Regulation of Polyadenylation Factors by Sumoylation
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Regulation of Polyadenylation Factors by Sumoylation
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
RNA Maturation
5’ UTR 3’ UTR
5’ Cap
DNA
Pre-mRNA
AAAAAAAAAAAAAAAA
PolyadenylationSplicing
mRNA
Translation
Why add a PolyA Tail?
•Stability
•Transport to Cytoplasm
•Translation Initiation
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
Is Modification by Sumoylation Required for Polyadenylation?
3’ UTR AAUAAA GUCStFCPSF-73
SymplekinSymplekin
PAPAAAAAA
What is SUMO?
• Small Ubiquitin-related Modifier
•Just like phosphorylation
•Affects interactions of modified protein
•Does NOT result in protein degradation
NO!
Which SUMO is used?
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin
SUMO-1
SUMO-3
SUMO-2
There are 3 Types of SUMO:
Which SUMO is used?
Symplekin
Symplekin
SUMO-1 SUMO-3SUMO-2
FLAG +FLAG-Symplekin Plasmids HeLa Cells
FLAG Western Blot
Which SUMO is used?
Symplekin
SymplekinSUMO-1
SUMO-3
FLAG SymplekinFLAG SymplekinFLAGXNo SUMO
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
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
Pulldown Assay Symplekin?
SUMO-3 pulls down Symplekin
FIG 1B
WB FLAG
What about Endogenous Symplekin?
• CoIP proteins with Symplekin antibody
•HeLa nuclear extracts
•Stain for SUMO-3
What about Endogenous Symplekin?
SUMO-3 binds Endogenous Symplekin
FIG 1C
Which SUMO is used?
3’ UTR AAUAAA GUCStFCPSF-73
Symplekin
CPSF-73
Which SUMO is used?Symplekin
FLAG +FLAG-Symplekin Plasmids HeLa Cells
CPSF-73
SUMO-1 SUMO-3SUMO-2
FLAG Western Blot
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
Pulldown Assay: CPSF-73
• HeLa cells
•Cotransformed with His-SUMO-3 FLAG-CPSF-73 •Stain for FLAG
SUMO-3 pulls down CSPF-73
FIG 2B
Pulldown Assay: CPSF-73
FLAG antibody
CoIP: CPSF-73
• CoIP proteins with CPSF-73 antibody
•HeLa nuclear extracts
•Stain for SUMO-3
What about Endogenous CPSF-73?
SUMO-3 binds Endogenous CPSF-73
FIG 2C
SUMO-3 Modifies Symplekin and CPSF-73
Conclusion
Does Symplekin Interact with Other Sumoylation Components?
Sumoylation Overview
SAE1/2 Ubc9
SENP
E3
Target Protein
Do Our PolyA Proteins Interact with SENP?
E1 Activating EnzymeConjugating EnzymeE3 LigaseProtease
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
SENP2 Associates with Symplekin & CPSF-73?
Symplekin and CPSF-73 associate with SENP2
FIG 5D
Does Ubc9 siRNA result in REDUCED Sumoylation?
Sumoylation of Symplekin
SAE1/2
Ubc9E3
siRNA
Symplekin
Ubc9 siRNA Decrease Sumoylation
Ubc9 Knockdown = DECREASE Symplekin Sumoylation
FIG 6D
Conclusion
• SUMO-3 modifies Symplekin and CPSF-73
•Symplekin and CPSF- 73 bind SENP2
•Ubc9 RNAi REDUCES modification of Symplekin
Identify Residues Important for Sumoylation
Symplekin
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
Identifying Symplekin Sumoylated Residues
Symplekin
Wildtype
SUMO-3
FLAG
+ 293T Cells
FLAG Western Blot
SymplekinFLAG
SymplekinFLAG
K229R
K351R
Identifying Sumoylation Residues?
SymplekinSUMO-3
FLAG SymplekinFLAG SymplekinFLAGXNo SUMO
SUMO-3Sumoylated K Non- Sumoylated K
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*
What about function?
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
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
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
Does Sumoylation Affect 3’ End Processing?
Sumoylation Affect Cleavage?
SAE1/2
Ubc9E3
siRNA
Symplekin
Cleavage + Polyadenylation
Is Cleavage REDUCED by DECREASED Sumoylation?
Sumoylation and PolyA Cleavage
SVL RNAP32 labeled
SP6-SVL plasmid
SP6 RNA PolymeraseP32-GTP
Sumoylation and PolyA Cleavage
5’ Cleavage Product 3’ Cleavage Product
HeLa Cells
Nuclear Extracts
SVL RNAP32 labeled +
Promotes Cleavage
Ubc9
siRNA
VISUALIZE BY RADIOGRAPHY
Decrease Ubc9 Decrease Cleavage?
Sumoylation is important for 3’polyA Cleavage
FIG 6A
Sumoylation Affect Polyadenylation?
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
Ubc9 siRNA Decrease Polyadenylation
Polyadenylation is REDUCED when Sumoylation --|
FIG 6B
Does Reversing Sumoylation Affect 3’ Processing?
Increase SENP Decrease Polyadenylation?
SAE1/2
Ubc9
E3 PolyA Protein
Cleavage + PolyadenylationSENP
INCREASE SENP DECREASE POLYADENYLATION
Increase SENP2 Decrease Polyadenylation?
Increase SENP2 Decrease Polyadenylation
FIG 7C
Does Increasing SENP2 Affect Cleavage?
Increase SENP2 Decrease Cleavage?
FIG 7B
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
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
Take home messages
• Provides a mechanism for halting cellular translation
• Symplekin and polyadenylation are more affected
• 3’ RNA processing proteins are activated by sumoylation
HaH
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