Orientadores: Jörg Kobarg, José Andrés Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T04:43:04Z (GMT). No. of bitstreams: 1 Righetto_GermannaLima_M.pdf: 4383560 bytes, checksum: b0f7fd3a783153d8832bcf6f44d4b195 (MD5) Previous issue date: 2013 / Resumo: O processo de splicing do RNAm é responsável por orquestrar a junção de exons, criando uma grande diversidade de isoformas gênicas. Alterações nos componentes da maquinaria de splicing e, consequentemente, no processamento do pré-RNAm podem causar ou contribuir para uma infinidade de doenças, dentre elas o câncer. A proteína SF2/ASF foi o primeiro fator de splicing a ser caracterizado como proto-oncogênico, estando superexpresso em diferentes tipos de neoplasias. Sabe-se que a ativação desse fator é, principalmente, mediada por SR quinases conhecidas como splicing quinases e pertencentes à família das SRPKs. A quinase SRPK1, responsável pela fosforilação de SF2/ASF no citoplasma, tem conhecida superexpressão em leucemias. Já a quinase SRPK2, paráloga a SRPK1, possui relação já demonstrada com a proliferação de células leucêmicas e com sua diferenciação. Diante desse quadro, buscamos nesse estudo possíveis correlações entre a maquinaria de splicing e o câncer, dando enfoque à relação entre essa maquinaria e a leucemia. Para tanto, buscamos alterações no cDNA de SRPK2 e quantificamos a expressão das SR quinases SRPK1, SRPK2 e CLK1 em diferentes linhagens de leucemia. Além disso, avaliamos, usando o sistema de Exon Array (Affymetrix), o efeito da superexpressão do fator de splicing SF2/ASF em células de mamífero, buscando alterações globais no splicing dessas células capazes de explicar o caráter oncogênico do fator. Foram encontradas nesse estudo duas novas isoformas de SRPK2, isoladas a partir do cDNA das linhagens de leucemia estudadas. Também foi observada a expressão diferencial das SR quinases SRPK1 e SRPK2 nas linhagens leucêmicas de origem linfóide e mieloide, dando indícios sobre um possível papel divergente dessas quinases nos diferentes tipos de leucemia. Além disso, nas análises preliminares do conjunto de dados obtidos no experimento de Exon Array, foi possível traçar importantes considerações sobre seu caráter oncogênico. Esses dados preliminares do experimento de Exon Array, somados às demais alterações encontradas nas SR quinases, fornecem novas e interessantes pistas sobre a relação entre alterações na maquinaria de splicing e a oncogênese / Abstract: The mRNA splicing is the cellular process responsible for RNA edition, expanding the genome by the combination of gene exons. Mutations in components of this machinery may cause or contribute to a variety of diseases, including cancer. The SF2/ASF protein was the first splicing factor characterized as proto-oncogenic by its overexpression in diverse neoplasias. The cellular activation of this and other splicing factors are mainly dependent on specific kinases, known as splicing kinases and components of a SRPKs family. The SRPK1 kinase, responsible for the cytoplasmic phosphorylation of SF2/ASF, is overexpressed in leukemia. SRPK2, a paralog of SRPK1, is involved in leukemia cell proliferation and differentiation. In this study we searched for splicing machinery and cancer correlations, focusing in the relationship of this machinery and leukemia. In this study we searched for alterations in SRPK2 cDNA and quantified the expression of this kinase and SRPK1 and CLK1 in leukemia immortalized cells. Moreover, we analyzed using Exon Arrays (Affymetrix) the effect of SF2/ASF overexpression in global splicing of non-oncogenic cells, searching for alterations related to its oncogenic character. In this study we discovered two new isoforms of SRPK2 amplified through different leukemia cell lineages. We confirmed the differential expression of SRPK1 and SRPK2 kinases in lymphoid and myeloid leukemia lineages indicating a divergent correlation of these kinases in different leukemia types. In the Exon Array preliminary analysis we also observed important alterations in cellular gene expression. These data and the alterations found in SR kinases provide new and interesting clues about the relationship of splicing machinery alterations and oncogenesis / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

