Au cours de cette étude, nous avons isolé 4 ADNc codant pour des protéines activatrices putatives de l'APC/C de tomate : SlCCS52A, SlCCS52B, SlCDC20-1, et SlCDC20-2. Des données obtenues par RT-qPCR et par hybridation in situ révèlent des profils d'expression différents au niveau tissulaire mais également au cours du développement du fruit de tomate, suggérant que différentes protéines activatrices pourraient assurer la modulation spatio-temporelle de l'activité de l'APC/C chez la tomate. De plus, les transcrits du gène SlCCS52A s'accumulent plus particulièrement dans le fruit durant la phase d'expansion cellulaire, tandis que les transcrits du gène SlCCS52B sont plutôt présents durant les premiers stades de développement, caractérisés par une forte activité de division cellulaire. Afin de préciser le rôle de SlCCS52A et SlCCS52B dans le contrôle du cycle cellulaire et de l'endocycle chez la tomate, ces gènes ont fait l'objet d'une étude fonctionnelle. La réduction de l'expression de SlCCS52A entraîne une réduction de la taille des fruits et de la taille cellulaire, qui s'accompagne d'une diminution du niveau de ploïdie. La surexpression de ce gène modifie la cinétique de développement des fruits. La mise en place de l'endocycle est retardée, mais l'augmentation de la ploïdie est plus rapide et la croissance relative du fruit est alors plus importante. Enfin, la réduction de l'expression de SlCCS52B entraîne une augmentation de l'expression de SlCCS52A au niveau des fruits, suggérant l'existence de mécanismes compensatoires. L'ensemble de ces résultats montrent que SlCCS52A est impliqué dans la mise en place de l'endoréduplication chez la tomate, et participe au contrôle de l'expansion cellulaire. / In this study, we have isolated 4 cDNAs encoding putative proteins activating the APC/C in tomato: SlCCS52A, SlCCS52B, SlCDC20-1, and SlCDC20-2. Data obtained by RT-qPCR and in situ hybridization revealed different expression profiles in tissues but also during the development of tomato fruit, suggesting that different activator proteins could provide the spatio-temporal modulation of the APC/C activity in tomato. In addition, the SlCCS52A transcripts accumulate especially in the fruit during the cell expansion phase, while transcripts of the SlCCS52B gene are rather present during the early stages of development, characterized by a high activity of cell divisions. To clarify the role of SlCCS52A and SlCCS52B in cell cycle control and endocycle in tomato, we performed a functional analysis of these genes. Reducing the expression of SlCCS52A leads to reduced fruit size and cell size, accompanied by a decrease in the level of ploidy. The overexpression of this gene alters the kinetics of fruit development. The establishment of endocycle is delayed, but the increase in ploidy is faster and the relative growth of the fruit is much more important then. Finally, the reduced expression of SlCCS52B leads to an increased expression of SlCCS52A in fruit, suggesting the existence of compensatory mechanisms. All these results show that SlCCS52A is involved in the establishment of endoreduplication in tomato, and participates in the control of cell expansion.

Ubiquitin is a highly conserved 76 amino acid protein which is a unique and versatile signalling molecule. Ubiquitin can be attached by an isopeptide bond between its C-terminal diglycine to a lysine residue of a target substrate. However, it can also bind to itself though one of its own seven lysine residues allowing the formation of different chain types. These chains act as signals for different pathways, such as DNA damage repair, and in particular lysine-48 chains signal for proteins to be degraded via the proteasome by the ubiquitin proteasome system (UPS). This allows cells to control the concentration of proteins which is important in triggering cellular events, such as cyclin levels in cell division. Whilst old and incorrect proteins need to be removed so they do not interfere with normal processes. In order to recognise and ubiquitinate substrates an enzyme cascade has evolved. Ubiquitin is transferred from an ubiquitin activation enzyme (E1) to an ubiquitin conjugating enzyme (E2). The E2 which along with a ubiquitin ligase (E3) ubiquitinates a specific substrate. Research has focused on the E3 ligases since they are responsible for identifying substrates. One important ligase is the anaphase promoting complex/cyclosome (APC/C) which is responsible for faithful segregation of chromosome during mitosis. Failure to regulate this process can lead to aneuploidy, one of the main causes of cancer. It is therefore important to understand the function and regulation of APC/C and the UPS. This work firstly shows that four S. pombe kinases, Ssp2, Ppk9, Kin1 and Chk1 all contain a kinase associated 1 (KA1) or KA2 domain which they use to interact specifically with APC/C when it contained an unphosphorylated form of