Les insuffisances médullaires héréditaires (IMH) réunissent quatre syndromes principaux : le syndrome de Shwachman-Diamond, la Dyskératose Congénitale, l’anémie de Diamond Blackfan et l'anémie de Fanconi (FA) qui est le syndrome le plus fréquent. Alors que l'étiologie des autres IMH est reliée à des défauts de biogenèse des ribosomes ou d'homéostasie du nucléole, l'étiologie de la FA est considérée comme reposant principalement sur des défauts de réparation de l'ADN.La FA est un syndrome autosomique récessif rare qui inclut des défauts de développement, une prédisposition au cancer et des défauts hématologiques progressifs. Les patients présentent une pancytopénie précoce associée à une myélodysplasie qui les prédispose à la leucémie myéloïde aiguë. La sensibilité cellulaire aux agents pontants de l'ADN est la principale caractéristique qui distingue ce syndrome des autres IMH. La maladie est causée par des mutations homozygotes de la voie FANC-BRCA, qui réunit plus de vingt protéines nécessaires pour la réparation des pontages interbrins de l'ADN ainsi que pour la gestion du stress réplicatif et des conflits entre transcription et réplication. Parmi ces protéines, FANCA est retrouvée mutée chez 60% des patients atteints de FA.Dans ce travail, nous avons cherché à investiguer si la voie FANC ou la protéine FANCA étaient impliquées dans le fonctionnement du nucléole ou dans la biogenèse des ribosomes.Nous avons observé que les cellules déficientes en protéine FANCA ont une homéostasie nucléolaire altérée ainsi qu'une synthèse et une maturation des ARNr ralenties. Ces caractéristiques sont indépendantes de la signalisation des dommages de l'ADN mais sont à mettre en lien avec la présence de conflits entre la transcription et la réplication dans le nucléole. Nous avons montré que la déstructuration du nucléole mène à la stabilisation de la protéine p21 par la protéine nucléolaire NPM1. Egalement, nous avons montré que les ribosomes des cellules déficientes en protéine FANCA présentent un déséquilibre de Facteurs Eucaryotes d'Initiation de la traduction (EIFs) et de certaines isoformes de protéines ribosomales, telle que l'augmentation de la protéine RPL22L1, entrainant une baisse de la traduction.En conclusion, ce travail montre que le stress nucléolaire est impliqué dans l'étiologie de la FA, ce qui relie la FA aux autres IMH. Cette observation incite à étudier les relations entre la réponse aux dommages de l'ADN et le stress nucléolaire dans l'apparition de l'insuffisance médullaire. / Inherited bone marrow failure syndromes (iBMFs) group four main syndromes: Shwachman-Diamond syndrome, Dyskeratosis Congenita, Diamond Blackfan anemia and Fanconi anemia (FA), which is the most frequent one. Whereas the pathogenesis of the other iBMFs is linked to ribosomal biogenesis and nucleolar abnormalities, the pathogenesis of FA is considered to be mainly due to misrepaired DNA damage.The FA syndrome is a rare autosomic recessive disorder, which includes developmental defects, cancer predisposition and evolutive haematological alterations. The patients present an early pancytopenia associated with a progressive myelodysplasia that eventually predisposes them to acute myeloid leukemia. Cellular hypersensitivity to crosslinking agents is the main difference between this syndrome and other iBMFs. The pathology is due to homozygous mutations in the FANC-BRCA pathway, that groups more than twenty proteins necessary for interstrand crosslinks (ICls) repair, replication stress response and managing of conflicts between replication and transcription. Among them, the FANCA protein is mutated in 60% of the FA patients.In this work, we asked whether the FANC pathway or the FANCA protein might be involved in the nucleolar homeostasy or in ribosomal biogenesis.We observed that FANCA defective cells have an altered nucleolar homeostasy and a slowed ribosomal rRNA synthesis and processing, independently from DNA damage signalling but related to conflicts between replication and transcription in the nucleolus. We show that the destructuration of the nucleolus can lead to p21 stabilisation by the nucleolar protein NPM1. Also, we show that ribosomes in FANCA deficient cells present a misbalance of Eukaryotic translation Initiation Factors (EIFs), and of some ribosomal proteins isoforms, such as the increase of RPL22L1, leading to a translation slowdown.In conclusion, this study shows that nucleolar stress is involved in FA pathogenesis, which links FA to other iBMFs. This paves the way for the investigation of interplays between DNA damage and nucleolar stress in bone marrow failure onset.

