The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out. In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis. The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.

Senile plaques (Aβ) and neurofibrillary tangles (tau) are pathological hallmarks of Alzheimer’s disease (AD). If and how the formation of these deposits are mechanistically linked remains mainly unknown. In recent years, the focus has shifted from insoluble protein deposits to soluble aggregates of Aβ and tau. Protofibrils are large soluble Aβ oligomers which were linked to AD by the discovery of the Arctic AβPP mutation. Treatment of young tg-ArcSwe mice with an Aβ protofibril-selective antibody, mAb158, cleared protofibrils, prevented amyloid plaque deposition and protected cultured cells from protofibril-mediated toxicity. This suggests that Aβ protofibrils are necessary for the formation of Aβ deposits. Functional assessment of tg-ArcSwe mice in IntelliCage demonstrated hippocampal-dependent behavioral deficits such as memory/learning impairments, hyperactivity and perseverance behavior. Learning impairments did not correlate to Aβ-measures but to calbindin, which might be a good marker for Aβ-mediated neuronal dysfunction. Splicing of exon 10 in the tau gene differs between human and mouse brain. Exon 10 is part of the microtubule-binding domains which helps to maintain microtubule stability and axonal transport, functions vital to neuronal viability. Axonal transport dysfunction has been proposed as a common pathway of Aβ and tau pathogenesis in AD. Generation of a novel tau mouse model with absence of exon 10 led to age-dependent sensorimotor impairments which may relate to dysfunctions in cerebellum. No tau pathology was evident suggesting that a trigger of tau fibrillization e.g. a human Aβ or tau aggregate is needed. Generation of AβPPxE10 bitransgenic mice with no exon 10 showed lower Aβ plaque burden. Possibly changes in microtubule function lead to altered intracellular AβPP transport and Aβ production. Initiation of tau pathology in AβPPxE10 mice might require a certain type of Aβ-aggregates which is not produced or exist at too low concentration in transgenic mouse brain. In summary, the Aβ protofibril-selective antibody was found to be a promising treatment for AD. The IntelliCage system was proven to be useful for functional evaluation of AβPP mice. Exon 10 in tau was shown to affect sensorimotor functions and Aβ pathology in bitransgenic mice by mechanisms that deserve further investigation.

Through a genome wide study of spliceosome recruitment in Saccharomyces cerevisiae, we were able to identify a set of protein-coding genes that despite the fact that they contain no introns and their mRNA is not known to be spliced; their loci were occupied by the spliceosome. Bioinformatic analysis revealed the existence of splicing signals on these genes. Detailed analysis of BDF2, a representative gene, revealed that the spliceosome negatively regulates its mRNA levels through an unconventional one-step splicing reaction that cleaves BDF2 mRNA and targets the cleavage products for degradation. In an effort to clarify the mechanism of spliceosome recruitment to BDF2 locus, we identified that Bdf1, the redundant to Bdf2 factor, is required for the recruitment of the spliceosome at BDF2 and the subsequent down-regulation of its mRNA levels. The above led us to propose a new role for the spliceosome in the regulation of gene expression. Finally, we investigated the generality of this regulatory mechanism is S. cerevisiae and identified a set of genes which can be differentially spliced and whose physiological expression could be potentially regulated by the spliceosome.

With previous indication of the Group VIA phospholipase A2 (iPLA2β) enzyme regulating ER-stress induced apoptosis in β-cells by regulating the anti-apoptotic protein Bcl-xL via alternative splicing, our lab postulated iPLA2β to be utilizing a similar mechanism to regulate apoptosis in mice lung. Our previous lab work has shown implications of lung function compromise in iPLA2β-/- mice, and we speculated the cause to be due altered lung architecture stemming from the attenuation of apoptosis. The western blot analysis in this study suggested that iPLA2β is involved in the regulation of Bcl-xL, but the mRNA ratios of the splice variants suggested that alternative splicing is not the mechanism iPLA2β is utilizing for the regulation in our animal models. Additionally, the observation and assessment of the lung morphology of the iPLA2β-/- and wild type mice suggested that iPLA2β does not play an integral role in lung morphology.

