Le génome du bananier (Musa sp.) est envahi par un nombre important de séquences de Banana streak virus (BSV), virus à ADN double brin de la famille des Caulimoviridae qui n'a aucune étape d'intégration au génome hôte au cours de son cycle de multiplication. La majorité de ces intégrations eBSV (endogenous BSV) est défective mais certaines sont restées fonctionnelles et peuvent être à l'origine de particules virales suite à des stress. L'objectif de ce travail de thèse est de préciser si les eBSV sont maintenus ou non dans le génome de Musa balbisiana des bananiers et d'étudier les conséquences évolutives que cela engendre. Nous avons tout d'abord caractérisé les eBSV pour trois espèces de BSV (Banana streak goldfinger virus (BSGFV), Banana streak obino l'ewai virus (BSOLV), Banana streak imove virus (BSImV) présents dans le génome du bananier modèle M. balbisiana cv Pisang Klutuk Wulung (PKW). Nous avons montré que les intégrations eBSGFV et eBSOLV étaient di-alléliques avec un seul allèle fonctionnel à chaque fois, contrairement à eBSImV qui est mono-allélique et pour lequel nous n'avons pas pu identifier l'allèle à l'origine de l'infection. Leur contexte génomique d'intégration diffère avec une co-localisation d'eBSGFV et d'eBSOLV sur le chromosome 1 et d'eBSImV sur le chromosome 2. Ces résultats nous ont permis de développer les outils moléculaires nécessaires à la caractérisation de ces trois eBSV dans la diversité de M. balbisiana. Cette caractérisation a révélé la diversité de structures des eBSV et éclairé une partie encore inconnue de la phylogénie de l'espèce M. balbisiana. Dans un second temps nous avons étudié les mécanismes de régulation des eBSV. Ce travail a porté sur les mécanismes d'ARN interférent pouvant expliquer le maintien des eBSV dans le génome des bananiers. Cette analyse révèle que les eBSV sont effectivement sous contrôle d'un mécanisme de type ARNi et la forte production de petits ARNs de 24nt ciblant les eBSV suggère qu'il s'agit d'un silencing au niveau transcriptionnel (TGS). En parallèle, nous avons aussi recherché les mécanismes mis en place par les bananiers non-porteurs d'eBSV en cas d'infection afin de connaître les défenses constitutives des bananiers face à une attaque virale BSV. Nous avons, sur la base de ces résultats, proposé un modèle de régulation des eBSV et des BSV et discuté de l'impact que ces mécanismes auraient pu avoir sur l'évolution des eBSV. L'ensemble des données de ce travail ont permis de préciser les étapes évolutives qu'ont connues les eBSV dans le génome du bananier, expliquant le maintien que l'on observe aujourd'hui. / The nuclear genome of banana plants is invaded by numerous viral sequences of banana streak virus (BSV), a DNA virus belonging to the family Caulimoviridae which does not require integration for its replication. These endogenous BSV (eBSV) are mostly defective; however, some can release a functional viral genome following activating stresses. The objectives of this work were to identify if the eBSV are maintained or not in the M. balbisiana genome and to study the impacts of this on the evolution of banana plants. First, we characterized three functional eBSV sequences present within the Musa balbisiana cv PKW genome: (Banana streak goldfinger virus (BSGFV) ; Banana streak obino l'ewai virus (BSOLV) ; and, Banana streak imove virus (BSImV). We show that eBSOLV and eBSGFV are di-allelic with just one functional allele contrary to eBSImV which are mono-allelic and for which we cannot reveal the functional allele. Their genomic areas of integration are different and we also observe that eBSOLV and eBSVGFV are both on chromosome 2 whereas eBSImV is on chromosome 1. These results allowed us to develop the molecular tools required for the characterization of these 3 functional eBSVs within the diversity of M. balbisiana. This characterization has revealed the structural diversity of eBSV and has thus clarified previously unresolved details of M. balbisiana phylogeny. Secondly, we studied the regulatory mechanism of eBSV expression. This work investigated if RNA interference (RNAi) mechanisms could explain the maintenance of eBSV in the Musa genome. Our analyses have shown that, as expected, eBSV was under the control of RNAi mechanisms and the strong production of 24nt small RNAs that target eBSV suggests that Transcriptional Gene Silencing (TGS) was involved in this control. In parallel, we investigated the mechanisms implicated in the anti-viral defense during a BSV infection on a banana plant without eBSV in order to understand the constitutive defense of banana plants. On the basis of these results we have proposed a regulation model of eBSV and BSV and we discuss the impact of silencing regulation on eBSV evolution. Data accumulated during this work have clarified several steps in the co-evolutionary history of Musa sp. and eBSV and explain the maintenance of eBSVs in Musa genomes that we observe today.

