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
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
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http://www.fsma.org/FSMACommunity/Photos/Fa
milyPhotos/index.cfm?ID=3086&TYPE=1172
http://www.fsma.org/FSMACommunity/Photos/FamilyP
hotos/index.cfm?ID=2675&TYPE=1172
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• There is no cure
http://www.fsma.org/FSMACommunity/Photos/memorialphoto
s/index.cfm?ID=2740&TYPE=1203
http://www.mda.org/publications/fa-sma-qa.html
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?
Lefebvre, S., et al. (1995). Cell.
• Mutations and deletions of
the Survival of Motor
Neurons (SMN1) Gene
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– Found in more than 98% of
SMA patients
Functional
Protein
http://www.fsma.org/Research/DrugDiscovery/ParatekProgramDetails/
SMN Deficiency
SMN Deficiency in SMA
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Lefebvre, S., Burlet, P. , Liu, Q. Bertrandy,S., Clermont, O. , Munnich, A., Dreyfuss, G., & Melki, J. (1997). Nat Genet.
• SMA patients have reduced levels of SMN
• Severity of SMA phenotypes is inversely correlated with the
amount of SMN protein in cells
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?
Gem
Cytoplasm
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Liu, Q., & Dreyfuss, G. (1996). The EMBO Journal.
Found in nuclear structures called gems and
in the cytoplasm
The SMN Complex
Antibody for
Protein A
• SMN forms a
complex with
Protein A
another protein,
SIP1
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Protein B
Cell Extract
Add Probe
for Protein B
Detect
Protein B
Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell.
The SMN Complex
e SMN C
Inter a
plex
o f t he SMN C o
ed Fun
in RNA
x a n d its
Met a
and s
P Ass e
G e min4
SMN
As
S m Pr o
N
Gemin4
Pre G Gemin3
e min3
G e min5
Gemin5
In total, SMN is in a complex
& GA R1
with seven additional proteins
P Ass e mbly N
(identified as Gemins2-8)
G
e min8
min7
br
R
e
G
Gemin7
SMN
elicaseRNA
A H
anscription
G e min6
Gemin8
A Splicing
G e min2
Carissimi, C., Saieva, L., Baccon, J., Chiarella, P.,
Maiolica, A., Sawyer, A., et al. (2006). J Biol Chem,
281(12), 8126-8134.
Tr
Gemin6
Gemin2
Question 2
What are the functions of the
SMN protein?
Alternative Splicing
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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
• Splicing is mediated by the spliceosome, which consists of
snRNPs
• snRNPs are comprised of snRNAs and Sm proteins
Sm
Proteins
Forms
Fu n cti on sin Pre-mRNA
Part of
Splicing
snRNP
Spliceosome
snRNA
Will, C. L., & Luhrmann, R. (2001). Curr Opin Cell Biol.
Functions of SMN:
Binding to Components of snRNP
• SMN associates
with spliceosomal
snRNAs directly
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Yong, J., Golembe, T. J., Battle, D. J., Pellizzoni, L., & Dreyfuss, G.
(2004). Mol Cell Biol.
Functions of SMN:
Binding to Components of snRNP
Wash
Sm Protein
(B, D1, D2, D3,
E, F, or G)
• SMN binds to Sm
proteins directly
Remove
proteins
bound
to beads
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Beads coated
with SMN
Liu, Q., Fischer, U., Wang, F., & Dreyfuss, G. (1997). Cell.
Functions of SMN
Sm
Proteins
Forms
Fu n cti on sin Pre-mRNA
Part of
Splicing
snRNP
Spliceosome
snRNA
Functions of SMN:
snRNP Assembly
Gemin4
Sm
Proteins
SMN
snRNP
snRNA
Pellizzoni, L., Yong, J., & Dreyfuss, G. (2002). Science.
• The SMN complex is sufficient for snRNP assembly
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.
Functions of SMN:
snRNP Assembly
• Capacity for snRNP
assembly is directly
correlated with the
amount of SMN
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Wan, L., Battle, D. J., Yong, J., Gubitz, A. K., Kolb, S. J., Wang,
J., et al. (2005). Mol Cell Biol.
