Based On Comparative Genomics And Mrna High-throughput Sequencing Of Alternative Splicing Of Exon Evolutionary Studies

Alternative splicing (AS) is an important regulatory mechanism universally existing in eukaryotic genomes, and is associated with various types of physiological and pathological activities. AS exon is the basic element for alternative splicing evolution, characterized by the change of exon in terms of AS type, AS regulatory motif, expression level or corresponding protein domain between different species. The birth and evolution of AS exon not only increased the genome working efficiency, but also facilitated gene's gain-of-function and species phenotypic diversity at large. This makes AS exon evolution an interesting field of research in both molecular genetics and evolutionary biology.In recent years, the increasing number of genome projects, development of comparative genomics and application of high-throughput sequencing technology has made large scale AS exon evolution analysis possible. In this article, we combined Solexa sequencing in multiple species, real-time PCR with comparative genomics to investigate AS exon evolution and its biological significance at various levels.First, we used about 100 original perl scripts and multiple bioinformatic softwares to construct Alternative Splicing Exon Evolution Analyzer (ASEEA) system. ASEEA covers over 200,000 human exons (together with orthorlogous exons in 5 species) from Ensembl and integrates various types of information including exon selective pressure, protein domain, ESE (Exon Splicing Enhancer), repeat sequence and exon expression level in different species (derived from Solexa data), which can facilitate our understanding of the common features for AS exon evolution. Some of the perl scripts may also be helpful in future AS analysis using high-throughput sequencing.Using ASEEA, we first systematically summarized the common features for AS exon evolution by combining comparative genomics and high-throughput sequencing. Our results demonstrated that AS exons were less conserved than their constitutive counterparts. Among AS exons, older exons usually have lower Ka/Ks value, higher expression level, less repeat sequence, and are more inclined to have bio-functions. This trend illustrated the evolutionary path from a newly recruited exon to a fixed exon with conserved biological significance. We also believed that insertion of repeats and positive selection events may have played crucial roles during such process. Besides, we also found that during evolution the contribution of AS exon to gene's gain-of-function has been on the rise. Alternative splicing was believed to be an important reason for phenotypic difference among higher mammals, and the relative expression level of AS exons seemed to be increasing during evolution. In addition, we also discovered certain evolutionary feature for non-cassette AS exons (esp. intron rentions).Second, we used ASEEA to analyze 2006 cancer genes in Memorial Sloan-Kettering Cancer Center Database. Our results showed that AS exons in cancer genes followed the common rules for AS exon evolution. We reported that cancer gene exons were more conserved during evolution by having higher exon age, more selective pressure, more ESE weighted density, higher expression level and more overlapped protein domains. This implied that cancer genes were more strictly regulated in terms of AS, and were more selective when recruiting new exons. In addition, we also constructed a MySQL based cancer gene AS exon evolution database (OncoAS) and selected a number of exons/genes with potentially interesting evolutionary events.Finally, we selected VEGFA and PPAR-gamma from OncoAS for in-depth analysis. In VEGFA, we discovered exon8b-containing anti-angiogenic AS variant in mouse, and rabbit. We found out that the relative expression level of this variant has been increasing during evolution. Exon8b has experienced a minor form exon (in lower mammals) to major form exon (in human) conversion and was associated with increasing ESE weighted density. Our results indicated that the introduction of exon8b and exon6 (another AS exon) were important steps for the formation of VEGFA regulatory mechanism, and had profound impact on the cellular distribution and functionality of VEGFA. In PPAR-gamma, we discovered that four AS exons were at various stage of exon evolution. ExonB, the only coding AS exon in PPAR-gamma, was still in rapid evolution. ExonB may have experienced positive selection in rodents and may have gained bio-function related to fat storage. These two studies have made examples for future AS exon analysis using OncoAS Database.