Contribution de U2AF1, NCBP1 et eIF4A3 dans le contrôle du maintien de la pluripotence et le devenir cellulaire.Les mécanismes de maturation du transcrit primaire peuvent profondément affecter la diversité et la fonction des protéines produites à partir d’un gène unique dans le but de mettre en place un programme complexe impliqué dans le maintien de pluripotence et/ou l’initiation de la différenciation des cellules souches humaines. Les réseaux transcriptionnels régulant la pluripotence et la différenciation ont été intensément étudiés contrairement au rôle de l’épissage alternatif dans ces mécanismes, rôle qui pour le moment reste mal compris et pour lequel il n’existe que très peux d’exemples de groupes de gènes subissant un changement général de variant d’épissage aboutissant à la modification du devenir cellulaire. Notre objectif est d’identifier les composés essentiels du spliceosome qui sont impliqués dans le maintien de la pluripotence et la différenciation précoce dans les trois feuillets embryonnaires et d’explorer leurs rôles dans ces processus. Via l’analyse de données de séquençage d’ARN nous avons identifié plusieurs facteurs d’épissage différentiellement exprimés entre les cellules souches et les trois feuillets embryonnaires. Parmi ces facteurs nous focaliserons notre étude sur les facteurs préférentiellement surexprimés dans les cellules souches, qui par conséquent devraient y jouer un rôle primordial. Les candidats sélectionnés, U2AF1, NCBP1 et eIF4A3 ont été déplétés dans des cellules souches en utilisant un système shRNA inductible puis une analyse de séquençage ARN à haut débit a été effectuée pour comprendre les changements du transcriptome induits par ces déplétions. La déplétion d’U2AF1 entraine un changement majeur de l’expression de gènes impliqués dans le développement alors que la déplétion de NCBP1 et eIF4A3 entraine un changement d’expression de gènes impliqués dans le métabolisme, le remodelage de la chromatine et le développement. Des analyses complémentaires ont permis de mettre en lumière une régulation transcriptionnelle et post-transcriptionnelle des gènes différentiellement exprimés dans les conditions étudiées. L’épissage alternatif a pour ça part été modifié par les trois déplétions de manière individuelle. Un programme d’épissage tissu spécifique a été associé à chaque candidat et les conséquences de chaque programme seront décrites au niveau du contrôle qualité de l’ARNm et de la synthèse protéique.Nos résultats construisent une nouvelle vision concernant le rôle des composés essentiels du spliceosome dans le contrôle du devenir cellulaire à travers la modulation de l’épissage alternatif. Cet apport ajoute une nouvelle variable au contrôle de l’expression des gènes et permettra de mieux comprendre les mécanismes du développement précoce et de la diversité tissulaire. / Contribution of U2AF1, NCBP1 and eIF4A3 to the control of pluripotency maintenance and cell fate determination.Alternative pathways for processing the primary transcript can profoundly affect the diversity and function of the protein products that are generated from a single gene to set up complex programs involved in pluripotency and/or differentiation of human Embryonic Stem Cells (hESCs). While transcriptional networks regulating pluripotency and differentiation has been intensively studied, the role of Alternative Splicing (AS) in this process is not yet completely understood and clear examples of concerted switching of multiple genes from one isoform to another have not been demonstrated. Our goal is to identify Core Spliceosomal Factors (CSF), involved in the control of pluripotency maintenance, early differentiation into the three germ layers, and to explore their role in these processes. By RNA-Seq data analysis, we have identified several splicing factors that are differentially expressed between pluripotent stem cells and the three of the germ layers. Among these identified candidates, we focused on the factors that are more highly expressed in pluripotent stem cells, thereby they play a specific role in pluripotency maintenance. The selected candidates, U2AF1, NCBP1 and eIF4A3 were depleted in pluripotent stem cells using inducible shRNA system and RNA-Seq analyzes have been performed to understand transcriptomic changes induced by these depletions. U2AF1 depletion causes a major switch of developmental genes expression, while NCBP1 and eIF4A3 depletions regulate the expression of genes involved in metabolism, chromatin remodeling and development. Further analysis highlighted a transcriptional and post-transcriptional regulation of differentially expressed genes. Alternative Splicing (AS) were shown to be affected by both depletions. A tissue specific AS program was associated to each of the candidates and the consequences of these changes on mRNA quality control and protein synthesis will be described.Our results build a new idea regarding the role of Core Spliceosomal Factors in cell fate control trough the modulation of AS. This knowledge adds a new layer of gene expression control and will allow a better understanding of early development mechanisms and tissue diversity.

