Saccharomyces cerevisiae is an excellent experimental model organism to study various biological processes owing to its versatile genetics, biochemistry, and standard laboratory conditions. S. cerevisiae shows distinct biological responses under nutritional starvation conditions. S. cerevisiae undergoes dimorphic transition from a unicellular yeast form to a multicellular pseudohyphae (Gimeno et al., 1992) under nitrogen starvation, but in the complete absence of a fermentable carbon source, it undergoes gametogenesis called sporulation (Mitchell, 1994). While the signal transduction cascades and regulatory controls under nutritional starvation conditions are studied to great extent, the role of S. cerevisiae core RNA polymerase II (pol II) is not much understood. S. cerevisiae core RNA pol II consists of 12 subunits (Woychik and Hampsey, 2002), which is organized into a ten-subunit core and the Rpb4/7 subcomplex (Edwards et al., 1991). Rpb4/7 subcomplex is known to play important roles in stress survival (Choder 2004; Sampath and Sadhale, 2005.). S. cerevisiae rpb4 null diploid strains show reduced sporulation levels but exhibits a predisposition to pseudohyphal morphology (Pillai et al., 2003). Overexpression of Rpb7 partially rescues some of these defects (Sharma et al., 1999; Sheffer et al., 2001). Rpb7 is a highly conserved protein but Rpb4 is the least conserved amongst all RNA pol II subunits at the sequence level. Rpb4 and Rpb7 also affect different cellular functions, which are not directly dependent on each other. (a) Relative levels of RNA pol II subunits Rpb4 and Rpb7 differentially affect starvation response in Saccharomyces cerevisiae S. cerevisiae rpb4 null diploid strains show reduced sporulation levels as compared to wild type but exhibits pseudohyphal predisposition. Overexpression of RPB7 partially rescues the sporulation defect but results in an exaggeration of the pseudohyphae phenotype. We generated S. cerevisiae strains expressing different levels of Rpb4 and Rpb7 proteins in the same strains and analyzed their effect on sporulation and pseudohyphal morphology. We observed that sporulation is dependent on Rpb4 because sporulation level gradually increases with an increase in the Rpb4 protein level in the strain. Rpb7 reduces sporulation level but enhances pseudohyphal exaggeration in a dose-dependent manner. Rpb4 is dominant over Rpb7 in both the starvation responses because strain expressing an equimolar ratio of Rpb4 and Rpb7 protein exhibits RPB4+ phenotypes. (b) Domainal organization of Saccharomyces cerevisiae Rpb7 orthologs reflects functional conservation Rpb7 orthologs are known in eukaryotes and archaebacteria. The primary structure of Rpb7 is conserved. We chose Rpb7 orthologs from Candida albicans, Schizosaccharomyces pombe and Homo sapiens sapiens to investigate whether Rpb7 orthologs are also functionally conserved. We observed that all the orthologs tested are functionally conserved because they can complement the absence of RPB7 in S. cerevisiae. However, we uncovered functional differences amongst Rpb7 orthologs with respect to its function in rpb4 null strain and ess1 ts strain. Furthermore, we made N and C-terminal chimeric RPB7 constructs from these orthologs with S. cerevisiae Rpb7. These chimeras also can replace ScRpb7 in S. cerevisiae. However, functional differences were observed with each chimera pair in rpb4 null strain and ess1 ts strain, showing that the N and C-terminal domains of Rpb7 protein can be genetically dissected. The genetic observation on the domainal organization of Rpb7 orthologs is strengthened by the crystal structure of Rpb7 (Armache et al., 2005), which shows that Rpb7 is structurally organized into an N terminal RNP domain and a C terminal OB fold domain. (c) The Rpb7 subunit of Candida albicans RNA polymerase II induces lectin-mediated flocculation in Saccharomyces cerevisiae The Rpb7 ortholog of C. albicans is a conserved functional ortholog of ScRpb7. We observed that CaRpb7 induces Ca2+-dependent flocculation and agar-invasive growth in S. cerevisiae. CaRpb7 overexpression induces very high transcript levels of FLO1 and FLO11. We believe that the observed flocculation and agar-invasive phenotypes are due to Flo1 and Flo11 respectively, because Flo1 and Flo11 contribute mainly to cell-cell adhesion while Flo11 contributes mainly to cell-substrate adhesion (Verstrepen and Klis, 2006; Lo et al., 1998; Guo et al., 2000). Pathway analysis revealed that CaRpb7-induced flocculation is dependent on Mss11 transcriptional activator. Two-hybrid analysis revealed that CaRpb7 does not physically interact with transcriptional repressors known to repress FLO gene transcription, however genetic analysis revealed that CaRpb7 is epistatic to the repressor Sfl1. Rpb7 orthologs possess conserved domains with potential RNA binding ability (Orlicky et al., 1999) and ScRpb7 is known to play in mRNA stability (Lotan et al., 2007). The possibility of CaRpb7 specifically affecting the stability of FLO gene transcripts is being pursued.

