’hypothèse que les modifications post-traductionnelles de Tem1 pourraient jouer un rôle dans la régulation du MEN. Il a été montré que les résidus Y40 et Y45 sont phosphoryles in vivo. Afin de disséquer le rôle de ces résidus nous les avons mutés en phénylalanines. Ces mutations peuvent complémenter la létalité induite par la délétion de TEM1, suggérant que ce mutant conserve les fonctions essentielles de Tem1. Par ailleurs, la cinétique de progression du cycle cellulaire du mutant est la même que celle du type sauvage, signifiant que la perte de phosphorylation sur Tem1 ne semble pas agir sur la sortie de mitose. De plus, l’allèle mutant n’affecte pas la localisation aux SPBs de Tem1 ni celle de sa « GTPase-activating protein » Bub2/Bfa1 durant le cycle cellulaire. Bien que l’activité GTPasique de la protéine Tem1-Y40F,Y45F soit réduite in vitro, les mutations ne causent pas des défauts de SPOC in vivo et le mutant répond efficacement au mauvais alignement de fuseau mitotique en s’arrêtant en anaphase. Tous ces résultats nous suggèrent que la perte de phosphorylation de Tem1 n’affecte pas les fonctions de fin de mitose de cette GTPase. Par contre, nous avons découvert que la phosphorylation de Tem1 est requise pour la localisation asymétrique de Kar9 sur les SPBs, ainsi que pour l’alignement correct du fuseau mitotique durant la métaphase (la distribution de Kar9 est plus symétrique dans les cellules TEM1-Y40F,Y45F et que le fuseau mitotique n’est pas aligné correctement). Nous cherchons alors à trouver quelle kinase phosphoryle Tem1 et régule son activité. Les kinases potentielles sont la protéine Swe1 (la seule vraie kinase phosphorylant les tyrosines dans la levure) ainsi que la kinase Mps1 (kinase qui contrôle la duplication des SPBs). Nous développons actuellement des outils nous permettant de vérifier l’implication de ces deux candidats. Mots clés : Tem1, Kar9, cycle cellulaire, Mitotic Exit Network (MEN), Spindle Position Checkpoint (SPOC), phosphorylation on tyrosines. / In the budding yeast Saccharomyces cerevisiae a faithful mitosis requires positioning of the mitotic spindle along the mother-bud axis to ensure proper chromosome segregation. This is achieved by two distinct but functionally redundant mechanisms that require the APC (adenomatous polyposis coli)-like protein Kar9 and dynein (Dyn1), respectively. During metaphase, Kar9 localizes asymmetrically on the mitotic spindle, with a prominent accumulation on astral microtubules emanating from the old spindle pole body (SPB – i.e. the yeast equivalent of the centrosome) that is normally directed towards the bud. In case of spindle misalignment, a surveillance mechanism called Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis, thereby providing the time necessary to correct spindle alignment. The main target of the SPOC is the small GTPase Tem1, which activates a signal transduction cascade called Mitotic Exit Network (MEN) that drives cells out of mitosis and triggers cytokinesis. Tem1 is localized at SPBs, with an increasingly asymmetric pattern during the progression from metaphase to anaphase, when Tem1 is concentrated on bud-directed old SPB. Recent data have implicated MEN components also in the regulation of Kar9 localization at SPBs and in setting the right polarity of SPBs inheritance during metaphase. In particular, Kar9 localizes more symmetrically in MEN mutants than in wild type cells and this leads to spindle orientation and SPB inheritance defects (i.e. with the new SPB being oriented towards the bud). A key question emerging from these data is how MEN activity is regulated to promote proper Kar9 localization and spindle positioning in metaphase, while being restrained until telophase for what concerns its mitotic exit and cytokinetic functions. We hypothesised that Tem1 post-translational modifications might be relevant for this control and for this reason we have been focusing on the role of Tem1 phosphorylation. Tem1 was found in a wide phosphoproteomic study to be phosphorylated on two tyrosines (Y40 and Y45) located at its N-terminus. We constructed a non-phosphorylatable mutant, TEM1-Y40F,Y45F, where the two phosphorylated tyrosines were mutated to phenylalanine. This mutant allele was able to rescue the lethality caused by TEM1 deletion, suggesting that it retains all its the essential functions. The kinetics of cell cycle progression of TEM1-Y40F,Y45F cells was similar to that of wild type cells, suggesting that lack of Tem1 phosphorylation is unlikely to affect mitotic exit. In addition, the TEM1-Y40F,Y45F allele did not affect the