of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...

In eukaryotic interphase cells, heterochromatin mostly localizes either at the nucleolar periphery or at the nuclear lamina. Genome localization studies are crucial due to evidence that spatial organization of the genome affects gene function. Nucleolus-associated domains (NADs) are mainly heterochromatic regions that have been mapped only in a handful of mouse and human somatic cells, and in plants. The extent to which changes in NAD localization occur during cellular differentiation remains unknown. In this thesis, we characterize a map of genome-wide NADs in F121-9 mouse embryonic stem cells (mESCs). We identified NADs by deep sequencing chromatin associated with biochemically purified nucleoli and using NADfinder software to call NAD peaks. F121-9 NADs are mostly comprised of genomic regions with inactive or lowly transcribed genes and overlap extensively with lamina-associated domains (LADs) and regions with late replication timing. Similar to somatic mouse embryonic fibroblasts (MEFs), where NADs have been previously characterized by our laboratory, F121-9 mESCs display abundant “Type I” NADs. This subset of NADs frequently associates with nuclear lamina and nucleolar periphery and resembles constitutive heterochromatin. Compared to MEFs, F121-9 mESCs have fewer “Type II” NADs; this subset of NADs is frequently found at the nucleolar periphery but not at the nuclear lamina. mESC NADs are also less enriched in H3K27me3 modified regions compared to MEF NADs. This suggests that Polycomb complex-mediated facultative vii heterochromatin expansion is part of NAD maturation during cellular differentiation. Comparison of MEF and mESC NADs also revealed enrichment of developmentally regulated genes in NADs specific to these cell types. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. Our F121-9 mESC NAD studies identified distinct features of stem cell NADs and will facilitate future studies of genome organization changes during mammalian development.

The nucleolus is a plurifunctional organelle with dynamic protein exchange involved in diverse aspects of cell biology. Additionally, the nucleolus has been shown to have a role in the replication of numerous viruses, which includes HIV-1. Several groups have reported HIV-1 vRNA localization within the nucleolus. Moreover, it has been demonstrated the HIV-1 Rev protein localizes to the nucleolus and interacts with nucleolar proteins, including NPM1. Despite evidence for a nucleolar involvement during replication, a functional link has not been demonstrated. I investigated whether introncontaining vRNAs have a Rev-mediated nucleolar localization step prior to export. Furthermore, I examined whether NPM1 mediates Rev nucleolar localization, participates in Rev function, and/or post-transcriptional events during viral replication. I used coupled RNA fluorescence in situhybridization and indirect immunofluorescence to visualize intron-containing vRNA relative to the nucleolus in the absence or presence of Rev expression. An RNAi-based approach was used to examine the role of NPM1 in Rev function and viral replication in cell lines and primary human macrophages. My research findings support a model for a Rev-independent nucleolar localization step of introncontaining vRNA prior to export. Intriguingly, my results also suggest NPM1 does not participate in Rev nucleolar localization or Rev-mediated vRNA export, as previously proposed. Rather, my findings support a novel role for NPM1, the cytoplasmic localization and utilization of a select class of Rev-dependent vRNAs. Collectively, my findings provide novel insight for a functional role of the nucleolus and NPM1 in HIV-1 replication, which enhances our current understanding of HIV-1 biology.

No description available.

C-terminal mutations of the phosphoprotein nucleophosmin (NPM) are the most frequent genetic aberration detected in adult acute myeloid leukemia (AML). I focused on characterization of type A, B and E of AML-related C-terminal mutations. The plasmids bearing fluorescently labeled wild type or mutated NPM have been constructed to characterize mutation-induced changes in the localization of NPM. Mammalian cell lines HEK293T, HeLa and NIH 3T3 were used for production of the chimeric proteins. The intracellular localization of the mutated forms of NPM was analyzed by immunofluorescence staining and fluorescence microscopy of the living cells. The localization of the mutNPM type A and B was almost identical and predominantly cytoplasmic, while mutNPM type E was detected in nucleolus and cytoplasm simultaneously. However localization of the mutated forms was greatly influenced by the used cell line. It has been demonstrated that the exogenous NPM interacts with the endogenous NPM and that they mutually affect their intracellular localization due to heterooligomer formation. Detailed analysis of the relationship between the C-terminal mutations and the localization of the mutated NPM improves understanding of specific mutation effect on the formation and progression of AML and also specifies its prognostic...