SETMAR est un gène chimérique constitué d’un domaine SET (codant des fonctions d’histone méthylase) et du domaine MAR (ayant conservé certaines fonctions de la transposase HsMAR1). Des études ont montré que les deux domaines sont biologiquement actifs et sont impliqués dans la stabilité et/ou dans la régulation de l’expression du génome humain. La littérature suggère que l’expression de SETMAR est plus forte dans les cellules cancéreuses que dans les cellules saines. Notre hypothèse de travail est que la protéine SETMAR est surexprimée en conditions pathologiques, permettant aux cellules de franchir les points de contrôle du cycle cellulaire, contribuant ainsi à augmenter l’instabilité génétique. Notre objectif est d’étudier la régulation de l’expression de SETMAR et son implication dans l’oncogenèse, gliale en particulier. / SETMAR is a chimeric gene consisting of a SET domain (encoding methylase histone functions) and a MAR domain (having retained some of the of the HsMAR1 transposase functions). Studies have shown that the two domains are biologically active and are involved in the stability and / or in the regulation of the human genome expression. The literature suggests that SETMAR expression is higher in cancer cells than in normal cells. Our working hypothesis is that SETMAR protein is overexpressed in pathological conditions, allowing cells to overcome the cellular cycle checkpoints, helping to increase the genetic instability. Our goal is to study the regulation of the SETMAR expression and its involvement in oncogenesis, glial in particular.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

Le genre des Deltaretrovirus est composé des virus de la leucose bovine (BLV) et des virus T-lymphotropes de primates (PTLV-1, -2, -3 et -4) qui regroupent les virus humains T-lymphotropes (HTLV-1, -2, -3 et -4) et les virus simiens apparentés (STLV-1, -2, -3 et -4). Seules les infections par les PTLV-1 et BLV sont associées à des pathologies : une lymphoprolifération maligne appelée ATLL chez l'homme et le singe ou une maladie neurologique appelée HAM/TSP chez l'homme infectés par les PTLV-1 et une lymphoproliferation maligne chez les bovins infectés par BLV. L'infection par HTLV-2 n'est associée qu'à une lymphocytose et le pouvoir pathogène d'HTLV-3 n'est pas caractérisé à ce jour. Les lentivirus, parmi lesquels on trouve les agents étiologiques du SIDA VIH-1 et VIH-2, et les Deltaretrovirus, sont des rétrovirus complexes. Ils codent donc, en plus des protéines structurales et enzymatiques, pour des protéines régulatrices ainsi que pour des protéines auxiliaires qui seront au centre des travaux présentés. Chez HTLV-1 et BLV, les protéines auxiliaires jouent un rôle primordial dans l'infectiosité in vivo. Or ces protéines n'avaient pas encore été décrites chez les PTLV-3. Leur caractérisation composait donc le premier objectif de ces travaux de thèse. Nous avons ainsi identifié les ARN messagers codant 3 nouvelles protéines putatives in vitro. Nous avons étudié les caractéristiques de ces protéines, notamment leur rôle dans le cycle des PTLV-3 in vitro et leur expression in vivo. Dans un second travail, nous avons essayé de comprendre si les différents domaines fonctionnels déjà identifiés dans la protéine Vpx, une protéine auxiliaire de VIH-2 influençaient sa capacité à interagir avec le facteur de restriction cellulaire SAMHD-1. Nous avons voulu déterminer le compartiment cellulaire dans lequel Vpx induisait la dégradation de SAMHD-1 et la cinétique de ce phénomène, qui permet à ce virus de se répliquer dans les lignées myéloïdes. Ces travaux apportent des éléments nouveaux dans la compréhension du rôle des protéines auxiliaires sur la régulation fine du cycle rétroviral et l'échappement au système immunitaire inné / Deltaretroviruses include bovine leukemia viruses (BLV) and primate Tlymphotropic viruses (PTLV-1, -2, -3 and -4) which are composed of human Tlymphotropic (HTLV-1, -2, -3 and -4) and of their simian counterparts (STLV-1, -2, -3 and -4). PTLV-1 and BLV are the only ones associated to pathologies: a lymphoproliferative disorder named ATLL in humans and non human primates and a neurological disorder named HAM/TSP in humans in the case of PTLV-1 and a Bmalignant lymphoproliferation in BLV infected cattle. HTLV-2 has not been associated with any