In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

In Arabidopsis, the F-box gene family encodes a large number of proteins postulated to act as substrate selectors for proteasome-mediated protein degradation. Recent reports document the importance of F-box proteins in developmental and metabolic signaling. Our microarray analyses of inflorescences of the brevipedicellus(bp) mutant indicate several F-box proteins are upregulated, suggesting that BP represses these genes in wild type plants to condition normal inflorescence development. We undertook analyses to examine the function of these proteins and their contribution to the pleiotropic phenotypes of bp. Yeast-2-hybrid screens revealed that the F-box protein At1g80440 binds to phenylalanine ammonia lyase-1(PAL1), the gateway enzyme of phenylpropanoid metabolism. Transgenic lines driven by the 35S cauliflower mosaic virus were attained but could not be propagated, suggesting a fatal phenotype. BP driven F-box expression results in phyllotaxy defects, manifest as alterations in the emergence of inflorescence and floral meristems in the axils of some cauline leaves.

Aspergillus fumigatus ist die häufigste Ursache für Lungeninfektionen in immunsuppri-mierten Patienten. Virulenzfaktoren sind häufig an Kontrollmechanismen für Entwick-lung gekoppelt, welche im verwandten Modellorganismus Aspergillus nidulans entdeckt wurden. Diese Arbeit präsentiert die Charakterisierung des F-box Proteins Fbx15 in A. fumigatus, welches einen starken Einfluss auf die Entwicklung in A. nidulans hat. Die Deletion von fbx15 resultierte in starken Wachstumsdefekten unter vielen Stress induzie-renden Bedingungen, welche klassische Virulenz Faktoren beinhalten, wie erhöhte Tem-peratur, oxidativer Stress und Aminosäuremangel, während das Wachstum unter Stan-dardbedingungen nicht beeinflusst war. Oxidativer Stress induziert eine transiente Erhöhung der fbx15 Expression, welche nach 40 Minuten zu einer dreifach erhöhten Pro-teinmenge führte. Fbx15 ist ein stabiles F-box Protein mit einer Halbwertszeit von 90 Minuten. Generell funktionieren F-box Proteine als Substratadapter für SCF-E3-Ubiquitin-Ligasen. Fbx15 liegt unter normalen Bedingungen phosphoryliert vor und in-teragiert mit der Skp1/A Untereinheit des SCF-Komplexes, vorzugsweise in kleineren Subpopulationen im Zytoplasma. Phosphoryliertes Fbx15 wird bevorzugt in SCF-Komplexe eingebaut. Oxidativer Stress führt zu einer schnellen Dephosphorylierung von Fbx15. Fbx15 Varianten, welche nicht phosphoryliert werden können, interagieren mit Skp1/A primär im Kern. Fbx15 rekrutiert drei Untereinheiten des COP9-Signalosoms und Proteine welche in Transkription, Translation, Signalübertragung, Morphologie oder Stoffwechsel involviert sind. Fbx15 bindet die Ssn6/F Untereinheit des konservierten Ssn6/SsnF-Tup1/RcoA Co-Repressors und wird für dessen Kernlokalisation benötigt. Dephosphoryliertes Fbx15 interagiert mit Ssn6/F im Kern und eine Fbx15-Ssn6/F be-dingte Genrepression wird für die Reduzierung der Gliotoxin-Biosynthese benötigt. fbx15 Deletionsstämme sind nicht in der Lage immunsupprimierte Mäuse in einem Model für invasive Aspergillose zu infizieren, was eine essentielle Funktion von Fbx15 für die Viru-lenz bestätigt. Diese Arbeit zeigt, dass Fbx15 nicht nur Teil von SCF-E3-Ubiquitin-Ligasen sein kann, sondern eine zweite neue molekulare Funktion aufweist, welche die physische Interaktion mit der Co-Repressor Untereinheit Ssn6/F und dessen Lokalisa-tionskontrolle beinhaltet. Diese duale Funktion resultiert in einer essentiellen Funktion von Fbx15 für die Kontrolle der oxidativen Stressantwort, des Sekundärmetabolismus und der Virulenz im opportunistischen Humanpathogen A. fumigatus.