SPB localization of Tem1 and its regulatory GTPase-activating protein Bub2/Bfa1 during the cell cycle. Moreover, although the Tem1-Y40F,Y45F mutant protein showed reduced GTPase activity in vitro, it did not cause SPOC defects in vivo and could efficiently respond to spindle mispositioning. Altogether, these results suggest that lack of Tem1 phosphorylation does not affect the late mitotic functions of the GTPase. In contrast, we found that Tem1 phosphorylation is required for Kar9 asymmetry at SPBs and proper spindle positioning during metaphase. Indeed, TEM1-Y40F,Y45F cells display a more symmetric pattern of Kar9 distribution at SPBs in this cell cycle stage, as well as spindle position and orientation defects. We are currently investigating if Tem1 phosphorylation also regulates the pattern of SPB inheritance. Finally, an important question that we are trying to answer is “what is the kinase that phosphorylates Tem1?” The best candidates are the wee1-like kinase Swe1, which is the only true tyrosine kinase of budding yeast, and Mps1, a dual-specificity protein kinase controlling SPB duplication. While we are developing specific tools to study Tem1 phosphorylation and ultimately identify its promoting kinase, we gained preliminary data suggesting that both kinases might be involved in spindle positioning.

Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:24:49Z No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:25:15Z (GMT) No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Made available in DSpace on 2014-11-26T16:25:15Z (GMT). No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Células-tronco (CT) são células autorrenováveis e não especializadas, com o potencial de diferenciação multidirecional. Células-tronco de tecido adiposo (CT-TA) são um tipo de células-tronco adultas multipotentes, de fácil isolamento e cultura. Nos últimos anos, CT-TA têm mostrado grande potencial para engenharia de tecidos e terapias baseadas em células. Apesar do interesse em aplicações clínicas deste tipo de célula, os mecanismos moleculares fundamentais a sua autorrenovação e diferenciação ainda não foram completamente elucidados. miRNAs são pequenos RNAs não-codificadores, com 21-25 nucleotídeos de comprimento, que tem se mostrado como importantes reguladores da expressão gênica em nível póstranscricional. miRNAs podem atuar por meio de clivagem direta de mRNAs alvo ou através da repressão da tradução, dependendo da complementaridade entre o mRNA e a sequência do miRNA. Perfis de miRNAs de CT adultas sugerem que estes pequenos reguladores podem contribuir para as propriedades intrínsecas das CT. Para entender melhor os mecanismos de ação dos miRNAs em CT-TA, miRNAs associados ao polissomos de CT-TA foram isolados durante a diferenciação celular. Procurando miRNAs reguladores das etapas iniciais de diferenciação ou envolvidos na autorrenovação de CT, as culturas de células foram induzidas a diferenciação adipogênica durante 72 h. O lisado celular foi submetido à ultracentrifugação em gradiente de sacarose para separar monosomos, polissomos e fração livre de ribossomos. O RNA total associado aos ribossomos foi extraído, os fragmentos de RNA (<200 nt) foram enriquecidos e a seleção de tamanho de fragmentos de RNA apropriados ocorreu durante a preparação das amostras para o sequenciamento em larga escala. As amostras foram sequenciadas utilizando a plataforma SOLiD ™, e as frações polissomais de culturas Não Induzida e 72h de indução foram comparadas e dezesseis miRNAs foram identificados. miRNAs encontrados em um trabalho prévio do grupo foram adicionados a esses dados, e sete miRNAs (hsamiR-29b-1-5p, hsa- miR-29c-5, hsa-miR-30c-5p, hsa-miR-143-5p, hsa-miR-210-3p, hsa-miR-210- 5p e hsa-miR-6775- 5p) foram testados por RT-qPCR para confirmar a expressão diferencial, sendo que um deles (hsa-miR-210-5p) mostrou diferença estatisticamente significativa. / Stem cells (SC) are self-renewing and non-specialized cells with the potential of multi-directional differentiation. Adipose Stem Cell (ADSC) is a type of multipotent adult stem cell, easy to isolate and culture. In the past few years, hADSCs have shown great potential for tissue engineering and cell-based therapies. Despite the interest in clinical applications of this kind of cell, the molecular mechanisms underlying their self-renewal and differentiation have yet to be fully elucidated. miRNAs are small noncoding RNAs, 21-25 nucleotides in length, that have been shown to be important regulators of posttranscriptional gene expression. miRNAs can act through direct cleavage of