disease so far and the pathogenic potential of HTLV-3 remains unknown. Lentiviruses, including HIV-1 and -2 the AIDS etiological agents, and Deltaretroviruses, are complex retroviruses. Therefore, in addition to structural and enzymatic proteins they encode regulatory proteins and also auxiliary proteins, the main subject of this work. HTLV-1 and BLV auxiliary proteins play key roles in viral infection in vivo. Whether the genome of PTLV-3 encodes such proteins was not determined yet. Therefore their characterization was the first goal of my PhD work. We identified in vitro messenger RNAs encoding 3 new putative proteins. Their impact on the PTLV-3 viral life cycle in vitro and their expression in vivo were then investigated. As a second part of this work, we examined the relationship existing between the Vpx HIV-2 auxiliary protein and its ability to interact with a restriction factor named SAMHD-1. Vpx induces SAMHD-1 degradation and the kinetic of such degradation allows the virus to replicate in myeloid cells. Altogether, these projects provide new insights into the understanding of the roles played by retroviral auxiliary proteins in connection with a tight regulation of viral life cycle and an escape from innate immunity

Chez Arabidopsis thaliana, les gènes d’ARNr 5S sont transcrits par l’ARN polymérase III qui fait intervenir plusieurs facteurs de transcription dont TFIIIA qui reconnaît spécifiquement le promoteur interne de ces gènes et permet ainsi le recrutement de l’ensemble du complexe de transcription. Le gène TFIIIA est composé de sept exons dont le troisième, résultant de l’exonisation d’une molécule d’ARNr 5S, est épissé de façon alternative produisant ainsi deux transcrits. Le transcrit ES, qui ne contient pas cet exon, code pour la protéine TFIIIA pleine longueur et fonctionnelle. Le transcrit EI, dans lequel l’exon « 5S-like » est maintenu, est reconnu et dégradé par la voie NMD (Non-Mediated Decay). L’exon « 5S-like » a, en effet, la particularité de contenir un codon stop prémature qui en fait une cible de la voie NMD. Lors de l’étude de l’expression des transcrits ES et EI ainsi que celle de l’accumulation de la protéine TFIIIA au cours du développement, nous avons montré que le taux de la protéine TFIIIA fonctionnelle est soumis à plusieurs niveaux de régulation. En effet, la production de la protéine TFIIIA pleine longueur est le résultat de la synthèse du transcrit ES et de sa traduction mais également de l’efficacité du clivage protéolytique de la protéine. Lors de la maturation de la graine, l’accumulation croissante de protéine TFIIIA résulte d’une augmentation des quantités de transcrits ES couplée à une diminution du clivage protéolytique. Dans les premiers jours du développement, la protéine TFIIIA n’est détectée qu’après le 4ème jour, suite à la diminution du clivage. Sa présence est corrélée au remodelage de la chromatine de l’ADNr 5S. La combinaison de ces deux mécanismes permet ainsi la production de TFIIIA et de son produit de transcription l’ARNr 5S en fonction des besoins de la plante. / In Arabidopsis thaliana, 5S rRNA genes are transcribed by RNA polymerase III. TFIIIA, specifically required for transcription of these genes, binds to the internal control region of the 5S rRNA genes and allows the assembly of the full transcription complex pol III. The TFIIIA gene consists of seven exons, the third of which results in the exonisation of one 5S rRNA molecule. This exon “5S-like” is alternatively skipped or included to produce either of two transcript isoforms. The ES transcript encodes the fully functional TFIIIA protein. The EI transcript, which contains the exon “5S-like”, is a target of the NMD pathway (Non-Mediated Decay). Indeed, the exon “5S-like” contains a premature stop codon, which is recognized by this RNA decay pathway. During the study of the ES and EI transcripts expression and the TFIIIA protein accumulation throughout the plant development, we show that TFIIIA functional protein levels are under control of many regulation steps. Actually the production of the full-length TFIIIA protein results from production and translation of the ES transcript but also from the proteolytic cleavage efficiency of TFIIIA protein. During the seed maturation, the strong accumulation of TFIIIA results from an increase in ES amounts and a proteolytic cleavage decrease. After the fourth day post-germination, TFIIIA protein is detected because the proteolytic cleavage decreases. TFIIIA presence is correlated with 5S rDNA chromatin reorganization. The combination of these two mechanisms allows TFIIIA production and its transcription product 5S rRNA according to the plants needs.

