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Master of Science / Department of Computing and Information Sciences / William H. Hsu / Alternative splicing is a mechanism for generating different gene transcripts (called iso- forms) from the same genomic sequence. Finding alternative splicing events experimentally is both expensive and time consuming. Computational methods in general, and EST analy- sis and machine learning algorithms in particular, can be used to complement experimental methods in the process of identifying alternative splicing events. In this thesis, I first iden- tify alternative splicing exons by analyzing EST-genome alignment. Next, I explore the predictive power of a rich set of features that have been experimentally shown to affect al- ternative splicing. I use these features to build support vector machine (SVM) classifiers for distinguishing between alternatively spliced exons and constitutive exons. My results show that simple, linear SVM classifiers built from a rich set of features give results comparable to those of more sophisticated SVM classifiers that use more basic sequence features. Finally, I use feature selection methods to identify computationally the most informative features for the prediction problem considered.

Chapter 1 of this work focuses on alternative splicing of titin as a proof of concept therapy for treating diastolic dysfunction and restrictive filling in a genetic murine model (Ttn^(ΔIAjxn)). The Ttn^(ΔIAjxn) mouse has increased strain on the spring region of titin and acts as a mechanical analogue of the titin-based increase in passive myocardial stiffness found in patients with heart failure and preserved ejection fraction (HFpEF). HFpEF is a complex disease characterized by diastolic dysfunction, exercise intolerance, and concentric hypertrophic remodeling. Approximately half all of heart failure patients suffer from diastolic dysfunction, however, no effective therapy exists for treating this pervasive syndrome. Titin, the largest known protein and molecular spring in the heart, has emerged as a prime candidate for therapeutic targets aimed at restoring compliance to the sarcomere in order to improve diastolic function. Titin has two main cardiac isoforms that are regulated by alternative splicing; the smaller N2B isoform (~3.0 MDa) and the larger more compliant N2BA isoform (~3.3 MDa). Diastolic stiffness of the left ventricle is dependent upon the N2BA:N2B isoform ratio. In the first half of this work, we modified these two primary isoforms by inhibiting the known titin splicing factor Rbm20. We demonstrate that Rbm20 reduction restores diastolic function, improves exercise tolerance and attenuates afterload induced pathologic remodeling of the left ventricle in Ttn^(ΔIAjxn) mice.The work in chapter 2 is focused on studies using the previously published N2B knock out (KO) murine model. The N2B spring element found in cardiac titin's I-band region has been proposed as a sensor and signaling "hot spot" in the sarcomere. This study investigates the role of titin's cardiac specific N2B element as a mechano-sensor for stress and strain induced remodeling of the heart. The N2B KO mouse was subjected to a variety of stressors including transverse aortic constriction (TAC), aortocaval fistula (ACF), chronic swimming, voluntary running and isoproterenol stimulation. Our data revealed that the N2B element is essential in preload stimulated cardiac hypertrophy as well as remodeling due to beta-adrenergic stress. Cardiac hypertrophy is a common maladaptive feature of heart failure patients and the mechanical triggers that determine pathologic growth are not well understood. My work in the N2B KO mouse reveal titin's important role in cardiac remodeling.

Alternative splicing, the process by which a single gene may code for similar but different proteins, is an important process in biology, linked to development, cellular differentiation, genetic diseases, and more. Genome-wide analysis of alternative splicing patterns and regulation has been recently made possible due to new high throughput techniques for monitoring gene expression and genomic sequencing. This thesis introduces two algorithms for alternative splicing analysis based on large microarray and genomic sequence data. The algorithms, based on generative probabilistic models that capture structure and patterns in the data, are used to study global properties of alternative splicing. In the first part of the thesis, a microarray platform for monitoring alternative splicing is introduced. A spatial noise removal algorithm that removes artifacts and improves data fidelity is presented. The GenASAP algorithm (generative model for alternative splicing array platform) models the non-linear process in which targeted molecules bind to a microarray’s probes and is used to predict patterns of alternative splicing. Two versions of GenASAP have been developed. The first uses variational approximation to infer the relative amounts of the targeted molecules, while the second incorporates a more accurate noise and generative model and utilizes Markov chain Monte Carlo (MCMC) sampling. GenASAP, the first method to provide quantitative predictions of alternative splicing patterns on large scale data sets, is shown to generate useful and precise predictions based on independent RT-PCR validation (a slow but more accurate approach to measuring cellular expression patterns). In the second part of the thesis, the results obtained by GenASAP are analysed to reveal jointly regulated genes. The sequences of the genes are examined for potential regulatory factors binding sites using a new motif finding algorithm designed for this purpose. The motif finding algorithm, called GenBITES (generative model for binding sites) uses a fully Bayesian generative model for sequences, and the MCMC approach used for inference in the model includes moves that can efficiently create or delete motifs, and extend or contract the width of existing motifs. GenBITES has been applied to several synthetic and real data sets, and is shown to be highly competitive at a task for which many algorithms already exist. Although developed to analyze alternative splicing data, GenBITES outperforms most reported results on a benchmark data set based on transcription data.

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.

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.

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.

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