Chez Saccharomyces cerevisiae, les souches mutantes pour Rrd1, une protéine qui possède une activité de peptidyl prolyl cis/trans isomérase, montrent une résistance marquée à la rapamycine et sont sensibles au 4-nitroquinoline 1-oxide, un agent causant des dommages à l’ADN. PTPA, l’homologue de Rrd1 chez les mammifères, est reconnu en tant qu’activateur de protéine phosphatase 2A. Notre laboratoire a précédemment démontré que la surexpression de PTPA mène à l’apoptose de façon indépendante des protéines phosphatase 2A. La fonction moléculaire de Rrd1/PTPA était encore largement inconnue au départ de mon projet de doctorat. Mes recherches ont d’abord montré que Rrd1 est associé à la chromatine ainsi qu’à l’ARN polymérase II. L’analyse in vitro et in vivo par dichroïsme circulaire a révélé que Rrd1 est responsable de changements au niveau de la structure du domaine C-terminal de la grande sous-unité de l’ARN polymérase II, Rpb1, en réponse à la rapamycine et au 4-nitroquinoline 1-oxide. Nous avons également démontré que Rrd1 est requis pour modifier l’occupation de l’ARN polymérase II sur des gènes répondant à un traitement à la rapamycine. Finalement, nous avons montré que suite à un traitement avec la rapamycine, Rrd1 médie la dégradation de l’ARN polymérase II et que ce mécanisme est indépendant de l’ubiquitine. La dernière partie de mon projet était d’acquérir une meilleure connaissance de la fonction de PTPA, l’homologue de Rrd1 chez les mammifères. Nos résultats montrent que le «knockdown» de PTPA n’affecte pas la sensibilité des cellules à différentes drogues telles que la rapamycine, le 4-nitroquinoline 1-oxide ou le peroxyde d’hydrogène (H2O2). Nous avons également tenté d’identifier des partenaires protéiques pour PTPA grâce à la méthode TAP, mais nous ne sommes pas parvenus à identifier de partenaires stables. Nous avons démontré que la surexpression de la protéine PTPA catalytiquement inactive n’induisait pas l’apoptose indiquant que l’activité de PTPA est requise pour produire cet effet. Finalement, nous avons tenté d’étudier PTPA dans un modèle de souris. Dans un premier lieu, nous avons déterminé que PTPA était exprimé surtout au niveau des tissus suivants : la moelle osseuse, le thymus et le cerveau. Nous avons également généré avec succès plusieurs souris chimères dans le but de créer une souris «knockout» pour PTPA, mais l’allèle mutante ne s’est pas transférée au niveau des cellules germinales. Mes résultats ainsi que ceux obtenus par mon laboratoire sur la levure suggèrent un rôle général pour Rrd1 au niveau de la régulation des gènes. La question demeure toujours toutefois à savoir si PTPA peut effectuer un rôle similaire chez les mammifères et une vision différente pour déterminer la fonction de cette protéine sera requise pour adresser adéquatement cette question dans le futur. / In Saccharomyces cerevisiae, mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to rapamycin and show sensitivity to the DNA damaging agent 4-nitroquinoline 1-oxide. PTPA, the mammalian homolog of Rrd1, has been shown to activate protein phosphatase 2A. Our laboratory previously found that overexpression of PTPA leads to apoptosis independently of PP2A. At the outset of my thesis work, the molecular function of Rrd1/PTPA was largely unknown. My work has shown that Rrd1 is associated with the chromatin and interacts with RNA polymerase II. In vitro and in vivo analysis with circular dichroism revealed that Rrd1 mediates structural changes of the C-terminal domain of the large subunit of RNA pol II, Rpb1, in response to rapamycin and 4-nitroquinoline 1-oxide. Consistent with this, we demonstrated that Rrd1 is required to alter RNA pol II occupancy on rapamycin responsive genes. We also showed that upon rapamycin exposure Rrd1 mediates the degradation of RNA polymerase II and that this mechanism is ubiquitin-independent. Another part of my work was to gain insight into the function of PTPA, the mammalian counterpart of Rrd1. PTPA knockdown did not affect sensitivity to rapamycin, 4-nitroquinoline 1-oxide or H2O2. We also attempted to find protein interaction partners for PTPA using tandem affinity purification, but no stable partners for PTPA were found. We also