target mRNAs or through translational repression, depending of complementary pairing between the mRNA and miRNA sequence.miRNA profile of adult SCs suggests that these small regulators can contribute to the intrinsic properties of SCs. To better understand the mechanisms of action of miRNAs in hADSCs, we isolate miRNAs associated to polysomes of hADSC during cellular differentiation. Looking for miRNAs regulators of early steps of differentiation or involved in ADSC self-renewing, cell cultures were induced to adipogenic differentiation for 72 h. The cell lysate was submitted to ultracentrifugation on a sucrose gradient to separate monosomes, polysomes and the fraction free of ribosomes. The total RNA associated to ribosomes was extracted and the RNA fragments (<200 nt) were enriched and the size selection of appropriate RNA fragments occurred during the preparation of samples for deep sequencing. The samples were sequenced using SOLiD™ platform, and polysomal fraction of cell cultures non induced and 72h of induction were compared and sixteen miRNAs were identified. miRNAs found in a previous work of our group were added to these data and seven miRNAs (hsa-miR-29b-1-5p, hsa-miR-29c-5, hsa-miR-30c-5p, hsa-miR- 143-5p, hsa-miR-210-3p, hsa-miR-210-5p e hsa-miR-6775-5p) were tested by RTqPCR to confirm differential expression, and one of them (hsa-miR-210-5p) showed statistical significant difference.

Bisher wurde davon ausgegangen, dass der IKK-komplex durch Regulation des Transkriptionsfaktors NF-kappaB die stressinduzierte Expression von Zielgenen steuert. Im Rahmen der hier vorgelegten Dissertation konnte jedoch gezeigt werden, dass der IKK-Komplex unabhängig von seiner Rolle in der NF-kappaB-Aktivierung die Stabilität einer Vielzahl von mRNAs kontrolliert. Mittels der Kombination von Ko-Immunopräzipitationsstudien und SILAC-MS konnte die induzierte Interaktion der regulatorischen Untereinheit des IKK-Komplexes IKKgamma mit dem Gerüstprotein EDC4 (Enhancer of Decapping 4) nachgewiesen werden. EDC4 ist eine essentielle Komponente sogenannter zytoplasmatischer „Processing Bodies“ (P-Bodies). Diese fungieren als Depots für die Speicherung von mRNAs, aber auch als Orte der mRNA-Degradation und der miRNA-vermittelten Repression spezifischer Zielgene. Die Interaktion von IKKgamma mit EDC4 konnte durch verschiedene Stimuli induziert werden. Dazu zählen DNA-Schäden durch Doppelstrangbrüche, aber auch die Aktivierung von Oberflächenrezeptoren durch TNFalpha und IL-1beta. EDC4 dient darüber hinaus als Substrat der Kinase IKKbeta. Mittels Massenspektrometrie und Kinaseassays konnten vier IKK-abhängige Phosphorylierungsstellen identifiziert werden. Die IKK-vermittelte Phosphorylierung von EDC4 ist essentiell für die Regulation von mRNAs und die damit verbundene Bildung der zytoplasmatischen P-Bodies. Diese Befunde konnten sowohl in stabilen induzierbaren Zelllinien, mittels transienter Transfektion und durch den Gebrauch von Kinaseinhibitoren in primären als auch in Krebszelllinien bestätigt werden. mRNA-Stabilitätsassays und eine RNA-Seq Analyse bestätigten die stressinduzierten Änderungen in den Halbwertszeiten spezifischer Transkripte und offenbarten einen gemeinsamen Regulationsmechanismus des IKK-Komplexes mit EDC4. / The IKK complex is deemed to regulate gene expression through the activation of the transcription factor NF-kappaB. Here I describe an NF-kappaB-independent function of the IKK complex in regulating mRNA stability across different cell types and stimuli. A SILAC-MS screen for interaction partners of the regulatory subunit IKKgamma revealed an inducible interaction with Enhancer of mRNA Decapping 4 (EDC4). EDC4 is an essential component of cytoplasmic processing bodies (P-bodies). P-bodies function as sites of mRNA storage, degradation and miRNA-mediated silencing. Interaction between IKKgamma and EDC4 can be induced by various stimuli, including DNA damage, TNFalpha and IL-1beta. EDC4 was identified as a novel IKK substrate and four IKKbeta phosphorylation sites were determined by mass spectrometry and in kinase assays. Stable inducible cell lines, transient transfection and kinase inhibitors were used in different human cancer and in primary cell lines and demonstrated that phosphorylation of EDC4 by IKK is essential for formation of P-Bodies in response to numerous stimuli. mRNA stability assays confirmed stress-induced changes in the half-life of target mRNAs and revealed common regulation of mRNA stability by IKK and EDC4. The transcriptome-wide reach of this joint regulation was assessed via RNA-Seq analysis.

