journal club lecture
TRANSCRIPT
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Regulation of Polyadenylation Factors by Sumoylation
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Mechanisms of Gene RegulationDNA
RNA
Protein
All three points are subject to regulation
Transcription
RNA
Post-translation
Transcription FactorsPromoter Sequences
SplicingRNA editing
PhosphorylationMethylation
Polyadenylation
Sumoylation
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RNA Maturation
5’ UTR 3’ UTR
5’ Cap
DNA
Pre-mRNA
AAAAAAAAAAAAAAAA
PolyadenylationSplicing
mRNA
Translation
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Why add a PolyA Tail?
•Stability
•Transport to Cytoplasm
•Translation Initiation
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Polyadenylation: How it happens
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin
• Cleavage
Cleavage Polyadenylation Specific Factor
Cleavage Stimulation Factor
Two Step Process:
• Polyadenylation
PolyAdenylation Polymerase
Symplekin
PAPAAAAAA
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Is Modification by Sumoylation Required for Polyadenylation?
3’ UTR AAUAAA GUCStFCPSF-73
SymplekinSymplekin
PAPAAAAAA
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What is SUMO?
• Small Ubiquitin-related Modifier
•Just like phosphorylation
•Affects interactions of modified protein
•Does NOT result in protein degradation
NO!
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Which SUMO is used?
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin
SUMO-1
SUMO-3
SUMO-2
There are 3 Types of SUMO:
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Which SUMO is used?
Symplekin
Symplekin
SUMO-1 SUMO-3SUMO-2
FLAG +FLAG-Symplekin Plasmids HeLa Cells
FLAG Western Blot
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Which SUMO is used?
Symplekin
SymplekinSUMO-1
SUMO-3
FLAG SymplekinFLAG SymplekinFLAGXNo SUMO
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Which SUMO is used:Symplekin
Symplekin
SUM
O-1
SUM
O-3
SUM
O-2
FLAG
No SUM
O
FLAG AB
SUMO-3 Modifies Symplekin
FIG 1A
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PullDown Assay: Symplekin
• 293T cells
•Cotransfected with His-Tagged SUMO-3 Flag-Symplekin
•Pulldown on a Ni column via His-Tag
•Visualize on a Western Blot via FLAG tag
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Pulldown Assay Symplekin?
SUMO-3 pulls down Symplekin
FIG 1B
WB FLAG
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What about Endogenous Symplekin?
• CoIP proteins with Symplekin antibody
•HeLa nuclear extracts
•Stain for SUMO-3
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What about Endogenous Symplekin?
SUMO-3 binds Endogenous Symplekin
FIG 1C
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Which SUMO is used?
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin
CPSF-73
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Which SUMO is used?Symplekin
FLAG +FLAG-Symplekin Plasmids HeLa Cells
CPSF-73
SUMO-1 SUMO-3SUMO-2
FLAG Western Blot
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Which SUMO is used:CPSF-73
CPSF-73
SUM
O-3
FLAG
FLAG AB
SUMO-3 Modifies CPSF-73
No SUM
O
SUM
O-2
SUM
O-1FIG 2A
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Pulldown Assay: CPSF-73
• HeLa cells
•Cotransformed with His-SUMO-3 FLAG-CPSF-73 •Stain for FLAG
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SUMO-3 pulls down CSPF-73
FIG 2B
Pulldown Assay: CPSF-73
FLAG antibody
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CoIP: CPSF-73
• CoIP proteins with CPSF-73 antibody
•HeLa nuclear extracts
•Stain for SUMO-3
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What about Endogenous CPSF-73?
SUMO-3 binds Endogenous CPSF-73
FIG 2C
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SUMO-3 Modifies Symplekin and CPSF-73
Conclusion
Does Symplekin Interact with Other Sumoylation Components?
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Sumoylation Overview
SAE1/2 Ubc9
SENP
E3
Target Protein
Do Our PolyA Proteins Interact with SENP?
E1 Activating EnzymeConjugating EnzymeE3 LigaseProtease
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Pulldown Assay: SENP2 Associate with Symplekin & CPSF?
HeLa Cells
Incubated Overnight
GST GST-SENP2
OR
Glutathione Beads
Nuclear Extracts
Antibodies to Detect Target Protein Binding
Y Y
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SENP2 Associates with Symplekin & CPSF-73?
Symplekin and CPSF-73 associate with SENP2
FIG 5D
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Does Ubc9 siRNA result in REDUCED Sumoylation?