a subunit called Cut9. Yeast two hybrid and native far Westerns demonstrated that the KA domains interact with the APC/C co activator Slp1. Phosphorylation assays showed that three of these kinases phosphorylated a ~30kDa band of the APC/C complex which was shown to be Mad2, an important subunit of the APC/C inhibitor complex the mitotic checkpoint complex (MCC). These finding suggest a new role for KA contain kinases as regulators of APC/C activity. Future studies to identify the residues of Mad2 which are phosphorylated by these kinases, as well as the binding site of Slp1 that the KA domains recognise, would provide a more detailed understanding of the molecular mechanisms involved in regulating APC/C activity. Secondly, this study investigated the role of the ubiquitin associated (UBA) domains in the S. pombe shuttle factor Rhp23. This protein can recognise the proteasome via an ubiquitin like (UBL) domain and ubiquitin chains via one of two UBA domains: an internal UBA1 and a C-terminal UBA2. To dissect the different functions of these two UBA domains point mutations were made that abolished the domains ability to recognise ubiquitin without altering the protein structure. The minimal domains and full length domains were tested in vitro and in vivo. These surprising results showed that the domains act differently in isolation when compared to the full length protein. They also demonstrate that the UBA1 domain is responsible for ubiquitin recognition in Rhp23, whilst the UBA2 domain appears to have little to no binding ability.

Les centrosomes sont les principaux centres organisateurs des microtubules dans les cellules animales, impliqués dans la division, la motilité, la polarité cellulaire. Ils participent à l'élaboration du fuseau mitotique, qui permet la séparation des chromosomes dans les cellules filles. Dans les neuroblastes de drosophile en interphase, un des deux centrosomes maintient son activité et sa position apicale dans la cellule, alors que l'autre est inactivé et se déplace vers le pôle basal. La duplication des centrioles est initiée par la kinase Plk4 une seule fois par cycle cellulaire. Toute dérégulation des niveaux de Plk4 conduit à un défaut du nombre de centrosomes, à l'origine de pathologies comme le cancer et la microcéphalie. Pendant ma thèse, j'ai étudié les rôles et régulations de Plk4 in vivo dans les neuroblastes de drosophile. J'ai montré un nouveau rôle de Plk4 dans l'établissement de l'asymétrie des centrosomes durant l'interphase. Plk4 favorise un comportement basal des centrosomes en inhibant la nucléation des microtubules et l'ancrage au pôle apical. Plk4 régule négativement la localisation du co-activateur de l'APC/C, Fizzy-related, que j'ai identifié comme un régulateur positif de l'activation du centrosome. APC/C est une E3 ubiquitine-ligase, qui cible les protéines régulant le cycle cellulaire vers la dégradation. J'ai montré que Plk4 interagit avec ce complexe in vivo. Des mutations du motif de liaison à l'APC/C conduisent à la stabilisation de Plk4 et à une dérégulation de son accumulation au centrosome au début de l'interphase. Mon étude a donc démontré que dans les neuroblastes Plk4 coordonne la duplication des centrioles et le cycle des centrosomes. / The centrosome is the main microtubule-organizing centre of animal cells with important roles in cell division, motility and polarity. In cycling cells, upon duplication, two centrosomes form the mitotic spindle, the apparatus that physically segregates the chromosomes into the daughter cells. In Drosophila neural stem cells of the larval brain, called neuroblasts, during interphase, one centrosome stays active and static at the apical side of the cell, while the other one is inactive and moves toward the basal side of the cell. Centriole duplication, which occurs only once per cell cycle, is initiated by the Polo-like kinase 4 (Plk4). Deregulation of Plk4 levels leads to alteration in centrosome number, a defect that can cause diseases such as cancer and microcephaly. During my PhD I studied the role/s and regulation of Plk4 in vivo in Drosophila neuroblasts. I found that Plk4 plays an important role in establishing centrosome asymmetry during interphase. Plk4 promotes centrosome basal-like behaviour, through inhibition of MT nucleation and centrosome apical anchorage. Plk4 negatively regulates the centrosomal localization of the APC/C co-activator Fizzy-related (Fzr) that I identified as a positive regulator of centrosome activation. The APC/C complex is an E3 ubiquitin-ligase that targets cell-cycle-related proteins to degradation. I showed that APC/C and Plk4 interact in vivo. Mutations in the APC/C binding motif lead to stabilization of Plk4 that presents unscheduled accumulation at the centrosome in early interphase neuroblasts.In conclusion, my study demonstrates that in neuroblasts, the kinase Plk4 couples centriole duplication and centrosome cycles.

