Les plantes sont ancrées au sol pendant la majorité de leur cycle de vie et doivent donc constamment adapter leur croissance et leur métabolisme aux stress abiotiques. Ainsi, la subsistance des plantes dépend de leur capacité à réguler rapidement l’expression des gènes afin d’adapter leur physiologie à l’environnement. L’expression d’un gène peut être contrôlé à plusieurs niveaux; transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel.De nombreux processus cellulaires vitaux tels que la réplication de l’ADN, la transcription, la synthèse protéique, et la dégradation des protéines, sont régulés par les signaux environnementaux. Des études chez la levure, la drosophile et les animaux ont montré que la protéine kinase TOR (Target Of Rapamycin) est impliquée dans le contrôle de la croissance cellulaire et de la prolifération en réponse à différents signaux tels que les nutriments, les acides aminés, les hormones et les facteurs de croissance. Chez Arabidopsis thaliana, TOR est nécessaire au développement de l’embryon et de l’endosperme. De plus, des modifications du niveau de protéine AtTOR affectent la croissance végétative et la reproduction.Le principal objectif de cette thèse est de caractériser les mécanismes qui contrôlent l’expression de AtTOR en déterminant les éléments de régulation situés sur le la région 5’ non traduite (5′UTR) de l’ARNm de AtTOR, puis de manipuler ces éléments de régulation afin d étudier leur rôle. Nous avons choisi de nous focaliser sur la région 5′UTR de AtTOR, et sur une microORF (uORF) située en amont de l’ORF principale de AtTOR. Il s’agit de la première tentative d’étude de la régulation de l’expression de TOR par ces éléments chez les eucaryotes.Trois constructions chimériques ont été réalisées pour cette étude et transformée transitoirement est de manière stable dans des plantes. La première construction (contrôle positif) incluse le promoteur de AtTOR, la région 5′UTR, le premier intron et le début du premier exon fusionné au gène rapporteur GUS. La seconde construction (microORF mutée) est présente une mutation du codon start de la microORF (ATG changé en TTG). Enfin, la troisième construction (5′UTR délétée) contient la même séquence que le contrôle positif mais sans la région 5′UTR. Ces constructions ont également été placée sous le contrôle du promoteur 35S au lieu du promoteur de AtTOR afin d’étudier un lien éventuel entre la 5′UTR et la microRF et le promoteur de AtTORNos résultats indiquent une régulation généralement négative exercée par la 5′UTR, et dans une moindre mesure par la microORF, sur l’expression de AtTOR. Cette régulation semble avoir lieu au niveau transcriptionnel ou au niveau de la stabilité de l’ARNm, mais pas au niveau de la traduction. En effet, les modifications du niveau de transcrit GUS sont suivie d’un changement équivalent de l’activité GUS. De plus, nous avons observé que l’auxine et le sucrose ont un effet positif sur l’expression de AtTOR. Dans le cas de l’auxine, cet effet semble lié à la présence de la région 5′UTR de AtTOR.D’autres études de la fonction de la région 5’UTR et de la microORF de AtTOR, ainsi que de leur relation avec d’autres éléments régulateurs localisée dans le promoteur de AtTOR, permettront de mieux comprendre comment ces éléments régulateurs contrôlent finement l’expression de AtTOR. / Land plants are anchored in one place for most of their life cycle and therefore must constantly adapt their growth and metabolism to abiotic stresses. Thus, plants’ subsistence depends on their ability to regulate rapidly gene expression in order to adapt their physiology to their environment. The expression of a gene can be controlled at many levels, including transcription, post-transcription, translation, and post-translation.Many vital cellular processes like DNA replication, transcription, protein synthesis, and protein degradation are regulated by environmental signals. Studies in yeast, Drosophila, and mammals showed that the target of rapamycin (TOR) protein is involved in control of cell growth and cell proliferation in response to different types of environmental signals such as nutrients, amino acids, hormones, and growth factors. In Arabidopsis thaliana, TOR is necessary for both embryo and endosperm development in, and changes of TOR protein level affect both vegetative and reproductive growth.The main purpose from this thesis is to highlight the mechanisms that control AtTOR expression at the post-transcriptional level through determination of the possible regulatory elements within the 5′ untranslated region (5′UTR) or the first intron of AtTOR mRNA itself, and