Das grundlegende Problem in der Transplantationsimmunologie ist es, die Langzeitakzeptanz eines fremden (allogen) Organs zu erreichen, ohne die sonstige Immunkompetenz des Empfängers zu beeinträchtigen. Die Induktion einer solchen spenderspezifischen Toleranz würde eine Alternative zum Langzeiteinsatz von Immunsuppressiva darstellen. Deswegen versucht man, während der Transplantation die Aktivierung der für die Abstoßung entscheidenden T-Helferzellen zu unterdrücken, bis eine Akzeptanz des Spenderorgans etabliert ist. Wichtig für eine Aktivierung der T-Zellen ist das für alle T-Helferzellen typische Zelloberflächenmolekül CD4. Antikörper gegen CD4 können in Tiermodellen eine Transplantattoleranz induzieren. Ein besonderes Interesse gilt der Charakterisierung der genauen Mechanismen dieser induzierten Transplantatakzeptanz, da diese noch wenig verstanden sind. Der von uns verwendete nicht-depletierende Maus-anti-Ratten-CD4mAk (RIB5/2) besitzt im allogenen Nierentransplantationsmodell der Ratte eine hohe toleranzinduzierende Wirkung und erzielt eine permanente Transplantatakzeptanz bei >80% der Empfängertiere. In dieser Arbeit wurde versucht, die Effekte dieses monoklonalen Antikörpers auf die T-Zellaktivierung näher zu untersuchen. Ausdruck der blockierten T-Zellaktivierung ist eine verminderte T-Zell-Proliferation und die Reduzierung der Synthese von TH-1-Effektorzytokinen, welche eine zelluläre Immunantwort fördern. Zu diesen für die Abstoßung gefährlichen Th-1-Effektorzytokinen gehören Interleukin 2 (=IL-2, Hauptwachstumsfaktor aktivierter T-Zellen) und Interferon gamma (=IFNgamma, wichtiger Aktivator von APC''s). Während die IL-2 Produktion vollständig verhindert wird, ist die Alloantigen-induzierte IFNgamma mRNA Expression nicht reduziert. Allerdings kommt es unter dem Einfluss des Antikörpers nicht zur IFNgamma Proteinsekretion. Wird jedoch das fehlende IL-2 ersetzt, kann sowohl die defekte Proliferation als auch die posttranskriptionelle Blockade der IFNgamma Produktion wieder aufgehoben werden. Das spiegelt sich auch in vivo wieder, da rekombinantes IL-2 auch hier den Toleranzstatus brechen kann. In dieser Arbeit konnte ein Kandidat dieser IFNgamma Translationskontrolle ermittelt werden. Zusätzlich wurde das Kochaperon p23, Teil eines Hsp90-Komplexes, in unsere Untersuchungen miteinbezogen, da es als ein differentiell reguliertes Gen in allogenen und anti-CD4mAk-behandelten T-Zellen identifiziert wurde. Hsp90 stabilisiert z.B. Kinasen, die wichtige Mediatoren der Signaltransduktion sind. p23 könnte aufgrund seiner Funktion als Kofaktor von Hsp90 an der Regulierung dieser Kinasen beteiligt sein, ist jedoch bisher kaum im Zusammenhang mit T-Zellaktivierung analysiert worden. Meine Untersuchungen ergaben, dass die p23 Expression ebenfalls durch den anti-CD4mAk reduziert wird. Da die Proliferation/p23 und die IFNgamma Synthese IL-2-abhängig reguliert werden, wurden IL-2-induzierte Signalwege auf ihre Relevanz für Proliferation und IFNgamma Regulation hin untersucht. Die Aufklärung der molekularen Mechanismen der anti-CD4mAk-Behandlung auf die T-Zellaktivierung soll mit dazu beitragen, Grundlagen für ein besseres Verständnis des Abstoßungsprozesses und damit Transplantatfunktions-Monitoring zu schaffen. / The major problem in transplantation immunology is the development of long-term donor-specific nonresponsiveness without reduction of the normal recipient immunocompetence. A tolerant state would obviate the need for continuing immunosuppressive therapy. One level of immunosuppression for inducing graft acceptance involves antibodies specific for T-cells of the recipient leading to donorspecific tolerance (e.g. by using of anti-CD4 monoclonal antibodies = aCD4mAb). CD4+ T cells play a predominant role in the cascade of immune processes following transplantation of foreign tissues. The anti-rat CD4 mAb RIB5/2 is very potent in inducing allo-specific tolerance to renal and heart allografts in rat recipients. Here I investigated the molecular mechanisms underlying anti-CD4 antibody mediated inhibition of allo-specific T cell activation and how this is antagonised by exogenous IL-2. IL-2 acts as growth factor for antigen-activated T cells by inducing