O nucléolo é uma organela nuclear responsável pela produção dos ribossomos, através das Regiões Organizadoras do Nucléolo (NORs). Espécies que possuem mais de um par de cromossomos contendo NORs terão, obrigatoriamente, pelo menos um par ativo, sendo as demais NORs funcionais de acordo com a demanda celular. O mecanismo de compensação de dose é visualizado e bem estabelecido em híbridos interespecíficos, conhecido como dominância nucleolar, com a inativação de NORs de um dos parentais por outras homeólogas ativas que as dominam. A arquitetura da cromatina nas NORs e o controle da sua expressão foram estudados com o objetivo de se entender os mecanismos envolvidos no fenômeno da dominância nucleolar em espécies diplóides que possuem múltiplas NORs. A espécie modelo utilizada neste estudo foi Crotalaria juncea (Leguminosae-Papilionoideae), caracterizada por conter 2n=2x=16, e NORs no braço curto do cromossomo 1, sendo este o principal organizador do nucléolo, e no braço longo do cromossomo 4 adjacente à heterocromatina centromérica, sendo este um sítio adicional (sítio menor) e de expressão facultativa, previamente determinada. Nas raízes de C. juncea sincronizadas, observou-se que a nucleologênese tem seu início durante o final da telófase, em que os 4 sítios de genes ribossomais podem ter atividade e formar até 4 nucléolos, os quais tendem a se fundir durante a interfase. A Hibridação in situ fluorescente (FISH) permitiu estudos da arquitetura da cromatina, com a visualização dos territórios cromossômicos, onde a cromatina não está organizada de forma aleatória dentro do núcleo, e consequentemente o rDNA 45S dentro do nucléolo. Observou-se também que todos os sítios de rDNA 45S possuem diferença no tamanho do arranjo repetitivo. Assim sendo, a hierarquia de dominância está de acordo com o tamanho de cada arranjo (sítio), e estes são ativados de acordo com a demanda celular. As análises das modificações nas histonas mostraram que a H3K9Met1 apresentou marcas fracas no nucléolo, enquanto no restante da cromatina nuclear sua marcação foi intensa. Já a H3K9Met2 apresentou marcação fortemente associada à cromatina presente no nucléolo, com alguns pequenos pontos heterocromáticos dispersos no núcleo. Pela observação entende-se que ambas metilações controlam diferentes tipos de heterocromatinas, ou seja, a H3K9Met2 controla principalmente heterocromatinas associadas aos genes ribossomais, e a H3K9Met controla heterocromatinas não associadas ao rDNA. O rDNA é hiperacetilado dentro do nucléolo para a H3K14. Não foi observada marcação nucleolar para H4K8ac, mas pôde ser observadas regiões hiperacetiladas em outras regiões da cromatina. A metilação do DNA esteve diretamente associada à diferentes níveis de organização da cromatina das NORs. As heterocromatinas adjacentes ao nucléolo apareceram fortemente metiladas, enquanto a cromatina distendida dentro do nucléolo apresentou marcação dispersa, com algumas regiões mais fortemente marcadas, onde a cromatina apresentava-se mais condensada e provavelmente não associados com a cromatina ativa. As fibras estendidas permitiram uma análise de alta resolução, onde foi possível observar que regiões não metiladas apareciam intercaladas entre grandes regiões fortemente metiladas, sugerindo que estas regiões hipometiladas estão, possivelmente, associadas com as alças de transcrição dentro do nucléolo. 12 Esses resultados contribuem para o entendimento sobre o controle genético e epigenético na arquitetura da cromatina ribossomal, bem como seu controle na expressão dos genes ribossomais no genoma das plantas. / The nucleolus is a nuclear organelle responsible for the ribosomes production, by Nucleolus Organizer Regions (NORs). Species presenting more than one chromosome pair with NORs should present, one pair expressing the genes, at least; while the other pairs expressing their genes accordingly to cellular demand. Dosage compensation mechanism is visualized and well established of interspecific hybrids as a well-described phenomena named nucleolar dominance, where a NOR from one parental could lead to inactivation of a NOR from the other parental which is dominated. The chromatin architecture and expression of the NORs were studied to address the mechanism involved in the nucleolar dominance of diploid species containing multiple sites of 45S rDNA. The model species used in the present study was the crop Crotalaria