La régulation de l’expression des gènes est ce qui permet à nos cellules de s’adapter à leur environnement, de combattre les infections ou, plus généralement, de produire la quantité exacte de protéine nécessaire pour répondre à un besoin spécifique. Parmi les joueurs les plus importants dans cette régulation de l’expression des gènes on retrouve les microARN (miARN). Ces petits ARN de 22 nucléotides sont présents chez la majorité des espèces multicellulaires et sont responsables du contrôle direct de plus de 30% des gènes exprimant des protéines chez les vertébrés. La famille de miARN lethal-7 (let-7) est composée de miARN parmi les plus connus et ayant des fonctions cruciales pour la cellule. La régulation du niveau des miARN let-7 est essentielle au bon développement cellulaire. La biogenèse de ces miARN, du transcrit primaire jusqu’à leur forme mature, est régulée principalement par Lin28, une protéine pluripotente très conservée. Cette protéine est composée d’un domaine cold shock (CSD) et de deux domaines de liaison au zinc. C’est grâce à ces domaines de liaison à l’ARN que Lin28 peut lier et inhiber la maturation des miARN let-7. L’objectif de cette thèse est de caractériser l’interaction entre Lin28 et le microARN précurseur let-7g afin de mieux comprendre le rôle de cette protéine dans l’inhibition de la biogenèse du miARN. À l’aide de techniques biochimiques et biophysiques, nous avons d’abord défini les principaux déterminants de l’interaction entre Lin28 et la boucle terminale du miARN précurseur let-7g (TL-let-7g). Nous avons conclu que le domaine C-terminal de Lin28, composé d’un motif riche en lysines et arginines ainsi que de deux motifs de liaison au zinc, permet à la protéine de lier spécifiquement et avec haute affinité un renflement riche en guanine conservé chez les précurseurs de la famille let-7. Aussi, parce que la séquence et la spécificité de liaison à l’ARN de ce domaine C-terminal sont semblables à celles de la protéine NCp7 du VIH, nous avons défini ce dernier comme le domaine NCp7-like de Lin28. Par la suite, nous avons caractérisé la multimérisation de trois protéines Lin28 sur la boucle terminale de pre-let-7g. Ceci a permis de réconcilier d’apparentes contradictions retrouvées dans la littérature actuelle concernant les sites de liaison de Lin28 lors de sa liaison aux miARN précurseurs. Nous avons identifié trois sites de liaison à haute affinité sur TL-let-7g qui sont liés dans un ordre précis par trois protéines Lin28. Lors de la formation du complexe multimérique, le CSD permet une déstabilisation de l’ARN, ce qui rend accessible plusieurs sites de liaison. Le domaine NCp7-like permet plutôt un assemblage ordonné de la protéine et facilite la liaison initiale de cette dernière. Ces nouveaux résultats rendent possible la mise au point d’un nouveau modèle de l’interaction entre Lin28 et le miARN précurseur let-7g. En conclusion, les études réalisées dans cette thèse apportent une meilleure compréhension des mécanismes moléculaires impliqués dans la régulation post-transcriptionnelle d’une importante famille de miARN et permettront de guider les futures études dans le domaine de recherche en pleine effervescence qu’est celui de la biogenèse des miARN. / The regulation of gene expression is what allows our cells to adapt to their environment, to fight infections or, more generally, to express the appropriate level of proteins to meet a specific need. The microRNAs (miRNAs) are among the most important players in the regulation of gene expression. These small RNAs of 22 nucleotides are present in most multicellular species and are responsible for the direct control of more than 30% of protein-expressing genes in vertebrates. The miRNA lethal-7 (let-7) family consist of some of the most studied miRNAs and plays crucial roles in the cell. The appropriate regulation of the let-7 miRNAs level is essential for proper cellular development. The biogenesis of these miRNAs, from the primary transcript to their mature form is mainly regulated by Lin28, a highly-conserved pluripotent protein. This protein is composed of a cold shock domain (CSD) and two zinc-binding domains. These RNA-binding domains allow Lin28 to bind and inhibit the maturation of the let-7 miRNA. The objective of this thesis is to characterize the interaction between the Lin28 protein and the let-7g miRNA precursor to better understand the role of this protein in the inhibition of miARN biogenesis. Using biochemical and biophysical techniques, we first identified the main determinants of the interaction between Lin28 and the terminal loop of the precursor miRNA let-7g (TL-let-7g). We concluded that the C-terminal domain of Lin28, composed of a lysine-rich and arginine-rich motif in addition to two zinc-binding motifs, is sufficient to bind with high affinity a conserved guanine-rich bulge located on the TL-let-7g. In addition, because the sequence and RNA-binding specificity of this C-terminal domain are similar to those of the HIV protein NCp7, we defined this region as the NCp7-like domain of Lin28. Subsequently, we characterized the multimerization of three Lin28 proteins on the terminal loop of pre-let-7g. This study helped to reconcile apparent contradictions found in the current literature regarding the Lin28-binding sites on miRNA precursors. We identified three high-affinity binding sites on TL-let-7g that are bound in a stepwise manner by the three Lin28 proteins. As part of the formation of the multimeric complex, both RNA-binding domains of Lin28 play an important role. The CSD destabilizes the RNA and this exposes several binding sites, whereas the NCp7-like domain allows an orderly protein assembly and facilitates the initial binding of the protein. These results lead us to propose a new model for the interaction between Lin28 and pre-let-7g. In conclusion, these studies provide a better understanding of the molecular mechanisms involved in the post-transcriptional regulation of an important family of miRNAs and will help guide future projects in the expanding research area of miRNA biogenesis.

