Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2013-01-24T16:40:43Z No. of bitstreams: 1 lysangela_r_alves_ioc_bcm_0005_2010.pdf: 12423469 bytes, checksum: 674a41c28ac9c8cf38b2ee15fb9b891b (MD5) / Made available in DSpace on 2013-01-24T16:40:43Z (GMT). No. of bitstreams: 1 lysangela_r_alves_ioc_bcm_0005_2010.pdf: 12423469 bytes, checksum: 674a41c28ac9c8cf38b2ee15fb9b891b (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / Em tripanossomatídeos a regulação da expressão gênica ocorre principalmente em nível pós-transcricional. Acredita-se que a estabilidade do mRNA e o acesso aos polissomos sejam fortemente regulados, permitindo ao Trypanosoma cruzi uma rápida adaptação à diferentes condições ambientais as quais está exposto durante seu ciclo de vida. A regulação pós-transcricional requer uma associação entre mRNAs e determinadas proteínas formando complexos ribonucleoprotéicos (mRNPs). Nosso objetivo foi investigar a dinâmica de associação entre os mRNAs e proteínas, isolando e caracterizando proteínas e complexos protéicos ligados a mRNAs poliA+ das frações polissomal e pós-polissomal de epimastigotas em fase exponencial de crescimento e epimastigotas sujeitos a estresse nutricional. As amostras obtidas foram analisadas por espectrometria de massas (LC-MS/MS) e posteriormente comparadas. Nós identificamos 542 proteínas e dentre essas 24 estavam presentes em todas as frações analisadas, enquanto que outras eram exclusivas de uma fração específica: epimastigota frações polissomal (0,37%) e pós-polissomal (2,95%); estresse frações polissomal (13,8%) e pós-polissomal (40,78%). Proteínas sabidamente envolvidas com metabolismo de mRNA foram identificadas, sendo que esse resultado é importante para confirmar a confiabilidade da nossa técnica de isolamento das mRNPs. Essa abordagem em larga escala possibilitou uma análise mais completa da composição dos mRNPs e a dinâmica durante o estresse nutricional em T. cruzi. A partir dos dados obtidos nós selecionamos 6 proteínas para caracterização: fator de elongação 1-alfa (EF1-), proteína de ligação a RNA com domínio dedo de zinco (ZF-211.70), proteína de ligação a RNA com domínio cold shock (CD-33.60), prostaglandina F 2 alfa sintase (PF2 S), prostaglandina F sintase (PFS) e uma proteína hipotética com domínio de fator de replicação A (Hip -11.150). Os anticorpos contra as proteínas EF1-, ZF-211.70, PF2S e PFS apresentaram reatividade específica com uma proteína única de tamanho esperado, já as proteínas CD-33.60 e Hip-11.150 apresentaram um reatividade baixa ou inexistente em extratos de epimastigotas e por isso não foram utilizadas para os ensaios posteriores. Ensaios de imunofluorescência, sedimentação de polissomos em gradiente de sacarose e expressão ao longo do ciclo de vida nos permitiu uma caracterização inicial das proteínas selecionadas, etapas importantes para aprofundarmos o estudo na regulação de expressão gênica em T. cruzi. / Gene regulation is mainly posttranscriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Posttranscriptional regulation requires the association between mRNAs and some proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poli(T) beads to isolate and characterize proteins and protein complexes bound to poli -A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC -MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and postpolysomal (2.95%) fractions; stress polysomal (13.8%) and postpolysomal (40.78% ) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi. From the results obtained we selected six proteins for characterization: elongation factor 1-alpha (EF1-), zinc finger RNA binding protein (ZF-211.70), RNA binding protein with a cold-shock domain (CD-33.60), prostaglandin F 2 alfa synthase (PF2S), prostaglandin F synthase (PFS) and a hypothetical protein with a replication factor domain (Hip-11.150). The antibodies produced against EF1-, ZF-211.70, PF2S and PFS recognized a specific protein of expected size in epimastigote protein extracts; however, the CD-33.60 and Hip-11.150 antibodies did not recognized a specific protein and they were not used for further experiments. Immunofluorescence assays, polysome profile in sucrose density gradient and the expression pattern through the parasite life cyle with the selected proteins allowed us a preliminary characterization and further studies will help to elucidate the posttranscriptional regulation and the formation of RNA regulons in T. cruzi.

Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

Au cours de la transcription, plusieurs facteurs sont assemblés sur les ARN messagers pour former des Ribonucléoparticules de messagers (mRNPs), et contrôler leur maturation, leur stabilité et leur devenir dans le cytoplasme. Afin d’assurer la production de protéines fonctionnelles, la cellule dispose de plusieurs mécanismes de régulation et de contrôle de qualité assurant la fidélité de l’information génétique transmise au niveau ARN messager et protéine.Chez la levure Saccharomyces cerevisiae, un ensemble de protéines associées au pore nucléaire, incluant la SUMO protéase Ulp1, a été impliqué dans un contrôle de qualité des mRNPs régulant leur export vers le cytoplasme. Ces données suggéraient que l’export des ARN messagers pourrait être contrôlé par la modification post-traductionnelle par le polypeptide SUMO d’un ou de plusieurs effecteurs au sein des mRNPs. Afin de mieux comprendre ces processus, nous avons combiné plusieurs approches visant à identifier ces protéines SUMOylées. En particulier, nous avons mis en place un crible protéomique visant à identifier les protéines dont l’association sur les mRNPs dépend d’Ulp1. Ce crible nous a permis de mettre en évidence une régulation par Ulp1 de l’assemblage du complexe THO sur les ARN messagers. Ce complexe, recruté sur les gènes et les mRNPs, est connu pour contribuer à l’efficacité de la transcription, prévenir l’instabilité génétique liée à la formation d’hybrides ADN matrice – ARN messager (dénommés R-loops) et permettre l’export des mRNPs. En combinant l’analyse biochimique de différentes catégories de mRNPs à des expériences d’immunoprécipitation de l’ARN, nous avons montré que l’activité de la SUMO-protéase Ulp1 est nécessaire à l’association du complexe THO sur différents ARN messagers. De plus, nous avons montré que le complexe THO est SUMOylé sur le domaine C-terminal de sa sous-unité Hpr1, et que Ulp1 régule cette modification. Enfin, cet événement de SUMOylation du complexe THO régule son association avec les mRNPs. L’analyse fonctionnelle de mutants affectant la SUMOylation du complexe THO révèle que des défauts de SUMOylation de ce complexe compromettent ses fonctions dans la transcription sans affecter l’export. De manière intéressante, nous avons observé que la présence d’un intron sur des rapporteurs LacZ diminue la sensibilité de leur expression à des inactivations ou des défauts de SUMOylation du complexe THO. Ce phénotype entraine une augmentation relative des niveaux d’ARN pré-messagers dans ces mutants, un phénomène rendant compte de la fuite cytoplasmique apparente d’ARN non épissés précédemment observée dans le mutant ulp1. L’ensemble de ces données caractérise pour la première fois un rôle de la SUMOylation dans le contrôle de l’assemblage et du devenir cellulaire des mRNPs. / During transcription, several factors associate with mRNA to form messenger Ribonucleoparticles (mRNPs), thereby controlling their processing, their stability, and their cytoplasmic fate. To ensure the production of functional proteins from these mRNAs, eukaryotic cells contain numerous regulatory and quality control systems in order to prevent aberrant mRNP accumulation and export.In the yeast Saccharomyces cerevisiae, several nuclear pore associated proteins, including the SUMO isopeptidase Ulp1, have been involved in a mRNP quality control regulating their nuclear export. These data suggested that post-translational modification by SUMO of one or several mRNP components could regulate mRNA export. In order to understand the molecular mechanisms underlying this process, we undertook several approaches to identify these SUMOylated factors. In particular, we have set up a proteomic screen to identify mRNP components whose assembly onto mRNPs depends on Ulp1 activity.This proteomic survey revealed an Ulp1-dependent regulation of THO complex assembly to mRNPs. This complex, recruited to transcribed genes and mRNPs, is known to regulate transcription elongation by preventing DNA-RNA hybrids formation (termed R-loops), and mRNP export. Through a combination of proteomic analysis of mRNPs assembled in Ulp1 mutant cells, with RNA / chromatin immunoprecipitation experiments, we demonstrate that Ulp1 controls specifically the recruitment of the THO complex within mRNPs. SUMOylation analysis further reveals that Ulp1 targets the THO complex subunit Hpr1 on its C-terminal domain for deSUMOylation. We further show that this SUMOylation event regulates THO complex association within mRNPs. Finally, functional analysis reveal that impaired deSUMOylation of the THO complex do not affect mRNP export, but disturbs expression of LacZ reporter genes, a phenotype classically associated with THO complex dysfunction. Intriguingly, the transcriptional effect of inactivation or impaired deSUMOylation of the THO complex on LacZ expression is alleviated by the presence of an intron, providing a molecular basis for previously reported pre-mRNA leakage phenotypes. Our data therefore unravels for the first time a function of SUMO in the control of mRNP assembly contributing to proper mRNP homeostasis.

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