juncea (Leguminosae-Papilionoideae) characterized by 2n=2x=16 chromosomes, being the main NOR mapped into chromosome 1 short arm and presenting an additional site (minor site) in the chromosome 4 long arm adjacent to a centromeric heterochromatin and facultatively expressed. Synchronized meristematic root tip cells determined to nucleologenesis starts during the late-telophase, often expressing every ribosomal gene sites, when up to four nucleoli could be observed and these become merged during interphases. FISH allowed nucleolar chromatin architecture be accessed revealing distinct chromosomal domains (territories), suggesting a non-random distribution of the 45S rDNA, even between homologous chromosomes, into the nucleolus. The 45S rDNA sites from both chromosome pairs 1 and 4 of C. juncea showed differences in their array sizes. The differences in the 45S rDNA array sizes and the order of loci expression suggest a hierarchy of dominance, a feature of nucleolar dominance; being the small RONs activated only on demand. Immunodetection of histone modifications showed different patterns to methylation distribution across the chromatin as a whole; where H3K9Met1 was found mainly distributed along the nuclear chromatin without an evident signal into nucleolus, while H3K9Met2 was detected as conspicuous dots in the nuclear chromatin and highly accumulated into the nucleolus. The results indicate different control on heterochromatin establishment and maintenance, being the modifications specific to certain chromosomal regions. Indeed, H3K9Met is a key component in the nucleolus chromatin architecture and expression. The chromatin inside the nucleolus showed a high accumulation of H3K14ac, with a weak fluorescent signal along the nucleus; on the other hand H4K8ac showed a strong signal homogenously distributed across the nuclear chromatin, but without evident signals inside the nucleolus. DNA methylation was directly associated with different levels of chromatin organization of the NORs. The heterochromatic regions associated to RON are highly methylated, while the chromatin inside the nucleolus showed weaker signals, with some bright spots probably in condensed regions and related to chromatin inactivity. Extended DNA fiber allowed a higher resolution mapping that revealed long methylated regions intermingled by nomethylated ones, being the last probably associated to transcriptional loops of rRNA genes into the nucleolus. The results presented herein contributes to a better understand about the nucleolar chromatin architecture and the genetic and epigenetic control of the ribosomal genes expression on plant genomes.

O nucléolo é uma organela subnuclear formada pela atividade transcricional dos genes ribossomais 18S-5.8S-26S (rDNA 45S) e consequente biogênese dos ribossomos. A atividade destes genes resulta na região organizadora do nucléolo (NOR), na forma de uma constrição secundária em cromossomos metafásicos. As constrições secundárias se condensam progressivamente durante a mitose e se descondensam ao final da telófase quando a reestruturação do nucléolo se inicia. Genomas que apresentam mais de um locus de rDNA 45S deve apresentar, obrigatoriamente, pelo menos um par de NORs, enquanto os demais loci poderão ou não serem expressos. O controle da expressão dos genes ribossomais e a formação da cromatina nucleolar são modulados por eventos epigenéticos. Embora alguns pontos sobre o funcionamento dos genes ribossomais e a formação do nucléolo estejam bem estabelecidos, questões como o padrão de condensação da cromatina nucleolar durante a mitose, o padrão de funcionamento de sítios adicionais de genes ribossomais, o papel das modificações epigenéticas na dinâmica da cromatina nucleolar e na expressão do rDNA 45S e o mecanismo de herança dos genes ativos, permanecem abertas. A espécie Crotalaria juncea (Leguminosae-Papilionoideae), com 2n=2x=16 cromossomos, que possui um locus de rDNA 45S no braço curto do cromossomo 1, que sempre forma constrição secundária, e um sítio adicional com atividade facultativa no braço curto do cromossomo 4, é um excelente modelo para o estudo destas questões. No contexto apresentado, foram estudadas a dinâmica de condensação das NORs durante o ciclo celular e sua correlação com a atividade dos genes ribossomais, incluindo o locus adicional, e ainda o papel da metilação da citosina do DNA durante estes processos. Os