demonstrated that overexpression of a catalytically inactive PTPA mutant did not induce apoptosis, indicating that PTPA activity is required to produce this effect. Finally, we attempted to study PTPA in a mouse model. We first determined that PTPA was expressed in a tissue-specific manner and was most abundant in bone marrow, thymus and brain. We pursued creation of a knockout mouse and successfully generated chimeras, but the mutated allele was not transmitted to the germline. My data and other data from our laboratory regarding the yeast work suggest a general role for Rrd1 in regulation of gene transcription. Whether PTPA has a similar function in mammalian cells remains unknown, and a different vision of what the protein does in mammalian cells will be required to adequately address this question in the future.

No description available.

Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T17:37:36Z (GMT). No. of bitstreams: 1 Vaz_ThaisHaline_D.pdf: 13126521 bytes, checksum: 672f3c7b0345ee333ab24793e624068d (MD5) Previous issue date: 2008 / Resumo: A resposta individual das células está na base da resistência do organismo à infecção viral. O principal mecanismo de resistência envolve a participação de inúmeros genes da via de sinalização dos interferons. Vários estudos vêm sendo conduzidos em larga escala para identificar genes que respondem aos mais variados tratamentos, assim como clusters gênicos relacionados a determinadas enfermidades, como a leucemia. A função do produto de muitos destes genes ainda não foi caracterizada. Numa ampla revisão destes artigos identificamos a proteína KIAA0082/ISG95 respondendo a interferon, à infecção pelo vírus da hepatite C (HCV), ao tratamento celular com oligodeoxinucleotídeos CpG, fazendo parte de um cluster de genes relacionados à leucemia e sendo super-expressa em linfócitos T ativados. Embora não possua função conhecida, esta proteína apresenta quatro domínios que indicam uma possível atividade relacionada ao metabolismo de RNA. Neste trabalho demonstramos que o promotor do gene ISG95 responde à estimulação por interferon num sistema repórter em células Vero. As atividades bioquímicas de ISG95 foram determinadas usando a proteína recombinante expressa em células de inseto Sf9. ISG95 interage com RNA e com S-adenosilmetionina, possuindo também atividade de metiltransferase in vitro. Ensaios de localização sub-celular demonstraram sua distribuição nuclear. Além disso, através do método duplo-híbrido de levedura e de ensaio de co-imunoprecipitação, foi possível identificar sua interação com o domínio C-terminal (CTD) da RNA polimerase II, o que é consistente com sua localização nuclear e com a função predita para o domínio WW localizado na extremidade C-terminal de ISG95. Os resultados indicam que ISG95 é parte da via de resposta a interferon e tem função associada possivelmente a eventos de processamento de prémRNA mediados pelo domínio CTD da RNA polimerase II / Abstract: A major mechanism of cellular resistance to viral invasion involves genes from the interferon signaling pathway, called ISGs (interferon stimulated genes). Global transcriptional profiling studies have linked increased expression of ISG95 (KIAA0082) to response to interferon treatment and to viral infection, suggesting that it may be part of the cellular defense against viral replication. In this work, we shown that the ISG95 promoter can drive interferoninduced transcription of a reporter gene in Vero cell cultures. The biochemical functions of ISG95 were assessed using recombinant protein. ISG95 shows RNA- and S-adenosyl-methionine binding and protein methyltransferase activity in vitro. ISG95 interacts with the C-terminal domain of RNA polymerase II, which is consistent with its nuclear localization and with the predicted function of the WW domain found in the C-terminal region of ISG95. The results presented in this work indicate that ISG95 is part of the interferon response pathway and functions in the pre-mRNA processing events mediated by the C-terminal domain of the RNA polymerase II / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Des études antérieures suggèrent que les complexes SAGA et TFIID sont des facteurs jouant un rôle complémentaire