Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.Author Summary: In mammalian genomes, a single gene can be alternatively spliced into multiple isoforms which greatly increase the functional diversity of the genome. In the human, more than 95% of multi-exon genes undergo alternative splicing. It is hard to computationally differentiate the functions for the splice isoforms of the same gene, because they are almost always annotated with the same functions and share similar sequences. In this paper, we developed a generic framework to identify the ‘responsible’ isoform(s) for each function that the gene carries out, and therefore predict functional assignment on the isoform level instead of on the gene level. Within this generic framework, we implemented and evaluated several related algorithms for isoform function prediction. We tested these algorithms through both computational evaluation and experimental validation of the predicted ‘responsible’ isoform(s) and the predicted disparate functions of the isoforms of Cdkn2a and of Anxa6. Our algorithm represents the first effort to predict and differentiate isoforms through large-scale genomic data integration.