<p>The Anaphase Promoting Complex/Cyclosome (APC/C) is a RING E3 ligase that plays essential roles both within and outside of the cell cycle. At the onset of anaphase, the APC/C targets cyclin B and securin for degradation, initiating chromosome separation and mitotic exit. Regulation of APC/C activity is critical for a functional cell cycle, and this is largely mediated by cytostatic factor (CSF) activity and the Spindle Assembly Checkpoint (SAC). </p> <p>Prior to fertilization, vertebrate eggs are arrested in metaphase of meiosis II by CSF activity, a key component of which is the APC/C inhibitor Emi2. Although the roles and regulation of Emi2 in maintaining CSF arrest have been extensively studied, its function during the oocyte maturation process, especially at the meiosis I to meiosis II (MI-MII) transition, was not well understood. Studies presented in this dissertation characterize an Emi2-mediated auto-inhibitory loop of the APC/C that provides the molecular basis of a critical biochemical event during the MI-MII transition--the partial degradation of cyclin B. In brief, phosphorylation of the Emi2 N-terminus by Cdc2/cyclin B targets it for proteasomal degradation in meiosis I (MI). During anaphase of MI, the APC/C triggers its own inactivation by degrading cyclin B, therefore stabilizing its inhibitor, Emi2. The timely inactivation of APC/C activity prevents the complete inactivation of Cdc2 kinase, which is crucial for prohibiting S phase onset and parthenogenetic activation of the oocytes.</p> <p>To better understand the regulation of the APC/C, a number of the studies presented here are aimed at identifying the mechanism for Emi2 inhibition of the APC/C. Many APC/C inhibitors have been reported to function as "pseudosubstrates", which inhibit the APC/C by preventing substrate binding. After carefully examining the ubiquitin reactions mediated by the APC/C in vitro, we have found that it is the last step in the ubiquitylation process, where ubiquitin is transferred from a charged E2 to the substrate, that is targeted by Emi2. In addition, biochemical studies have also revealed that Emi2 itself has RING-dependent ligase activity and this activity enables it to inhibit the APC/C in a sub-stoichiometrical manner. </p> <p>Although the ultimate goal for both CSF activity and the SAC signaling pathway is APC/C inhibition, a much more complicated regulatory network is known to control SAC. Previous researches in our lab have identified Xnf7 to be an APC/C inhibitor that is required for the SAC pathway in Xenopus egg extract. In an effort to characterize the human Xnf7 homolog, we have found that Trim39, a protein that has been implicated in apoptosis regulation, is required for the SAC pathway in RPE cells. Like Emi2, both Xnf7 and Trim39 are RING E3 ligases whose activity is essential for their function. Interestingly, the ligase activity of both proteins appears to be regulated by the checkpoint. While we continue to characterize the roles and regulation of both Trim39 and Xnf7 in the SAC, future investigations into the mechanisms that underlie APC/C inhibition by all the three E3 ligases--Emi2, Xnf7 and Trim39--would be of great interest.</p> / Dissertation

Post-translational modification of proteins with ubiquitin regulates many aspects of cell physiology, including protein degradation. A uniform polyubiquitin chain that is linked through Lys48 has been widely accepted as central for recognition and destruction by the 26S proteasome. Work in more recent years has demonstrated that the repertoire of proteolytic signals may encompass chains of other linkage types, including Lys11-linked ubiquitin chains and short assemblies of mixed linkage. In this dissertation I examine whether catalysis mediated by the Anaphase-Promoting Complex/Cyclosome (APC/C) is dependent on polyubiquitination and whether the proteolytic machinery exerts a requirement for specific ubiquitin linkages to efficiently degrade cyclin B1. In chapter II, I describe a novel method in which Xenopus cell-cycle extracts are made largely dependent on exogenous ubiquitin by inhibiting ubiquitin recycling, allowing us to evaluate the relative contribution of distinct ubiquitin linkages in APC/C-mediated ubiquitination and degradation. Utilizing this approach, in chapter III, I found that the conjugation of single ubiquitin moieties to multiple lysine residues in cyclin promotes efficient degradation of cyclin B1 in mitotic Xenopus extracts. Lysine11-ubiquitin chain-formation becomes essential to proteasomal targeting only when the number of available lysine residues in cyclin B1 is restricted. Analysis in a reconstituted system revealed that APC/C catalyzes multiple monoubiquitination with rapid kinetics and species bearing four or more monoubiquitins on distinct lysines are recognized by ubiquitin receptors. These multiply monoubiquitinated species are rapidly degraded by purified proteasomes. In chapter IV, I examine the role of distinct E2 enzymes in APC/C-dependent proteolysis. I demonstrate that the chain-extending E2 UBE2S and long Lys11-linked ubiquitin assemblies are dispensable for cyclin B1 degradation, but become increasingly important with restriction of the number of ubiquitination sites. Our findings support a model where through attachment of monoubiquitin to multiple lysine residues, and possibly elaboration of some short chains, UBCH10, or possibly members of the UBC4/5 family, cooperate with the APC/C to generate a robust proteolytic signal on cyclin B1.

