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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