Ce travail de thèse porte sur l’étude des ARN polymérases ARN dépendant impliquées dans le RNA silencing chez Arabidopsis thaliana. Durant ma thèse, la recherche d'interacteurs des RDR, parmi des protéines impliquées dans le RNA silencing, a permis la détection d'interaction entre RDR6 et SDE3, RDR6 et SGS3, mais aussi entre SDE3 et SGS3 en Co-IP et BiFC. Une co-localisation de ces protéines a été observée lorsqu'elles sont produites transitoirement dans des cellules épidermales de N. benthamiana.Un crible d’une banque d’ADNc d’A. thaliana par double hybride de levure, a permis d’isoler des interacteurs potentiels de RDR6. Deux interacteurs potentiels, AtUAP56-1 et U2B’’, sont impliqués dans l’épissage des précurseurs des ARNm. Un effet sur le RNA silencing dans des mutants de l’épissage en 3’ des ARNm était connu et nous avons confirmé l’interaction entre RDR6 et AtUAP56-1 par BiFC. L’étude de lignées mutantes pour AtUAP56-1 a donc été initiée.Une étude biochimique de RDR6 et de RDR2 a été réalisée. Des formes recombinantes de RDR2 et RDR6 ont été produites de façon transitoire dans des feuilles de N. benthamiana, et une étude comparative de RDR2 et RDR6 a été réalisée. Les deux RDR sont actives sur des matrices ARN et ADN, et montrent in vitro une activité amorce-indépendante. De plus, nous avons détecté pour la première fois une activité amorce-dépendante de RDR6 et RDR2. Ces résultats apportent de nouvelles données biochimiques qui sont en accord avec les études menées in vivo et enrichissent les modèles actuels du RNA silencing. / The aim of this work was to study RNA-dependent RNA polymerases involved in RNA silencing in Arabidopsis thaliana. During my thesis, the search for RDR interactors among proteins involved in RNA silencing allowed the detection of interactions between RDR6 and SDE3, RDR6 and SGS3, and also between SDE3 and SGS3 using Co-IP and BiFC. In addition, the co-localisation of these proteins was observed when produced transiently in epidermal cells of N. Benthamiana.A screen of an A. thaliana cDNA library by yeast two hybrid allowed us to identify some putative new RDR6 interactors. Two putative RDR6 interactors, AtUAP56 and U2B’’, are known to be involved in pre-miRNA splicing. Furthermore, a link between pre-mRNA 3’ splicing and RNA silencing was previously reported. We also confirmed the interaction between AtUAP56-1 and RDR6 by BiFC. An investigation of A. thaliana of AtUAP56-1 mutants has been initiated.Recombinant RDRs were produced transiently in N. Benthamiana, and a biochemical comparative study of RDR2 and RDR6 performed. We found that RDR2, like RDR6, has a de novo polymerase activity on DNA and RNA templates, and for both RDRs we also showed, for the first time, a primer-dependant synthesis of dsRNA from RNA template. These findings provide important new insights into our understanding of the molecular mechanisms of RNA silencing amplification in Arabidopsis.