through manipulation of these regulatory elements to study their precise role. We have chosen to focus on the small upstream open reading frame (uORF) as well as the 5′UTR region. This is the first attempt to study the regulation of TOR kinase expression in eukaryotes through these small uORF or the sequence of 5′ untranslated region (5′UTR).To achieve this purpose, three chimeric constructs have been established and transformed in Nicotiana benthamiana leaves and Arabidopsis thaliana plants. The first construct (the positive control) contains the AtTOR promoter, the 5′UTR, the first intron, and the beginning of the second exon fused to the GUS reporter gene. The second construct (mutated uORF) have the same sequence as the positive control construct except the start codon of uORF was changed from ATG to TTG. The third construct (deleted 5′UTR) have the same sequence as the positive control construct without the 5′UTR. These constructs have also been placed under the activity of CaMV 35S promoter instead of AtTOR promoter to investigate whether there is a link between the 5′UTR/or uORF and the promoter.Our work show an overall negative regulation exerted by the 5′UTR and, to a lesser extent, by the uORF on AtTOR gene regulation. This regulation is likely at the level of transcription or mRNA stability, since the changes in GUS transcript level was followed by the same changes in GUS activity. In addition we found that external inducers like auxin or sucrose exert a positive effect on AtTOR expression. This effect appears somehow linked to the presence of the 5′UTR of AtTOR mRNA.Greater insight into the molecular mechanisms of AtTOR 5′UTR/or uORF function and its relationship with other regulatory elements located in AtTOR promoter will be required to understand how these regulatory elements work either individually or in combination to achieve the fine and accurate regulation of their gene expression.

O gene do Retardo Mental do X Frágil (FMR1) possui 17 éxons e seu transcrito primário pode sofrer splicing alternativo, havendo, entre outros eventos, possibilidade de exclusão ou inclusão do éxon 12. O produto da expressão do FMR1, a proteína do retardo mental do X frágil (FMRP), possui papéis importantes no sistema nervoso central, atuando como repressora da tradução de RNAm em espinhas dendríticas e controlando a síntese de proteínas envolvidas na função sináptica. Entre dois domínios centrais do tipo KH presentes na FMRP, o segundo (KH-2) é responsável pela interação da proteína aos polissomos. O domínio KH-2 é codificado pelos éxons 9 a 13 do FMR1 e possui a alça variável mais longa já observada entre proteínas humanas, que é codificada pelos éxons 11 e 12. A inclusão do éxon 12 no RNAm do FMR1 causa uma extensão em fase dessa alça variável do KH-2 da FMRP. Estas isoformas apresentam expressão significativa em neurônios cortico-cerebrais e cerebelares do rato, no primeiro mês pós-natal. Este trabalho baseia-se em resultados prévios do grupo de pesquisa, em que se identificaram sequências curtas no íntron 12 do FMR1, com potencial para agir como acentuadores de splicing. Baseando-nos na hipótese de que essas sequências constituem elementos transcritos que se ligam a fatores proteicos do núcleo celular, potencialmente reguladores do splicing do pré-RNAm do FMR1, realizamos ensaios de precipitação por afinidade com extratos nucleares de córtex cerebral de rato e transcritos do loco, biotinilados. Análises por espectrometria de massas revelaram enriquecimento de proteínas nucleares, contendo domínios de ligação a RNA, principalmente aquelas relacionadas à regulação e processamento de pré-RNAm, sobretudo o splicing / Fragile X Mental Retardation 1 gene (FMR1) comprises 17 exons. Its primary transcript is subject to alternative splicing, allowing for the possibility of exon 12 inclusion or skipping, among other events. The product of FMR1 gene expression, fragile X mental retardation protein (FMRP), has important roles in the central nervous system, acting as a translational repressor in dendritic spines, thus controlling the synthesis of proteins involved in synaptic function. FMRP has two central KH domains. One of them (KH-2) is responsible for its interaction with polysomes. The KH-2 domain is encoded by FMR1 exons 9 to 13. It contains the longest variable loop ever observed among human KH-containing proteins, which is encoded by FMR1 exons 11 and 12. Exon-12 inclusion in FMR1 mRNA causes an in-frame extension of FMRP KH-2 domain variable loop. These isoforms appear significantly expressed in cortico-cerebral and cerebellar neurons of the rat in the first