L'objectif de mon travail est de comprendre le rôle de Rrp6, une exoribonuclease 3'-5', dans l'expression des gènes. Dans ce but, j'ai utilisé le promoteur du virus de l'immunodéficience humaine (VIH-1) comme modèle d'étude de la régulation des gènes chez les mammifères. En utilisant ce modèle dans le chapitre 1 des résultats, nous avons montré l'existence d'un nouveau mécanisme de répression de l'expression des gènes dépendant de l'ARN qui requiert les actions combinées de Rrp6 et du microprocesseur. A la suite de ce travail, nous avons caractérisé les complexes de protéines associés à Rrp6 qui contribuent à cette répression de la transcription (résultats - chapitre 2). Ces deux études suggèrent un rôle de Rrp6 dans la répression de la transcription au niveau du promoteur du VIH-1 mais aussi sur certains gènes cellulaires. Au cours des études présentées dans le chapitre 1, nous avons observé une forte diminution de l'expression de la protéine Dicer dans les cellules déplétées de Rrp6. Dicer est un élément central de la régulation de la maturation des microARN (miRNA) et donc joue un rôle important dans tous les processus cellulaires qui sont régulés par les miRNA, incluant de nombreux processus biologiques et physiologiques. Ainsi, il est important de connaitre les voies de régulation de Dicer. Dans le chapitre 3 des résultats, nous décrivons un nouveau mécanisme de régulation de Dicer par Rrp6. En effet, nos résultats montrent que Rrp6 est nécessaire pour un epissage efficace de l'ARNm de Dicer. Nos travaux décrivent un nouveau role de Rrp6 dans des processus cellulaires distincts: transcription et splicing / The objective of my doctoral work was to understand the role of a 3' to 5' exoribonuclease, Rrp6, in gene expression. I used the Human Immunodeficiency Virus (HIV-1) promoter as a model to study gene regulation in mammalian cells. Using this model, in Result-chapter 1, we demonstrated a novel mechanism of RNA-dependent transcriptional gene silencing that depends on the cooperative activities of Rrp6 and microprocessor. Following this study, we characterized the Rrp6-containing complex that contributes to the transcriptional silencing at HIV-1 promoter (Result-chapter 2). These two studies suggest a role for Rrp6 in transcriptional repression at the HIV-1 promoter and also at a subset of cellular genes. During the course of our studies presented in chapter 1, we observed a dramatic decrease of Dicer protein level in the cells depleted of Rrp6. Dicer is a central regulator of microRNA (miRNA) maturation and therefore exerts an important role in all cellular processes that are regulated by miRNAs, including diverse biological and physiological processes. Thus, it is important to know how Human Dicer1 is regulated. In Result-chapter 3, we describe a new regulatory mechanism of Dicer1 expression by Rrp6. Indeed, our results demonstrate that Rrp6 is required for efficient splicing of Dicer1 mRNA. Our work describes a novel role for Rrp6 in distinct cellular processes: transcription and splicing.

Eukaryotic genes contain non-coding sequences - introns that are removed during pre-mRNA splicing by the spliceosome. The spliceosome is composed of five snRNPs (U1, U2, U4/U6 and U5) which assemble on pre-mRNA in a step-wise manner and together with additional non-snRNP proteins catalyse splicing. Mutations in splicing factors can cause severe diseases, for example a point missense mutation (called AD29) in hPrp31 (U4/U6 snRNP specific protein) induces retinitis pigmentosa, disease often leading to complete blindness. In this PhD thesis we show that the hPrp31 AD29 mutant is unstable and is not properly incorporated into spliceosomal snRNPs. In addition, the expression of the mutant protein reduces cell proliferation, which indicates that it interferes with cellular metabolism (likely splicing) and could explain the induction of retinitis pigmentosa. Next, we focus on a role of nuclear environment in pre-mRNA splicing. It was shown that new U4/U6·U5 snRNPs are preferentially assembled in non-membrane nuclear structure - Cajal body. Here we expand this finding and provide evidence that Cajal bodies are also important for U4/U6·U5 snRNP recycling after splicing. In addition, we analyzed a role of chromatin and particularly histone acetylation modulates in splicing regulation. Using inhibitor of...

Bioinformática estrutural de proteínas modificadas por eventos de splicing alternativo / Structural Bioinformatics of Proteins modified by Alternative Splicing

The identification of alternative splicing in the human genome elucidated the potential to several enduring genomic questions. Not only could this phenomenon explain why organism complexity was not at all correlated with the genome size, or explain how an organisms could be affected by experience and environment at the molecular level, but it was perhaps the most flexible and adaptive regulatory mechanism identified to date. While the pathogenic aberrations of this mechanism have generally been readily investigated and identified as potential therapeutic targets, its meditative or advantageous instances have largely not been considered. Initiated exon skipping has been shown to have therapeutic effects in Muscular Dystrophy animal models and even in vitro human muscle cells (Aartsma-Rus, Annemieke, et al, Human Molecular Genetics 2003, McClorey, G., et al, Neuromuscular disorders, 2006). However, the consideration that this process may be occurring endogenously in human cells and contributing to other complex diseases has remained largely ignored. In this work, we have undertaken the first large-scale statistical examination of alternatively spliced variants between the tissues of diseased and normal patients. We hypothesize that there are endogenous alternative splicing events occurring in these tissues that purposefully mediate mutative damage and contribute to the differentiation between diseased and healthy phenotypes. By integrating data from several different sources and employing statistic and machine learning models, we have identified significant differences in gene characteristics between canonical and spliced variants correlated with changes in clinical outcomes. We conclude that this evidence supports our hypothesis that alternative splicing can be positively driven to mediate genetic damage. Expression of these genetically damaged and canonically spliced variants is clearly implicated in diseased tissue and poor clinical outcomes.

"Host resolution of viral infection is dependent upon components of the innate and acquired immune system. The mammalian protein Apobec3 plays an important role as part of the immune system’s innate defenses through its modification of reverse transcribed viral DNA. Recently, Apobec3 was found to directly inhibit HIV-1 and HBV replication through deaminating newly transcribed deoxycytidine residues to deoxyuridine. The ability of mouse and simian Apobec3 variants to inhibit human retroviruses and vice versa highlights the utility of analyzing cross-species homologues. To better understand this editing enzyme, differentially pathogen-susceptible inbred mice were used as an experimental model. The purpose of this project is to examine the effects of murine Apobec3 (muA3) alternative splicing on its DNA-editing characteristics. Three distinct Apobec3 isoforms were isolated from pathogen-susceptible BALB/cByJ (“C”) inbred mice, and two Apobec3 isoforms came from pathogen-resistant C57BL/6ByJ (“Y”) mice. The five muA3 isoforms were cloned, sequenced, and expressed from a constitutive promoter in a haploid Saccharomyces cerevisia strain. MuA3 DNA-editing activity was measured via the CAN1 forward mutation assay. The five isoforms studied in this project were discovered to be strain-specific. One isoform from each mouse strain mutated the yeast CAN1 locus significantly. Additionally, both muA3 isoform mRNAs derived from the pathogen-resistant Y mice were found to persist at a higher level (2.7 -12.4 fold) than any of the C mouse isoforms. This suggests that the absence of exon 5 or some other signal in the Y mice may influence transcript stability. Evidence also suggests that the murine Apobec3 start codon is actually 33bp upstream of its reference start, with implications for previous research performed using muA3. Sequencing analysis of genomic DNA revealed the presence of a 4bp insertion in a region of BALB/cByJ muA3 which may have disrupted an intronic splicing enhancer signal. Furthermore, a novel BALB/cByJ Apobec3 isoform was characterized. This is the first report of strain-specific processing with regard to muA3."

Orientador: Lucia Galvão de Albuquerque / Coorientador: Daniel Guariz Pinheiro / Coorientador: Maria Inês Tiraboschi Ferro / Banca: Larissa Fernanda Simielli Fonseca / Banca: Alexéia Barufatti Grisólia / Baanca: Poliana Fernanda Giachetto / Resumo: O sequenciamento do RNA (RNA-Seq) é uma das abordagens utilizadas para identificar transcritos correspondentes a genes candidatos que provocam variações em vias metabólicas, resultando em diferentes fenótipos. Uma vez que, área de olho de lombo (AOL) e o conteúdo de gordura intramuscular (GI) são características poligênicas, muitos genes e mecanismos que induzem as diferenças no crescimento e desenvolvimento muscular, bem como nos índices de conteúdo de GI da raça Nelore, ainda são desconhecidos. Portanto, o objetivo foi estudar o transcriptoma do músculo de bovinos da raça Nelore, com o propósito de identificar genes e eventos de splicing alternativos diferencialmente expressos (DEGs e DAS) associados à característica de carcaça (AOL) e carne (conteúdo de GI), por meio do RNA-Seq. Para tanto, um total de 80 animais foram sequenciados e fenotipados para AOL e conteúdo de GI (mensurados pelo método químico - que avalia lipídios totais). Os resultados foram apresentados nos capítulos 2, 3 e 4. No capítulo 2, foram selecionados para análise de expressão diferencial, 15 animais com maiores e 15 animais com menores AOL. Após as análises, 288 genes foram diferencialmente expressos (q-value ≤0,05), dos quais 182 foram superexpressos e 106 foram reprimidos no grupo de animais com maiores AOL em comparação com o grupo com menores AOL. Genes pertencentes a famílias da actina, miosina, colágeno, integrina, transportador de soluto, ubiquitina e kelch-like foram diferencialmente expressos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: RNA sequencing (RNA-Seq) is an approach for screening the expression of functional candidate genes and identifying important molecular mechanisms creating variation in pathways that result in different tissue phenotypes. Since ribeye muscle area (REA) and intramuscular fat content (IF) are polygenic traits, many genes and mechanisms that induce differences in muscle growth and development, as well as IF content of the Nelore cattle still unknown. The aim was to study the muscle transcriptome of the Nelore cattle with the purpose of identifying differentially expressed genes (DEGs) and differentially expressed alternative splicing events (DAS) associated with the carcass (REA) and meat (IF) traits, through of the RNA-seq approach. Therefore, a total of 80 animals were sequenced and phenotyped for REA and IF contet (measured by the chemical method - which evaluate total lipids). The results were presented in chapters 2, 3 and 4. In chapter 2, 15 animals with highest REA and 15 animals with lowest REA were selected for differential expression analysis. Upon analysis, 289 DEGs were identified (q-value ≤0.05): 183 upregulated and 106 downregulated in the highest REA group. Genes belonging to important families, such as actin, myosin, collagen, integrin, solute carrier, ubiquitin, and kelch-like, were among the DEGs. Gene set enrichment analysis showed that many of the significantly enriched gene ontology (GO) terms are closely associated with muscle development, growth, and degrad... (Complete abstract click electronic access below) / Doutor