In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes, and various other chromatin proteins. Chromatin structure varies along the chromosome and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (silent or transcriptionally repressed). In the budding yeast, Saccharomyces cerevisiae, there are four canonical transcriptionally silent regions, namely, the HMR, the HML (cryptic mating loci), the telomeres and the RDN1. Silencing at the HM loci and the telomeres is very well characterized. The repressive structure at the HMR spans around 3.5 Kb and extends between the two silencers E and I. It is well established that silencing in HMR is due to a specialized chromatin organization brought about by Orc1p, Rap1p, Abf1p and Sir proteins. Following recruitment, the Sir proteins spread along the DNA to form a repressive chromatin domain believed to arise from the deacetylation of amino-terminal tails of histones H3 and H4 by Sir2p (an NAD dependent deacetylase) and the interaction of Sir3p and Sir4p with the histones. The bi-directional spreading of silencing at HMR is restricted by barrier or boundary elements that flank the silencers. A tRNAThr gene in the right boundary of HMR acts as a strong barrier. Mutations in the promoter of this tRNA gene (tDNA) or in RNA polymerase III subunits/ transcription factors weaken the barrier activity of this tDNA. The barrier activity of this tDNA is also dependent on histone acetyltransferases like Sas2p and Gcn5p. Silencing in HML is uniformly high between the silencers E and I and falls sharply outside I. Recently, barriers to HML silencing have been discovered. A 0.71Kb sequence near E, which maps to the upstream activating sequence of YCL069W, acts as a robust barrier to spread of HML silencing. This is effectively the left boundary of silent HML. The right boundary maps to the promoter of CHA1 gene though silencing is believed to terminate at HML-I. An unusual form of silencing occurs at the RDN1, which contains 100-150 copies of tandemly repeated rRNA genes. Some RNA polymerase II transcribed genes integrated within the array are silenced by a Sir2p dependent mechanism whereas genes driven by RNA polymerases III and I are transcriptionally active. Though all the three forms of silencing (RDN1, HM and telomere) require Sir2p, RDN1 silencing differs from the others in its relative strength and factors responsible for repression. Several trans-acting factors required for RDN1 silencing are known. However, it is still unclear as to what limits the spread of RDN1 heterochromatin into neighbouring essential genes. RDN1 silencing spreads unidirectionally in its left hand side sequence. However, the zone of RDN1 heterochromatin does not engulf the essential gene, ACS2, which is present ~3 kb away from NTS1. This implies that there is a mechanism by which rDNA heterochromatin is contained. There could be several ways by which this is accomplished. Firstly, the cell could be critically maintaining the levels of Sir2p, the protein required for silencing at all the four silenced loci, such that silencing in the left flank of RDN1 does not spread beyond 300 bp of NTS1 (Buck et al, 2002). There is a ~2.5 kb gene free intervening sequence between NTS1 of the rDNA array and the Ty1 LTR, in which interval Sir2p level could fall below the threshold mark required for causing repression. In fact Buck et al. have demonstrated that Sir2p is bound to upto 1.5 kb from the NTS1 in the left flank but there is no accompanying silencing of the mURA3 reporter in these regions (1200L and 2000L), suggesting that the level of Sir2p at these sequences could be lower than the threshold required for initiation of silencing. Secondly, there could be cis-acting boundary elements or barriers as in the case of HMR, which prevents the propagation of RDN1 silencing. The third option is that termination of RNA polymerase I transcription at the terminator sites automatically halts the spread of rDNA silencing since Buck et al. have demonstrated that progression of rDNA heterochromatin is dependent on RNA pol I transcription. This however, does not seem to be the case as deletion of both the terminator sites within NTS1 does not lengthen the zone of silencing. Finally, there could be an euchromatin organizing center further from the array, which creates an “open” chromatin configuration required to confront the Sir2p mediated condensed chromatin. The balance of these two opposing activities, much like that at the telomeres, could set up a molecular boundary for containing rDNA silent chromatin. We have attempted to identify whether there are any sequences in the unique left flank of RDN1 that can act as a heterochromatin barrier. Towards that end we tested four overlapping fragments from NTS1 of RDN1 to the promoter of ACS2 for boundary activity in a quantitative mating