month after birth. We have previously identified short sequences within FMR1 intron 12 that may potentially act as splicing enhancers. Our study is based on the hypothesis that those sequences when transcribed should bind to nuclear protein factors that may function as FMR1 exon 12 pre-mRNA splicing regulators. To initiate an experimental approach to test that hypothesis, we conducted affinity precipitation assays with rat cerebral cortex nuclear extracts and biotinylated transcripts. Mass spectrometry analyses disclosed proteins that have been described to be enriched in the cell nucleus, contain RNA-binding domains, and be functionally related to pre-mRNA processing, notably splicing

Viele biologische Prozesse im Verhalten von ganzen Organismen, aber auch in den Prozessen und der biochemischen Zusammensetzung von Zellen zeigen einen zirkadianen Rhythmus, also einen Rhythmus mit einer Periode von etwa 24 Stunden. Diese 24-Stunden-Rhythmen sind in der Genexpression auf allen Ebenen zu finden: von der Tran- skriptionsinitiation bis zur Proteindegradation. Auf Transkriptebene, zirkadiane mRNA-Produktion und mRNA-Abundanz ist umfassend gemessen. Auf der anderen Seite, zirkadiane posttranskriptionelle Regulation ist weit weniger verstanden. In dieser Arbeit untersuche ich, wie bisher ungemessene, posttranskriptionelle Prozesse die rhythmischen Eigenschaften der Genexpression beeinflussen. Dazu beschreibe ich die Lebensstadien eines Bio-Moleküls mit einem Modell-Motiv, einer einfachen Differentialgleichung mit zeitabhängigen, rhythmischen Raten. Als erstes diskutiere ich die Einschränkungen von Phase und Amplitude zirkadianer Transkripte, die nur von konstanter PTR beeinflusst werden. Bei vielen gemessenen Transkripten sind diese Einschränkungen verletzt. In diesen Fällen muss es eine rhythmische PTR geben. Ich untersuche, welche rhythmische PTR diese Fälle erklären können und führe einen statistischen Test ein, der auf unbeobachtete, rhythmische PTR testet. Durch die Analyse zweier Datensätze von Mausleber und -niere finde ich, dass 18% aller zirkadianen Gene in Niere und 34% in Leber rhythmisch posttranskriptionell reguliert sind. Im zweiten Teil analysiere ich weitere Aspekte von PTR in einem Hypothesen-getriebenen Ansatz. Ich zeige, dass Spleißen mit einem Rhythmus von 24 Stunden 12 Stunden-Rhythmen in der Abundanz von mRNA erzeugen kann. Als nächstes schlage ich ein Modell vor, das rhythmische Degradation von Mitgliedern der zirkadianen Uhr beschreibt. Schließlich erweitere ich das Modell-Grundmotiv zu einer partiellen Differentialgleichung (PDG), die das “Altern” von Molekülen beschreibt. / In almost all organisms on Earth, many behavioral, physiological, and biochemical activities oscillate with a circadian rhythm, a rhythm with a period of about 24 hours. In gene expression, the 24-hour-rhythm can be found on all stages: from transcription initiation to protein degradation. On the transcript level, circadian mRNA production and mRNA abundance are comprehensively charted through numerous genome-wide high throughput studies. Circadian post-transcriptional regulation, however, is less well understood. In this thesis, I will investigate how unobserved post-transcriptional processes influence rhythmic properties of gene expression. To this end, I quantify the life-stages of biomolecules using one modeling motif, a simple ordinary differential equation describing production and degradation with time-dependent rhythmic rates. This basic modeling motif is systematically varied to examine and discuss various influences of post-transcriptional regulation (PTR) on circadian mRNA expression. I first discuss the restrictions of rhythmic phase and amplitude of circadian transcripts influenced by non-rhythmic PTR. For many genes these restrictions are violated and we have to assume the existence of a rhythmic PTR. I discuss which rhythmic PTR can explain these findings and further introduce a statistical test to quantify the extent of unobserved rhythmic PTR. Analyzing two data sets on mouse liver and kidney, I find that 18% of circadian genes in kidney and 34% in liver are under rhythmic post-transcriptional control. In a second part, I analyze more specific aspects of PTR in a hypothesis-driven approach. Firstly, I find that splicing with a rhythm of 24 hours is able to generate 12-hour rhythms in abundance of mature mRNA. Secondly, I propose and analyze a model to investigate rhythmic degradation of core clock genes. And finally, I extend the core modeling motif to a partial differential equation (PDE) model that accounts for the “aging” process of molecules.