La méiose est la division cellulaire qui aboutit à la production de gamètes haploïdes. Lors de la méiose, un unique évènement de réplication est suivi de deux divisions afin de réduire la ploïdie. Lors de ces deux divisions, la cohésion entre chromatides sœurs est éliminée de façon séquentielle pour permettre la succession de deux ségrégations de chromosomes équilibrées. La progression du ‘’cycle méiotique’’ est contrôlée par des régulateurs communs à la mitose et à la méiose mais également par des mécanismes nécessitant des protéines spécifiques à la méiose. L’objectif de de mon travail de thèse était de décrypter les mécanismes moléculaires permettant l’enchainement de deux divisions équilibrées pour la production de gamètes haploïdes. Nous avons pu montrer que la protéine OSD1 inhibait l’APC/C pour permettre la progression méiotique. Nous avons également mis en évidence un réseau fonctionnel, comprenant OSD1, CYCA1;2/TAM et TDM, indispensable à trois étapes clés de la progression méiotique chez Arabidopsis ; la transition prophase-méiose I, la transition méiose I-méiose II et la sortie de méiose. Ces travaux ont également permis de caractériser chez Arabidopsis les deux paralogues de Shugoshin, qui sont des protéines conservées et impliquées dans la protection de la cohésion centromérique. Nous avons également identifié Patronus comme un nouveau protecteur de la cohésion centromérique en méiose. Les résultats obtenus suggèrent que Patronus est un régulateur de l’APC/C qui permet d’empêcher l’élimination de la cohésion centromérique en interkinèse méiotique. / Meiosis is a specialized type of cell division that generates haploid gametes. At meiosis, two divisions follow a single DNA replication event leading to ploidy halving. A stepwise sister chromatids cohesion release also occurs to allow the two successive balanced rounds of chromosome segregation. In addition to general cell-cycle regulators, meiosis requires specific proteins. The aim of this thesis was to understand the molecular mechanisms leading to two successive balanced chromosome segregations. We show that OSD1 promotes meiotic progression through APC/C inhibition and we identified a functional network between OSD1, CYCA1;2/TAM and TDM in Arabidopsis. This functional network controls three key steps of meiotic progression; the prophase-meiosis I transition, the meiosis I-meiosis II transition and the meiosis exit. In addition, we characterized the two Arabidopsis thaliana Shugoshin paralogs, which are conserved proteins involved in sister chromatid cohesion protection. We also identified Patronus, an uncharacterized protein, as a novel protector of meiotic centromeric cohesion. We suggest that Patronus is a novel APC/C regulator that prevents cohesins release during meiotic interkinesis. This work identified two APC/C regulators essential for meiosis in Arabidopsis thaliana.

