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.

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

“…These arrays contain probes for thousands of exon-exon junctions that are subject to alternative splicing. The arrays were analyzed for splicing changes by two previously described methods: MADS+ and OmniViewer (Sugnet et al 2006;Shen et al 2010). Each analysis method identified a large set of digitoxin-responsive alternative splicing events, including many changes in cassette exon inclusion (608 repressed exons, 132 enhanced exons by OmniViewer analysis) as well as other changes (Supplemental Fig.…”

“…This procedure generates a ''sepscore'' for each probed alternative event that assesses change in splicing pattern normalized by gene expression. When coupled with a low statistical false-discovery rate (q < 0.001), this score can characterize splicing changes that range from quite large (absolute sepscore > 1.0) to small but consistent (absolute sepscore between 0.3 and 0.5) (Sugnet et al 2006).…”

Identification of the Expressed Form of Human Cytosolic Phospholipase A2β (cPLA2β)

“…The reason for the predominance of unspliced cPLA 2 ␤ cDNA is not known, but it raised the possibility that multiple splice variants exist. Increasing evidence indicates that alternate splicing and exon skipping are major contributors to protein diversity in humans; therefore, it is important to identify the endogenously expressed protein variants in cells and tissues (37)(38)(39)(40)(41)(42)(43). In this study, we describe the identification and characterization of endogenous cPLA 2 ␤ protein, which is derived from a novel splice variant of the cPLA 2 ␤ gene.…”

Global analysis of mRNA splicing

“…Thus, most versions of these arrays include probes within constitutive exons so that transcripts can first be scored as present or absent from the sample, then assessed for AS based on junction probes. Splice junction arrays have proven valuable in functional classification of RNA processing mutants in yeast (Clark et al 2002;Burckin et al 2005) and definition of tissuespecific splicing patterns and regulatory motifs in mammalian cells (Johnson et al 2003;Pan et al 2004;Sugnet et al 2006;Fagnani et al 2007). The latter studies have produced the recurring observation that transcripts regulated at the level of alternative splicing comprise a distinct population from those regulated at the level of transcript abundance.…”