dans la transcription par l’ARN polymérase II. Chez S. cerevisiae, environ 10% des gènes semblent dépendants de SAGA alors que TFIID aurait un rôle dominant sur 90% du génome. De nouvelles approches m’ont permis de montrer que SAGA est recruté sur les régions régulatrices d’une majorité de gènes, indépendamment de leur classification. Des analyses d’ARN nouvellement-synthétisés ont également démontré que l’inactivation des complexes SAGA ou TFIID, par mutation ou déplétion inductible de leurs sous-unités, altèrent la transcription de pratiquement tous les gènes par l’ARN polymérase II. L’acétyltransférase Gcn5 et la sous-unité Spt3 agissent de façon synergique au sein du complexe SAGA pour stimuler le recrutement de TBP et la transcription par l’ARN polymérase II. Ces données indiquent que les complexes SAGA et TFIID agissent comme des cofacteurs généraux, étant nécessaires pour la synthèse de quasiment tous les ARNm et ayant des effets équivalents sur les gènes précédemment décrits comme dominés par SAGA ou par TFIID. / Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II (RNA Pol II) transcription with about 10% of genes in S. cerevisiae dependent on SAGA. The remainder 90% of the genome would be regulated by TFIID. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We observed that SAGA maps to regulatory elements of most genes, irrespective of previous designations of SAGA- or TFIID-dominated genes. Additionally, disruption of either SAGA or TFIID through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly-synthesized RNA or RNA Pol II chromatin immunoprecipitation. We also found that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that both SAGA and TFIID act as general cofactors required for essentially all RNA Pol II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.

ARS2 is a stable component of the nuclear cap-binding complex (CBC) and is critical for RNA Polymerase II transcript processing. As such, ARS2 functions in numerous RNA Polymerase II transcript processing events, which happen co-transcriptionally from initiation to termination, and post-transcriptionally during maturation and export into the cytoplasm. Developmentally, ARS2 is essential for stem cell maintenance and differentiation during embryogenesis and in neural stem cells. Two major questions in the field were: 1) how does ARS2 function in stem cell maintenance and/or differentiation? and 2) how does ARS2 distinguish between disparate RNA classes and processing complexes? In chapter 2, I show that ARS2 is required for the proliferation and cell fate decisions of progenitors in the mouse retina. Specifically, ARS2 knockdown delays cell cycle progression and leads to premature cell cycle exit. Additionally, ARS2 knockdown increases the proportion of cells expressing rod photoreceptor marker Nrl, and decreases Müller glial marker expression. Similarly, knockdown of FLASH, an essential component for replication-dependent histone transcript processing and cell cycle progression, increases the proportion of cells expressing the Nrl reporter, suggesting ARS2’s role in histone processing is contributing to cell cycle progression and fate specification in the developing retina. In chapter 3, I used bioinformatics analysis and homology modeling to classify four structural domains of mammalian ARS2, including a newly identified RNA recognition motif (RRM), and performed mutagenesis to assess their functions. The unstructured C-terminus is required for interaction with the CBC, the Mid domain is implicated in binding DROSHA, which is required for microRNA biogenesis, while the zinc finger and RRM are involved in binding FLASH. Moreover, the zinc finger is required for interacting with RNA. Collectively, this work establishes a model where ARS2 acts as a scaffold, using multiple domains to interact with distinct processing complexes in a mutually exclusive manner. It is also the first study describing the requirements of ARS2 in the developing retina. Understanding the molecular mechanisms governing progenitor proliferation and cell fate specification is crucial in order to design therapies for retinal degenerative diseases. / Graduate / 0487

