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SMN Deficiency in SMA: Splicing Gone Awry. Gideon Dreyfuss Howard Hughes Medical Institute, University of Pennsylvania School of Medicine [Played by Stephanne Levin]. Road Map. Background on Spinal Muscular Atrophy. 2. The Survival of Motor Neurons Gene Causes SMA.

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Smn deficiency in sma splicing gone awry

SMN Deficiency in SMA: Splicing Gone Awry

Gideon Dreyfuss

Howard Hughes Medical Institute, University of

Pennsylvania School of Medicine

[Played by Stephanne Levin]


Road map
Road Map

  • 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
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/FamilyPhotos/index.cfm?ID=3086&TYPE=1172

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

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

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


Spinal muscular atrophy sma1
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
What Causes SMA?

Lefebvre, S., et al. (1995). Cell.

  • Mutations and deletions of the Survival of Motor Neurons (SMN1) Gene

    • Found in more than 98% of SMA patients

Functional Protein

http://www.fsma.org/Research/DrugDiscovery/ParatekProgramDetails/

SMN Deficiency


Smn deficiency in sma
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
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
Question 1

  • How and where does SMN exist within the cell?


Where is smn within the cell
Where is SMN within the Cell?

Gem

Found in nuclear structures called gems and in the cytoplasm

Cytoplasm

Liu, Q., & Dreyfuss, G. (1996). The EMBO Journal.


The smn complex

Antibody for Protein A

Protein A

Protein B

Cell Extract

The SMN Complex

  • SMN forms a complex with another protein, SIP1

Add Probe for Protein B

Detect Protein B

Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell.


The smn complex1

a

n

d

s

As

S

m Pr

o

Gemin4

P Ass

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G

e

min4

N

Pre

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Gemin3

G

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min3

G

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min5

Gemin5

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Gemin7

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H

anscription

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Gemin8

G

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Gemin2

Gemin6

The SMN Complex

Inter

a

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SMN

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SMN

C

plex

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n RNA

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SMN

In total, SMN is in a complex with seven additional proteins (identified as Gemins2-8)

G

e

min8

A Splicing

Carissimi, C., Saieva, L., Baccon, J., Chiarella, P., Maiolica, A., Sawyer, A., et al. (2006). J Biol Chem, 281(12), 8126-8134.


Question 2
Question 2

  • What are the functions of the SMN protein?


Alternative splicing
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.


Snrnps

Spliceosome

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.


Functions of smn binding to components of snrnp

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 associates with spliceosomal snRNAs directly


Functions of smn binding to components of snrnp1

Wash Dreyfuss, G. (2004).

Sm Protein

(B, D1, D2, D3, E, F, or G)

Remove proteins bound to beads

Beads coated with SMN

Functions of SMN: Binding to Components of snRNP

  • SMN binds to Sm proteins directly

Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell.


Functions of smn

Spliceosome Dreyfuss, G. (2004).

Functions of SMN


Functions of smn snrnp assembly
Functions of SMN: Dreyfuss, G. (2004). snRNP Assembly

  • The SMN complex is sufficient for snRNP assembly

G

e

min4

SMN

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


Functions of smn snrnp assembly1
Functions of SMN: Dreyfuss, G. (2004). 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.


Functions of smn snrnp assembly2
Functions of SMN: Dreyfuss, G. (2004). 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.


Functions of smn1

Spliceosome Dreyfuss, G. (2004).

Functions of SMN


Pre mrna splicing
Pre-mRNA Splicing Dreyfuss, G. (2004).

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


Functions of smn pre mrna splicing
Functions of SMN: Dreyfuss, G. (2004). pre-mRNA Splicing

  • SMN mutant inhibits pre-mRNA splicing in vitro

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


Functions of smn2

a Dreyfuss, G. (2004).

n

d

s

As

S

m Pr

o

Gemin4

P Ass

e

G

e

min4

N

Pre

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Gemin3

G

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min3

G

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min5

Gemin5

&

GA

R1

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P Ass

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mbly

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min7

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Gemin7

SMN

RNA

H

anscription

T

r

G

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min6

Gemin8

Spliceosome

G

e

min2

Gemin2

Gemin6

Functions of SMN

The SMN Complex

Functions in

Part of

Binds

Pre-mRNA Splicing

  • SMN binds to snRNA and Sm proteins directly

  • SMN mediates snRNP assembly

  • SMN is required for proper splicing


Question 3
Question 3 Dreyfuss, G. (2004).

