journal club

1. Regulation of Polyadenylation Factors
by Sumoylation

2. Mechanisms of Gene Regulation
DNA
RNA
Protein
All three points are subject to regulation
Transcription
RNA
Post-translation
Transcription Factors
Promoter Sequences
Splicing
RNA editing
Phosphorylation
Methylation
Polyadenylation
Sumoylation

3. RNA Maturation
5’ UTR 3’ UTR
5’ Cap
DNA
Pre-mRNA
AAAAAAAAAAAAAAAA
PolyadenylationSplicing
mRNA
Translation

4. Why add a PolyA Tail?
•Stability
•Transport to Cytoplasm
•Translation Initiation

5. Polyadenylation: How it happens
3’ UTR AAUAAA GU
CStF
CPSF-73
Symplekin
• Cleavage
Cleavage Polyadenylation Specific Factor
Cleavage Stimulation Factor
Two Step Process:
• Polyadenylation
PolyAdenylation Polymerase
Symplekin
PAP
AAAAAA

6. Is Modification by Sumoylation Required for
Polyadenylation?
3’ UTR AAUAAA GU
CStF
CPSF-73
Symplekin
Symplekin
PAP
AAAAAA

7. What is SUMO?
• Small Ubiquitin-related Modifier
•Just like phosphorylation
•Affects interactions of modified protein
•Does NOT result in protein degradation
NO!

8. Which SUMO is used?
3’ UTR AAUAAA GU
CStF
CPSF-73
Symplekin
SUMO-1
SUMO-3
SUMO-2
There are 3 Types of SUMO:

9. Which SUMO is used?
Symplekin
Symplekin
SUMO-1 SUMO-3SUMO-2
FLAG
+FLAG-Symplekin Plasmids HeLa Cells
FLAG Western Blot

10. Which SUMO is used?
Symplekin
Symplekin
FLAG SymplekinFLAG SymplekinFLAG

11. Which SUMO is used:Symplekin
SymplekinFLAG
FLAG AB
SUMO-3 Modifies Symplekin
FIG 1A

12. 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

13. Pulldown Assay Symplekin?
SUMO-3 pulls down Symplekin
FIG 1B
WB FLAG

14. What about Endogenous Symplekin?
• CoIP proteins with Symplekin antibody
•HeLa nuclear extracts
•Stain for SUMO-3

15. What about Endogenous Symplekin?
SUMO-3 binds Endogenous Symplekin
FIG 1C

16. Which SUMO is used?
3’ UTR AAUAAA GU
CStF
CPSF-73
Symplekin
CPSF-73

17. Which SUMO is used?
Symplekin
FLAG
+
FLAG-Symplekin Plasmids HeLa Cells
CPSF-73
SUMO-1 SUMO-3SUMO-2
FLAG Western Blot

18. Which SUMO is used:CPSF-73
CPSF-73FLAG
FLAG AB
SUMO-3 Modifies CPSF-73
FIG 2A

19. Pulldown Assay: CPSF-73
• HeLa cells
•Cotransformed with His-SUMO-3
FLAG-CPSF-73
•Stain for FLAG

20. SUMO-3 pulls down CSPF-73
FIG 2B
Pulldown Assay: CPSF-73
FLAG antibody

21. CoIP: CPSF-73
• CoIP proteins with CPSF-73 antibody
•HeLa nuclear extracts
•Stain for SUMO-3

22. What about Endogenous CPSF-73?
SUMO-3 binds Endogenous CPSF-73
FIG 2C

23. SUMO-3 Modifies Symplekin and CPSF-73
Conclusion
Does Symplekin Interact with Other
Sumoylation Components?

24. Sumoylation Overview
SAE1/2 Ubc9
SENP
E3
Target Protein
Do Our PolyA Proteins Interact with SENP?
E1 Activating EnzymeConjugating EnzymeE3 LigaseProtease