assay. We have found that of all the four fragments tested, only a 0.427 kb tRNAGln-Ty1 LTR fragment, which is present 2.4 Kb from the NTS1 acts as a robust barrier in this assay. Further mapping revealed that the barrier activity of this sequence resides in the tRNAGln gene and that its activity is orientation-independent. tDNAs are transcribed by RNA polymerase III from internal promoters termed Box A and Box B. It has been shown for the HMR-tRNAThr that the transcriptional potential of the tDNA is crucial for its barrier function. Mutations in genes encoding various subunits of the RNA polymerase III complex, or transitions in the conserved bases within Box B known to disrupt transcription complex assembly and subsequent transcription, abrogate the barrier activity of HMR-tRNAThr. Similarly, loss of transcriptional ability of the tRNAAla in the centromere of S. pombe also abolishes its barrier activity, enforcing the fact that RNA polymerase III transcription is a decisive factor for a tDNA barrier. Contrary to the above observations, we report that barrier activity of tRNAGln is very negligibly dependent on RNA polymerase III mediated transcription. Mating assays done with the RNA pol III mutants and promoter point mutants, G18C and C55G in boxes A and B respectively, underline the fact that for this tDNA barrier, RNA pol III driven transcription is dispensable. We also show by RT-PCR analysis that in the C55G tRNAGln mutant there is loss of transcription as expected, whereas other wild type copies of tRNAGln are transcribed. Studies with another tDNA barrier, TRT2-tRNAThr, yielded similar results, again emphasizing the point that transcription through the tDNA, which leads to nucleosome displacement and therefore barrier activity, may not be applicable for all tDNA barriers. Acetylation of amino terminal tails of histones is known to influence the epigenetic state of chromatin. Addition of acetyl moiety to histones H3 and H4 initiates a cascade of events, which involves recruitment of a host of other chromatin modifiers to the target sequence, and ultimately culminates in the formation of an euchromatin-favouring environment. As reported for the HMR right boundary, we find that barrier activity of tRNAGln depends on two histone acetyl transferase complexes, SAS-I (comprised of Sas2p, Sas4p and Sas5p) and SAGA (contains Gcn5p HAT). Contrary to the HMR boundary, the barrier activity of tRNAGln is independent of two other nucleoplasmic HATs, NuA3 (Sas3p being the HAT) and NuA4 (Esa1p is the HAT). Barrier function of TRT2-tRNAThr also depends on HATs. Therefore it appears that requirement of HATs for boundary activity is a conserved theme, albeit with differential effects at different barrier sequences. We next attempted to determine the function of tRNAGln in its natural location on chromosome XII. As mentioned earlier, RDN1 silencing spreads upto ~0.3 kb in its left flanking sequence. However, Sir2p occupancy has been observed till 1.5 kb although there is no silencing of reporter genes observed beyond 0.3 kb of NTS1. This lead us to speculate that there could be a boundary sequence in the left flank that stops silencing, or a euchromatin-organizing element, which counters the propagation of silencing by a long-range effect. Since over expression of Sir2p extends the domain of silencing from 0.3 kb to 2.0 kb and the tRNAGln is present at 2.3 kb from NTS1, it was a good candidate for a heterochromatin barrier/ euchromatiniser. However, deletion of tRNAGln does not affect the zone of RDN1 silencing as is evident from our cell viability assays (which is a measure of the expression of the essential gene ACS2 situated further to the left of tRNAGln). Deletion of SAS2 and GCN5, factors that are required for barrier activity of tRNAGln in mating assays, also have no effect on the extent of spreading of RDN1 silencing in normal or Sir2p over expression conditions. Together, these observations imply that in situ, tRNAGln does not act as a barrier or an element with long-range euchromatin inducing properties. It still remains unclear as to what contains RDN1 silencing. It is possible that the cell critically monitors the level of Sir2p in order to maintain boundaries of silencing at the rDNA locus. Telomeres also nucleate the formation of silenced domain which spreads along the subtelomeric region upto ~ 2Kb. The key players in the formation of telomeric heterochromation are the Sir proteins, Sir2p, Sir3p and Sir4p, Rap1p, yKu complex and ORC. Protein-protein interactions between the telosome and the subtelomeric repeat bound silencing proteins create a domain of core heterochromatin that spreads in the adjacent sequences. While Sir2p deacetylates H4K16, Sir3p interacts with the hypoacetylated histone tails and helps in the spreading of the repressive chromatin structure. As