1. SMN Deficiency in SMA: Splicing Gone Awry Gideon Dreyfuss Howard Hughes Medical Institute, University of Pennsylvania School of Medicine [Played by Stephanne Levin]

2. Road Map 1.Background on Spinal Muscular Atrophy 2. The Survival of Motor Neurons Gene Causes SMA 3. What is the SMN Complex and Where is It in the Cell? 4. Functions of SMN 5. How does SMN Deficiency Cause SMA? 6. Conclusion and Future Research Microsoft Clip Art

3. Spinal Muscular Atrophy (SMA) Affects 1 in 6000 newborns Leading genetic cause of infant mortality Results in death of ~50% of affected children by age 2 There is no cure http://www.fsma.org/FSMACommunity/Photos/FamilyP hotos/index.cfm?ID=2675&TYPE=1172 http://www.fsma.org/FSMACommunity/Photos/Fa milyPhotos/index.cfm?ID=3086&TYPE=1172 http://www.mda.org/publications/fa-sma-qa.html http://www.fsma.org/FSMACommunity/Photos/memorialphoto s/index.cfm?ID=2740&TYPE=1203

4. Spinal Muscular Atrophy (SMA) Causes degeneration of motor neurons in spinal cord Leads to: –Loss of voluntary muscle movement –Muscle weakness –Muscular atrophy Purves, D., Augustine, G., Fitzpatrick, D., Hall, W., LaMantia, A., McNamara, J., & White, L. (Eds.). (2008). Neuroscience (4th ed.). Sunderland: Sinauer Associates, Inc.

5. What Causes SMA? SMN Deficiency http://www.fsma.org/Research/DrugDiscovery/ParatekProgramDetails/ Mutations and deletions of the Survival of Motor Neurons (SMN1) Gene –Found in more than 98% of SMA patients Lefebvre, S., et al. (1995). Cell. Functional Protein

6. SMN Deficiency in SMA SMA patients have reduced levels of SMN Severity of SMA phenotypes is inversely correlated with the amount of SMN protein in cells Lefebvre, S., Burlet, P., Liu, Q. Bertrandy,S., Clermont, O., Munnich, A., Dreyfuss, G., & Melki, J. (1997). Nat Genet.

7. Research Questions 1. How and where does SMN exist within the cell? 2. What are the functions of the SMN protein? 3. How does SMN deficiency cause SMA?

8. Question 1 How and where does SMN exist within the cell?

9. Where is SMN within the Cell? Found in nuclear structures called gems and in the cytoplasm Liu, Q., & Dreyfuss, G. (1996). The EMBO Journal. Cytoplasm Gem

10. The SMN Complex SMN forms a complex with another protein, SIP1 Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell. Protein B Protein A Cell Extract Antibody for Protein A Add Probe for Protein B Detect Protein B

11. The SMN Complex Intera of theSMNCox and its edFun in RNAMeta G e SMNCplex A Splicing emin8 SMN Carissimi, C., Saieva, L., Baccon, J., Chiarella, P., Maiolica, A., Sawyer, A., et al. (2006). J Biol Chem, 281(12), 8126-8134. In total, SMN is in a complex with seven additional proteins (identified as Gemins2-8) N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin8 Gemin4 Gemin3 Gemin5 SMN Gemin2 Gemin7 Gemin6

12. Question 2 What are the functions of the SMN protein?

13. Alternative Splicing http://content.nejm.org/content/vol347/issue19/images/large/10f2.jpeg One gene  Multiple proteins Ast, Gil (2005). The alternative genome. Scientific American, 292 (4), 40-47.

14. snRNPs Splicing is mediated by the spliceosome, which consists of snRNPs snRNPs are comprised of snRNAs and Sm proteins Will, C. L., & Luhrmann, R. (2001). Curr Opin Cell Biol. Spliceosome

15. Functions of SMN: Binding to Components of snRNP SMN associates with spliceosomal snRNAs directly Yong, J., Golembe, T. J., Battle, D. J., Pellizzoni, L., & Dreyfuss, G. (2004). Mol Cell Biol.

16. Functions of SMN: Binding to Components of snRNP SMN binds to Sm proteins directly Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell. Beads coated with SMN Sm Protein (B, D1, D2, D3, E, F, or G) Wash Remove proteins bound to beads

17. Functions of SMN Spliceosome

18. Functions of SMN: snRNP Assembly The SMN complex is sufficient for snRNP assembly Pellizzoni, L., Yong, J., & Dreyfuss, G. (2002). Science. Gemin4 SMN

19. Functions of SMN: snRNP Assembly SMN is associated with snRNPs throughout their assembly pathway Massenet, S., Pellizzoni, L., Paushkin, S., Mattaj, I. W., & Dreyfuss, G. (2002). Mol Cell Biol.

20. Functions of SMN: snRNP Assembly Capacity for snRNP assembly is directly correlated with the amount of SMN Wan, L., Battle, D. J., Yong, J., Gubitz, A. K., Kolb, S. J., Wang, J., et al. (2005). Mol Cell Biol.

21. Functions of SMN Spliceosome

22. Pre-mRNA Splicing http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980408f.htm

23. Functions of SMN: pre-mRNA Splicing SMN mutant inhibits pre-mRNA splicing in vitro Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). Cell.

