Alternative Splicing and its Significance
The vast majority of human genes are broken up into segments (exons) that must be spliced together following transcription of the gene into a precursor mRNA (pre-mRNA) (Figure 1). Up to seventy six percent of human genes express multiple mRNAs by alternative splicing of their pre-mRNAs (Johnson et al. 2003) in which exons are joined in different patterns. As a result, individual genes express multiple mRNAs that are identical except for discrete regions of variability (Figure 1). Some genes contain multiple alternatively spliced regions and express hundreds or even thousands of different mRNAs (Graveley 2001). Eighty percent of the time the variability is within the coding region resulting in the expression of different protein isoforms (Modrek and Lee 2002). For many genes, alternative splicing directs expression of functionally divergent protein isoforms according to cell-specific regulation (based on differentiated cell type, developmental stage, gender, or in response to an external signal) (Black 2003; Faustino and Cooper 2003). Alternative splicing can alter the function of proteins by removing or adding specific domains (nuclear localization signals, transcription activation domains, DNA or RNA binding domains, trans-membrane domains), post-translation modification sites, or by causing substantial changes in protein structure by altering even just a few residues (Davletov and Jimenez 2004).
Therefore, alternative splicing not only contributes to an extremely diverse human proteome that is expressed from a relatively small number of genes but it also allows regulated expression of these protein isoforms in response to a wide range of cues.
Alternative splicing also generates variability within untranslated regions of mRNAs and affects gene expression by adding or removing mRNA elements that regulate translation efficiency, mRNA stability, or intracellular localization. In addition, up to 30% of alternative splicing changes introduce a premature termination codon (PTC) that lead to mRNA by the nonsense mediated decay (NMD) surveillance mechanism (Figure 1). So cells can also utilize splicing for ON / OFF regulation of gene expression (Lewis et al. 2003).
Since all types of proteins (structural, enzymatic, regulatory) are expressed as splice variants, the impact of alternative splicing is huge and largely uncharacterized on a genomic scale (Lareau et al. 2004). Sex determination in Drosophila is determined by a hierarchy of alternative splicing events in which splicing of an upstream mediator generates an isoform that regulates splicing of the downstream targets (Black 2003). Some genes involved in regulating apoptosis do so by generating anti- or pro-apoptotic isoforms via alternative splicing (Wu et al. 2003). Splicing and its disruption has a major impact on causing human disease and modifying its severity or susceptibility (Wang and Cooper, 2007). In the Cooper lab we study myotonic dystrophy, a disease in which a microsatellite expansion in one region of the genome has a trans -dominant effect on regulation of a subset of alternatively spliced pre-mRNAs (see Projects, Mechanism of myotonic dystrophy pathogenesis ). The effect is to reverse developmentally regulated shifts such that embryonic isoforms are expressed in adult tissues (Faustino and Cooper 2003, Ranum and Cooper, 2006). We have demonstrated how the inappropriate expression of embryonic isoforms in adult tissues results in specific symptoms of the disease (Philips et al. 1998; Savkur et al. 2001; Charlet-B. et al. 2002).
Regulation of alternative splicing has a huge biological impact, yet there is a great deal to learn about a range of issues pertaining to splicing regulation. Cis-acting elements that are required for cell-specific splicing and some of the proteins that bind to them have been identified in several experimental systems. The question remains how binding of the regulatory proteins to their cognate elements communicates to the basal splicing machinery to use or skip a regulated alternative splice site. In most cases, splicing is clearly not regulated by a single cell-specific protein binding to a single auxiliary cis-element. Rather, regulated splicing follows the paradigms of regulated transcription in which assembly of multicomponent complexes determine whether the basal machinery is recruited or repressed.
On a genomic level, microarray analysis of alternative splicing is revealing a huge degree of regulation in different cell types and during development (Blencowe 2006). As for transcription, regulation is highly coordinated by modulation of the activities of multiple regulators and involve a network of signaling events (see Projects, Mechanisms of coordinated splicing regulation. Many of the known alternative splicing regulators are themselves expressed as alternatively spliced isoforms and there is an established potential for cross- and auto-regulation ( Lareau et al. 2007, Ni et al. 2007) . Several factors that regulate splicing have been identified. How they act and the signaling events that modulate their activities are just beginning to be understood (see Projects, Mechanism of cell-specific alternative splicing).
- Black, D.L. 2003. Mechanisms of Alternative Pre-Messenger RNA Splicing. Annu Rev Biochem 27: 27-48.
- Blencowe, B. J. 2006. Alternative splicing: new insights from global analyses. Cell 126:37-47.
- Charlet-B., N., Savkur, R.S., Singh, G., Philips, A.V., Grice, E.A., and Cooper, T.A. 2002. Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 10: 45-53.
- Davletov, B. and Jimenez, J.L. 2004. Sculpting a domain by splicing. Nat Struct Mol Biol 11: 4-5.
- Faustino, N.A. and Cooper, T.A. 2003. Pre-mRNA splicing and human disease. Genes Dev 17: 419-437.
- Graveley, B.R. 2001. Alternative splicing: increasing diversity in the proteomic world. Trends Genet 17: 100-107.
- Johnson, J.M., Castle, J., Garrett-Engele, P., Kan , Z., Loerch, P.M., Armour, C.D., Santos , R., Schadt, E.E., Stoughton , R., and Shoemaker, D.D. 2003. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302: 2141-2144.
- Lareau, L.F., Green, R.E., Bhatnagar, R.S., and Brenner, S.E. 2004. The evolving roles of alternative splicing. Curr Opin Struct Biol 14: 273-282.
- Lareau, L. F., M. Inada, R. E. Green, J. C. Wengrod, and S. E. Brenner. 2007. Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature 446:926-929.
- Lewis, B.P., Green, R.E., and Brenner, S.E. 2003. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 100: 189-192.
- Modrek, B. and Lee, C. 2002. A genomic view of alternative splicing. Nat Genet 30: 13-19.
- Ni, J. Z., L. Grate, J. P. Donohue, C. Preston, N. Nobida, G. O'Brien, L. Shiue, T. A. Clark, J. E. Blume, and M. Ares, Jr. 2007. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev. 21:708-718.
- Philips, A.V., Timchenko, L.T., and Cooper, T.A. 1998. Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 280: 737-741.
- Ranum, L. P., and T. A. Cooper. 2006. RNA-Mediated Neuromuscular Disorders. Ann. Rev. Neuroscience 29: 259-277.
- Savkur, R.S., Philips, A.V., and Cooper, T.A. 2001. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat. Gen. 29: 40-47.
- Wu, J.Y., Tang, H., and Havlioglu, N. 2003. Alternative pre-mRNA splicing and regulation of programmed cell death. Prog Mol Subcell Biol 31: 153-185.
- Wang, G. S., and T. A. Cooper. 2007. Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet 8:749-761.
- Xu, Q., Modrek, B., and Lee, C. 2002. Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. Nucleic Acids Res 30: 3754-3766.