Abstract
Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, ‘skipping’, exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon–intron boundary establishment leading to skipping events.
Highlights
Alternative splicing (AS) refers to various mechanisms of post-transcriptional gene regulation in higher eukaryotes generating many unique transcripts from a single genecoding region
Comparing our results to the annotated exon-skipping database for the human genome, which contains 39 232 events and was downloaded from the MISO website, we found only $30% of our events overlapped previously annotated skipping events, with many unique skipping events being newly detected by our method
The trans-acting pre-mRNA-binding proteins, often called SFs, include splicing activators that promote the usage of a particular splice site and splicing repressors that reduce the usage of a particular site. We examined how these exonic-splicing enhancers (ESEs)’s and intronic-splicing enhancers (ISEs) might distribute around splice sites within skipping-isoform-dominated group (SIDG) and isoformdominated group (IIDG) exonskipping-event locations based on identification of their recognition motifs, and asked how their positional patterns could affect splicing outcomes
Summary
Alternative splicing (AS) refers to various mechanisms of post-transcriptional gene regulation in higher eukaryotes generating many unique transcripts from a single genecoding region. Non-protein-coding sequences (introns) in pre-mRNA molecules are excised by the spliceosome machinery and protein-coding exons are joined together to form mature mRNA molecules. AS events result in mRNAs in which exons have been reconnected with variable inclusion, exclusion and ordinal positioning, and greatly increase the diversity of proteins that can be encoded by the genome. In humans, >90% of multi-exon genes undergo AS [1,2].
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