Orientadores: Íscia Teresinha Lopes Cendes, Cristiane de Souza Rocha / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T01:10:50Z (GMT). No. of bitstreams: 1 BrumattiGoncalves_KatiaCristiane_M.pdf: 3380943 bytes, checksum: e87dbdc3a9db8349ec00b7148c98fd7b (MD5) Previous issue date: 2015 / Resumo: O campo da bioinformática associada à Next Generation Sequencing (NGS) ainda está em estado imaturo. A técnica de microarray tem sido muito utilizada nas últimas décadas em estudos de níveis de expressão de genes, porém essa técnica possui limitações. Sequenciamento de RNA (RNA-Seq) tem vantagens sobre as abordagens atuais, pois permite que o transcriptoma inteiro seja pesquisado com alto rendimento, fazendo com que RNA-Seq seja útil para estudar transcriptomas complexos, além disso, permite a análise de splicing alternativo. Muitas ferramentas têm sido desenvolvidas para abordar diferentes aspectos da análise de dados em RNA-Seq, e sua análise é um desafio constante. Nesse contexto, o objetivo deste estudo foi utilizar métodos de bioinformática para a análise de expressão gênica utilizando dados de RNA-Seq. Para isso, foram utilizados dados brutos obtidos em dois experimentos diferentes: a) utilizando animais normais, na qual a análise comparativa foi realizada da região do hipocampo (CA1, CA2 e CA3) e giro denteado, e b) utilizando animais tratados com pilocarpina e animais controle. Na análise dos dois experimentos, foram encontrados 3 genes (Nnat, Sv2b e Neurod6) em comum que tem diferença na expressão, ambos genes tem envolvimento no sistema nervoso central. Na análise de splicing alternativo, a ferramenta MISO (Mixture of Isoforms) comparado ao pipeline utilizado em Cuffdiff, gerou resultados melhores e mais detalhados, já que a ferramenta também realiza a quantificação dos transcritos, e com seus resultados foram descobertos 6 transcritos (Arpp21, Gria1, Gria2, Nrxn1, Dclk1 e Rtn1) em comum nas regiões do hipocampo, que tem alta expressão em giro denteado. Atualmente, existem diversos softwares em ascensão para análise diferencial, porém, o pipeline utilizado neste trabalho é ainda uma das principais ferramentas para análise de RNA-Seq, por usar algoritmos confiáveis e permitir flexibilização das análises quando necessário. Este estudo apresentou uma proposta de pipeline para a análise de expressão diferencial e identificação de splicing alternativo, para dados obtidos através de tecnologia de sequenciamento RNA-Seq. Foram identificados 5760 transcritos considerados significativamente expressos, e sugere que 6 transcritos sejam decorrentes de splicing alternativo / Abstract: The field of bioinformatics associated with Next Generation Sequencing (NGS) is still in an immature state. The microarray technique has been widely used in recent decades in studies of gene expression levels, but this technique has limitations. Sequencing RNA (RNA-Seq) has advantages over current approaches because it allows the whole transcriptome is researched with high yield, making RNA-Seq be useful for studying complex transcriptomes, moreover, allows the analysis of alternative splicing. Many tools have been developed to aproach different aspects of data analysis in RNA-Seq, and its analysis is a constant challenge. In this context, the objective of this study was to use bioinformatics methods for gene expression analysis using RNA-Seq data. For this, the raw data obtained in two different experiments were used: a) using normal animalsin which was made a comparative analysis of the hippocampus (CA1, CA2 and CA3) and dentate gyrus, and b) using pilocarpine treated animals and animals control. In the analysis of two experiments, were found three genes (NNAT, Sv2b and Neurod6) in common that there is a difference in the expression, both of genes is involved in the central nervous system. In alternative splicing analysis, MISO (Mixture of Isoforms) tool compared to the pipeline used in Cuffdiff, gave better and more detailed