Les différents mécanismes de régulation posttranscriptionnelle de l’expression des gènes sont de plus en plus reconnus comme des processus essentiels dans divers phénomènes physiologiques importants, comme la prolifération cellulaire et la réponse aux dommages à l’ADN. Deux des protéines impliquées dans ce type de régulation sont Staufen1 (Stau1) et Staufen2 (Stau2). Elles sont des protéines de liaison à l’ARN double brin qui contribuent au transport de l’ARN messager (ARNm), au contrôle de la traduction, à l’épissage alternatif et sont responsables de la dégradation de certains ARNm spécifiques. Les protéines Staufen peuvent en effet s’associer à des ARNm bien précis, d’autant plus que, majoritairement, Stau1 et Stau2 ne se retrouvent pas en complexe avec les mêmes cibles. De nombreuses évidences récentes montrent l’implication de divers mécanismes de régulation posttranscriptionnelle dans la réponse aux dommages à l’ADN, plusieurs protéines de liaison à l’ARN y participant d’ailleurs. De façon importante, cette réponse dicte un ou plusieurs destin(s) à la cellule qui doit réagir à la suite de dommages à l’intégrité de son ADN: réparation de l’ADN, arrêt de la prolifération cellulaire, apoptose. Nous avons donc fait l’hypothèse que l’expression de Stau1 et/ou de Stau2 pourrait être affectée en réponse à un stress génotoxique, ce qui pourrait avoir comme conséquence de moduler l’expression et/ou la stabilité de leurs ARNm cibles. De même, notre laboratoire a récemment observé que l’expression de Stau1 varie pendant le cycle cellulaire, celle-ci étant plus élevée jusqu’au début de la mitose (prométaphase), puis elle diminue alors que les cellules complètent leur division. Par conséquent, nous avons fait l’hypothèse que Stau1 pourrait lier des ARNm de façon différentielle dans des cellules bloquées en prométaphase et dans des cellules asynchrones. D’un côté, en employant la camptothécine (CPT), une drogue causant des dommages à l’ADN, pour traiter des cellules de la lignée de cancer colorectal HCT116, nous avons observé que seule l’expression de Stau2 est réduite de façon considérable, tant au niveau de la protéine que de l’ARNm. L’utilisation d’autres agents cytotoxiques a permis de confirmer cette observation initiale. De plus, nous avons constaté que l’expression de Stau2 est touchée même dans des conditions n’engendrant pas une réponse apoptotique, ce qui suggère que cette déplétion de Stau2 est possiblement importante pour la mise en place d’une réponse appropriée aux dommages à l’ADN. D’ailleurs, la surexpression de Stau2 conjointement avec le traitement à la CPT entraîne un retard dans l’induction de l’apoptose dans les cellules HCT116. Nous avons aussi montré que la diminution de l’expression de Stau2 est due à une régulation de sa transcription en réponse au stress génotoxique, ce pourquoi une région minimale du promoteur putatif de Stau2 est nécessaire. Également, nous avons identifié que le facteur de transcription E2F1, couramment impliqué dans la réponse aux dommages à l’ADN, peut contrôler l’expression de Stau2. Ainsi, E2F1 permet une augmentation de l’expression de Stau2 dans des cellules non traitées, mais cette hausse est abolie dans des cellules traitées à la CPT, ce qui suggère que la CPT pourrait agir en inhibant l’activation transcriptionnelle de Stau2 par E2F1. Enfin, nous avons observé que certains ARNm associés à Stau2, et codant pour des protéines impliquées dans la réponse aux dommages à l’ADN et l’apoptose, sont exprimés différemment dans des cellules traitées à la CPT et des cellules non traitées. D’un autre côté, nous avons identifié les ARNm associés à Stau1 lors de la prométaphase, alors que l’expression de Stau1 est à son niveau le plus élevé pendant le cycle cellulaire, grâce à une étude à grande échelle de micropuces d’ADN dans des cellules HEK293T. Nous avons par la suite confirmé l’association entre Stau1 et certains ARNm d’intérêts, donc codant pour des protéines impliquées dans la régulation de la prolifération cellulaire et/ou le déroulement de la mitose. Une comparaison de la liaison de ces ARNm à Stau1 dans des cellules bloquées en prométaphase par rapport à des cellules asynchrones nous a permis de constater une association préférentielle dans les cellules en prométaphase. Ceci suggère une augmentation potentielle de la régulation de ces ARNm par Stau1 à ce moment du cycle cellulaire. Les données présentées dans cette thèse indiquent vraisemblablement que la régulation posttranscriptionnelle de l’expression génique contrôlée par les protéines Staufen se fait en partie grâce à