the expression of cell cycle proteins and also enhances the expression of cytokines, e.g. IFNgamma in T cells. IFNgamma profoundly affects a variety of immune responses, including activation of antigen presenting cells. Anti-CD4 treatment, in vivo and in vitro, completely abrogated IL-2 production by alloreactive T cells. In contrast, anti-CD4 treated allo-activated T cells showed similar IFNgamma mRNA expression as untreated allo-activated T cells, but did not secrete any protein. Thus, the anti-CD4 antibody cannot prevent IFNgamma mRNA expression but is interfering with posttranscriptional mechanisms controlling IFNgamma production during allo-activation of T cells. The investigations revealed a candidate of these IFNgamma translation control. Additionally I investigated the heat shock protein 90 (Hsp90)-associated cochaperone p23. p23 upregulation during T cell activation is also abrogated by anti-CD4 treatment. Hsp90 chaperoning is critical for proper folding, stabilization and trafficking of a number of cellular signaling proteins as e.g. kinases. Hsp90-kinase complexes play an important role in T-cell signal transduction and little is known about the importance or even regulation of Hsp90-cochaperones like p23 during T-cell activation. I analysed the regulation of p23 and downstream effects on different kinases involved in T-cell signaling. These findings are supposed to contribute to a better understanding of the mechanisms underlying tolerance induction.

<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>

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.

All cellular RNAs contain numerous chemically modified nucleosides, but the largest number and the greatest variety are found in transfer RNA (tRNA). These modifications are posttranscriptionally introduced by modification enzymes during the complex process of tRNA maturation. The function of these modified nucleosides is not well known, but it seems that when present in the anticodon region, they play a direct role in increasing translational efficiency and fidelity, while modifications outside the anticodon region would be involved in the maintenance of the structural integrity of tRNA. Among the naturally occurring nucleoside modifications, base and ribose methylations are by far the most frequently encountered. They are catalyzed by tRNA methyltransferases (MTases), using generally the S-adenosyl-L-methionine (AdoMet) as methyl donor. Most of the knowledge about tRNA MTases comes from studies on bacterial and eukaryal model organisms, and very few informations are available about tRNA methylation in Archaea, particularly for thermophilic and hyperthermophilic Archaea whose GC-rich tRNAs are difficult to sequence. Nevertheless, some works on tRNA hydrolyzates from thermophiles or hyperthermophiles highlighted the presence of numerous methylated nucleosides. Furthermore, it has been shown that the only sequenced tRNA from an hyperthermophilic Archaea, the initiator methionine tRNA (tRNAiMet) from the Sulfolobus acidocaldarius, contains ten modified nucleosides, nine of them bearing a methylation on the base, on the ribose or on both base and ribose.<p>Of special interest is the modified nucleoside found at position 9 of this tRNA. It is an adenosine derivative, but the exact nature of the modification is unknown. In the yeast S. cerevisiae, some tRNAs with a guanosine at this position are methylated by the MTase Trm10p to form m1G9 (126). Since Trm10p-related proteins are found in hyperthermophilic archaea, such a homolog could be responsible for modification at position 9 of S. acidocaldarius tRNAiMet. In this work, we showed indeed that the Trm10p-related protein Saci_1677p from S. acidocaldarius methylates position 9 of tRNAs, but is specific for position N1 of adenosine, forming m1A rather than m1G. Interestingly, we demonstrated that Tk0422p from T. kodakaraensis, the euryarchaeal homolog to Saci_1677p, is the first tRNA MTase presenting a broadened nucleoside recognition capability, methylating both position N1 of A and of G to form m1A and m1G at position 9 of tRNAs. <p>This unique tRNA (m1A-m1G) MTase activity was further studied on one hand by site-directed mutagenesis