Les protéines de liaison à l’ARN PUMILIO 1 et 2 (PUM1/2) exercent un rôle central dans le maintien des cellules souches chez les Invertébrés en se fixant, en association avec des partenaires protéiques, sur la région 3’ UTR de certains ARNm, régulant ainsi leur devenir. A ce jour, le rôle de PUM1/2 dans les cellules souches/progénitrices hématopoïétiques (CSPHs) a été peu étudié. La perte de la coordination entre auto-renouvellement et différenciation des CSPHs peut aboutir à des hémopathies chez l'Homme, d’où la nécessité de comprendre les mécanismes sous-jacents. Notre équipe a mis en évidence, par une approche de shARN, que l’invalidation des protéines PUM1/2 dans les CSHs humaines et murines conduit à une réduction de leur expansion, associée à une apoptose accrue et un arrêt du cycle cellulaire en phase G0/G1, et aussi à une perte du potentiel clonogénique in vitro et du potentiel de reconstitution in vivo. L’objectif de notre travail a consisté à : a/ évaluer les effets de la surexpression de PUM1/2 dans les CSPHs, b/ déterminer l’implication de PUM1/2 dans les processus leucémiques, c/ étudier les mécanismes moléculaires responsables de l’activité de PUM1/2 en identifiant les cibles et les partenaires protéiques par une approche de protéomique globale. Nos résultats suggèrent qu’une surexpression modérée de PUM1 (2/3 fois) dans les cellules CD34+ limite la perte du potentiel clonogénique alors qu’une expression plus élevée (5/10 fois et plus) est toxique. L’analyse de l’expression de PUM1/2 par RT-qPCR dans les échantillons de Leucémies Aigue Myeloïdes (LAM) (GOELAMSthèque) montre une augmentation significative dans les échantillons les plus immatures (LAM0-2) comparés aux contrôles sains. La perte de PUM1/2 par shARN dans les cellules primaires de leucémies ainsi que dans des lignées issues de différents processus leucémiques réduit fortement leur survie. La recherche des partenaires associés à PUM par spectrométrie de masse a permis de découvrir Argonaute2 et MOV10 (tous les 2 impliqués dans la machinerie des miRNA), ainsi que des protéines de liaison aux ARNs, ELAV1 déjà connue pour son implication dans le maintien des CSH murines et IMP3, impliqué dans de nombreux cancers et dans la régulation du cycle cellulaire. L’invalidation de IMP3 ou ELAV1 dans les CSPHs conduisent, in vitro, aux mêmes effets observés avec la perte du PUM 1/2, une diminution de l’expansion avec une augmentation de l’apoptose, et la perte du potentiel clonogénique. Enfin, nous avons identifié FoxP1 (Forkhead box P1) comme nouvelle cible directe de PUM1/2, dont le rôle est encore très peu décrit dans l’hématopoïèse. L’étude fonctionnelle de FoxP1 sur les CSPHs par shARN mime les effets observés avec les facteurs PUM1/2. De plus, la surexpression de FoxP1 restaure partiellement les activités antiprolifératives et pro-apoptotiques générées par les shPUM1/2. Enfin, le profil d’expression de FoxP1 dans les LAM corrèle avec le profil d’expression de PUM1/2. Nos résultats confirment le rôle majeur joué par les protéines PUM1/2 en partie via la régulation positive de FoxP1 qui contribue au maintien les CSPHs normales et pathologiques. / Pumilio 1 and 2 (PUM1/2) RNA-binding proteins exert a central role in stem cell maintenance among Invertebrates by binding the 3'UTR of mRNA targets in association with protein partners, thus regulating mRNA stability/translation. Nothing is known regarding normal and pathologic hematopoietic stem and progenitor cells (HSPCs). Loss of coordination between self-renewal and differentiation of HSPCs can lead to leukemia in humans, hence the need to understand the mechanisms. Our team has highlighted the fundamental role played by the post-transcriptional regulators Pumilio (PUM) 1/2 on normal HSPC properties. By a shRNA approach, PUM 1/2 knockdown in human and murine HSPCs leads to: a/ a reduced expansion associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase, b/ the loss of their clonogenic capacity and their in vivo reconstitution potential. The objective of our work is to: a/ evaluate the effects of PUM 1/2 overexpression in HSPC, b/ determine PUM1/2 involvement in leukemic processes; c/ investigate the molecular mechanisms responsible of PUM activity in HSPC by identifying protein targets and partners. Our results showed that a moderate overexpression of PUM1 (2 to 3 fold) in normal CD34+ HSPCs limits the loss of their clonogenic potential, while a higher expression (5 to 10 fold or more) is toxic. The expression analysis of PUM1/2 transcripts in Acute Myeloid Leukemia (AML) (GOELAMSthèque) showed a significant increase in the most immature samples (AML0-2) as compared to healthy controls. PUM1/2 knockdown by shRNA in AML cells significantly reduced their survival. The same effect was observed in cell lines from several leukemic processes. We identified various PUM-associated partners by mass spectrometry, Argonaute2 and MOV10 (involved in the miRNA machinery), and the RNA-binding proteins IMP3 (involved in several cancer and in cell cycle regulation) and HuR/ELAV1 (already known to be involved in murine HSPCs maintenance). IMP3 or ELAV1 knockdown in HSPCs in vitro lead to the same effect of a PUM1/2 invalidation, a decreased expansion with an increased apoptosis and the loss of clonogenic potential. Finally, we identify the forkhead box P1 (FOXP1) transcription factor as a new direct target up-regulated by PUM1 and PUM2. Functional study of FoxP1 knockdown by shRNA in HSPCs mimic PUM1/2 activities. Moreover, FOXP1 overexpression partially rescued shPUM antiproliferative and pro-apoptotic effects. Also, the PUM1/2 and FOXP1 expression levels in leukemic primary cells were measured by RT-qPCR and revealed a positive correlation. Our results reveal that PUM1/2 are direct positive regulators of FOXP1 which contributes to the maintenance of normal and leukemic HSPCs.

