Análise de splicing alternativo durante o processo de amadurecimento de frutos: aplicação em café e tomate

Abstract

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.