<p>Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint. </p><p>Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the <italic>Xenopus</italic> egg extract system, our laboratory has previously demonstrated that the RING domain-containing ubiquitin E3 ligase Xnf7 can inhibit Xenopus APC/C activity. In the thesis, we have identified TRIM39 as an Xnf7-related human regulator of the APC/C. Our study showed that TRIM39 restrains the ability of the APC/C to ubiquitylate Cyclin B in vitro and attenuates the degradation of Cyclin B and geminin when TRIM39 is incubated in cell lysates. Notably, it has been reported that TRIM39 activity is responsible for the accumulation of the Bax-interacting protein (and activator) MOAP-1 following etoposide-induced DNA damage. Our data indicated that MOAP-1 is a novel APC/C substrate, and that the ligase activity of TRIM39 appears to be essential for preventing its degradation. We further demonstrated that decreased levels of the APC/C activator Cdh1 induces MOAP-1 protein accumulation, thereby promoting DNA damage-induced apoptosis in 293T, PC3 and H1299 cells. This study illustrates a potential function for the APC/C in DNA damage induced apoptosis and also demonstrates that TRIM39 regulates both the cell cycle and apoptosis via APC/C inhibition.</p><p>To extend our observations regarding the role for TRIM39 in APC/C regulation, we investigated effects on the cell cycle via real-time imaging microscopy. We found cells arrest at G1/S in TRIM39 depleted RPE cells, a cell line which is commonly used for cell cycle analysis. This arrest phenotype is not observed in 293T, PC3 and H1299 cells which bear mutant p53 alleles. Further analysis showed that TRIM39 depleted RPE cells upregulate many genes that function downstream of p53 activity, such as the cdk inhibitor p21--thus, arresting cells at G1/S and reducing proliferation. The reduced growth can be rescued by p53 knockdown. Mechanistically, TRIM39 interacts with p53 and promotes destruction of p53 by ubiquitylation. This ubiquitylation is independent of the activity of the most intensively studied p53-directed E3 ligase, MDM2; depletion of both MDM2 and TRIM39 has a synergistic effect on p53 accumulation. This elevated p53 leads to more apoptosis in cancer cells bearing wildtype p53. Consequently, TRIM39 depletion might be employed as a combination treatment with MDM2 inhibitor, such as nutlin-3a, to stimulate tumor cell death.</p><p>In the thesis, we have found TRIM39 inhibits both the APC/C and p53. Both are essential regulators of cell cycle and apoptosis. Moreover, we have determined that the inhibitory activity of TRIM39 requires its E3 ligase activity. Future experiments will be directed towards investigating how TRIM39 protein stability and ligase activity are regulated to understand more fully the physiological situations in which TRIM39 is able to exert its ability to modulate the cell cycle and apoptosis. I will also discuss some preliminary data regarding changes in TRIM39 ligase activity induced by Chk1 and changes in TRIM39 protein abundance regulated by polo-like kinase 1(Plk1). Chk1 and Plk1 are essential kinases for cell cycle checkpoint and progression. Connecting Chk1 and Plk1 to TRIM39 may provide a more thorough understanding of TRIM39's ability to control the APC/C inhibition and p53 ubiquitylation in response to cell cycle or cell damage cues. Since the APC/C and p53 both can regulate cell cycle and apoptosis, further investigations into the involvement of TRIM39 in the life-or-death decision will be of great interest.</p> / Dissertation

La régulation post-transcriptionnelle joue un rôle de premier plan dans le contrôle fin de l’expression génique en permettant une modulation de la synthèse de protéines dans le temps et l’espace, en fonction des besoins de la cellule. Ainsi, des protéines reconnaissant des éléments d’ARN présents sur des transcrits peuvent influencer toutes les étapes de leur existence, soit leur épissage, leur export nucléaire, leur localisation subcellulaire, leur traduction et leur dégradation. Staufen1 (Stau1) est un membre de la famille des protéines liant l’ARN double-brin qui contribue à la régulation post-transcriptionnelle par son implication dans des mécanismes qui vont promouvoir l’épissage alternatif, le transport, la dé-répression de la traduction et l’induction de la dégradation d’ARN messagers (ARNm) spécifiques. L’identité des cibles potentielles de Stau1 est maintenant connue puisqu’une étude à l’échelle du génome a montré que la protéine s’associe à près de 7% du transcriptome des cellules HEK293T. Ces ARNm se classent dans un large éventail de catégories fonctionnelles, mais il est tout de même intéressant de noter qu’une grande proportion d’entre eux code pour des protéines reliées au métabolisme cellulaire et à la régulation de processus cellulaires. En considérant toutes ces informations, nous avons émis l’hypothèse que les différentes activités de Stau1 puissent être modulées afin de contrôler adéquatement l’expression des transcrits liés par la protéine. Dans la mesure où certains ARNm faisant partie des complexes définis par la présence de Stau1 codent pour des régulateurs clés de la prolifération cellulaire, nous