25. 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

26. SENP2 Associates with Symplekin & CPSF-73?
Symplekin and CPSF-73 associate with SENP2
FIG 5D

27. Does Ubc9 siRNA result in REDUCED
Sumoylation?

28. Sumoylation of Symplekin
SAE1/2
Ubc9
E3
siRNA
Symplekin

29. Ubc9 siRNA Decrease Sumoylation
Ubc9 Knockdown = DECREASE Symplekin Sumoylation
FIG 6D

30. Conclusion
• SUMO-3 modifies Symplekin and CPSF-73
•Symplekin and CPSF- 73 bind SENP2
•Ubc9 RNAi REDUCES modification of Symplekin

31. Identify Residues Important for
Sumoylation
Symplekin

32. Identify Symplekin Sumoylation Residues
Western Blot of FLAG-Symplekin
QCKEEPKEEK VVKTESVLIK RRLSAQGQAI
DDSDSTLKKM KLEPNLGEDD EDKDLEPGP
WT K229
WT 351
Sumoylation occurs on K residues42 K residues in Symplekin6 (Hydrophobic)K*XE/DPCR Mutagenesis KR
R
R

33. Identifying Symplekin Sumoylated Residues
Symplekin
Wildtype
SUMO-3
FLAG
+ 293T Cells
FLAG Western Blot
SymplekinFLAG
SymplekinFLAG
K229R
K351R

34. Identifying Sumoylation Residues?
SymplekinFLAG SymplekinFLAG SymplekinFLAG
Sumoylated K Non- Sumoylated K

35. Sumoylation Decreased in K->R Mutants?
SymplekinFLAG
FLAG AB
K229 and K351 are IMPORTANT for Sumoylation
FIG 3A*

36. What about function?

37. Symplekin Sumoylation and Cell Viability
3’ UTR AAUAAA GU
CStFCPSF-73
Symplekin
Symplekin
PAP
AAAAAA
Rationale
Polyadenylation is important for cell viabilityMutation of K residues on Symplekin = REDUCED SUMO
Without SUMO No PolyA addition
SUMO Required for PolyA addition

38. 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

39. Is Sumoylation Required for Cell Viability?
Relative # of
Cells
Suggests Sumoylation Important for Cell Viability
FIG 4A

40. Does Sumoylation Affect 3’ End
Processing?

41. Sumoylation Affect Cleavage?
SAE1/2
Ubc9
E3
siRNA
Symplekin
Cleavage + Polyadenylation

42. Is Cleavage REDUCED by DECREASED
Sumoylation?

43. Sumoylation and PolyA Cleavage
SVL RNAP32 labeled
SP6-SVL plasmid
SP6 RNA PolymeraseP32-GTP

44. Sumoylation and PolyA Cleavage
5’ Cleavage Product 3’ Cleavage Product
HeLa Cells
Nuclear Extracts
SVL RNAP32 labeled
+
Promotes Cleavage
Ubc9
siRNA
VISUALIZE BY RADIOGRAPHY

45. Decrease Ubc9 Decrease Cleavage?
Sumoylation is important for 3’polyA Cleavage
FIG 6A

46. Sumoylation Affect Polyadenylation?

47. 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

48. Ubc9 siRNA Decrease Polyadenylation
Polyadenylation is REDUCED when Sumoylation --|
FIG 6B

49. Does Reversing Sumoylation Affect 3’
Processing?

50. Increase SENP Decrease Polyadenylation?
SAE1/2
Ubc9
E3 PolyA Protein
Cleavage + Polyadenylation
SENP
INCREASE SENP DECREASE POLYADENYLATION

51. Increase SENP2 Decrease Polyadenylation?
Increase SENP2 Decrease Polyadenylation
FIG 7C

52. Does Increasing SENP2 Affect
Cleavage?

53. Increase SENP2 Decrease Cleavage?
FIG 7B

54. 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

55. 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

56. Take home messages
• Provides a mechanism for halting cellular translation
• Symplekin and polyadenylation are more affected
• 3’ RNA processing proteins are activated by sumoylation

57. HaH
Have you hugged your advisor today?Happy Valentines Day!!!Pesky Questions are discouraged during
today’s talk