Experimental validation of tappAS: a computational framework to assess the functional impact of alternative splicing in a neural differentiation context
[EN] Alternative splicing (AS) is a regulatory mechanism of gene expression that contributes to proteomic diversity by increasing the number of mRNA species that are transcribed from a single gene. As a result, varieties or isoforms of the same protein may present different structure, location and f...
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| Tipo de recurso: | tesis de maestría |
| Fecha de publicación: | 2019 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | inglés |
| OAI Identifier: | oai:riunet.upv.es:10251/142186 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/142186 |
| Access Level: | acceso abierto |
| Palabra clave: | Splicing alternativo TappAS RNAseq Isoformas NPCs OPCs MTNs Alternative splicing (AS) Isoforms MICROBIOLOGIA Máster Universitario en Biotecnología Biomédica-Màster Universitari en Biotecnologia Biomèdica |
| Sumario: | [EN] Alternative splicing (AS) is a regulatory mechanism of gene expression that contributes to proteomic diversity by increasing the number of mRNA species that are transcribed from a single gene. As a result, varieties or isoforms of the same protein may present different structure, location and function. In addition, the variability in the expression rates of these isoforms can play an important role in the differentiation and in the determination of cell fate, in the appearance of diseases and can be used for the characterization of different cellular populations within a single one. TappAS is a Java application developed for the analysis of RNA-seq data at the gene and isoform level. It provides a set of tools to study the AS but also its functional implication, ie if this AS has any effect on a domain relevant to the functionality of the protein. The objective of this work is the validation of TappAS to reveal the effectiveness, specificity and sensitivity of the application to detect and evaluate expression rates at the isoform level. According to the literature, the levels of AS are particularly high in the nervous system, so that tappAS was evaluated in vitro in the context of the cellular differentiation of the nervous system. To this end, cultures of progenitor neural cells (NPCs) were established and cell differentiations were made from NPCs to oligodendrocyte progenitor cells (OPCs) and motor neuron progenitors (MTNs). Subsequently, samples of RNA and proteins were collected at different times of differentiation to experimentally evaluate expression levels by RT-qPCR and Western Blot. The performed in silico analysis in our model have predicted AS events in genes involved in mitochondrial activity and dynamics (OMA1, MUL1) that could provide functional heterogeneity and energy supply throughout the differentiation process. The results obtained by RT-qPCR revealed the predicted isoforms and the gene expression rates obtained were similar to the theoretical data provided by TappAS. The knowledge of the mitochondrial quality control pathways and the abnormal mitochondrial dynamics and distribution could ultimately shed light on the effect of mitochondrial dysfunction in the development of neurological pathologies. |
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