La transcription est la première étape de l’expression des gènes. Chez les eucaryotes, la transcription par l’ARN polymérase II (Pol II) est un processus hautement régulé. Elle commence par la fixation d’activateurs spécifiques sur des régions régulatrices. Cela permet le recrutement de co-activateurs suivi des facteurs généraux de la transcription (GTFs) et de l’ARN polymérase II pour former le complexe de préinitiation (PIC). Le Médiateur est un complexe co-activateur essentiel à ce processus. Chez la levure Saccharomyces cerevisiae, il est composé de 25 sous-unités dont 10 sont essentielles à la viabilité. Son rôle principal est d’intégrer les signaux de régulation pour les transmettre aux composants du PIC. On connait aujourd’hui un certain nombre de fonctions du Médiateur. Néanmoins, sa complexité et la présence de sous-unités essentielles compliquent la compréhension détaillée de son mécanisme de fonctionnement in vivo. Au cours de ma thèse, je me suis intéressé aux sous-unités essentielles Med10 et Med7, toutes deux appartenant au module du milieu du Médiateur, peu étudié jusqu’à présent. Nous avons construit une collection de mutants thermosensibles de ces deux sous-unités chez la levure S. cerevisiae. Nous avons caractérisés ces mutants par différentes approches de biologie moléculaire, biochimie et génomique fonctionnelle. L’étude de la sous-unité Med10 nous a permis de mettre en évidence in vivo un lien fonctionnel entre le Médiateur et TFIIB, un GTF essentiel au recrutement de la Pol II. Nous avons ainsi identifié les sous-unités Med14 et Med10 qui sont en contact avec TFIIB. Nos analyses de ChIP-seq montrent que le module du milieu et Med10, en particulier, est requis pour la formation correcte du PIC sur l’ensemble du génome. Ces données nous ont également permis de montrer que le Médiateur influence la formation du PIC en relation avec l’architecture des promoteurs en termes de présence de boîtes TATA, d’occupation des nucléosomes et leur dynamique. Ce travail nous a permis une meilleure compréhension du rôle du Médiateur dans l’activation de la transcription et donné des informations mécanistiques sur la façon dont l’interaction entre le Médiateur et TFIIB (et les autres GTFs) ainsi que l’architecture des promoteurs mènent à une régulation gène-spécifique. / Transcription is the first step of gene expression. In eukaryotes, messenger RNA (mRNA) transcription is a highly regulated process. Transcription begins with the binding of a specific transcription factor on a DNA regulatory sequence. This enable the recruitment of co-activators, followed by general transcription factors (GTFs) and RNA polymerase II (Pol II) to form preinitiation complex (PIC). Mediator is a co-activator complex which is essential in this process. In yeast Saccharomyces cerevisiae, Mediator is composed of 25 subunits, among which 10 are essential for cell viability, organized into four distinct modules. The main role of this complex is to transmit regulatory signal to PIC components. Although Mediator has been the subject of a large numbers of studies, its complexity prevents the detailed understanding of how it acts in vivo. During my PhD, I focused my work on the study of the two essential subunits Med7 and Med10. Both of these subunits belong to the middle module, poorly studied so far. We obtained a collection of temperature-sensitive mutants of Med7 and Med10 in yeast S. cerevisiae. We used different molecular biology and functional genomics to characterize these mutants. The work on Med10 subunit enabled us to highlight in vivo a functional link between Mediator and TFIIB, one of the GTFs. Notably, we have shown a new contact between Med14 subunit and TFIIB. Our ChIP-seq analysis shows that Mediator middle module, and in particular Med10 subunits, is crucial for PIC assembly genome-wide. These data also permit us to show that Mediator influence PIC formation in relation to promoter architecture. Taken together, these results indicates that Mediator in crucial to orchestrate the incorporation of the different proteins into the PIC. This work permit us to improve our understanding of how functional interplay between Mediator, TFIIB, other GTFs, and the promoter architecture leads to gene-specific transcription.

