Alteration of correct splicing patterns by disruption of an exonic splicing enhancer may be a frequent mechanism by which point mutations cause genetic diseases. Spinal muscular atrophy results from the lack of functional survival of motor neuron 1 gene (SMN1), even though all affected individuals carry a nearly identical, normal SMN2 gene. SMN2 is only partially active because a translationally silent, single-nucleotide difference in exon 7 causes exon skipping. Using ESE motif-prediction tools, mutational analysis and in vivo and in vitro splicing assays, we show that this single-nucleotide change occurs within a heptamer motif of an exonic splicing enhancer, which in SMN1 is recognized directly by SF2/ASF. The abrogation of the SF2/ASF-dependent ESE is the basis for inefficient inclusion of exon 7 in SMN2, resulting in the spinal muscular atrophy phenotype.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Emery, A.E. Population frequencies of inherited neuromuscular diseases—a world survey. Neuromuscul. Disord. 1, 19–29 (1991).
Lefebvre, S., Burglen, L., Frezal, J., Munnich, A. & Melki, J. The role of the SMN gene in proximal spinal muscular atrophy. Hum. Mol. Genet. 7, 1531–1536 (1998).
Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).
Jablonka, S., Rossoll, W., Schrank, B. & Sendtner, M. The role of SMN in spinal muscular atrophy. J. Neurol. 247 (Suppl 1), I37–I42 (2000).
Liu, Q. & Dreyfuss, G. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 15, 3555–3565 (1996).
Meister, G., Buhler, D., Pillai, R., Lottspeich, F. & Fischer, U. A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs. Nature Cell Biol. 3, 945–949 (2001).
Pellizzoni, L., Kataoka, N., Charroux, B. & Dreyfuss, G. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell 95, 615–624 (1998).
Pellizzoni, L., Charroux, B., Rappsilber, J., Mann, M. & Dreyfuss, G. A functional interaction between the survival motor neuron complex and RNA polymerase II. J. Cell Biol. 152, 75–85 (2001).
Monani, U.R. et al. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum. Mol. Genet. 8, 1177–1183 (1999).
Lorson, C.L., Hahnen, E., Androphy, E.J. & Wirth, B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl Acad. Sci. USA 96, 6307–6311 (1999).
Lorson, C.L. & Androphy, E.J. An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum. Mol. Genet. 9, 259–265 (2000).
Monani, U.R., Coovert, D.D. & Burghes, A.H. Animal models of spinal muscular atrophy. Hum. Mol. Genet. 9, 2451–2457 (2000).
Schrank, B. et al. Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc. Natl Acad. Sci. USA 94, 9920–9925 (1997).
Hsieh-Li, H.M. et al. A mouse model for spinal muscular atrophy. Nature Genet. 24, 66–70 (2000).
Monani, U.R. et al. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(−/−) mice and results in a mouse with spinal muscular atrophy. Hum. Mol. Genet. 9, 333–339 (2000).
Muro, A.F. et al. Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display. Mol. Cell. Biol. 19, 2657–2671 (1999).
Watakabe, A., Tanaka, K. & Shimura, Y. The role of exon sequences in splice site selection. Genes Dev. 7, 407–418 (1993).
Cáceres, J.F. & Krainer, A.R. Mammalian pre-mRNA splicing factors. in Eukaryotic mRNA Processing (ed. Krainer, A.R.) 174–212 (IRL Press, Oxford, 1997).
Blencowe, B.J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem. Sci. 25, 106–110 (2000).
Graveley, B.R., Hertel, K.J. & Maniatis, T. A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers. EMBO J. 17, 6747–6756 (1998).
Tanaka, K., Watakabe, A. & Shimura, Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol. Cell. Biol. 14, 1347–1354 (1994).
Coulter, L.R., Landree, M.A. & Cooper, T.A. Identification of a new class of exonic splicing enhancers by in vivo selection. Mol. Cell. Biol. 17, 2143–2150 (1997).
Liu, H.X., Zhang, M. & Krainer, A.R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12, 1998–2012 (1998).
Schaal, T.D. & Maniatis, T. Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences. Mol. Cell. Biol. 19, 1705–1719 (1999).
Liu, H.X., Chew, S.L., Cartegni, L., Zhang, M.Q. & Krainer, A.R. Exonic splicing enhancer motif recognized by human SC35 under splicing conditions. Mol. Cell. Biol. 20, 1063–1071 (2000).
Liu, H.X., Cartegni, L., Zhang, M.Q. & Krainer, A.R. A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nature Genet. 27, 55–58 (2001).