a result telomere proximal genes are silent whereas the ones further away are expressed. There is a gradient of acetylation of histone H4, with the hypoacetylated histones at the telomeric ends and the hyperacetylated ones distant from the telomere. Recently it has been shown that this gradient is maintained by the concerted and antagonistic actions of Sir2p and Sas2p. In a sas2Δ strain Sir3p spreads to ~15 kb in the subtelomeric regions and there is increase in the levels of hypoacetylated histones. Though the molecular mechanism by which telomeric silencing is restrained is beginning to be understood, it remains unanswered whether there are any cis-acting sequences, capable of recruiting euchromatin-inducing factors such as Sas2p, near the telomeres. We have identified a RNA polymerase II driven gene, AAD3, in the subtelomeric region of chromosome III that has robust anti-silencing activity. Deletion mapping revealed that only 0.381 kb in the 5′ portion of the gene (excluding the promoter) is sufficient for barrier activity and that this property is orientation-independent (henceforth referred to as TEL-B). The barrier acivity of TEL-B depends strongly on Sas2p and Esa1p but not on Gcn5p and Sas3p, and is independent of cohesin. Previous investigations have shown that acetylation of H4K16 by Sas2p at subtelomeric regions of chromosome VI leads to deposition of HTZ1 in the nucleosome and its subsequent acetylation by Esa1p of NuA4. All these events together are required to contain the onslaught of telomeric core heterochromatin on neighbouring active regions. Since barrier activity of TEL-B depends on Sas2p and Esa1p, it is possible that TEL-B has the potential to act as a bona fide barrier in situ in its endogenous context. Our hypothesis is further cemented by the observation that there is a physical association between Sas2p, the molecule at the top of the entire cascade of events, with TEL-B by yeast one hybrid analysis. Further experiments will shed light on the role of this sequence in its natural location. In summary, I have identified and characterized two different barrier sequences in S. cerevisiae. Not many barriers are known in budding yeast and there is extensive ongoing research dedicated to understand the mechanism(s) of barrier function. In chapter I of my thesis I present a review of current literature regarding silencing barriers in yeast and other systems. In chapter II I have outlined a detailed characterization of a tDNA barrier element, tRNAGln, present near the silenced rDNA array on chromosome XII. My work addresses the various models for barrier activity and their applicability to the tRNAGln barrier. I have also attempted to understand the role of this tDNA in its natural location on the chromosome with respect to limitation of RDN1 silencing. In chapter III I have described an intensive study of a RNA polymerase II transcribed gene, AAD3, present near the right telomere of chromosome III, which acts as a robust barrier to silencing. I have attempted to answer which mechanism(s) is/are operational at this sequence so as to endow it with barrier potential. My studies with the two barrier elements highlight novel trans-acting factors required for barrier function, differential and selective requirements of certain factors for different barriers, and provide a mechanistic view of the boundary activity of these sequences.

Une fraction non négligeable de protéines qui contrôlent la dynamique de la chromatine et la transcription est conservée au cours de l'évolution chez les eucaryotes. Ces protéines se retrouvent dérégulées dans de nombreuses maladies, dont les cancers. Dans cette étude, nous avons exploité la purification de deux protéines associées à la chromatine pour étudier de nouveaux acteurs impliqués dans la réduction au silence (ou silencing) de la transcription au sein de l'hétérochromatine et/ou de l'euchromatine chez la levure Schizosaccharomyces pombe, un modèle de référence pour la biologie de la chromatine.Mmi1 est un facteur de liaison à l'ARN capable de guider la formation d'hétérochromatine facultative sur des gènes méiotiques. Parmi les protéines partenaires de Mmi1, nous nous sommes intéressés à Ccr4-Not, un complexe multifonctionnel, conservé de la levure à l'Homme, important pour la maturation de l'extrémité 3' des ARNs et pour le contrôle de l'expression des gènes. Nos travaux montrent que Ccr4-Not est également nécessaire pour le dépôt de la marque H3K9 méthylée aux gènes cibles de Mmi1, ainsi que pour le silencing de la transcription au sein de l'hétérochromatine constitutive, indépendamment de Mmi1.En parallèle, nous avons étudié deux nouveaux partenaires potentiels de RITS (RNA-Induced Transcriptional Silencing), un complexe nécessaire à la formation de l'hétérochromatine et l'inactivation de gène. Ces partenaires