ARMC5 est une protéine qui contient sept motifs Armadillo répétitifs organisés en tandem et un domaine BTB. Nous avons observé que cette protéine était fortement exprimée dans les organes lymphoïdes, les glandes surrénales et le cerveau. Les souris avec une délétion d’Armc5 (souris KO) étaient de petite taille, et présentaient une diminution de la prolifération et la différenciation des lymphocytes T. L’absence d’ARMC5 entraînait une déficience de la réponse immunitaire médiée par les lymphocytes CD4+ et CD8+ dans les modèles expérimentaux d’encéphalomyélite auto-immune et d’infection au virus de la chorioméningite lymphocytaire, respectivement. Par la suite, plusieurs études ont révélé que la mutation ARMC5 était associée à l’hyperplasie macronodulaire bilatérale primitive des surrénales (HMBPS), qui représente une cause rare du syndrome de Cushing. Nous avons ensuite confirmé que l’hyperplasie des glandes surrénales s’était développée chez les souris KO âgées, et qu’elle s’accompagnait d’une légère augmentation des taux sériques de glucocorticoïdes. Comme ARMC5 ne présentait pas d’activité enzymatique, il était probable qu’elle faisait appel à d’autres protéines pour exercer sa fonction. Nous avons identifié plusieurs protéines qui se liaient à ARMC5, et plus particulièrement le complexe ARMC5/Cullin3 qui formait une ubiquitine ligase (E3) spécifique de la sous-unité RPB1 de l’ARN polymérase II. ARMC5 contrôlait le processus d’ubiquitination de RPB1 qui, par conséquent, s’accumulait dans plusieurs organes majeurs : les glandes surrénales, les ganglions lymphatiques, le cerveau, les poumons, le foie, etc. chez la souris KO. Ces résultats démontrent un rôle clé de l’ubiquitine ligase dans la dégradation de la protéine RPB1. Une accumulation similaire a également été observée dans les tissus hyperplasiques des surrénales provenant de patients atteints d’HMBPS et porteurs de la mutation ARMC5, ce qui souligne la pertinence clinique de nos résultats de recherche fondamentale dans les maladies humaines. Un défaut de dégradation de RPB1 augmentait le pool d’ARN polymérase II. Par ailleurs, nous avons identifié un groupe de gènes fortement surexprimés dans les glandes surrénales déficientes en ARMC5, parmi lesquels figurent les gènes effecteurs qui seraient impliqués dans l’hyperplasie des surrénales chez les souris KO et l’HMBPS chez les patients porteurs de la mutation ARMC5. Finalement, nous avons montré que la délétion ou la mutation d’Armc5 augmentait considérablement le risque des anomalies du tube neural chez les souris et les humains. Chez les patients souffrant de myéloméningocèle, nous avons constaté neuf différentes mutations faux-sens délétères, dont une diminuait l’interaction entre ARMC5 et RPB1. L’augmentation du pool d’ARN polymérase II dans les cellules précurseurs neurales (CPN), causée par la délétion ARMC5, influençait un groupe particulier de gènes, dont certains (p. ex. Folh1) seraient susceptibles de participer au développement du tube neural. En résumé, l’association ARMC5 et Cullin3 forme un complexe E3 qui cible RPB1 provoquant son ubiquitination et sa dégradation. En absence d’un tel mécanisme, on observe une perturbation de l’homéostasie de l’ARN polymérase II, qui mène à une diminution de la réponse immunitaire médiée par lymphocytes T, le développement d’HMBPS et un risque accru d’anomalies du tube neural. / ARMC5 protein contains seven tandem Armadillo repeats and one BTB domain. We observed that Armc5 was highly expressed in the lymphatic organs, adrenal glands, and brain. Armc5 knockout (KO) mice were small in size and exhibited compromised T cell proliferation and differentiation. The absence of ARMC5 resulted in an impairment of the CD4 + cell- and CD8 + cell-mediated immune response in the experimental autoimmune encephalomyelitis model and lymphocytic choriomeningitis virus infection model, respectively. Subsequently, several studies revealed that ARMC5 mutations were related to primary bilateral macronodular adrenal hyperplasia (PBMAH), which is a rare cause of Cushing’s syndrome. We then confirmed that adrenal gland hyperplasia was indeed developed in aged Armc5 KO mice with mildly increased serum glucocorticoid levels. Since ARMC5 did not exhibit enzymatic activity, its function likely depends on the interaction with other proteins. We identified several proteins that binds to ARMC5, most notably ARMC5 binding to Cullin3, forming a ubiquitin ligase (E3) specific for RNA polymerase II subunit I (RPB1). ARMC5 regulated the ubiquitination of RPB1, and its deletion resulted in RPB1 accumulation in major organs (e.g., adrenal glands, lymph nodes, brain, lung, and liver), indicating the critical role of this E3 in RPB1 degradation. A similar accumulation was also found in hyperplasia tissues from adrenal glands of PBMAH patients carrying ARMC5 mutations, underscoring the clinical relevance of our basic research findings in human disease. Defective degradation of RPB1 led to an enlarged RNA polymerase II (Pol II) pool. In addition, we have identified a group of genes strongly upregulated in KO adrenal glands, including the effector genes which would be involved in adrenal gland hyperplasia in Armc5 KO mice and PBMAH patients carrying ARMC5 mutation. Finally, we have shown that deleting or mutating Armc5 significantly augments the risk of neural tube defects in mice and humans. In patients with myelomeningocele, we found nine deleterious missense mutations in ARMC5, one of which weakened the interaction between ARMC5 and RPB1. The enlarged Pol II pool in Armc5 KO neural precursor cells (NPCs) influenced a particular group of genes, some of which (e.g., Folh1) are thought to be involved in the development of the neural tube. In summary, ARMC5 and CUL3 form an E3 complex, which targets RPB1 causing its ubiquitination and degradation. In the absence of such a mechanism, there is a disturbance of RNA polymerase II homeostasis, which leads to a decrease in the T cell-mediated immune response, the development of PBMAH and an increased risk of neural tube defects.