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Sumoylation of Symplekin
SAE1/2
Ubc9E3
siRNA
Symplekin
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Ubc9 siRNA Decrease Sumoylation
Ubc9 Knockdown = DECREASE Symplekin Sumoylation
FIG 6D
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Conclusion
• SUMO-3 modifies Symplekin and CPSF-73
•Symplekin and CPSF- 73 bind SENP2
•Ubc9 RNAi REDUCES modification of Symplekin
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Identify Residues Important for Sumoylation
Symplekin
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Identify Symplekin Sumoylation Residues
Western Blot of FLAG-Symplekin
QCKEEPKEEK VVKTESVLIK RRLSAQGQAI
DDSDSTLKKM KLEPNLGEDD EDKDLEPGP
WT K229
WT 351
SUMO-3
SUMO-3
Sumoylation occurs on K residues42 K residues in Symplekin6 (Hydrophobic)K*XE/DPCR Mutagenesis KR
R
R
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Identifying Symplekin Sumoylated Residues
Symplekin
Wildtype
SUMO-3
FLAG
+ 293T Cells
FLAG Western Blot
SymplekinFLAG
SymplekinFLAG
K229R
K351R
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Identifying Sumoylation Residues?
SymplekinSUMO-3
FLAG SymplekinFLAG SymplekinFLAGXNo SUMO
SUMO-3Sumoylated K Non- Sumoylated K
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Sumoylation Decreased in K->R Mutants?
wt sym
plekin
wt sym
plekin
No SUM
O
No SUM
O
SymplekinFLAG
FLAG AB
K229 and K351 are IMPORTANT for Sumoylation
K229
symple
kinK2
29/3
51 sy
mplekin
K351
symple
kinFIG 3A*
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What about function?
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Symplekin Sumoylation and Cell Viability
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin Symplekin
PAPAAAAAA
Rationale
Polyadenylation is important for cell viability Mutation of K residues on Symplekin = REDUCED SUMO
Without SUMO No PolyA addition
SUMO Required for PolyA addition
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Symplekin Sumoylation and Cell Viability
+
HeLa Cells
Count Cells
WT 5’ UTR Symplekin+
+K>R 5’ UTR Symplekin
siRNA Symplekin
siRNA Symplekin
siRNA Symplekin
Directed against the 5’ UTR
Only endogenous Symplekin targeted
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Is Sumoylation Required for Cell Viability?
Relative # of Cells
Empt
y Vec
tor
Symple
kin RN
Ai
Symple
kin RN
Ai + W
T
Symple
kin RN
Ai + M
T
Suggests Sumoylation Important for Cell Viability
FIG 4A
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Does Sumoylation Affect 3’ End Processing?
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Sumoylation Affect Cleavage?
SAE1/2
Ubc9E3
siRNA
Symplekin
Cleavage + Polyadenylation
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Is Cleavage REDUCED by DECREASED Sumoylation?
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Sumoylation and PolyA Cleavage
SVL RNAP32 labeled
SP6-SVL plasmid
SP6 RNA PolymeraseP32-GTP
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Sumoylation and PolyA Cleavage
5’ Cleavage Product 3’ Cleavage Product
HeLa Cells
Nuclear Extracts
SVL RNAP32 labeled +
Promotes Cleavage
Ubc9
siRNA
VISUALIZE BY RADIOGRAPHY
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Decrease Ubc9 Decrease Cleavage?
Sumoylation is important for 3’polyA Cleavage
FIG 6A
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Sumoylation Affect Polyadenylation?
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Sumoylation and PolyA Cleavage
5’ Cleavage Product 3’ Cleavage Product
HeLa Cells
Nuclear Extracts
Pre-Cleaved SVL RNAP32 labeled +
Promote Polyadenylation
Ubc9
siRNA
VISUALIZE BY RADIOGRAPHY
AAAAAAA
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Ubc9 siRNA Decrease Polyadenylation
Polyadenylation is REDUCED when Sumoylation --|
FIG 6B
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Does Reversing Sumoylation Affect 3’ Processing?
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Increase SENP Decrease Polyadenylation?
SAE1/2
Ubc9
E3 PolyA Protein
Cleavage + PolyadenylationSENP
INCREASE SENP DECREASE POLYADENYLATION
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Increase SENP2 Decrease Polyadenylation?
Increase SENP2 Decrease Polyadenylation
FIG 7C
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Does Increasing SENP2 Affect Cleavage?
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Increase SENP2 Decrease Cleavage?
FIG 7B
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What We’ve Learned• Polyadenylation Complex is modified by SUMO-3
- CPSF-73- Symplekin
• Conserved Lysines in proteins of the Polyadenylation Complex are required for sumoylation
- Symplekin K229/K351
• Polyadenylation Complex is bound by SENP a SUMO protease -CPSF-73 -Symplekin
•Loss of Ubc9 reduces SUMO modification of -Symplekin
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What We’ve Learned
• Inhibiting Sumoylation decreases 3’ processing activity - Cleavage
- Polyadenylation
• Reversing Sumoylation is decreases 3’ processing activity - Cleavage
- Polyadenylation
• Inhibiting Sumoylation is detrimental to cell viability
•Loss of Ubc9 reduces SUMO modification of -Symplekin
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Take home messages
• Provides a mechanism for halting cellular translation
• Symplekin and polyadenylation are more affected
• 3’ RNA processing proteins are activated by sumoylation
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HaH
Have you hugged your advisor today?Happy Valentines Day!!!Pesky Questions are discouraged during today’s talk