Les organismes bactériens sont en contact direct avec leur environnement et doivent donc constamment s’acclimater aux variations de celui-ci. Pour cela, plusieurs leviers de régulations peuvent être actionnés. Récemment, la régulation post-transcriptionnelle par les ARN régulateurs a été proposée comme un mécanisme de régulation rapide et peu coûteux pour la cellule. Chez le phytopathogène Dickeya dadantii, la régulation de la virulence a quasi exclusivement été étudiée au niveau transcriptionnel et l’implication des ARN régulateurs dans la virulence reste très peu connue. Pour cela, nous avons tout d’abord étudié le rôle des chaperons à ARN dans la pathogénie de D. dadantii et mis en évidence leur implication dans de nombreux facteurs de virulence comme la production d’enzyme de dégradation de la paroi végétale. Puis, nous avons développé une nouvelle méthode d’identification d’ARN à partir de données RNA-seq. Cette méthode a été développée pour tirer profit des séquençages réalisés en paired-end, permettant de séquencer les deux extrémités d’un transcrit. Son évaluation dans sa capacité à détecter de manière précise des ARN connus a montré une performance supérieure aux méthodes de détection existantes. Enfin, cette nouvelle méthode a été appliquée sur des données de séquençage de petits transcrits. Cette analyse nous a permis d’identifier plus d’un millier d’ARN régulateurs potentiels, dont plusieurs pourraient être impliqués dans la régulation de la virulence. Ces travaux ont donc permis de mettre en lumière l’existence d’une régulation post-transcriptionnelle chez D. dadantii et de proposer des pistes concernant les acteurs et mécanismes concernés / Bacterial organisms are directly exposed to environmental conditions and have to respond to environmental stress. To do so, several regulation network are known. Recently, post transcriptional regulation with small RNAs was suggested to be a fast and cheap in energy regulation mechanism. In the phytopathogen Dickeya dadantii, investigations on pathogenic process mostly focused on its control by transcriptional regulators. Knowledge of post-transcriptional regulation of the virulence factors is still in its infancy.To this end, we first studied the impact of RNA chaperones in the virulence of D. dadantii and showed that they were involved in the regulation of several virulence factors, like production of cell wall degrading enzyme. Then, we developed a new method to detect sRNAs from paired-end bacterial RNA-seq data. This method take paired end sequencing into account, which allow the sequencing of the both ends of each fragment. A comparative assessment showed that this method outperforms all the existing methods in terms of sRNA detection and boundary precision. Finally, this method was applied to sequencing data. With this analysis, more than one thousand sRNAs has been detected, with the identification of several candidates potentially involved in virulence.Thereby, this work highlight the existence of post-transcriptionnal regulation in D. dadantii and suggest candidates and mechanisms involved in this regulation

Les rhabdomyosarcomes embryonnaires et aléolaires (RMS) appartiennent aux tumeurs des tissus mous les plus fréquentes chez les enfants dont elles représentent 2/3 des cas. Plusieurs données suggèrent que la dérégulation des cellules progénitrices du muscle squelettique pourrait jouer un rôle dans l'émergence des cellules de RMS qui ont aussi bien perdu le contrôle de la régulation de la prolifération cellulaire que la capacité à se différencier.Néanmoins les mécanismes de développement des RMS restent à caractériser. La famille des IMPs et notamment IMP-2, protéines liant les ARN, sont à la fois fortement exprimées dans le muscle en régénération in vivo mais aussi dans les cellules de RMS.Au cours de ma thèse, j’ai pu mettre en évidence le rôle d’IMP-2 dans la motilité des cellules de RMS et dans les cellules musculaires ainsi que dans le contrôle de l’intégrité du cytosquelette de microtubules (MTs) et dans le remodelage des adhésions focales. En effet, IMP-2 est impliqué à la fois dans la régulation de l’expression de MuRF-3, une protéine lié àla stabilisation des MTs et de Pinch-2, un important