of residues potentially important for the catalytic activity of Tk0422p enzyme, and on the other hand by determining the importance of the pH on the efficacy of the methylation reaction. Indeed, protonation state of atom N1 of A and G differs at physiological pH (N1 of G being protonated contrary to N1 of A), and we showed that m1G formation was increased with increasing pH. This could reflect the need of the enzyme to deprotonate G to be able to catalyze de methyltransfer. We showed also that the activity of the two archaeal enzymes (Saci_1677p and Tk0422p) present different dependence toward the structure of tRNA, the euryarchaeal Tk0422p requiring the intact tRNA structure while its crenarchaeal counterpart Saci_1677p being able to modify some truncated tRNAs.<p>Finally, some attempts to unveil the in vivo function of these enzymes, as well as their enzymatic mechanisms were undertaken, but these experiments are very preliminary and underline the needs for the development of genetic tools applicable to Archaea./ Tous les ARN cellulaires contiennent des nucléosides modifiés chimiquement, mais ce sont les ARNt qui en contiennent la plus grande variété et la plus grande proportion. Ces modifications sont introduites post-transcriptionnellement par des enzymes de modification durant le processus complexe de maturation des ARNt. Parmi les nucléosides modifiés, les méthylations de bases ou de riboses sont les plus fréquemment rencontrées. Elles sont catalysées par des ARNt méthyltransférases (MTases) utilisant pour la plupart de la S-adenosyl-L-methionine (AdoMet) comme donneur de méthyle. <p>La plupart des connaissances relatives aux ARNt MTases provient d’études sur des organismes modèles eucaryotes et bactériens, et peu de choses sont connues en ce qui concerne les archées, plus particulièrement les archées thermophiles et hyperthermophiles dont les ARNt GC riches sont difficiles à séquencer. Néanmoins, des travaux sur des hydrolysats d’ARNt de thermophiles et hyperthermophiles ont mis en évidence la présence d’un grand nombre de nucléosides modifiés. De plus, le seul ARNt d’archée hyperthermophile séquencé à ce jour, l’ARNtiMet de S. acidocaldarius contient 10 nucléosides modifiés, essentiellement par méthylation de la base, du ribose, ou des deux à la fois. Le nucléoside présent en position 9 de cet ARNt porte une modification chimique de nature encore inconnue. Or, chez la levure S. cerevisiae, certains ARNt possédant une guanosine à cette position sont méthylés par la MTase Trm10p pour former la 1-méthylguanosine. Etant donné qu’il existe une protéine apparentée à Trm10p chez les archées hyperthermophiles, celle-ci pourrait être responsable de la modification trouvée en position 9 de l’ARNtiMet de S. acidocaldarius. Dans ce travail, nous avons montré qu’effectivement la protéine Saci_1677p de la crénarchée S. acidocaldarius, orthologue à Trm10p, modifie la position 9 des ARNt, mais catalyse la formation de 1-methyladénosine (m1A) plutôt que de m1G dans les ARNt. De façon intéressante, nous avons montré que chez l’euryarchée T. kodakaraensis, l’enzyme Tk0422p homologue à Saci_1677p est capable de méthyler à la fois une adénosine et une guanosine en position 9 des ARNt. A notre connaissance, cette enzyme est la première ARNt MTase présentant une capacité élargie de reconnaissance de substrat.<p>Le présent travail a contribué à la caractérisation fonctionnelle et structurale de ces deux enzymes archéennes, et a permis d’améliorer la connaissance générale de la machinerie de modification des ARNt d’archées.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le glioblastome (GBM) est la tumeur primitive du cerveau la plus fréquente et la plus agressive chez l’adulte. Le mauvais pronostic de cette pathologie peut être expliqué par la présence de cellules résistantes aux traitements à l’origine des rechutes appelées cellules souches de glioblastome (CSG). Caractérisées par une plasticité cellulaire, elles sont capables de s’adapter aux environnements défavorables à leur survie. Ainsi, malgré des traitements multimodaux agressifs, les bénéfices de la prise en charge du GBM restent très modestes ; il est donc nécessaire de mieux caractériser ces cellules