Trypanosoma cruzi é o agente causador da Doença de Chagas, que segundo a OMS, atinge oito milhões de pessoas principalmente na América Latina, causando danos à saúde pública, juntamente com um impacto econômico negativo. Durante o processo de infecção, uma variedade de eventos celulares ocorre apenas pelo simples contato do parasito com a célula hospedeira, levando a modificações no metabolismo celular e alterações morfológicas. O parasita é capaz de modular respostas celulares e imunológicas da célula hospedeira para sua própria sobrevivência. Além do que, pode alterar compartimentos celulares como o número e tamanho de nucléolos, sugerindo que a presença do parasita poderia estar interferindo na maquinaria nuclear. Porém, pouco se conhece sobre a organização nuclear da célula hospedeira quando infectada por Trypanosoma cruzi. O objetivo deste estudo foi de investigar pela primeira vez os compartimentos nucleares das células hospedeiras durante o curso da infecção por T. cruzi. Células LLC-MK2 foram infectadas com T. cruzi e reações de imunofluorescência indireta foram realizadas utilizando anticorpos e marcadores específicos para proteínas nucleares. As análises das imagens de microscopia confocal e quantificação das fluorescências pelo ImageJ mostraram padrões distintos nos compartimentos nucleares quando comparadas com células não infectadas. Corpos de Cajal e Speckles sofrem alterações quando a célula está infectada e isso depende do ciclo celular do parasita. Neste trabalho também foi investigado através de quantificação de imagem e immunoblotting o comportamento das Ribonucleoproteínas A1 e A2B1 durante a parasitemia. Estas análises demonstram que o T. cruzi pode modular a célula hospedeira quando infectada a favor de sua sobrevivência, promovendo alterações na dinâmica dos compartimentos nucleares durante o seu ciclo celular. Esse estudo inédito poderá auxiliar a compreender a biologia do parasita e sua interação com a célula hospedeira e desta maneira contribuir na busca de possíveis alvos terapêuticos / Trypanosoma cruzi is the causal agente of Chagas disease, that affects about eight million people mostly in Latin America according to the WHO, causing damage to public health and a negative economic impact. During infection, a variety of signaling processes occur after contact of the parasite to the host cell, what can lead to metabolic modifications as well morphological alterations in both cells. The parasite can modulate host cell cellular and immunological responses for its own survival. In addiction, the presence of T. cruzi can modify the nuclear compartments such as nucleoli, suggesting that the presence of the parasite could be interfering with the nuclear machinery. However, little is know about the nuclear organization when the host cell is infected with Trypanosoma cruzi. This study aimed to investigate for the first time the nuclear compartment of host cells infected by T. cruzi using specific antibodies and fluorescent markers for nuclear compartments, in order to investigate the morphological and functional changes in the nucleus of the host cell. Using LLC-MK2 cells infected with T. cruzi, we performed indirect immunofluorescence using distinct nuclear antibodies. Confocal microscopy analysis of infected cells showed pattern variations in the nuclear compartments when compared to uninfected cells. Cajal bodies and Speckles suffer alterations when the cell is infected and it is related to the parasite life cycle. In this work we also investigated by image quantification and immunoblotting the behavior of Ribonucleoproteins A1 and A2B1 during infection. These evidences support the idea that T. cruzi can modulate host cell response to ensure its own survival during the infection, promoting changes in the dynamics of the nuclear compartments. This unpublished data may help to understand the biology of the parasite and its interaction with the host cell and thus contributing to seek for potential therapeutic targets

Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase. / It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.