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
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