results, as the tool also performs the quantification of transcripts, and their results were found 6 transcripts (Arpp21, Gria1, Gria2, Nrxn1, Dclk1 and Rtn1) in common in the regions of the hippocampus, which has high expression in the dentate gyrus. Currently, there are various software on the rise for differential analysis, however, the pipeline used in this work is still one of the main tools for RNA-Seq analysis, by using reliable algorithms and allow flexibility of analyzes when necessary. This study showed a pipeline proposed for the analysis of differential expression, and alternative splicing of identification data obtained for RNA-Seq sequencing technology. 5760 transcripts considered significantly expressed were identified, and suggests that 6 transcripts are derived from alternative splicing / Mestrado / Fisiopatologia Médica / Mestra em Ciências

O splicing alternativo é o processo pelo qual diversos transcritos podem ser gerados a partir de um único gene, sendo de extrema importância para diversidade do repertório transcricional e proteico. Diferentes variantes de splicing são expressas entre os diferentes tecidos e estágios de desenvolvimento garantindo o funcionamento normal da célula, portanto, qualquer alteração neste padrão pode resultar no aparecimento de doenças. Neste contexto, o objetivo deste trabalho foi o estabelecimento de metodologias para identificação de variantes de splicing em câncer de mama sob influência do oncogene ERBB2, o qual é um marcador de mau prognóstico altamente expresso em cerca de 30% dos tumores de mama. Foram estabelecidas duas estratégias para construção de bibliotecas de cDNA. A construção de bibliotecas de cDNA enriquecidas para splicing alternativo, baseada na formação e captura de moléculas de heteroduplexes em combinação com a amplificação de RNAm, foi realizada a partir de RNA total de linhagem celular de mama e a partir de um grupo de cinco amostras tumorais, todas com alta expressão de ERBB2. Foram identificadas 79 possíveis variantes de splicing alternativo em câncer de mama, das quais 18 foram selecionadas para validação por RT-PCR. Foi obtida uma taxa de vallidação de 94% e foram identificadas duas novas variantes de splicing alternativo. A regulação da expressão mediada por ERBB2 de três variantes de splicing foi confirmada por duas metodologias distintas, eletroforese em chip e estratégia baseada na ligação de sondas específicas, que revelou desbalanço de expressão entre as variantes, demonstrando a influência do oncogene na regulação de variantes de splicing. A segunda abordagem utilizada, foi a construção de bibliotecas de cDNA para avaliação do transcriptoma total, utilizando sequênciamento de alto desempenho. Foram utilizados RNA total de duas linhagens celulares de mama que diferem apenas na expressão do gene ERBB2. Foram identificadas 2.865 novas variantes de splicing, das quais 20, que reportaram a identificação de um novo éxon, foram selecionadas para validação, com uma taxa de validação de 90%. Seis destas variantes apresentaram aumento de expressão na linhagem com alta expressão de ERBB2. Além disso, foi detectado um enriquecimento de algumas categorias de variantes na linhagem celular com alta expressão de ERBB2, reforçando a influência do oncogene na regulação do splicing alternativo, podendo resultar em variantes de splicing associadas a este grupo de câncer de mama, que podem ser candidatas a marcadores moleculares. / Alternative splicing is a process, by which many differente transcripts can be generated by one single gene, significantly expanding the transcriptional and proteomic diversity. Different splicing variants are generated among different transcripts and developmental stages, assuring normal cell function. Therefore, alterations in the splicing process can lead to diseases outcome. In this context, the aim of this study was the establishment of methodologies for the identification of alternative splicing in breast cancer influenced by ERBB2 oncogene, which is a poor prognostic molecular marker, highly expressed in 30% of human breast cancer. Two strategies were established for the construction of cDNA libraries. Alternative enriched splicing libraries, based on heteroduplex capture combined with mRNA amplification, were constructed from total RNA from a cell line and also from