resultados demonstram que a cromatina da região organizadora do nucléolo segrega em um estado descondensado durante a mitose, na forma de constrição secundária, ou seja, tal estrutura não se condensa durante a metáfase e não volta a se distender no início da telófase. Aparentemente, o que causa correlações equivocadas entre a atividade nucleolar e a observação morfológica da constrição secundária na metáfase é a contração forçada da cromatina da NOR causada por agentes antimitogênicos. Este modelo de segregação em um estado aberto pode ser explicado pela descrição de diversas proteínas que permanecem diretamente ligadas ou indiretamente associadas à região da NOR durante a mitose, funcionando como uma barreira física para a compactação. Ambos os sítios, principais e adicionais, do rDNA 45S presentes em Crotalaria juncea apresentam atividade transcricional, embora o locus do cromossomo 4 mostre atividade facultativa. Ao contrário do que foi anteriormente proposto, uma vez ativo, o locus adicional permanece descondensado durante todo o ciclo mitótico, seguindo o mesmo comportamento dos sítios principais. As constrições secundárias e a cromatina nucleolar são hipermetiladas em nível citológico, independentemente de sua atividade. A aparente hipometilação observada no rDNA 45S em cromossomos mitóticos e núcleos interfásicos se deve ao menor grau de compactação da região organizadora do nucléolo e, consequentemente, à baixa densidade de cromatina. / The nucleolus is a subnuclear organelle formed as a result of transcriptional activity of ribosomal RNA genes 18S-5.8S-26S (45S rDNA) and subsequent ribosome biogenesis. This activity forms the nucleolar organizing region (NOR) as a secondary constriction in metaphase chromosomes. The secondary constrictions progressively condense during mitosis and decondense at the end of telophase, when nucleoli start to reassemble. Genomes presenting more than one 45S rDNA locus must have at least one pair of NOR bearing chromosomes, while other loci may be expressed or not. Ribosomal gene expression and nucleolar chromatin assembly are modulated by specific epigenetic events. Although some topics related to rDNA gene activity and nucleolus formation are well understood, questions such as the behavior of nucleolar chromatin condensation during mitosis, standard functions associated with rDNA additional sites, role of epigenetic modifications in nucleolar chromatin and 45S rDNA expression processes, and inheritance mechanism of active genes, remain to be solved. Crotalaria juncea (Leguminosae - Papilionoideae) has 2n=2x=16 chromosomes and carries a 45S rDNA locus at the short arm of chromosome 1, always presenting a secondary constriction, and an additional site with facultative activity at the short arm of chromosome 4, being an excellent model to resolve these questions. Thus, this study aimed to study NOR condensation dynamics during the cell cycle and its correlation with ribosomal gene activity, including the additional locus, while analyzing the role of rDNA cytosine methylation during this process. The results show that NOR chromatin segregate in a decondensed way throughout mitosis, as a secondary constriction. In other words, this structure does not condense during metaphase and the NOR is not reassembled at the beginning of telophase. Misinterpretations relating nucleolar activity with morphological observations of secondary constrictions, appear to be induced by the artificial contraction of NOR chromatin caused by antimitotic drugs. This segregation model in an open state may be supported by strong diversity of proteins that are maintained attached to NORs during mitosis, serving as a physic barrier for condensation. Both principal and additional 45S rDNA sites of C. juncea are transcriptionally active, although the additional locus in chromosome 4 presented facultative activity depending upon ribosomal request. Unlike what was previously proposed, once the additional site is activated, it remains in an open configuration throughout the cell cycle, similarly to principal site behavior. Secondary constrictions and nucleolar chromatin are hypermethylated at cytological level, regardless of their activity. The seeming hipomethylated state of 45S rDNA in interphase nucleus and mitotic chromosomes is due to a lower compaction level of nucleolar organizing regions and subsequent low chromatin density.