avons voulu examiner si l’expression de la protéine varie au cours du cycle de division cellulaire. Nous avons montré que l’abondance de Stau1 est maximale en début de mitose et qu’elle diminue ensuite lorsque les cellules complètent la division cellulaire. Nous avons ensuite découvert que cette baisse d’expression de Stau1 en sortie de mitose dépend du complexe promoteur d’anaphase/cyclosome (APC/C). En soutien à l’idée que Stau1 soit une cible de cette ubiquitine ligase de type E3, nous avons de plus démontré que Stau1 est ubiquitiné et dégradé par le protéasome. Ce contrôle des niveaux de Stau1 semble important puisque la surexpression de la protéine retarde la sortie de mitose et entraîne une diminution importante de la prolifération cellulaire. Par ailleurs, nous avons supposé que les différentes fonctions de Stau1 puissent également être sujettes à une régulation. Compte tenu que les activités de nombreuses protéines liant l’ARN peuvent être contrôlées par des modifications post-traductionnelles telles que la phosphorylation, nous avons voulu tester la possibilité que Stau1 soit phosphorylé. L’immunopurification de Stau1 et son analyse par spectrométrie de masse nous a permis d’identifier trois phosphosites dans la protéine. L’évaluation du rôle de ces événements de phosphorylation à l’aide de mutants phoshomimétiques ou non-phoshorylables a révélé que la modification de Stau1 pourrait compromettre son association à la protéine UPF1. Comme cette interaction est nécessaire pour déstabiliser les transcrits liés par Stau1, nos résultats suggèrent fortement que la fonction de Stau1 dans la dégradation d’ARNm est régulée négativement par sa phosphorylation. Toutes ces données mettent en lumière l’importance des modifications post-traductionnelles telles que l’ubiquitination et la phosphorylation dans la modulation de l’expression et des fonctions de Stau 1. Somme toute, il est vraisemblable que ces mécanismes de contrôle puissent avoir un impact significatif sur le destin des ARNm liés par Stau1, particulièrement dans un contexte de progression dans le cycle cellulaire. / Post-transcriptional regulation plays a major role in the fine tuning of gene expression by allowing a modulation of protein synthesis in space and time, according to cellular requirements. For instance, proteins recognizing RNA elements on transcripts can influence all the steps of their existence, such as their splicing, nuclear export, subcellular localization, translation and degradation. Staufen1 (Stau1) is a member of the double-stranded RNA-binding protein family that contributes to the post-transcriptional regulation of gene expression by its involvement in mechanisms that promote alternative splicing, transport, de-repression of translation and decay of specific messenger RNAs (mRNAs). The identity of potential Stau1 targets is now known as genome-wide analyses have shown that the protein is associated with about 7% of the HEK293T cell transcriptome. Although these mRNAs are classified in a broad range of functional categories, a large proportion of them code for proteins related to cellular metabolism and regulation of cellular processes. Considering all this information, we hypothesized that the different activities of Stau1 may be modulated in order to control appropriately the expression of Stau1-bound mRNAs. Since some of the mRNAs that are part of Stau1-containing complexes encode key regulators of cell proliferation, we wanted to examine whether Stau1 expression fluctuates during the cell division cycle. We showed that Stau1 abundance peaks at the onset of mitosis and then decreases as cells complete division. We then found that Stau1 down-regulation in mitosis exit is mediated by the anaphase promoting complex/cyclosome (APC/C). To support the idea that Stau1 is a target of this E3-ubiquitin ligase, we further demonstrated that Stau1 is ubiquitinated and degraded by the proteasome. The importance of controlling Stau1 levels during the cell cycle is underscored by the observation that its overexpression delays mitotic exit and impairs cell proliferation. Furthermore, we speculated that Stau1 different functions may also be regulated. In the view that the activities of numerous RNA-binding proteins can be controlled by post-translational modifications such as phosphorylation, we tested the possibility that Stau1 is phosphorylated. Mass spectrometry analysis of immunopurified Stau1 allowed the identification of three phosphosites in this protein. Assessment of the role of these phosphorylation events using phosphomimetic or non-phosphorylatable mutants revealed that Stau1 phosphorylation may compromise its association with Upf1. Because this interaction is necessary to elicit the destabilisation of Stau1-bound RNAs, our results strongly suggest that Stau1 function in mRNA decay is negatively regulated by its phosphorylation. Collectively, these data highlight the importance of post-translational modifications such as ubiquitination and phosphorylation in the modulation of Stau1 expression and functions. Overall, the mechanisms that control Stau1 are likely to have a significant impact on the fate of Stau1-bound mRNAs, especially in the context of cell cycle progression.