five tumor samples, all of them presenting high ERBB2 expression. Seventy nine putative splicing variants were identified and 18 of them were selected for RT-PCR validation. A high validation level was obtained (94%) and two novel alternative splicing variants were identified. ERBB2 mediated regulation was confirmed for three variants by two distinct methodologies, electrophoresis on a chip and probe specific ligation approach. The alteration in the expression balance of variants suggests the influence of the oncogene in the splicing pattern regulation. The second strategy was the construction of cDNA libraries for global transcriptome analysis based on deep sequencing. Total RNA from two mammary epithelial cell lines expressing different ERBB2 levels were used and 2,865 novel splicing variants were identified. Twenty novel events reporting the inclusion of novel exons were selected for RT-PCR validation with 90% validation rate. Six variants presented higher expression in the cell line with high levels of ERBB2. Moreover enrichment in splicing events was detected in the ERBB2 high expressing cell line, supporting the ERBB2 influence in alternative splicing regulation, possibly resulting in splicing variants associated to this subgroup of cancer that can be tested as molecular markers.

Os genes eucarióticos, em sua maioria, são divididos em exons e introns, requerendo processamento do RNAm para remover as sequências intrônicas e juntar os exons (splicing). As bordas exon/intron são definidas por sítios de splice que normalmente são reconhecidos com alta fidelidade, gerando os mesmos RNAms processados a cada vez. Apesar desse reconhecimento preciso, tem sido observada a junção de exons de maneiras alternativas (splicing alternativo), foco de muitos estudos recentes devido à sua importância em vários processos biológicos. Este processamento alternativo do RNAm pode ser principalmente de três tipos: exclusão de exon, no qual um exon pode ser incluído ou não no RNAm maduro; uso alternativo de sítios de splice, resultando em exons mais longos ou mais curtos e retenção de intron, o tipo menos estudado, no qual uma sequência intrônica é mantida no RNAm maduro. Um dos aspectos cruciais no entendimento de splicing alternativo é conhecer os mecanismos que levam à geração de diferentes transcritos. Coerente com a importância dos sítios de splice no splicing de RNAms, a retenção de intron parece ser causada por falha no reconhecimento daqueles que são sub-ótimos. Como os sítios de splice são reconhecidos aos pares ao se estabelecer uma ponte através de exons ou introns, dependendo de qual é mais curto, uma falha no reconhecimento de um exon ou de um intron leva a diferentes tipos de splicing alternativo (exclusão de exon ou retenção de intron, respectivamente). Desta forma, acredita-se que a ocorrência de retenção de intron esteja também associada a uma falha no reconhecimento de introns curtos. Embora estudos de introns retidos individuais tenham abordado estas questões, poucas análises sistemáticas de grandes quantidades de dados foram conduzidas sobre as características gerais que levam à retenção de intron. Para este fim, realizamos uma análise de bioinformática de sequências do genoma e transcriptoma (RNAm) humanos armazenadas em formato de computador. Para realizar as análises computacionais, desenvolvemos um sistema de anotação de splicing alternativo completo. Particionamos os eventos de retenção de intron identificados em sequências expressas pelo nosso sistema de anotação em dois grupos, com base na abundância relativa das duas isoformas (um grupo de eventos com <50% e outro com >50% de transcritos retendo o intron) e comparamos características relevantes. Verificamos que uma maior frequência de retenção de intron em humano está associada a sítios de splice mais fracos, genes com introns mais curtos e maior nível de expressão gênica, e menor densidade de um conjunto de elementos inibitórios exônicos e do promotor de splicing intrônico GGG. Os dois grupos apresentaram eventos conservados em camundongo, nos quais os introns retidos também eram curtos e apresentavam sítios de splice mais fracos. Embora nossos resultados tenham confirmado que sítios de splice mais fracos estão associados à retenção de intron, eles mostram que uma fração não-desprezível dos eventos