Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

Abstract

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. Alternative splicing expands the protein-coding potential of genes and genomes. RNAs copied from a gene can be spliced differently to produce distinct proteins under regulatory influences that arise during development or upon environmental change. These authors present a global analysis of alternative splicing in the mouse, using microarray measurements of splicing from 22 adult tissues. The ability to measure thousands of splicing events across the genome in many tissues has allowed the capture of co-regulated sets of exons whose inclusion in mRNA occurs preferentially in a given set of tissues. An examination of the sequences associated with exons whose expression is regulated in brain or muscle as compared to other tissues reveals extreme conservation of intron sequences nearby the regulated exon. These conserved regions contain sequence motifs likely to contribute to the regulation of alternative splicing in brain and muscle cells. The availability of global gene expression data with splicing level resolution should spur the development of computational methods for detecting and predicting alternative splicing and its regulation. In addition, the authors make strong predictions for biological experiments leading to the identification of components and their mechanisms of action in the regulation of splicing during mammalian development.