agiraient à l'interface entre la régulation de la chromatine et de la transcription. Le premier partenaire est l'histone chaperonne Spt6. Une caractérisation initiale entreprise sur Spt6 a montré son rôle crucial dans le silencing des gènes à l'hétérochromatine constitutive et facultative. Le second partenaire est Abo1, une histone chaperonne putative et homologue à la protéine humaine ATAD2, une protéine exprimée dans de nombreuses tumeurs et considérée comme une cible prometteuse pour le traitement de certains cancers, bien qu'à ce jour il n'y ait que peu d'information disponible sur sa fonction moléculaire. Nous avons dans un premier temps montré qu'Abo1 est nécessaire pour le silencing de la transcription au sein de l'hétérochromatine constitutive. Cependant, l'analyse du transcriptome des cellules abo1Δ a montré qu'Abo1 est également nécessaire au silencing transcriptionel de nombreux gènes codant et non-codant localisés dans l'euchromatine. Par la suite, nous avons purifié Abo1 et identifié par spectrométrie de masse le réseau des protéines qui lui est associé. Cette approche protéomique a montré qu'Abo1 est connectée à de nombreuses protéines impliquées dans le contrôle de la transcription, comme des histones chaperonnes et des complexes de remodelage ATP-dépendant de la chromatine. Enfin, nous avons montré que le défaut de croissance sévère observé dans les cellules abo1Δ est complètement rétabli par l'expression de ATAD2 humain. Ce dernier résultat indique que la caractérisation fonctionnelle d'Abo1, entreprise dans la levure, a le potentiel de fournir des informations importantes sur la fonction moléculaire non seulement d'Abo1, mais aussi d'ATAD2 et de son lien avec les cancers.En résumé, nos résultats permettent une meilleure compréhension de la fonction de trois acteurs impliqués dans le silencing de la transcription chez la levure fissipare. De plus, la caractérisation plus approfondie d'Abo1 pourrait grandement contribuer à élucider la fonction d'ATAD2 et de son rôle dans les cancers. / A sizeable fraction of proteins controlling chromatin dynamics and transcription are conserved throughout eukaryotes and are deregulated in many diseases, including cancer. In this study, we exploited the purification of two chromatin-associated proteins to characterize new actors in the context of euchromatic and/or heterochromatic gene silencing in Schizosaccharomyces pombe, a reference model for the biology of chromatin.Mmi1 is an RNA binding factor that can guide the formation of facultative heterochromatin assembly at meiotic genes. Among new proteins interacting with Mmi1, we examined the function of Ccr4-Not, which is a conserved multifunctional complex processing 3'ends of RNAs and regulating gene expression. We found that Ccr4-Not is also required for the deposition of H3K9 methylation mark at Mmi1 target genes and for gene silencing in a Mmi1-independent manner at constitutive heterochromatin.In parallel, we studied two new potential partners of RITS (RNA-Induced Transcriptional Silencing), a complex required for heterochromatin formation and gene silencing. Both partners are believed to act at the interface between chromatin and transcription regulation. The first one is the histone chaperone Spt6. An initial functional characterization conducted on this protein showed its implication in gene silencing, both at constitutive and facultative heterochromatin. The second one is Abo1, a putative histone chaperone which is homologue to human ATAD2 protein, a male germ factor ectopically expressed in many tumors and considered as a promising target for cancer therapy, although little is known about its molecular function. We first showed that Abo1 is necessary for proper heterochromatin gene silencing at constitutive heterochromatin. However, transcriptomic analysis of abo1∆ cells further extended Abo1's function in gene silencing to protein-coding and non-coding regions within euchromatin. In addition, we purified Abo1 and identified by mass spectrometry the network of its associated proteins. This proteomic approach showed that Abo1 is connected to several chromatin- and transcription-linked proteins, such as histone chaperones and ATP-dependent chromatin remodeling complexes. Finally, we demonstrated that the severe growth defect observed in abo1Δ is completely rescued by the expression of human ATAD2. This later finding indicates that the functional characterization of Abo1 in yeast has the potential to provide important insights into the molecular function not only of Abo1, but also of the cancer linked ATAD2 protein.Altogether, our results permitted a better understanding of three actors involved in chromatin-based gene silencing in fission yeast. In addition, a further characterization of Abo1 may contribute elucidating the function of ATAD2 and its role in cancer.