médiateur de l’adhésion cellulaire. / The RNA-binding proteins IMPs (IGF-II mRNA binding protein) first discovered in rhabdomyosarcoma cells (RMS) are expressed during embryonic development but their expression is decreased in adult tissues.We showed that IMPs and particularly IMP-2 are strongly expressed in mouse myoblatsts, during early regeneration of skeletal muscle in vivo and in and RMS. IMP-2 loss of function experiments using siRNA have shown that IMP-2 is necessary for microtubules stability(MTs), cell motility and invasion of myoblasts and RMS.Expression of IMP-2 specifically increases MTs stability by an enrichment of detyrosinated tubulin Glu-tubulin. Detyrosination is indispensable for myogenic differentiation and plays substantial role in tumor growth. Additionaly, MTs stabilization play an important role in focal adhesion remodeling, in cytoskeleton integrity, cell adhesion and cell motility.To get new insight into molecular mechanism underlying the function of IMP-2 in MTs stability and cell motility, full ranscriptome analysis was performed between IMP-2 knockdown (KD) myoblasts and control myoblatsts. We have further shown that IMP-2 controls the mRNA levels of many important mediators of cell adhesion such as PINCH-2, as well as multiple cytoskeleton remodeling, such as MuRF-3.We have identified a number of functionally relevant protein partners of IMP-2.Moreover subsequent RNAi screens have revealed the importance of IMP-2 regulated transcripts involved in cell motility and cell adhesion In conclusion, we show that IMP-2 dependent regulation of mRNA such as MuRF3 and PINCH2 largely contributes to the motility –deficient in IMP-2 KD cells. Moreover these results indicate clearly, that further analysis of IMP2 protein partners and RNA targets regulated by IMP-2 will help to characterized the function of IMP-2 and to propose a model of IMP-2 transcriptional regulation of gene expression in myoblasts and RMS cells.

O iodo em excesso promove o efeito Wolff-Chaikoff. Oligominerais já foram descritos como potenciais reguladores da expressão de proteínas. Tornou-se interessante avaliar se o iodo interferiria com a expressão do mRNA da NIS, em curtos períodos de tempo. Foram realizados, em ratos e células (de 30 min24h), estudos de expressão, comprimento de cauda poli-A e recrutamento para polissomos, do mRNA de NIS. Observou-se, in vivo e in vitro, que o excesso de iodo promoveu diminuição da expressão e do comprimento da cauda poli-A do mRNA de NIS, em todos os períodos estudados, além de promover menor recrutamento deste mRNA para os polissomos. A diminuição da cauda poli-A do mRNA de NIS pode ter aumentado sua instabilidade/degradação e também ter sido responsável por uma menor eficiência de tradução deste transcrito. Conclui-se que: (a) o iodo regula pós-transcricionalmente a expressão gênica da NIS, sendo fundamental nos processos que norteiam o efeito Wolff-Chaikoff e (b) oligoelementos têm relevância na regulação da expressão de proteínas relacionadas ao seu transporte. / Iodide in excess exerts the Wolff-Chaikoff effect. It is described that some minerals can regulate the expression of proteins. This study aimed to investigate if the iodide could modify the expression of NIS mRNA, in short periods of time. Rats and cells, divided in time-groups of 30 min up to 24h, were used in studies of expression, poly-A tale length and polysomal profile of NIS mRNA. Both in vivo and in vitro studies showed that the iodide treatment promoted a reduction in the expression and the poly-A length of NIS mRNA, in all time-groups, and decreased its recruitment to the polysomes. It is possible that the reduction of NIS mRNA poly-A tale length has increased the instability/degradation of this transcript, and impaired the translation efficiency of it. Concluding: a) the iodine exerts a post-transcriptional regulation of NIS mRNA expression, being essencial in the processes that guide the Wollf-Chaikoff effect; b) the oligoelements have an extremely important role in the expression regulation of proteins related to their transport.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

No description available.

The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.