la modulation de l’expression de Stau1 et de Stau2 en fonction des conditions cellulaires. Nous envisageons alors que cette variation de l’expression des protéines Staufen ait des conséquences sur des sous-ensembles d’ARNm auxquels elles sont liées et que de cette façon, elles jouent un rôle pour réguler des processus physiologiques essentiels comme la réponse aux dommages à l’ADN et la progression dans le cycle cellulaire. / The various mecanisms of post-transcriptional regulation of gene expression are more and more recognized as essential processes in diverse important physiological phenomenons, like cell proliferation and the DNA damage response (DDR). Two of the proteins implicated in this type of regulation are Staufen1 (Stau1) and Staufen2 (Stau2). They are double-stranded RNA binding proteins contributing to messenger RNA (mRNA) transport, translation control, alternative splicing and are responsible for the degradation of some specific mRNAs. The Staufen proteins are indeed able to associate with particular mRNAs. Interestingly, Stau1 and Stau2 predominantly form complexes with different targets. Recent evidences show the implication of various post-transcriptional regulation mecanisms in the DDR, moreover several RNA binding proteins are involved. Importantly, this response dictates one or several cell fates following damage to the integrity of the cell’s DNA: DNA repair, cell proliferation arrest, apoptosis. We hypothesized that Stau1 and/or Stau2 expression could be affected in response to genotoxic stress, which could consequently modulate the expression and/or the stability of their mRNA targets. Also, our laboratory has recently observed that Stau1 expression varies during the cell cycle. It is elevated up to the beginning of mitosis (prometaphase) and it decreases as cells complete their division. We therefore hypothesized that Stau1 could differentially bind mRNAs in cells blocked in prometaphasis and in asynchronous cells. On the one hand, by using camptothecin (CPT), a DNA damaging agent, to treat cells from the colorectal cancer cell line HCT116, we observed that only the expression of Stau2 is considerably reduced, both at the level of the protein and that of the mRNA. The use of other cytotoxic agents allowed us to confirm this initial observation. We also noted that Stau2 expression is down-regulated even in conditions that do not induce apoptosis, suggesting that the decrease in Stau2 expression may be required for a proper DDR. Indeed, Stau2 overexpression together with the CPT treatment causes a delay in apoptosis induction in HCT116 cells. We also showed that Stau2 down-regulation is due to the regulation of its transcription in response to the genotoxic stress, which necessitates a minimal region in Stau2’s putative promoter. Besides, we identified the E2F1 transcription factor, commonly implicated in the DDR, as a regulator of Stau2 expression. E2F1 thus stimulates an increase in Stau2 expression in non-treated cells, but this up-regulation is abolished in CPT-treated cells, which suggests that CPT could act by inhibiting Stau2 transcriptional activation by E2F1. Finally, we observed that some Stau2-associated mRNAs, which code for proteins implicated in the DDR and apoptosis, are differentially expressed in CPT-treated cells compared to non-treated cells. On the other hand, we identified Stau1-associated mRNAs during prometaphase, when Stau1 expression is at its highest level in the cell cycle, by performing a large-scale study using DNA microarrays in HEK293T cells. We subsequently confirmed the association between Stau1 and some mRNAs of interest, mainly coding for proteins involved in the regulation of cell proliferation and/or mitosis progression. A comparison of the association between Stau1 and these mRNAs in prometaphase-blocked cells with that in asynchronous cells allowed us to notice a preferential association in prometaphase-blocked cells. This suggests a potential increase of the regulation of these mRNAs by Stau1 at that point of the cell cycle. The data presented in this thesis indicate that in all likelihood the post-transcriptional regulation of gene expression controlled by the Staufen proteins happens in part thanks to the modulation of Stau1 and Stau2 expression according to the cellular conditions. We then contemplate that this fluctuation in Staufen proteins expression has consequences on mRNA subsets with which they associate, and that this may mean they have an important role to play in regulating essential physiological processes like DDR and cell cycle progression.

