Alternative splicing (AS) is a common posttranscriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. The release of the human genome draft revealed a much smaller number of genes than anticipated. Because of its potential role in expanding protein diversity, interest in alternative splicing has been increasing over the last decade. Although recent studies have shown that 94% human multiexon genes undergo AS, evolution of AS and thus its potential role in functional innovation in eukaryotic genomes remain largely unexplored. Here we review available evidence regarding the evolution of AS prevalence and functional role. In addition we stress the need to correct for the strong effect of transcript coverage in AS detection and set out a strategy to ultimately elucidate the extent of the role of AS in functional innovation on a genomic scale. 1. Introduction The first draft of the human genome sequence [1, 2] was unveiled in February 2001 and surprisingly it was shown to contain ~23000 genes, only a fraction of the numbers of genes originally predicted [3]. To put this into perspective, there are ~20,000 genes in the genome of the nematode C. elegans. The lack of an association between gene number and organismal complexity has resulted in an increased interest in alternative splicing (AS) given it has been proposed to be a major factor in expanding the regulatory and functional complexity, protein diversity, and organismal complexity of higher eukaryotes [4–6]. However, despite the best efforts of many research groups we still understand very little about the actual role played by AS in the evolution of functional innovation—here understood as the appearance of novel functional transcripts—underpinning the increased organismal complexity observed. Alternative splicing is a posttranscriptional process in eukaryotic organisms by which multiple distinct transcripts are produced from a single gene [4]. Previous studies using high-throughput sequencing technology have reported that up to 92%~94% of human multiexon genes undergo AS [7, 8], often in a tissue/developmental stage-specific manner [7, 9]. With the development and constant improvement of whole genome transcription profiling and bioinformatics algorithms, the ubiquity of AS in the mammalian genome began to become clear. The concept of one gene-one protein gave way as evidence mounted for the high percentage of AS incidence in nonhuman species [7, 8], such as fruit fly [10], Arabidopsis [11] and other eukaryotes [5]. Despite the
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