  • How does SMN deficiency cause SMA?


Smn deficiency in sma binding to sm proteins

a Dreyfuss, G. (2004).

n

d

s

As

S

m Pr

o

Gemin4

P Ass

e

G

e

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Pre

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Gemin3

G

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min3

G

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min5

Gemin5

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R1

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r

P Ass

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mbly

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Gemin7

SMN

RNA

H

anscription

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Gemin8

Spliceosome

G

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min2

Gemin2

Gemin6

SMN Deficiency in SMA: Binding to Sm Proteins

The SMN Complex

Functions in

Part of

Binds

Pre-mRNA Splicing


Smn deficiency in sma defective binding to sm proteins
SMN Deficiency in SMA: Defective Binding to Sm Proteins Dreyfuss, G. (2004).

  • 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

a Dreyfuss, G. (2004).

n

d

s

As

S

m Pr

o

Gemin4

P Ass

e

G

e

min4

N

Pre

-

Gemin3

G

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G

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min5

Gemin5

&

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min7

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RNA

H

anscription

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r

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Gemin8

Spliceosome

G

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Gemin2

Gemin6

SMN Deficiency in SMA: snRNP Assembly

The SMN Complex

Functions in

Part of

Binds

Pre-mRNA Splicing


Smn deficiency in sma decreased snrnp assembly
SMN Deficiency in SMA: Decreased snRNP Assembly Dreyfuss, G. (2004).

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

a Dreyfuss, G. (2004).

n

d

s

As

S

m Pr

o

Gemin4

P Ass

e

G

e

min4

N

Pre

-

Gemin3

G

e

min3

G

e

min5

Gemin5

&

GA

R1

b

r

P Ass

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mbly

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min7

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Gemin7

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RNA

H

anscription

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r

G

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min6

Gemin8

Spliceosome

G

e

min2

Gemin2

Gemin6

SMN Deficiency in SMA: Defective Splicing

The SMN Complex

Functions in

Part of

Binds

Pre-mRNA Splicing


Smn deficiency in sma defective splicing1
SMN Deficiency in SMA: Defective Splicing Dreyfuss, G. (2004).

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


The role of smn in sma

a Dreyfuss, G. (2004).

n

d

s

As

S

m Pr

o

Gemin4

P Ass

e

G

e

min4

N

Pre

-

Gemin3

G

e

min3

G

e

min5

Gemin5

&

GA

R1

b

r

P Ass

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mbly

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N

min7

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elicase

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Gemin7

SMN

RNA

H

anscription

T

r

G

e

min6

Gemin8

G

e

min2

Gemin2

Gemin6

The Role of SMN in SMA

The SMN Complex

Decreased Binding

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


Smn deficiency in sma defective splicing2
SMN Deficiency in SMA : Defective Splicing Dreyfuss, G. (2004).

  • 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.


Smn deficiency in sma defective splicing3
SMN Deficiency in SMA: Defective Splicing Dreyfuss, G. (2004).

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


The role of smn in sma1
The Role of SMN in SMA Dreyfuss, G. (2004).

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

Hypothesis:

Disruption of motor neuron development and survival

Cell type-specific defects in snRNP assembly and splicing

Low levels of SMN in motor neurons


Remaining questions
Remaining Questions Dreyfuss, G. (2004).

  • 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
Conclusions Dreyfuss, G. (2004).

  • 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
Acknowledgements Dreyfuss, G. (2004).

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.


References
References Dreyfuss, G. (2004).

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.


References1
References Dreyfuss, G. (2004).

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.


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