Genome-wide identification of intrasplicing events in the human transcriptome and hints to their regulatory potential

Abstract

Alternative splicing is one of the major regulators of both, transcriptome diversity and individual isoform abundance. Therefore regulation of alternative splicing is crucial and yet, due to the complexity of the human genome (23.000 genes, most of which can be alternatively spliced) diverse and multileveled. Identifying and understanding the scope and variety of splicing events is still an ongoing process. We established a novel pipeline to extract splicing events from specific RNA-sequencing datasets and identified numerous splicing events that did not span the entirety of the respective annotated intron but used intronic splice sites. These splicing events could be generally categorized into three groups: 5recursive (using the exonic splice donor and intronic acceptor), 3recursive (using an intronic splice donor and an exonic acceptor) and nested (using two intronic splice sites). Surprisingly, the splicing events we found occurred in introns of all lengths, but generally followed the abundance scheme of all introns, i.e. most were found in introns between 500 and 5000 bps. After confirmation of these splice events by conventional methodologies, we further analyzed the impact of intrasplicing on full intron removal. For this we established a luciferase-based reporter which showed that these intronic splicing steps can be beneficial, deleterious or neutral for full intron removal. Thus intrasplicing events can be crucial for determining the transcriptional output. This in part confirms recent findings on recursive splicing events in humans and other vertebrates and further uncovers an additional level of transcriptome regulation based on a yet undiscovered level of flexibility and regulation of splice site selection and its impact on gene expression.