We carried out the first analysis of alternative splicing complexity in human tissues using mRNA-Seq data. New splice junctions were detected in approximately 20% of multiexon genes, many of which are tissue specific. By combining mRNA-Seq and EST-cDNA sequence data, we estimate that transcripts from approximately 95% of multiexon genes undergo alternative splicing and that there are approximately 100,000 intermediate- to high-abundance alternative splicing events in major human tissues. From a comparison with quantitative alternative splicing microarray profiling data, we also show that mRNA-Seq data provide reliable measurements for exon inclusion levels.

Alternative splicing has a crucial role in the generation of biological complexity, and its misregulation is often involved in human disease. Here we describe the assembly of a 'splicing code', which uses combinations of hundreds of RNA features to predict tissue-dependent changes in alternative splicing for thousands of exons. The code determines new classes of splicing patterns, identifies distinct regulatory programs in different tissues, and identifies mutation-verified regulatory sequences. Widespread regulatory strategies are revealed, including the use of unexpectedly large combinations of features, the establishment of low exon inclusion levels that are overcome by features in specific tissues, the appearance of features deeper into introns than previously appreciated, and the modulation of splice variant levels by transcript structure characteristics. The code detected a class of exons whose inclusion silences expression in adult tissues by activating nonsense-mediated messenger RNA decay, but whose exclusion promotes expression during embryogenesis. The code facilitates the discovery and detailed characterization of regulated alternative splicing events on a genome-wide scale.

We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights into how the evolutionary history of exons can impact on their inclusion levels, and also information on how global regulatory properties of AS define tissue type. Like global transcription profiles, global AS profiles reflect tissue identity. Interestingly, we find that transcription and AS act independently on different sets of genes in order to define tissue-specific expression profiles. These results demonstrate the utility of our quantitative microarray platform and data for revealing important global regulatory features of AS.

Sequence-based analyses have predicted that ∼35% of mammalian alternative splicing (AS) events produce premature termination codon (PTC)-containing splice variants that are targeted by the process of nonsense-mediated mRNA decay (NMD). This led to speculation that AS may often regulate gene expression by activating NMD. Using AS microarrays, we show that PTC-containing splice variants are generally produced at uniformly low levels across diverse mammalian cells and tissues, independently of the action of NMD. Our results suggest that most PTC-introducing AS events are not under positive selection pressure and therefore may not contribute important functional roles. Alternative splicing (AS), the process by which exons in transcripts are joined in different combinations to generate multiple mRNA variants, represents an important mechanism for the expression of structurally and functionally distinct proteins from a limited number of genes (Graveley 2001;Black 2003). Regulation of AS plays critical roles in cell growth, differentiation, and cell death, and aberrant AS has been implicated in many human diseases (Smith and Valcarcel 2000;Caceres and Kornblihtt 2002;Cartegni et al. 2002). It has been estimated that at least 74% of human genes contain one or more alternative exons (Johnson et al. 2003), yet it is not known to what extent the resulting splice variants specify functionally relevant transcripts and proteins.Previous studies have shown that the introduction of premature termination codons (PTCs) in spliced transcripts can activate transcript degradation via the process of nonsense-mediated mRNA decay (NMD) (Hillman et al. 2004;Maquat 2004;Alonso 2005). NMD is important for the removal of nonfunctional PTC-containing transcripts (Mendell et al. 2004;Mitrovich and Anderson 2005). It has also been shown to function in the autoregulation of transcript levels of several RNAbinding proteins, including splicing factors (Morrison et al. 1997;Sureau et al. 2001;Wollerton et al. 2004; for review, see Lareau et al. 2004). In addition to a PTC, NMD requires a set of upstream frameshift (UPF) protein factors that associate with the spliced transcripts via interactions with a post-splicing exon junction complex Recent bioinformatics analyses of expressed sequence tag (EST) and cDNA sequence data have predicted that ∼35% of AS events have the potential to introduce PTCs that could elicit NMD of transcripts Lewis et al. 2003). This finding led to speculation that NMD activated by AS may represent a widely used mechanism by which gene expression is down-regulated (Hillman et al. 2004;Neu-Yilik et al. 2004;Alonso 2005;Lejeune and Maquat 2005), and it has also been proposed that the introduction of PTCs by AS serves as a mechanism for the tissue-specific regulation of gene expression (Hillman et al. 2004;Holbrook et al. 2004;Raes and Van de Peer 2005). However, no study has yet examined experimentally the actual levels of PTC-containing mRNA splice variants in normal cells and tissues, and it is not known to what extent AS f...

Background: Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene coexpression as a means to predict gene function.