Phospho-Dependant Regulation Of TFIP11 Splicing Factor

Sarah Hanache1, Amandine Duchemin1, Paul Peixoto2, Eric Hervouet2, Franck Dequiedt1 and Denis Mottet1

1Laboratory of Protein Signaling and Interactions (PSI), GIGA-Molecular Biology of Disease. Avenue de l’Hôpital 1, (B34) 4000 Sart-Tilman, University of Liège.2Laboratoire de Biochimie, groupe "Autophagy, EMT and antitumor T-cell immunity", UFR-ST, Université de Bourgogne Franche-Comté, INSERM UMR1098, Equipe TIM-C

AIM OF THIS STUDY1) Identification of kinases regulating TFIP11 phosphorylation

2) Identification of phospho-residues of TFIP11and the study of phosphorylation impact on TFIP11 activity.

CONCLUSIONTFIP11 is a phosphorylated protein. Two Important kinases, known to regulate the splicing program, were identified as TFIP11 interactants: CK2 and PRP4K.

These kinases have consensus phosphorylation sites on TFIP11 peptide sequence.

An interesting interaction has been found between TFIP11 and EFTUD2 and both proteins seem to regulate the same splicing program.

Preliminary PLA data shows that the inhibition of CK2 kinase modulates this TFIP11/EFTUD2 interaction.

RESULTS

Sarah.Hanache@student.ulg.ac.be

BACKGROUNDPre-mRNA splicing is a fundamental process in mammalian gene expression contributing to protein diversity. Splicing factors are frequently

phosphorylated by kinases. Such phosphorylation regulates their interactions with protein partners, and thus regulates splicing reactions.

Our lab demonstrated that the splicing factor TFIP11 controls cancer cell-cycle progression by regulating splicing of a specific subset of pre-

mRNA.

EFTUD2, a spliceosome GTPase, interacts and colocalizes with TFIP11

TFIP11 is a phosphoprotein showing specific phosphorylation bands in western blot. These bands decrease upon TFIP11 silencing and Calf lambda phosphatase (CIP) treatment.

Control Hela cells lysate

130 kDa95 kDa66 kDa

Cyto

sol

Nucle

opla

sm

Chro

mat

in

Cyto

sol

Nucle

opla

sm

Chro

mat

in

Control CIP treatment

130 kDa

95 and 90 kDa

66 Kda

130 kDa

95 kDa

Cyto

sol

Nucle

opla

sm

Chro

mat

in

Control siTFIP11

Cyto

sol

Nucle

opla

sm

Chro

mat

in

siGL3

Cyto

sol

Nucle

opla

sm

Chro

mat

in

TFIP11

Lamin A+C

TFIP11 TFIP11

Lamin A+C

TFIP11 peptide sequence has consensus and potential sites for PRP4K and CK2 (two kinases known to regulate the splicing activity)

PRP4K consensus phospho-sitesCK2 consensus phospho-sites

N-TIP NSLS

NLS

1 22 109 148 192 350 666 699 734 837

TFIP11

β-actin

IP Input(2,5%)

IgG TFIP

11

IgG

TFIP

11 (M

)

TFIP11 (R)

EFTUD2

DHX15

TFIP11 interacts and colocalizes with PRP4K and CK2 in the nuclear speckles

IP Input

(2,5%)

IgG

TF

IP11

IgG

TF

IP11

(M

)

TFIP11 (R)

DHX15

PRP4K

DAPI CK2 MergedTFIP11

DAPI PRP4K TFIP11 Merged

CK2-∝

EFTUD2

TFIP11DAPI

Merged

Ct siEFTDU2

P-LIS

ATFJP11/EFTDU

2DA

PIEFTD

U2

Dots/Cell

(±SEM)

0

1

P-LIS

A

Ct siEFTDU2IF

****

****

Dots/Cell

(±SEM)

1,5

Ct siDHX150

Ct siDHX15DH

X15

IFP-LIS

ATFJP11/D

HX15

DAPI

P-LIS

A

EFTUD2 and TFIP11 silencing lead to the same effects on cell cycle progression

HSC70

EF

TU

D2

siR

NA

#1

GL

3 si

RN

A

No

siR

NA

EF

TU

D2

siR

NA

#3

48H 96HHeLa

EF

TU

D2

siR

NA

#1

GL

3 si

RN

A

No

siR

NA

EF

TU

D2

siR

NA

#3

EFTUD2

NoSi-48H

SiEFTUD2#1-48H

SiEFTUD2#3-48H

SiGL3-48H

NoSi-96H

SiEFTUD2#1-96H

SiEFTUD2#3-96H

SiGL3-96H

0

20

40

60

80G1

S

G2

NoSi-48H

SiEFTUD2#1-48H

SiEFTUD2#3-48H

SiGL3-48H

NoSi-96H

SiEFTUD2#1-96H

SiEFTUD2#3-96H

SiGL3-96H

0

20

40

60

80G1

S

G2

EFTUD2 and TFIP11 silencing lead to the same intron retention on a subset of genes

-6

-5

-4

-3

-2

-1

0

Exon1-Intron1 Exon2-Intron2

Sororin RNA

n1

Sororin Intron Retention

Nosi siEFTUD2#1 siEFTUD2#3 SiGL3

-8.00

-7.00

-6.00

-5.00

-4.00

-3.00

-2.00

-1.00

0.00ALYREF intron retention

Nosi siEFTUD2 #1 siEFTUD2 #3 gl3

DAPI TFIP11 SC35 Merged Cytoplasm Nucleoplasm Chromatin

CK2-∝*

LaminA+C

MEK2

*Two different exposures