24. Functions of SMN N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin8 Gemin4 Gemin3 Gemin5 SMN Gemin2 Gemin7 Gemin6 Bind s The SMN Complex Spliceosome Part of Pre-mRNA Splicing Functions in SMN binds to snRNA and Sm proteins directly SMN mediates snRNP assembly SMN is required for proper splicing

25. Question 3 How does SMN deficiency cause SMA?

26. SMN Deficiency in SMA: Binding to Sm Proteins N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin8 Gemin4 Gemin3 Gemin5 SMN Gemin2 Gemin7 Gemin6 Bind s The SMN Complex Spliceosome Part of Pre-mRNA Splicing Functions in

27. SMN Deficiency in SMA: Defective Binding to Sm Proteins SMN mutants from SMA patients are defective in binding to Sm proteins in vivo Pellizzoni, L., Charroux, B., & Dreyfuss, G. (1999). Proc Natl Acad Sci.

28. SMN Deficiency in SMA: snRNP Assembly N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin8 Gemin4 Gemin3 Gemin5 SMN Gemin2 Gemin7 Gemin6 Bind s The SMN Complex Spliceosome Part of Pre-mRNA Splicing Functions in

29. SMN Deficiency in SMA: Decreased snRNP Assembly Wan, L., Battle, D. J., Yong, J., Gubitz, A. K., Kolb, S. J., Wang, J., et al. (2005). Mol Cell Biol. Cells of SMA patients have decreased SMN protein levels and decreased snRNP assembly

30. SMN Deficiency in SMA: Defective Splicing N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin8 Gemin4 Gemin3 Gemin5 SMN Gemin2 Gemin7 Gemin6 Bind s The SMN Complex Spliceosome Part of Pre-mRNA Splicing Functions in

31. SMN Deficiency in SMA: Defective Splicing Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). Cell.

32. The Role of SMN in SMA N N RNA H e and sAs P Asse &GAR1 P Assembly elicaseA anscription Gemin5 min7 Sm Pro Pre- br R Tr G Gemin6 Gemin4 Gemin3 Gemin2 Gemin 8 Gemin4 Gemin3 Gemin 5 SMN Gemin2 Gemin 7 Gemin 6 Decreased Binding The SMN Complex But how does defective splicing selectively cause degeneration of motor neurons?

33. SMN Deficiency in SMA : Defective Splicing SMN deficiency results in cell type-specific alterations in snRNA levels Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., et al. (2008). Cell.

34. SMN Deficiency in SMA: Defective Splicing Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., et al. (2008). Cell.

35. The Role of SMN in SMA But how does defective splicing selectively cause degeneration of motor neurons? Hypothesis: Cell type-specific defects in snRNP assembly and splicing Disruption of motor neuron development and survival Low levels of SMN in motor neurons

36. Remaining Questions 1. SMN deficiency and the selective death of motor neurons? 2. Does SMN have unique functions in motor neurons? 3. How can this knowledge help in the development of treatments for SMA?

37. Conclusions 1.SMN exists as a multi-protein complex in the cytoplasm and nucleus 2. SMN functions in: -snRNP assembly -Pre-mRNA splicing 3. SMN deficiency causes SMA by: - Interfering with snRNP assembly - Causing widespread defects in splicing

38. Acknowledgements I would like to thank all of the graduate students working in my lab and all of my collaborators who have contributed to my research. I would also like to thank Lake Forest College, my colleagues in BIO480, and Dr. DebBurman.

39. References Carissimi, C., Saieva, L., Baccon, J., Chiarella, P., Maiolica, A., Sawyer, A., et al. (2006). Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly. J Biol Chem, 281(12), 8126-8134. Charroux, B., Pellizzoni, L., Perkinson, R. A., Shevchenko, A., Mann, M., & Dreyfuss, G. (1999). Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems. J Cell Biol, 147(6), 1181-1194. Charroux, B., Pellizzoni, L., Perkinson, R. A., Yong, J., Shevchenko, A., Mann, M., et al. (2000). Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli. J Cell Biol, 148(6), 1177-1186. Fischer, U., Liu, Q., & Dreyfuss, G. (1997). The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell, 90(6), 1023-1029. Lefebvre,,Suzie, Burlet,,Philippe, Liu,,Qing, Bertrandy,,Solange, Clermont,,Olivier, Munnich,,Arnold, et al. (1997). Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet, 16(3), 265-269. Liu, Q., & Dreyfuss, G. (1996). A novel nuclear structure containing the survival of motor neurons protein. The EMBO Journal, 15(14), 3555-3565. Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell, 90(6), 1013-1021.

40. References Massenet, S., Pellizzoni, L., Paushkin, S., Mattaj, I. W., & Dreyfuss, G. (2002). The SMN complex is associated with snRNPs throughout their cytoplasmic assembly pathway. Mol Cell Biol, 22(18), 6533-6541. Pellizzoni, L., Charroux, B., & Dreyfuss, G. (1999). SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proc Natl Acad Sci, 96(20), 11167-11172. Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell, 95(5), 615-624. Pellizzoni, L., Yong, J., & Dreyfuss, G. (2002). Essential role for the SMN complex in the specificity of snRNP assembly. Science, 298(5599), 1775-1779. Wan, L., Battle, D. J., Yong, J., Gubitz, A. K., Kolb, S. J., Wang, J., et al. (2005). The survival of motor neurons protein determines the capacity for snRNP assembly: Biochemical deficiency in spinal muscular atrophy. Mol Cell Biol, 25(13), 5543-5551. Will, C. L., & Luhrmann, R. (2001). Spliceosomal UsnRNP biogenesis, structure and function. Curr Opin Cell Biol, 13(3), 290-301. Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., et al. (2008). SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 133(4), 585-600.