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.
At a glance
- Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008) et al.
- Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature Genet. 40, 1413–1415 (2008) , , , &
- Splicing in disease: disruption of the splicing code and the decoding machinery. Nature Rev. Genet. 8, 749–761 (2007) &
- Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813 (2008) &
- Decrypting the genome’s alternative messages. Curr. Opin. Cell Biol. 21, 377–386 (2009) &
- Alternative splicing: global insights. FEBS J. 277, 856–866 (2010) , &
- Expansion of the eukaryotic proteome by alternative splicing. Nature 463, 457–463 (2010) &
- Alternative splicing: new insights from global analyses. Cell 126, 37–47 (2006)
- Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336 (2003)
- Functional coordination of alternative splicing in the mammalian central nervous system. Genome Biol. 8, R108 (2007) et al.
- Inferring global levels of alternative splicing isoforms using a generative model of microarray data. Bioinformatics 22, 606–613 (2006) , , &
- Unusual intron conservation near tissue-regulated exons found by splicing microarrays. PLoS Comput. Biol. 2, e4 (2006) et al.
- A correlation with exon expression approach to identify cis-regulatory elements for tissue-specific alternative splicing. Nucleic Acids Res. 35, 4845–4857 (2007) et al.
- Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines. Nature Genet. 40, 1416–1425 (2008) et al.
- The splicing regulatory element, UGCAUG, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons. Nucleic Acids Res. 33, 714–724 (2005) , , , &
- Neuron-specific alternative splicing of nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron sequence. J. Biol. Chem. 271, 17613–17616 (1996)
- An RNA map predicting Nova-dependent splicing regulation. Nature 444, 580–586 (2006) et al.
- HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008) et al.
- The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream. Mol. Cell. Biol. 17, 4667–4676 (1997) &
- A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart. RNA 3, 996–1015 (1997) &
- Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment. Mol. Cell. Biol. 25, 879–887 (2005) &
- Target RNA motif and target mRNAs of the Quaking STAR protein. Nature Struct. Mol. Biol. 12, 691–698 (2005) &
- Intronic sequences flanking alternatively spliced exons are conserved between human and mouse. Genome Res. 13, 1631–1637 (2003) &
- Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007–1013 (2002) , , &
- Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev. 18, 1241–1250 (2004) &
- Inference of splicing regulatory activities by sequence neighborhood analysis. PLoS Genet. 2, e191 (2006) et al.
- Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements. PLoS Genet. 3, e85 (2007) , &
- Coevolutionary networks of splicing cis-regulatory elements. Proc. Natl Acad. Sci. USA 104, 18583–18588 (2007) , , &
- Conserved RNA secondary structures promote alternative splicing. RNA 14, 1463–1469 (2008) &
- 2006) Pattern Recognition and Machine Learning. (Springer,
- A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423 (1948)
- Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. Mol. Cell 13, 91–100 (2004) , , , &
- A unique intronic splicing enhancer controls the inclusion of the agrin Y exon. RNA 3, 1275–1288 (1997) , , , &
- Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats. Mol. Cell. Biol. 18, 3900–3906 (1998) &
- Caspase-2 pre-mRNA alternative splicing: identification of an intronic element containing a decoy 3′ acceptor site. Proc. Natl Acad. Sci. USA 98, 938–943 (2001) , , &
- Muscle-specific exonic splicing silencer for exon exclusion in human ATP synthase γ-subunit pre-mRNA. J. Biol. Chem. 277, 6974–6984 (2002) et al.
- A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG. EMBO J. 22, 905–912 (2003) et al.
- Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2. Genes Dev. 22, 2550–2563 (2008) et al.
- Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein. Cell 138, 898–910 (2009) et al.
- A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones. Genome Biol. 7, R1 (2006) et al.
- Function of quaking in myelination: regulation of alternative splicing. Proc. Natl Acad. Sci. USA 99, 4233–4238 (2002) , , &
- Structure of PTB bound to RNA: specific binding and implications for splicing regulation. Science 309, 2054–2057 (2005) et al.
- Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein. Mol. Cell. Biol. 20, 7463–7479 (2000) et al.
- A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons. RNA 11, 1825–1834 (2005) , , , &
- Mutation of PTB binding sites causes misregulation of alternative 3′ splice site selection in vivo . RNA 3, 764–778 (1997) , , &
- Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J. 19, 4362–4371 (2000) et al.
- Exportin 4 mediates a novel nuclear import pathway for Sox family transcription factors. J. Cell Biol. 185, 27–34 (2009) et al.
- Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int. J. Biochem. Cell Biol. 39, 2195–2214 (2007) , , , &
- An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell 135, 852–864 (2008) et al.
- RNACompete: Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. Nature Biotechnol. 27, 667–670 (2009) et al.
- Supplementary information (2.4M)
This file contains Supplementary Information and Data, Supplementary Figures 1-3, Supplementary Tables 1-2 and References.