L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella. / We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella.

Chez les eucaryotes, plusieurs protéines liant l'ARN ou RBPs agissent sur l'ARNm à différents niveaux, de l'épissage à la traduction. Récemment, un grand nombre d’ARN non-codant des protéines (npcRNAs) ont été identifiés chez les eucaryotes et ont été montré comme interagissant avec une variété de ribonucléoprotéines (RNP) pour contrôler l'expression des gènes au niveau post-transcriptionnel. Nous avons identifié une Nuclear-Speckle RBP (ou NSR) qui interagit avec le npcRNA, ENOD40, un lncARN qui s'accumule au cours de la formation des racines latérales et des nodules chez les légumineuses. Durant cette thèse nous avons analysé le rôle des NSR d’Arabidopsis thaliana ainsi que leur lien avec les npcARN.Deux gènes AtNSRs homologues existent chez Arabidopsis nommés NSRa et NSRb, ces gènes codent des protéines localisées dans des speckles nucléaires avec certaines protéines apparentées à l’épissage. Fait intéressant, les fusions AtNSR-GFP sont relocalisées dans des granules cytoplasmiques dans certaines cellules des racines différenciées ainsi que lors d’une co-expression éctopique de ENOD40. Le gène AtNSRb est régulé par l'auxine alors AtNSRa est constitutif. Les simples mutants Atnsr ne montrent pas de phénotype, mais la croissance des racines des doubles mutants est partiellement insensible à l'auxine, ce qui suggère une fonction redondante de ces protéines dans les racines. La localisation observée pour ces protéines nous a mené à explorer un rôle des NSRs dans l’épissage, nous avons donc analysé le profil d'épissage de 288 gènes en réponse à l'auxine chez Arabidopsis et comparé ces profils entre le WT et les mutants nsra/nsrb. Tout d’abord nous avons remarqué que l’épissage général ne variait pas, en revanche, l’analyse de 288 gènes alternativement épissés montre que le profil d'épissage de 77 gènes semble être modifié durant la réponse à l'auxine et 51 gènes nécessitent les protéines AtNSR pour ce changement. Afin de vérifier l’interaction des NSRs avec les cibles d’AS et avec les npcARN nous avons co-immunoprécipité les NSRs et nous avons identifié au moins 5 cible d’AS et 2 npcARN. L’expression de l’ARN ENOD40 ainsi que du partenaire npcARN module L’AS chez Arabidopsis. Dans un deuxième chapitre, nous avons exploré le rôle des NSRs dans le PTGS déclenché par un transgène contenant un intron ce qui nous a permis de lier l’épissage alternatif et le silencing. Nous proposons donc que les NSRs pourraient lier l’épissage alternatif et l’action des ARN non codants, notamment lors de la croissance de la racine. / In eukaryotes, several RNA binding proteins (RBPs) act on mRNA at various levels from splicing to translation. Recently a large number of non-protein coding RNAs (npcRNAs) have been identified in eukaryotes and shown to integrate into a variety of ribonucleoproteins (RNP) to control posttranscriptional gene expression. Our laboratory has identified a plant Nuclear-Speckle RBP (or NSR) that interacts with an npcRNA, ENOD40 that accumulates during lateral root and nodule formation in legumes. NSR is relocalised into a cytoplasmic RNP in the ENOD40-expressing cells. During this PhD, we have analysed the role of NSRs in Arabidopsis thaliana and its link with npcRNAs. Two AtNSR homologs from Arabidopsis thaliana, named AtNSRa and AtNSRb, code for proteins also localised in nuclear speckles together with certain splicing-related proteins. Interestingly, AtNSR-GFP fusions are relocalised into cytoplasmic granules in certain differentiated root cells and by ectopic expression of the ENOD40 RNA. The AtNSRb gene is regulated by auxin whereas AtNSRa is constitutive. Root growth and lateral root formation of double nsra/nsrb mutants is partially insensitive to auxin. The localisation of these proteins prompted us to explore roles in splicing. No defects in general splicing were observed however analysis of 288 alternatively spliced genes in WT and nsra/nsrb roots in response to auxin revealed 77 changes in splicing profiles in response to auxin from which 51 required AtNSRs. In order to validate the interaction of NSRs with alternatively spliced mRNAs and npcRNAs, we have co-immunoprecipitated NSRs and identified at least 5 interacting alternatively spliced mRNAs and 2 npcRNAs. Expression of the ENOD40 RNA or one interacting ncRNA modulate alternatively splicing in Arabidopsis. In a second chapter, we explored the role of NSRs in the modulation of PTGS triggered by intron-containing transgenes allowing us to link alternatively splicing and silencing. We propose that NSRs may link alternative splicing and the action of non-coding RNA, notably during root growth and development.