Meiosis is a specialized cell cycle, which generates haploid gametes from diploid parental cells. During meiosis one round of cohesion establishment during premeiotic DNA replication mediates two rounds of chromosome segregation. During meiosis I homologous chromosomes separate, whereas sister chromatids segregate during the second meiotic division without an intervening round of DNA replication. Both rounds of chromosome segregation are triggered by an ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC/C). APC/C-dependent destruction of securin/Pds1 is required to activate separase, a thiol protease that mediates chromosome segregation by cleavage of the cohesin complex. The first meiotic division is preceded by an extended prophase I, during which maternal and paternal chromatids undergo recombination. The persistence of cohesion during premeiotic S- and prophase I is essential for recombination and both meiotic nuclear divisions. In order to prevent premature loss of cohesion, the APC/C has to be inactivated during early meiosis. How the APC/C is kept inactive during premeiotic S- and prophase I was unknown. This question has been addressed by studying the APC/C subunit Mnd2 from the budding yeast Saccharomyces cerevisiae. This work demonstrates that Mnd2 is required for the persistence of cohesion during premeiotic S- and prophase I. Mnd2 prevents premature activation of the APC/C by the meiosis-specific substrate recognition factor Ama1. In cells lacking Mnd2, the APC/C-Ama1 enzyme triggers premature ubiquitin-dependent degradation of Pds1, which leads to premature separation of sister chromatids due to an unrestrained activity of separase. Thus, chromosome segregation during meiosis depends on both inhibition of a meiosis-specific APC/C and timely activation of APC/C- dependent proteolysis. / Die Meiose ist ein spezialisierter Zellzyklus, der zum Ziel hat haploide Gameten aus diploiden Vorläuferzellen zu produzieren. Dafür erfolgen nach der prä-meiotischen DNA Replikation zwei aufeinanderfolgende Kernteilungen. In der ersten meiotischen Teilung erfolgt die Trennung der homologen Chromosomen. In einer zweiten meiotischen Teilung werden dann die Schwesterchromatiden getrennt. Die Trennung der Chromosomen wird durch den Anaphase-Promoting Complex oder Cyclosome (APC/C), einer Ubiquitin Ligase, reguliert. Der APC/C initiiert den Abbau von Securin/Pds1, einem Inhibitor der Thiol-Protease Separase, welche für die Trennung der Chromosomen zum Beginn der Anaphase verantwortlich ist. In einer im Vergleich zur Mitose extrem langen meiotischen Prophase I findet Rekombination zwischen maternalen und paternalen Chromosomen statt. Für diesen Vorgang, sowie für die beiden folgenden meiotischen Teilungen, wird Kohäsion zwischen den Schwesterchromatiden benötigt. Ein frühzeitiger Verlust der Kohäsion führt zur frühzeitigen Trennnung der Schwesterchromatiden, wodurch aneuploide Gameten produziert werden können. Daher muss die Aktivität des APC/C während der meiotischen Prophase I inhibiert werden. Wie der APC/C während der Prophase I inaktiviert wird, war bisher unbekannt. Einsicht in dieses Problem ergab sich aus der Untersuchung der APC/C Untereinheit Mnd2 aus der Bäckerhefe Saccharomyces cerevisiae. Es wird gezeigt, dass Mnd2 für den Verbleib der Kohäsion zwischen den Schwesterchromatiden während der meiotischen S- und Prophase I benötigt wird. Während dieser Phase verhindert Mnd2 die frühzeitige Aktivierung der Meiose-spezifischen Form des APC/C-Ama1. In meiotischen Zellen, die kein Mnd2 besitzen, löst das APC/C-Ama1 Enzym die Ubiquitin-abhängige Zerstörung von Pds1 aus. Dies führt zu einer frühzeitigen Aktivierung von Separase, welches die Trennung der Schwesterchromatiden schon während der meiotischen S- und Prophase I zur Folge hat. Die korrekte Verteilung der Chromosomen hängt daher sowohl von der Inhibierung als auch der Aktivierung des APC/C ab.

No description available.