não pode ser explicada apenas por esta característica. Nossa análise sugere que elementos reguladores em cis provavelmente têm um papel na regulação da retenção de intron e também revelou características previamente desconhecidas que parecem influenciar sua ocorrência. Estes resultados salientam a importância de considerar o compromisso entre estas características na regulação da frequência relativa de retenção de intron. / Most eukaryotic genes are split in exons and introns, requiring mRNA processing to remove intervening sequences and join exons (splicing). Exon/intron borders are defined by splice sites that are normally recognized with high fidelity, yielding the same processed mRNA each time. Notwithstanding such precise recognition, alternative joining of exons has been observed (alternative splicing) and is the focus of many recent studies, due to its importance in several biological processes. This alternative mRNA processing can be mainly of three types: exon skipping, whereby an exon may be included or not in the mature mRNA; alternative use of splice sites, resulting in longer or shorter exons and intron retention, the least studied type whereby an intronic sequence is maintained in the mature mRNA. One of the key aspects in understanding alternative splicing is to know the mechanisms that lead to the generation of different transcripts. Coherent with the importance of splice sites in mRNA splicing, intron retention seems to be caused by failure in the recognition of those that are sub-optimal. As splice sites are recognized in pairs by bridging either exons or introns, depending on which is the shortest, failure to recognize an exon or an intron leads to different types of alternative splicing (exon skipping or intron retention, respectively). This way, the occurrence of intron retention is believed to be associated to failure in recognition of short introns also. Although studies on individual retained introns have addressed such issues, few systematic surveys of large amounts of data have been conducted on the general features leading to intron retention. To this end, we performed a bioinformatics analysis of human genome and transcriptome (mRNA) sequences stored in computer format. To perform the computational analyses we developed a complete alternative splicing annotation system. We partitioned intron retention events identified in expressed sequences by our annotation system in two groups based on the relative abundance of both isoforms (one group of events with <50% and another with >50% of transcripts retaining the intron) and compared relevant features. We found that a higher frequency of intron retention in human is associated to weaker splice sites, genes with shorter intron lengths and higher expression level, and lower density of a set of exonic inhibitory elements and the intronic splicing enhancer GGG. Both groups of events presented conserved events in mouse, in which the retained introns were also short and presented weaker splice sites. Although our results confirmed that weaker splice sites are associated to intron retention, they showed that a non-negligible fraction of events can not be explained by this feature alone. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating intron retention and also revealed previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of intron retention.

Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and biotic and abiotic stresses. Recent work showed that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various AS small-molecule inhibitors that perturb splicing and thereby provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Here, I show that the macrolide Pladienolide B (PB) and herboxidiene (GEX1A) inhibits both constitutive and alternative splicing, mimics an abiotic stress signal, and activates the abscisic acid (ABA) pathway in plants. Moreover, PB and GEX1A activate genome-wide transcriptional patterns involved in abiotic stress responses in plants. PB and GEX1A treatment triggered the ABA signaling pathway, activated ABA-inducible promoters, and led to stomatal closure. Interestingly, PB and GEX1A elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. This work establishes PB and GEX1A as potent splicing inhibitors in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.