<p>Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.</p><p>Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.</p><p>Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. <i>Trans</i>-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.</p><p>In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.</p>

One of the central themes of biology is to understand how individual cells achieve a high fidelity in gene expression. Each cell needs to ensure accurate protein levels for its proper functioning and its capability to proliferate. Therefore, complex regulatory mechanisms have evolved in order to render the expression of each gene dependent on the expression level of (all) other genes. Regulation can occur at different stages within the framework of the central dogma of molecular biology. One very effective and relatively direct mechanism concerns the regulation of the stability of mRNAs. All organisms have evolved diverse and powerful mechanisms to achieve this. In order to better comprehend the regulation in living cells, biochemists have studied specific degradation mechanisms in detail. In addition to that, modern high-throughput techniques allow to obtain quantitative data on a global scale by parallel analysis of the decay patterns of many different mRNAs from different genes. In previous studies, the interpretation of these mRNA decay experiments relied on a simple theoretical description based on an exponential decay. However, this does not account for the complexity of the responsible mechanisms and, as a consequence, the exponential decay is often not in agreement with the experimental decay patterns. We have developed an improved and more general theory of mRNA degradation which provides a general framework of mRNA expression and allows describing specific degradation mechanisms. We have made an attempt to provide detailed models for the regulation in different organisms. In the yeast S. cerevisiae, different degradation pathways are known to compete and furthermore most of them rely on the biochemical modification of mRNA molecules. In bacteria such as E. coli, degradation proceeds primarily endonucleolytically, i.e. it is governed by the initial cleavage within the coding region. In addition, it is often coupled to the level of maturity and the size of the polysome of an mRNA. Both for S. cerevisiae and E. coli, our descriptions lead to a considerable improvement of the interpretation of experimental data. The general outcome is that the degradation of mRNA must be described by an age-dependent degradation rate, which can be interpreted as a consequence of molecular aging of mRNAs. Within our theory, we find adequate ways to address this much debated topic from a theoretical perspective. The improvements of the understanding of mRNA degradation can be readily applied to further comprehend the mRNA expression under different internal or environmental conditions such as after the induction of transcription or stress application. Also, the role of mRNA decay can be assessed in the context of translation and protein synthesis. The ultimate goal in understanding gene regulation mediated by mRNA stability will be to identify the relevance and biological function of different mechanisms. Once more quantitative data will become available, our description allows to elaborate the role of each mechanism by devising a suitable model. / Ein zentrales Ziel der modernen Biologie ist es, ein umfassendes Verständnis der Genexpression zu erlangen. Die fundamentalen Prozesse sind im zentralen Dogma der Genexpression zusammengefasst: Die genetische Information wird von DNA in Boten-RNAs (mRNA) transkribiert und im Prozess der Translation von mRNA in Proteine übersetzt. Zum Erhalt ihrer Funktionalität und der Möglichkeit von Wachstum und Fortpflanzung muss in jeder Zelle und für jedes Gen die optimale Proteinkonzentration akkurat eingestellt werden. Hierzu hat jeder Organismus detaillierte Regulationsmechanismen entwickelt. Regulation kann auf allen Stufen der Genexpression erfolgen, insbesondere liefert der Abbau der mRNA-Moleküle einen effizienten und direkten Kontrollmechanismus. Daher sind in allen Lebewesen spezifische Mechanismen - die Degradationsmechanismen - entstanden, welche aktiv den Abbau befördern. Um ein besseres Verständnis von den zugrunde