Les complexes polyazaaromatiques de ruthéniumII, et en particulier le [Ru(bpy)3]2+, ont fait l’objet de nombreuses études fondamentales en photochimie et photophysique. Du fait de ses propriétés photophysiques, et entre autres grâce à son temps de vie de luminescence relativement long, le [Ru(bpy)3]2+ est devenu un composé modèle en photophysique. Dès les années 1970, et principalement grâce aux travaux de T.J. Meyer, la photophysique du [Ru(bpy)3]2+ a été étudiée en détail afin de permettre l’élaboration d’un modèle photophysique qui peut être valablement étendu aux autres complexes polyazaaromatiques de RuII. La caractérisation du complexe [Ru(bpy)2(dppz)]2+ et de ses interactions avec l’ADN a, elle, promu l’étude des complexes de RuII en présence de biomolécules et a encouragé la recherche pour l’utilisation de complexes de ruthénium comme photosondes en biochimie.<p>Dans ce cadre, le laboratoire de Chimie Organique et Photochimie de l’ULB s’est attaché au développement de complexes polyazaaromatiques de ruthéniumII se caractérisant par leur capacité à photoréagir avec certaines biomolécules. Ces complexes se caractérisent par l’utilisation de ligands fortement π-déficients, comme le 1,2,4,5,8-tétraazaphénanthrène (TAP). Nettement plus photooxydant que les complexes analogues au [Ru(bpy)3]2+, ces complexes photooxydants sont capables, sous irradiation, de donner lieu à un transfert d’électron depuis la base guanine de l’ADN vers le complexe excité. Les deux entités radicalaires ainsi formées peuvent ensuite réagir entre elles pour former un photoadduit au sein duquel un lien covalent lie irréversiblement un ligand TAP du complexe à la guanine.<p><p>Les travaux réalisés dans le cadre de cette thèse de doctorat s’inscrivent dans la poursuite de la recherche effectuée au sein du laboratoire autour de cette photoréaction. Deux axes majeurs ont été développés. Un premier axe de recherche a été dédié à l’étude fondamentale des propriétés photophysiques et photochimiques du photoadduit obtenu suite à la photoréaction du [Ru(TAP)3]2+ avec une base guanine. Cette étude photophysique fondamentale de l’adduit [Ru(TAP)2(TAP-GMP)] (présentée dans le deuxième chapitre) vise à caractériser sa photophysique afin de comprendre comment, sous irradiation, des biadduits entre un complexe de ruthénium et deux guanines sont observés, alors que les premières études réalisées sur les photoadduits indiquent que ceux-ci ne sont pas luminescents. Le second axe de recherche consiste en la mise au point de systèmes élaborés à base des complexes de ruthénium visant à contrôler leur photoréactivité dans un milieu biologique. Pour ce faire, les complexes de ruthénium photoréactifs ont été ancrés sur des molécules biologiques. D’une part, les complexes ont été conjugués sur des OAS, oligonucléotides anti-sens, afin de conférer aux conjugués résultants la possibilité de cibler une partie précise de l’ADN ou d’ARN, et mener, in fine, au blocage de l’expression d’un gène particulier. Ces conjugués ont déjà été étudiés par le passé dans notre laboratoire. Les résultats présentés ici (chapitre 3) permettent à la fois de mieux comprendre la photochimie des Ru-OAS en présence de leur cible spécifique, ainsi que de démontrer in vivo la validité de la stratégie de gene silencing envisagée depuis quelques années. D’autre part, des complexes de ruthénium ont été conjugués à des peptides ou plateformes en vue de leur permettre de pénétrer à l’intérieur des cellules (chapitre 4). Les complexes ne pouvant normalement pas traverser les membranes cytoplasmiques, nous avons démontré que l’ancrage de ceux-ci au peptide transvecteur TAT permet de les vectoriser dans le cytoplasme. Cette incorporation se fait vraisemblablement par endocytose. Lors de ces études, l’importance de la localisation finale du complexe au sein de la cellule a été mise en évidence. Afin de conférer une sélectivité de vectorisation dans des cellules données (pénétration active et selon la présence de récepteurs spécifiques à la surface membranaire), les complexes ont été ancrés sur une plateforme RAFT(RGD)4. Dans ce cas, nous avons démontré qu’une internalisation spécifique dans des cellules sur-exprimant l’intégrine αvβ3 est possible pour les conjugués Ru-RAFT(RGD)4. Finalement, des études ont été réalisées sur les complexes ancrés sur plateforme calixarènique. Les résultats présentés permettent de caractériser ces conjugués Ru-Calix afin d’orienter leur développement avant les études de vectorisation cellulaire. Grâce aux résultats obtenus, un design permettant aux complexes de conserver leur photoréactivité a pu être établi et servira pour les développements futurs. En sus de ces deux axes de recherche principaux, le premier chapitre de résultats et discussions porte quant à lui sur l’étude fondamentale des complexes [Ru(TAP)3]2+ et [Ru(TAP)2(phen)]2+ ;plus précisément, une étude complète du complexe [Ru(TAP)2(phen)]2+ dans l’acétonitrile et le butyronitrile en présence d’un composé calixarènique (développé dans l’équipe du Pr. Ivan Jabin) est présentée. Il appert que l’utilisation du calixarène permet de mettre en évidence des processus photophysiques et photochimiques complexes, qui n’avaient pas été détectés auparavant. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le beet necrotic yellow vein virus (BNYVV) est un phytovirus qui possède un génome segmenté à ARN de polarité positive. L’ARN3 viral renferme le domaine « core » qui contient une séquence de 20 nucléotides appelée « coremin », indispensable au mouvement systémique du virus chez Beta macrocarpa. L’ARN3 subit un processus de dégradation qui conduit à la formation d’un ARN non codant (ncRNA3) correspondant à son extrémité 3’. Ce dernier est stabilisé par la séquence « coremin » à son extrémité 5’. Grâce à l’outil génétique levure, l’exoribonucléase Xrn1 puis l’exoribonucléase XRN4 de plante ont été identifiées comme étant responsable de l’accumulation du ncRNA3 à partir d’ARN3. Nous avons démontré in vitro que l’accumulation de ncRNA3 est liée au blocage de Xrn1 par « coremin ». La protéine virale p14, un suppresseur du RNA silencing codée par l’ARN2, est aussi nécessaire au mouvement systémique du virus et interagit avec la séquence « coremin ». Nos travaux confirment que l’ARN3 est capable de complémenter partiellement un mutant allélique de p14 dans l’infection locale et systémique. Nos résultats mettent en évidence un effet de la protéine p14 sur la systémie du RNA silencing et sur une éventuelle cible cellulaire RDR6. / The beet necrotic yellow vein virus (BNYVV) is a multipartite positive-stranded RNA phytovirus. The RNA3 contains a « core » sequence in which resides the « coremin » motif of 20 nucleotides absolutely required for the viral systemic movement in Beta macrocarpa. The RNA3 undergoes a process that produces a noncoding RNA3 (ncRNA3), stabilized by « coremin » at its 5’ end. Using a yeast genetic approach, the exoribonuclease Xrn1 and plant XRN4 have been identified as being responsible for the ncRNA3 accumulation from RNA3 processing. In vitro, we showed that the ncRNA3 accumulation is due to the stalling of Xrn1 by “coremin”. The viral p14 protein, an RNA silencing suppressor encoded by the RNA2, is also required for the systemic movement and interacts with the “coremin” sequence. Our studies demonstrated the ability of RNA3 to partially complement an allelic p14 mutant in local and systemic infections. Our data highlighted an effect of the p14 protein on the RNA silencing movement and on the potential cellular target RDR6.

RNA interference (RNAi) is one of the key mechanisms that are involved in many biological processes such as control of plant gene expression, influence on chromatin arrangement or providing protection against invasive DNA or RNA transposons, viruses and transgenes. In plants, RNAi is triggered by double stranded RNA (dsRNA) that is cleaved by DICER LIKE (DCL) proteins to small RNAs (sRNAs). The size of these sRNAs is in range of 21 - 24 nucleotides (nt). Small RNA acts in the place of origin and they are also a mobile signal which in plants can move to a short distance through plasmodesmata and to a long distance trough phloem. sRNA and Argonaute (AGO) protein form RNA-induced silencing complex (RISC). Together, they recognize the target RNA molecule and contribute to an efficient RNAi phase which may be exhibited by gene silencing at posttranscriptional level (PTGS) or transcriptional level (TGS). The purpose of this study was to compare the effects of silencing constructs, witch in a controlled way differently trigger RNAi directed against the expression of the GFP reporter gene in the model organism Arabidopsis thaliana. Silencing constructs were placed under an inducible promoter activated by the presence of 17-β-estradiol (XVE system). They differed in the way of the dsRNA formation and in the...

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l’agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n’est retrouvé qu’en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l’aspect versatile de l’obtention de transcrits infectieux de ces différents clones, j’ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d’un promoteur constitutive végétal pour initier l’infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l’infection virale et étudier l’interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j’ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV. / The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.