Mazoyer, S. et al. A BRCA1 nonsense mutation causes exon skipping. Am. J. Hum. Genet. 62, 713–715 (1998).
Robberson, B.L., Cote, G.J. & Berget, S.M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol. Cell. Biol. 10, 84–94 (1990).
Mayeda, A. & Krainer, A.R. Mammalian in vitro splicing assays. Methods Mol. Biol. 118, 315–321 (1999).
Krainer, A.R., Conway, G.C. & Kozak, D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 4, 1158–1171 (1990).
Mayeda, A. & Krainer, A.R. Preparation of HeLa cell nuclear and cytosolic S100 extracts for in vitro splicing. Methods Mol. Biol. 118, 309–314 (1999).
Reed, R. & Chiara, M.D. Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking. Methods 18, 3–12 (1999).
Saito, I. & Sugiyama, H. Photoreactions of nucleic acids and their constituents with amino acids and related compounds. in Photochemistry and the Nucleic Acids Vol. 2 (ed. Morrison, H.) 317–340 (Wiley, New York, 1990).
Krawczak, M., Reiss, J. & Cooper, D.N. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum. Genet. 90, 41–54 (1992).
Teraoka, S.N. et al. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am. J. Hum. Genet. 64, 1617–1631 (1999).
Ars, E. et al. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum. Mol. Genet. 9, 237–247 (2000).
Hentze, M.W. & Kulozik, A.E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).
Graveley, B.R., Hertel, K.J. & Maniatis, T. SR proteins are 'locators' of the RNA splicing machinery. Curr. Biol. 9, R6–7 (1999).
Penalva, L.O., Lallena, M.J. & Valcárcel, J. Switch in 3′ splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation. Mol. Cell. Biol. 21, 1986–1996 (2001).
Newman, A.J. The role of U5 snRNP in pre-mRNA splicing. EMBO J. 16, 5797–5800 (1997).
Hofmann, Y., Lorson, C.L., Stamm, S., Androphy, E.J. & Wirth, B. Htra2-β1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc. Natl Acad. Sci. USA 97, 9618–9623 (2000).
Tacke, R., Boned, A. & Goridis, C. ASF alternative transcripts are highly conserved between mouse and man. Nucleic Acids Res. 20, 5482 (1992).
Rochette, C.F., Gilbert, N. & Simard, L.R. SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens. Hum. Genet. 108, 255–266 (2001).
Lim, S.R. & Hertel, K.J. Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3′ splice site pairing. J. Biol. Chem. 276, 45476–45483 (2001).
Andreassi, C. et al. Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum. Mol. Genet. 10, 2841–2849 (2001).
Zhang, M.L., Lorson, C.L., Androphy, E.J. & Zhou, J. An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA. Gene Ther. 8, 1532–1538 (2001).
Chang, J.G. et al. Treatment of spinal muscular atrophy by sodium butyrate. Proc. Natl Acad. Sci. USA 98, 9808–9813 (2001).
Hastings, M.L. & Krainer, A.R. Functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway. RNA 7, 471–482 (2001).
Zhu, J. & Krainer, A.R. Pre-mRNA splicing in the absence of an SR protein RS domain. Genes Dev. 14, 3166–3178 (2000).
Burge, C.B., Tuschl, T. & Sharp, P.A. Splicing of precursors to messenger RNAs by the spliceosome. in The RNA world II (eds Gesteland, R.F., Cech, T.A. & Atkins, J.F.) 525–560 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999).
We thank M. Hastings and J. Zhu for sharing reagents and for helpful comments on the manuscript. We are grateful to C. Lorson and E. Androphy for the pCITel plasmid and for helpful discussions. This work was supported by the National Institutes of Health (National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke), by Andrew's Buddies Corp., and by a postdoctoral fellowship from the Human Frontiers Science Program (to L.C.).
About this article
Cite this article
Cartegni, L., Krainer, A. Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30, 377–384 (2002). https://doi.org/10.1038/ng854
Combined treatment with the histone deacetylase inhibitor LBH589 and a splice‐switch antisense oligonucleotide enhances SMN2 splicing and SMN expression in Spinal Muscular Atrophy cells
Journal of Neurochemistry (2020)
Seminars in Cell & Developmental Biology (2019)
Frontiers in Genetics (2019)
Intraperitoneal delivery of a novel drug‐like compound improves disease severity in severe and intermediate mouse models of Spinal Muscular Atrophy
Scientific Reports (2019)
The FASEB Journal (2019)