afin de développer de nouvelles thérapies ciblant les CSG. Le rôle des mécanismes post-transcriptionnels de régulation de l’expression génique dans le maintien des cellules souches cancéreuses a été démontré dans différentes tumeurs. Cependant, peu de protéines de liaison à l'ARN (RBP), régulateurs clés de ces événements, ont été identifiés dans le contexte des CSG. Mon projet de thèse s’inscrit dans le cadre de l’étude de RBP régulant les propriétés d’auto-renouvèlement et de survie des CSG afin d’approfondir nos connaissances sur les mécanismes moléculaires qui contribuent à la formation ou récurrence des GBM. Dans un premier temps, j’ai cherché à définir un protocole permettant d’enrichir la population de CSG maintenues en conditions de culture non-adhérentes qui favorisent la formation de neurosphères et le maintien d’une hiérarchie cellulaire. Confrontée à une forte hétérogénéité de signatures moléculaires, j’ai choisi de prendre cette caractéristique en compte dans un second temps en menant une étude comparative du transcriptome de 5 modèles de cellules souches de glioblastome provenant de patients différents. Cette analyse inclut également des cellules différenciées in vitro à partir des CSG ainsi que des cellules souches neurales humaines. Grâce à une approche de séquençage de leur transcriptome, mes travaux de thèse ont permis d’identifier une famille de régulateurs post-transcriptionnels enrichis dans les CSG, les protéines nELAVL. Ces résultats ont pu être confirmés par l’analyse de leur expression protéique dans des modèles in vitro et dans des coupes de tumeurs provenant de différents patients atteints de GBM. Une co-expression des protéines nELAVL avec OLIG2 ou SOX2 a été observée confirmant ainsi leur association avec l’état souche. ELAVL4 correspond au membre de la famille nELAVL le plus réprimé lors de la différenciation des CSG. Des outils de modulation de son expression ont été développé en vue d’évaluer son rôle dans les CSG par des approches de perte d’expression ou de gain de fonction / Glioblastoma (GBM) is the most common and aggressive primary adult brain tumor. GBM dismal prognosis can be explained by the presence of treatment resistant cells responsible for tumor relapse known as glioblastoma stem cells (GSC). Characterized by cellular plasticity, they are able to adapt to hostile environments. Thus, despite aggressive multimodal treatments, GBM curative therapies have provided only a modest benefit; it is therefore necessary to better characterize these cells in order to develop new GSC targeted therapies. The role of posttranscriptional mechanisms of gene expression regulation in the maintenance of cancer stem cells has been demonstrated in different tumors. However, few RNA-binding proteins (RBPs), key regulators of these events, have been identified in GSC. My thesis project consists in identifying RBP that are critical for self-renewal and increased survival properties of GSC. The aim of this study is to deepen our knowledge of the underlying molecular mechanisms of GBM development or recurrence. At first, I established a protocol to enrich for GSC using nonadherent culture conditions that promote the formation of neurospheres comprising a cellular hierarchy. I chose to take into account the facing problem of GSC molecular heterogeneity in a second step and carried out a comparative study of the transcriptome of 5 glioblastoma stem cell cultures from different patients. This analysis also includes in vitro differentiated cells originating from GSC as well as human neural stem cells. Using a transcriptome sequencing approach, my thesis work has led to the identification of a family of post-transcriptional regulators enriched in GSC, nELAVL proteins. These results were confirmed by protein expression analysis in in vitro neurospheres and within tumor sections from different GBM patients. Co-expression of nELAVL proteins with OLIG2 or SOX2 was observed thus confirming their association with stemness. ELAVL4 repesents the most differentially expressed member of nELAVL family upon GSC differentiation. Knock-down and gain-offunction tools targeting ELAVL4 have been developed to further assess its roles in GSC maintenance