liegenden Prozessen zu erlangen, untersuchen Biochemiker die Degradationsmechanismen im Detail. Gleichzeitig erlauben moderne molekularbiologische Verfahren die simultane Bestimmung der Zerfallskurven von mRNA für alle untersuchten Gene einer Zelle. Aus theoretischer Perspektive wird der Zerfall der mRNA-Menge als exponentieller Zerfall mit konstanter Rate betrachtet. Diese Betrachtung dient der Interpretation der zugrunde liegenden Experimente, berücksichtigt aber nicht die fundierten Kenntnisse über die molekularen Mechanismen der Degradation. Zudem zeigen viele experimentelle Studien ein deutliches Abweichen von einem exponentiellen Zerfall. In der vorliegenden Doktorarbeit wird daher eine erweiterte theoretische Beschreibung für die Expression von mRNA-Molekülen eingeführt. Insbesondere lag der Schwerpunkt auf einer verbesserten Beschreibung des Prozesses der Degradation. Die Genexpression kann als ein stochastischer Prozess aufgefasst werden, in dem alle Einzelprozesse auf zufällig ablaufenden chemischen Reaktionen basieren. Die Beschreibung erfolgt daher im Rahmen von Methoden der stochastischen Modellierung. Die fundamentale Annahme besteht darin, dass jedes mRNA-Molekül eine zufällige Lebenszeit hat und diese Lebenszeit für jedes Gen durch eine statistische Lebenszeitverteilung gegeben ist. Ziel ist es nun, spezifische Lebenszeitverteilungen basierend auf den molekularen Degradationsmechanismen zu finden. In dieser Arbeit wurden theoretische Modelle für die Degradation in zwei verschiedenen Organismen entwickelt. Zum einen ist bekannt, dass in eukaryotischen Zellen wie dem Hefepilz S. cerevisiae mehrere Mechanismen zum Abbau der mRNA-Moleküle in Konkurrenz zueinander stehen. Zudem ist der Abbau durch mehrere geschwindigkeitsbestimmende biochemische Schritte charakterisiert. In der vorliegenden Arbeit wurden diese Feststellungen durch ein theoretisches Modell beschrieben. Eine Markow-Kette stellte sich als sehr erfolgreich heraus, um diese Komplexität in eine mathematisch-fassbare Form abzubilden. Zum anderen wird in Kolibakterien die Degradation überwiegend durch einen initialen Schnitt in der kodierenden Sequenz der mRNA eingeleitet. Des Weiteren gibt es komplexe Wechselwirkungen mit dem Prozess der Translation. Die dafür verantwortlichen Enzyme - die Ribosomen - schützen Teile der mRNA und vermindern dadurch deren Zerfall. In der vorliegenden Arbeit wurden diese Zusammenhänge im Rahmen eines weiteren spezifischen, theoretischen Modells untersucht. Beide Mechanismen konnten an experimentellen Daten verifiziert werden. Unter anderem konnten dadurch die Interpretation der Zerfallsexperimente deutlich verbessert und fundamentale Eigenschaften der mRNA-Moleküle bestimmt werden. Ein Vorteil der statistischen Herangehensweise in dieser Arbeit liegt darin, dass theoretische Konzepte für das molekulare Altern der mRNAs entwickelt werden konnten. Mit Hilfe dieser neuentwickelten Methode konnte gezeigt werden, dass sich die Komplexität der Abbaumechanismen in einem Alterungsprozess manifestiert. Dieser kann mit der Lebenserwartung von einzelnen mRNA-Molekülen beschrieben werden. In dieser Doktorarbeit wurde eine verallgemeinerte theoretische Beschreibung des Abbaus von mRNAMolek ülen entwickelt. Die zentrale Idee basiert auf der Verknüpfung von experimentellen Zerfallsmessungen mit den biochemischen Mechanismen der Degradation. In zukünftigen experimentellen Untersuchungen können die entwickelten Verfahren angewandt werden, um eine genauere Interpretation der Befunde zu ermöglichen. Insbesondere zeigt die Arbeit auf, wie verschiedene Hypothesen über den Degradationsmechanismus anhand eines geeigneten mathematischen Modells durch quantitative Experimente verifiziert oder falsifiziert werden können.

Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented. Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA. Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs. In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.

Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli. RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V. RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity. These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.

Indiana University-Purdue University Indianapolis (IUPUI) / My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.