Abstract
High-throughput methods such as EST sequencing, microarrays and deep sequencing have identified large numbers of alternative splicing (AS) events, but studies have shown that only a subset of these may be functional. Here we report a sensitive bioinformatics approach that identifies exons with evidence of a strong RNA selection pressure ratio (RSPR) —i.e., evolutionary selection against mutations that change only the mRNA sequence while leaving the protein sequence unchanged—measured across an entire evolutionary family, which greatly amplifies its predictive power. Using the UCSC 28 vertebrate genome alignment, this approach correctly predicted half to three-quarters of AS exons that are known binding targets of the NOVA splicing regulatory factor, and predicted 345 strongly selected alternative splicing events in human, and 262 in mouse. These predictions were strongly validated by several experimental criteria of functional AS such as independent detection of the same AS event in other species, reading frame-preservation, and experimental evidence of tissue-specific regulation: 75% (15/20) of a sample of high-RSPR exons displayed tissue specific regulation in a panel of ten tissues, vs. only 20% (4/20) among a sample of low-RSPR exons. These data suggest that RSPR can identify exons with functionally important splicing regulation, and provides biologists with a dataset of over 600 such exons. We present several case studies, including both well-studied examples (GRIN1) and novel examples (EXOC7). These data also show that RSPR strongly outperforms other approaches such as standard sequence conservation (which fails to distinguish amino acid selection pressure from RNA selection pressure), or pairwise genome comparison (which lacks adequate statistical power for predicting individual exons).
Highlights
Global analyses of alternative splicing (AS) have established its importance in protein diversity and gene regulation in higher eukaryotes [1,2]
Of the 21 coding region exons, two exons show evidence of dramatically increased RNA selection pressure ratio (RSPR) (RSPR values of 7 and 10 respectively) compared with the remaining exons (Figure 1). These two exons correspond to the well-studied N1 and C1 alternative exons, which have been shown to be regulated by PTB, NOVA2, hnRNP H and hnRNP A1 [45], and in turn control receptor desensitization [46] and NMDA receptor interactions [47]
We have presented an effective method for estimating RNA selection pressure within an individual exon, and have tested its predictions against a variety of empirical measures of functional alternative splicing, such as known NOVA-regulated exons, conserved alternative splicing, frame preservation, and tissuespecific splicing patterns
Summary
Global analyses of alternative splicing (AS) have established its importance in protein diversity and gene regulation in higher eukaryotes [1,2]. Alternative splicing is often regulated in a tissue-specific manner [5] and can undergo important changes in disease states such as cancer [6,7]. All of these illustrate that it is necessary to study the functional effects of alternative splicing to understand the complexity of biological system and human disease. A number of studies have shown that a significant fraction of these detected events are probably not functional [2,11,12] In this context, biologists need improved ways of distinguishing AS events that are likely to have important biological functions, before initiating costly experiments, such as high-throughput studies of regulation [13,14]
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