Characterization of evolutionarily conserved mammalian alternative splicing and alternative promoters

摘要

Recent studies suggest that surprisingly many mammalian genes have alternative splicing (AS) variants and alternative promoters (APs); however, their biological roles, and the characteristics that distinguish them from constitutive splicing and constitutive promoters (CPs), remain poorly understood. To help address these issues we have constructed and analyzed a large data set of AS variants evolutionarily conserved in human and mouse. In about one-fifth of cases, one isoform appears subject to nonsense-mediated mRNA decay (NMD), supporting the idea that a major role of AS is to regulate gene expression; a fourth of these NMD-inducing cases involve a conserved exon whose apparent sole purpose is to mediate destruction of the message when included. We explore sequence conservation likely related to splicing regulation, and find that the increased conservation that has been observed within AS exons primarily affects synonymous sites, suggesting that regulatory signals significantly constrain codon choice. We show that a lower frequency of the inclusion isoform relative to the exclusion isoform tends to be associated with weaker splice site signals, smaller exon size, and higher intronic sequence conservation, and provide evidence that all of these factors are under selection to control relative isoform frequencies.;We also constructed a large data set of evolutionarily conserved promoters, and used it to identify sequence features, functional associations, and expression patterns that differ by promoter type. The four promoter categories CpG-rich APs, CpG-poor APs, CpG-rich CPs and CpG-poor CPs each show characteristic strengths and patterns of sequence conservation, frequencies of putative transcription-related motifs, and tissue and developmental stage expression preferences. APs display substantially higher sequence conservation than CPs, and CpG-poor promoters than CpG-rich promoters. Among CpG-poor promoters, APs and CPs show sharply contrasting developmental stage preferences. We developed a discriminator to computationally predict promoter type, verified its accuracy through experimental tests that incorporate a novel method for deconvolving mixed sequence traces, and used it to find several new APs. The discriminator predicts that almost half of all mammalian genes have APs. This high frequency of APs, together with the strong purifying selection maintaining them, implies a crucial role in expanding the expression diversity of the mammalian genome.