Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: High-score SR protein motifs in exon 7 of SMN1 and SMN2.
Figure 2: Effect of point mutations on calculated SC35 and SF2/ASF motif scores.
Figure 3: Exon 7–skipping correlates with disruption of the proximal SF2/ASF heptamer motif.
Figure 4: The SMN1 SF2/ASF heptamer motif is a bona fide ESE.
Figure 5: SF2/ASF promotes inclusion of SMN1 exon 7 in vitro.
Figure 6: SF2/ASF binds directly to the high-score motif in SMN1 exon 7.
Figure 7: Model of SF2/ASF-dependent exon 7 inclusion in SMN1 and SMN2.

Similar content being viewed by others

References

  1. Emery, A.E. Population frequencies of inherited neuromuscular diseases—a world survey. Neuromuscul. Disord. 1, 19–29 (1991).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).

    Article  CAS  Google Scholar 

  4. Jablonka, S., Rossoll, W., Schrank, B. & Sendtner, M. The role of SMN in spinal muscular atrophy. J. Neurol. 247 (Suppl 1), I37–I42 (2000).

    Article  Google Scholar 

  5. Liu, Q. & Dreyfuss, G. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 15, 3555–3565 (1996).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

  12. Monani, U.R., Coovert, D.D. & Burghes, A.H. Animal models of spinal muscular atrophy. Hum. Mol. Genet. 9, 2451–2457 (2000).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Hsieh-Li, H.M. et al. A mouse model for spinal muscular atrophy. Nature Genet. 24, 66–70 (2000).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Watakabe, A., Tanaka, K. & Shimura, Y. The role of exon sequences in splice site selection. Genes Dev. 7, 407–418 (1993).

    Article  CAS  Google Scholar 

  18. 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).

    Google Scholar 

  19. Blencowe, B.J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem. Sci. 25, 106–110 (2000).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Tanaka, K., Watakabe, A. & Shimura, Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol. Cell. Biol. 14, 1347–1354 (1994).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Mazoyer, S. et al. A BRCA1 nonsense mutation causes exon skipping. Am. J. Hum. Genet. 62, 713–715 (1998).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. Mayeda, A. & Krainer, A.R. Mammalian in vitro splicing assays. Methods Mol. Biol. 118, 315–321 (1999).

    CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. 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).

    CAS  PubMed  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

  33. 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).

    Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. Hentze, M.W. & Kulozik, A.E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 307–310 (1999).

    Article  CAS  Google Scholar 

  38. Graveley, B.R., Hertel, K.J. & Maniatis, T. SR proteins are 'locators' of the RNA splicing machinery. Curr. Biol. 9, R6–7 (1999).

    Article  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. Newman, A.J. The role of U5 snRNP in pre-mRNA splicing. EMBO J. 16, 5797–5800 (1997).

    Article  CAS  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. Tacke, R., Boned, A. & Goridis, C. ASF alternative transcripts are highly conserved between mouse and man. Nucleic Acids Res. 20, 5482 (1992).

  43. 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).

    Article  CAS  Google Scholar 

  44. 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).

    Article  CAS  Google Scholar 

  45. 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).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Chang, J.G. et al. Treatment of spinal muscular atrophy by sodium butyrate. Proc. Natl Acad. Sci. USA 98, 9808–9813 (2001).

    Article  CAS  Google Scholar 

  48. 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).

    Article  CAS  Google Scholar 

  49. Zhu, J. & Krainer, A.R. Pre-mRNA splicing in the absence of an SR protein RS domain. Genes Dev. 14, 3166–3178 (2000).

    Article  CAS  Google Scholar 

  50. 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).

    Google Scholar 

Download references

Acknowledgements

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.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Adrian R. Krainer.

Rights and permissions

Reprints and permissions

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng854

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing