SMN Deficiency in SMA: Splicing Gone Awry Gideon Dreyfuss Howard Hughes Medical Institute,...

SMN Deficiency in SMA: Splicing Gone Awry

Gideon DreyfussHoward Hughes Medical Institute, University of

Pennsylvania School of Medicine

[Played by Stephanne Levin]

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

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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.



http://www.fsma.org/FSMACommunity/Photos/FamilyPhotos/index.cfm?ID=2675&TYPE=1172

http://www.fsma.org/FSMACommunity/Photos/FamilyPhotos/index.cfm?ID=3086&TYPE=1172



http://www.mda.org/publications/fa-sma-qa.html



http://www.fsma.org/FSMACommunity/Photos/memorialphotos/index.cfm?ID=2740&TYPE=1203

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.

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

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.



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?

Question 1

How and where does SMN exist within the cell?

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

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

The SMN Complex

Inter a of the SMN C o x a n d its

e d F u n in RNA Met a

G

e SMN C plex

A Splicing

e min8

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

a nd s As

P Ass e

& GA R1

P Ass e mbly

elicase A

anscription

G e min5

min7

S m Pr o

Pre -

b r

R

Tr

G

G e min6

G e min4

G e min3

G e min2

Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Question 2

What are the functions of the SMN protein?

Alternative Splicing



http://content.nejm.org/content/vol347/issue19/images/large/10f2.jpeg

One gene Multiple proteinsAst, Gil (2005). The alternative genome. Scientific American, 292 (4), 40-47.

snRNPs

• Splicing is mediated by the spliceosome, which consists of snRNPs

• snRNPs are comprised of snRNAs and Sm proteins

Part of

Pre-mRNA Splicing

Functions in Forms

snRNA

Sm Proteins

snRNP

Will, C. L., & Luhrmann, R. (2001). Curr Opin Cell Biol.

Spliceosome

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.

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

Functions of SMN

Part of

Pre-mRNA Splicing

Functions in Forms

snRNA

Sm Proteins

snRNP

Spliceosome

Functions of SMN: snRNP Assembly

• The SMN complex is sufficient for snRNP assembly

Pellizzoni, L., Yong, J., & Dreyfuss, G. (2002). Science.

Gemin4

snRNA

Sm Proteins

snRNP SMN


• SMN is associated with snRNPs throughout their assembly pathway

Massenet, S., Pellizzoni, L., Paushkin, S., Mattaj, I. W., & Dreyfuss, G. (2002). Mol Cell Biol.


• 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.



Functions of SMN

Part of

Pre-mRNA Splicing

Functions in Forms

snRNA

Sm Proteins

snRNP

Spliceosome

Pre-mRNA Splicing



http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980408f.htm

Functions of SMN: pre-mRNA Splicing

• SMN mutant inhibits pre-mRNA splicing in vitro QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). Cell.

Functions of SMN

N

N

RNA He

and s As P Asse

& GAR1

P Assembly elicase A anscription

Gemin5

min7

Sm ProPre-

brR

Tr

G

Gemin6

Gemin4 Gemin3

Gemin2 Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Forms

snRNA

Sm Proteins

snRNP

Binds

The SMN Complex

Spliceosome

Part ofPre-mRNA Splicing

Functions in

•SMN binds to snRNA and Sm proteins directly

•SMN mediates snRNP assembly

•SMN is required for proper splicing

Question 3

How does SMN deficiency cause SMA?

SMN Deficiency in SMA: Binding to Sm Proteins

N

N

RNA He

and s As P Asse

& GAR1


Gemin5

min7

Sm ProPre-

brR

Tr

G

Gemin6

Gemin4 Gemin3

Gemin2 Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Forms

snRNA

Sm Proteins

snRNP

Binds

The SMN Complex

Spliceosome


Functions in

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.



SMN Deficiency in SMA: snRNP Assembly

N

N

RNA He

and s As P Asse

& GAR1


Gemin5

min7

Sm ProPre-

brR

Tr

G

Gemin6

Gemin4 Gemin3

Gemin2 Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Forms

snRNA

Sm Proteins

snRNP

Binds

The SMN Complex

Spliceosome


Functions in

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

SMN Deficiency in SMA: Defective Splicing

N

N

RNA He

and s As P Asse

& GAR1


Gemin5

min7

Sm ProPre-

brR

Tr

G

Gemin6

Gemin4 Gemin3

Gemin2 Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Forms

snRNA

Sm Proteins

snRNP

Binds

The SMN Complex

Spliceosome


Functions in




Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). Cell.

The Role of SMN in SMA

N

N

RNA He

and s As P Asse

& GAR1

P Assembly elicase A

anscription

Gemin5

min7

Sm Pro

Pre-

brR

Tr

G

Gemin6

Gemin4

Gemin3

Gemin2 Gemin8

Gemin4

Gemin3

Gemin5

SMN

Gemin2

Gemin7

Gemin6

Defective Pre-mRNA

Splicing

Inability to Form

snRNA

Sm Proteins

snRNP

Decreased Binding

The SMN Complex

But how does defective splicing selectively cause degeneration of motor neurons?

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.








Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., et al. (2008). Cell.



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

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?

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

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.

ReferencesCarissimi, 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.

ReferencesMassenet, 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.

SMN Deficiency in SMA: Splicing Gone Awry Gideon Dreyfuss Howard Hughes Medical Institute,...

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