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Tiago Antônio de Oliveira Mendeshttp://lattes.cnpq.br/6003001459902104Eveline Teixeira Caixeta MouraGlória Regina FrancoJosé Miguel OrtegaMariana Lima Boroni Mar nsSérgio Vale Aguiar CamposJose Cleydson Ferreira Silvahttp://lattes.cnpq.br/2727952103969255Miquéias Fernandes2023-10-18T17:54:02Z2023-10-18T17:54:02Z2023-08-25http://hdl.handle.net/1843/59623https://orcid.org/0000-0002-6214-104XO Splicing Alternativo (AS) é um mecanismo cotranscricional que habilita o eucarioto estender seu proteoma mesmo com uma quantidade limitada de genes. A maquinaria celular realiza o AS pela combinação de regiões alternativas das isoformas, tanto em transcritos produtivos quanto em não produtivos. Os eventos de AS podem ser preditos com dados de RNASeq. Em plantas o evento mais frequente é o intron retention (IR) que pode ter efeito regulatório, por exemplo, ao inserir um códon de parada prematuro (PTC) que leva a degradação do transcrito pela via nonsense- mediated decay (NMD). Para estudar potenciais eventos de AS no processo biológico de amadurecimento de grãos de café e tomate conduzimos um estudo de diferencial AS (DAS) utilizando dados de RNASeq obtidos para experimento de expressão diferencial e o genoma de referência para identificar DAS. Para isso, nós desenvolvemos pipelines em Python afim de realizar uma curadoria automatizada dos 202 genes alvo que foram identificados pelo rMATS em 241 eventos de DAS nos contrastes de frutos verde com estágio intermediário de amadurecimento (yellow), e, maduro com yellow. Realizamos em seguida uma curadoria manual dos 241 eventos enriquecidos com a anotação do Interproscan5 que levou ao interesse de validar experimentalmente os genes Potassium channel AKT1 e Apyrase 7 com auxílio de PCR e qPCR. Os desafios identificados durante as análises de AS em dados de RNASeq nos induziram a desenvolver um aplicativo de interface amigável para explorar dados de DAS por RNASeq. O aplicativo foi desenvolvido composto de três módulos GeneAPPScript, GeneAPPServer, GeneAPPExplorer. O modulo GeneAPPScript é um wrapper poderoso que permite executar uma análise completa de DAS desde a obtenção dos dados em rede até a anotação funcional dos genes sob DAS. Esse modulo pode ser executado em distros Debian, como no ambiente Google Colaboratory (Colab) onde foi desenvolvido. Já o modulo GeneAPPServer é um backend Flask que permite integrar saídas de diferentes softwares de análise de DAS que geram dados em outputs tabulares. Usando o GeneAPPExplorer o usuário pode gerar dezenas de gráficos para visualizar graficamente importantes resultados implícitos nas tabelas técnicas exportadas pelos softwares de análises de DAS. Além disso pelo webapp o pesquisador tem acesso a tabelas enriquecidas de dados de anotação funcional, estrutural e atributos dos eventos. O GeneAPP tem potencial de contribuir na análise de AS em vários outros trabalhos depositados em bancos de dados públicos onde só foi analisado expressão diferencial no nível de gene, permitindo expandir o entendimento dos mecanismos moleculares e explorar o transcriptoma no nível de isoformas.Alternative Splicing (AS) is a co-transcriptional mechanism that enables the eukaryote to extend its proteome even with a limited number of genes. The cellular machinery performs AS by combining alternative regions of the isoforms in both productive and non-productive transcripts. AS events can be predicted using RNASeq data. In plants, the most frequent event is the retention of introns (IR) that can have a regulatory effect, for example, inserting a premature stop codon (PTC) that leads to degradation of the transcript through the nonsense-mediated decay (NMD) pathway. To study potential AS events in the biological process of coffee bean and tomato ripening, we conducted a DAS study using RNASeq data obtained for differential expression experiment and the referential coffee genome to identify differential AS (DAS). For this, we developed pipelines in Python to perform an automated curation of the 202 target genes that were identified with 241 DAS events by rMATS in the comparisons of early green fruits, intermediate yellow and final red ripening stage. We then carried out a manual curation of the 241 events enriched with the Interproscan5 annotation with further experimentally validating of Potassium channel AKT1 and Apyrase 7 genes under differential alternative expression during coffee grain ripening using conventional PCR and qPCR. Due to challenges identified during this analysis associated with the relationship of AS events and RNASeq data processing, we built an application of user- friendly APP to predict AS in RNASeq data. The application development is composed of three modules named GeneAPPScript, GeneAPPServer, GeneAPPExplorer. The GeneAPPScript module is a powerful wrapper that enables to perform a complete DAS analysis from obtaining network data to functional annotation of genes under DAS. This module can run on Debian distros, such as the Google Collaboratory (Colab) environment where it was developed. The GeneAPPServer module is a Flask backend that allows you to integrate outputs from different DAS analysis software that generate data in tabular outputs. Using GeneAPPExplorer, the user can generate dozens of graphs to graphically visualize important results implicit in technical tables exported by DAS analysis software. In addition, through the webapp, the researcher has access to tables enriched with functional and structural annotation data and event attributes. GeneAPP will contribute to the analysis of AS in several other works deposited in public databases where only differential expression at the gene level was analyzed, allowing further explanation when exploring the transcriptome at the isoform level.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em BioinformaticaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICASBioinformáticaCaféLycopersicon esculentumProcessamento AlternativoAmadurecimentoCaféSplicing AlternativoTomateWebappGeneappAnálise de splicing alternativo durante o processo de amadurecimento de frutos: aplicação em café e tomateAlternative splicing analysis during fruit ripening: application in coffee and tomatoinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese de Miquéias Fernandes.pdfTese de Miquéias Fernandes.pdfapplication/pdf14115276https://repositorio.ufmg.br/bitstream/1843/59623/1/Tese%20de%20Mique%cc%81ias%20Fernandes.pdfcd235e2bb6f1be3ee3555b348c4b736bMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-82118https://repositorio.ufmg.br/bitstream/1843/59623/2/license.txtcda590c95a0b51b4d15f60c9642ca272MD521843/596232023-10-18 14:54:02.87oai:repositorio.ufmg.br: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ório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2023-10-18T17:54:02Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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