Functions of SMN
Sm
Proteins
Forms
Fu n cti on sin Pre-mRNA
Part of
Splicing
snRNP
Spliceosome
snRNA
Pre-mRNA Splicing
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http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980408f.htm
Functions of SMN:
pre-mRNA Splicing
• SMN mutant inhibits
pre-mRNA splicing
in vitro
Pellizzoni, L., Kataoka, N., Charroux, B.,
& Dreyfuss, G. (1998). Cell.
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Functions of SMN
The SMN Complex
ands NGemin4
AsoPr
P Ass
e Sm
Gemin3
Pre
Gemin3
Gemin4
Gemin5
Gemin5
&GA
R1br
NR
P Ass
embly
Binds
Sm
Proteins
Part of
min7e G
SMN
Gemin7
RNA
elicase
AH
anscription
Tr
snRNP
Gemin8
Gemin6
Gemin6
Forms
Functions
in
Pre-mRNA
Splicing
Gemin2
Gemin2
Spliceosome
snRNA
•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
The SMN Complex
Gemin4
Gemin5
ands NGemin4
AsoPr
P Ass
e Sm
Gemin3
Pre
Gemin3
Gemin5
&GA
R1br
NR
P Ass
embly
Binds
Sm
Proteins
Part of
min7e G
SMN
Gemin7
RNA
elicase
AH
anscription
Tr
snRNP
Gemin8
Gemin6
Gemin6
Forms
Functions
in
Pre-mRNA
Splicing
Gemin2
Gemin2
snRNA
Spliceosome
SMN Deficiency in SMA:
Defective Binding to Sm Proteins
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Pellizzoni, L., Charroux, B., & Dreyfuss, G. (1999). Proc Natl Acad Sci.
• SMN mutants from SMA patients are defective in binding to
Sm proteins in vivo
SMN Deficiency in SMA:
snRNP Assembly
The SMN Complex
Gemin4
Gemin5
ands NGemin4
AsoPr
P Ass
e Sm
Gemin3
Pre
Gemin3
Gemin5
&GA
R1br
NR
P Ass
embly
Binds
Sm
Proteins
Part of
min7e G
SMN
Gemin7
RNA
elicase
AH
anscription
Tr
snRNP
Gemin8
Gemin6
Gemin6
Forms
Functions
in
Pre-mRNA
Splicing
Gemin2
Gemin2
snRNA
Spliceosome
SMN Deficiency in SMA:
Decreased snRNP Assembly
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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
The SMN Complex
Gemin4
Gemin5
ands NGemin4
AsoPr
P Ass
e Sm
Gemin3
Pre
Gemin3
Gemin5
&GA
R1br
NR
P Ass
embly
Binds
Sm
Proteins
Part of
min7e G
SMN
Gemin7
RNA
elicase
AH
anscription
Tr
snRNP
Gemin8
Gemin6
Gemin6
Forms
Functions
in
Pre-mRNA
Splicing
Gemin2
Gemin2
snRNA
Spliceosome
SMN Deficiency in SMA:
Defective Splicing
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Pellizzoni, L., Kataoka, N., Charroux, B., & Dreyfuss, G. (1998). Cell.
The Role of SMN in SMA
The SMN Complex
ands Gemin4
As o
P Ass
e NSm Pr
Gemin3
Pre
Gemin3
G emin4
Gemin5
Gemin5
&GA
R1 br
N R
P Ass
embly
Decreased
Binding
Sm
Proteins
min7 e
G
RNA
H
elicase
ASMN
Gemin7
Inability to
Form
snRNP
anscription Tr
Gemin6
Gemin8
Gemin2
Gemin6
Gemin2
snRNA
But how does defective splicing selectively
cause degeneration of motor neurons?
Defective
Pre-mRNA
Splicing
SMN Deficiency in SMA :
Defective Splicing
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QuickTime™ and a
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QuickTime™ and a
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Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., et al. (2008). Cell.
• SMN deficiency results in cell type-specific alterations
in snRNA levels
SMN Deficiency in SMA:
Defective Splicing
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are needed to see this picture.
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:
Low levels of SMN
in motor neurons
Cell type-specific
defects in snRNP
assembly and
splicing
Disruption of
motor neuron
development
and survival
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.
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.
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.