Inherited mutations in the BRCA2 gene predispose individuals to the development of breast and ovarian cancers. The BRCA2 protein plays a fundamental role in the repair of DNA double strand breaks by homologous recombination (HR). BRCA2 mediates the recruitment of the RAD51 recombinase to DNA damage sites, which in turn promotes homologous pairing and strand exchange during HR. It has been reported that increased BRCA2 mRNA levels correlate with poor cancer prognosis, and recently it has been shown that increased levels of BRCA2 suppress HR. As HR is regulated through the cell cycle and can only be employed during S and G2 phases of the cell cycle, in this study, the cell cycle-dependent regulation of BRCA2, as a key player of HR, was investigated. In this study I report that BRCA2 stability is regulated by the ubiquitin-proteasome system (UPS), which has become increasingly evident as an important regulator of DNA repair. In line with this, I found that BRCA2 can be ubiquitylated in vivo and that it interacts with proteins of the UPS. Interestingly, I observed that BRCA2 levels and its ubiquitylation status change during the cell cycle. Using a siRNA-based approach, I identified a candidate E3 ubiquitin ligase, the SCF^FBXW7 complex, which is also a known major cell cycle regulator. siRNA-mediated knockdown of FBXW7 led to stabilization of BRCA2 and overexpression of FBXW7 resulted in BRCA2 ubiquitylation in vivo. Furthermore, I have refined the regions that the SCF^FBXW7 interacts with on BRCA2, which likely occurs in a phosphorylation-dependent manner. Taken together, these observations suggest that BRCA2 stability is regulated by the UPS in a cell cycle-dependent manner, which may be an important regulatory mechanism for BRCA2 function.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Le système ubiquitine-protéasome joue un rôle central dans le contrôle de la progression du cycle cellulaire par la dégradation régulée de nombreuses protéines. Dans ce système, Fbw7 (aussi appelée Fbxw7, hCdc4, hAgo, Sel-10), est l'une des protéines F-box qui sert d'adaptateur de substrats pour l'une des plus importantes familles d'ubiquitine ligases : les complexes SCF (Skp1/Cullin/ F-box). Fbw7 assure la dégradation de plusieurs régulateurs positifs du cycle cellulaire : la cycline E, cMyc, c-Jun, Notch, Aurora A, mTOR, MCL1. En conséquence, l'altération des fonctions de Fbw7 conduit à des défauts de prolifération cellulaire, de différenciation et à de l'instabilité génomique. La mutation de Fbw7 dans les cancers entraîne une dérégulation de l'expression périodique cycline E qui n'est alors plus restreinte à la transition G1/S du cycle cellulaire. Nos résultats montrent qu'une isoforme, Fbw7, est exprimée dans les œufs de xénope matures arrêtés en métaphase II mais n'est pas fonctionnelle, expliquant la présence de grande quantité de cycline E dans les œufs à cette phase mitotique. Nous montrons que Fbw7 est maintenue inactive sous forme poly-ubiquitylée suite à sa phosphorylation par une PKC jusqu'à la fin des cycles embryonnaires rapides, au moment où la cycline E est brutalement dégradée. Nous montrons que la régulation négative de Fbw7 par PKC est conservée au cours des cycles cellulaires somatiques des cellules humaines, et contribue à l'expression périodique de la cycline E. Ces résultats mettent en évidence un nouveau mécanisme critique pour la régulation de Fbw7 au cours du cycle cellulaire et suggèrent que les fonctions de Fbw7 peuvent être altérées par une dérégulation de PKC, un phénomène observé dans de nombreux types de tumeurs humaines. / The ubiquitin-proteasome system plays a central role in the control of cell cycle progression through the regulated degradation of numerous critical proteins. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors. Fbw7 (also known as Fbxw7, hCdc4, hAgo, Sel-10) is one such F-box protein. It controls the stability and thus the levels of several positive regulators of the cell cycle, including cyclin E, cMyc, c-Jun, Notch, Aurora A, mTOR, Mcl1. As a consequence of its biological roles, alterations of the functions of Fbw7 lead to defects in cellular proliferation, differentiation and genetic instability. As seen in cancers, mutation of Fbw7 leads to deregulation of cyclin E expression, which is no more restricted to the G1-S phase boundary of the cell cycle. Here we report that Fbw7, although expressed in mature Xenopus eggs arrested in metaphase II, is not functional, explaining why cyclin E can be stockpiled in this mitotic-like phase. We found that, in these eggs as well as in early Xenopus embryos, Fbw7 is maintained under a PKC-dependent poly-ubiquitylated state until the end of the early rapid cleavage cycles where cyclin E is abruptly degraded. Importantly, we show that this PKC-dependent negative regulation of Fbw7 is conserved during human somatic cell cycles, resulting into the periodic expression of cyclin E. These findings reveal a novel mechanism critical for the temporal regulation of Fbw7 and suggest that the key functions of Fbw7 can be altered by PKC dysregulation, a mechanism known to occur in many types of human tumours.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.