Le repliement des protéines est un processus cellulaire crucial impliquant plusieurs protéines dont la calnexine, une chaperone du réticulum endoplasmique. Notre laboratoire et un autre groupe avons démontré que la calnexine est essentielle à la viabilité de la levure Schizosaccharomyces pombe. Dans le cadre d’études structure-fonction portant sur cette protéine, nous avons découvert un phénomène permettant la viabilité des cellules en absence de la calnexine. Cet état, nommé Cin pour calnexine independence, est induit par un mutant de la calnexine dépourvu du domaine central hautement conservé (Δhcd_Cnx1p). La caractérisation de l’état Cin a révélé plusieurs caractéristiques particulières telle la dominance, sa transmission de façon non-Mendélienne à la progéniture méïotique et sa transmission par des extraits protéiques dépourvus d’acides nucléiques. Toutes ces propriétés suggèrent donc que l’état Cin est médié via un élément de type prion. Le gène cif1+, pour calnexin independence factor, a été isolé lors de criblages visant à identifier des gènes impliqués dans l’état Cin. Il encode pour une protéine orpheline dont la surexpression induit de façon stable un état de viabilité en l’absence de la calnexine. Cet état diffère génétiquement et phénotypiquement de l’état Cin induit par le mutant Δhcd_Cnx1p préalablement caractérisé, ce qui suggère deux voies parallèles de signalisation du phénomène Cin. Une caractérisation exhaustive de Cif1p a permis de démontrer qu’il ne s’agissait pas du prion responsable de l’état Cin, malgré que cette protéine possède certaines propriétés typiques des prions in vitro. Finalement, Cif1p est une protéine nucléolaire dont la bonne localisation est essentielle à sa capacité à induire l’état Cin. Ceci suggère une interaction entre la fonction essentielle de la calnexine et une fonction exécutée dans le nucléole. Lors d’études visant à élucider la fonction cellulaire de Cif1p, il a été établi qu’elle interagissait avec certaines protéines de la grosse sous-unité du ribosome telle la protéine L3. Cependant, Cif1p ne co-sédimente pas avec des sous-unités ribosomales assemblées, des ribosomes ou des polysomes. De plus, des cellules contenant une délétion génomique de cif1 voient leur contenu en ribosomes perturbé lors de la phase stationnaire. Il semble donc que Cif1p joue un rôle dans la biosynthèse des ribosomes lors de la phase stationnaire. Ce rôle spécifique à cette phase de croissance coincide avec un clivage de la portion N-terminale de Cif1p, clivage qui a lieu lors de l’entrée des cellules en phase stationnaire. De plus, des études effectuées récemment dans notre laboratoire proposent que la calnexine joue un rôle important dans la signalisation de l’apoptose, et ce particulièrement en phase stationnaire. Ainsi, une voie impliquant Cif1p, sa fonction nucléolaire dans la biosynthèse des ribosomes en phase stationnaire, la calnexine et la médiation de l’apoptose semble se dessiner. D’autres travaux, notamment sur la fonction exacte de Cif1p, le rôle de son clivage et les autres composantes impliquées dans le phénomène Cin nous permettront de dessiner un portrait plus complet de cette voie cellulaire inédite. / Protein folding is a vital process that involves many proteins of the cell. One of them is calnexin, a chaperone of the endoplasmic reticulum. In the fission yeast Schizosaccharomyces pombe, calnexin is essential for survival of the cells. During structure-function studies on calnexin, our laboratory discovered a phenomenon allowing the viability of cells without this chaperone. This state, designated Cin for Calnexin INdependence, is induced by a calnexin mutant devoid of the highly conserved central domain (Δhcd_Cnx1p). Characterization of the Cin cells showed several exceptional properties such as dominance, non-Mendelian transmission and transmission via cell extracts devoid of nucleic acids of the Cin state. All these observations suggested that the Cin phenomenon is mediated via a prionic element. To identify genes implicated in the Cin state, genetic screens were performed. They led to the identification of the cif1+ gene, for calnexin independence factor. This gene encodes an orphan protein, the overexpression of which stably induces a state of viability in the absence of calnexin. Notably, this state is genetically and phenotypically distinct from the previously isolated Cin state arising from Δhcd_Cnx1p expression. This suggests the presence of two parallel pathways both able to signal the induction of the Cin phenomenon. The exhaustive characterization of Cif1p showed that it is not the prion solely responsible for the Cin state, although it displays prion-like properties in vitro. Finally, nucleolar localization of Cif1p is required to induce the Cincif1 state, thus suggesting an unexpected interaction between the vital cellular role of calnexin and a function of the nucleolus. While investigating Cif1p function in the cell, we observed that it interacts with ribosomal proteins of the large subunit, notably L3, but it does not sediment with assembled ribosomal subunits or whole ribosomes. However, cells containing a genomic deletion of cif1 also have a disrupted ribosome content during stationary phase. Altogether, these results suggest that Cif1p has a role in ribosomal biogenesis during stationary phase. This growth-phase specific role correlates with the occurence during stationary phase of a cleavage in the N-terminal part of Cif1p. Recent studies from our laboratory proposed that calnexin plays an important role in apoptosis signaling, especially in stationary phase. Thus, a pathway implicating Cif1p, its nucleolar function in ribosome biosynthesis in stationary phase, calnexin and apoptosis signaling is starting to emerge. However more studies, notably on the exact function of Cif1p, the role of its cleavage and the other proteins implicated in the Cin state will be necessary to draw the complete scheme of this unprecedented cellular pathway.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.