Phospho-Dependant Regulation Of TFIP11 Splicing Factor
Sarah Hanache
1, Amandine Duchemin
1, Paul Peixoto
2, Eric Hervouet
2, Franck Dequiedt
1and Denis Mottet
11Laboratory 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 STUDY
1) Identification of kinases regulating TFIP11 phosphorylation
2) Identification of phospho-residues of TFIP11and the study of phosphorylation impact on TFIP11 activity.
CONCLUSION
TFIP11 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
BACKGROUND
Pre-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 kDa 95 kDa 66 kDa Cy to so l Nu cl eo pl as m Ch ro m at in Cy to so l Nu cl eo pl as m Ch ro m at in
Control CIP treatment
130 kDa 95 and 90 kDa 66 Kda 130 kDa 95 kDa Cy to so l Nu cl eo pl as m Ch ro m at in Control siTFIP11 Cy to so l Nu cl eo pl as m Ch ro m at in siGL3 Cy to so l Nu cl eo pl as m Ch ro m at 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-sites CK2 consensus phospho-sites N-TIP NLS NSLS 1 22 109 148 192 350 666 699 734 837 TFIP11 β-actin IP Input (2,5%) Ig G TFIP11 Ig G TF IP 11 (M ) TFIP11 (R) EFTUD2 DHX15
TFIP11 interacts and colocalizes with PRP4K and CK2 in the nuclear speckles
IP Input (2,5%) IgG TF IP 11 IgG TFIP 11 ( M ) TFIP11 (R) DHX15 PRP4K
DAPI CK2 TFIP11 Merged
DAPI PRP4K TFIP11 Merged
CK2-∝ EFTUD2 TFIP11 DAPI Merged Ct siEFTDU2 P-LI SA TF JP 11 /E FT D U2 DA PI EF TD U 2 D ots /Ce ll (± SE M ) 0 1 P-LI SA Ct siEFTDU2 IF **** **** D ots /Ce ll (± SE M ) 1,5 Ct siDHX15 0 Ct siDHX15 DH X1 5 IF P-LI SA TF JP 11 /D H X1 5 DA PI P-LI SA
EFTUD2 and TFIP11 silencing lead to the same effects on cell cycle progression
HSC70 E F T U D 2 si R N A #1 G L 3 si R N A N o si R N A E F T U D 2 si R N A #3 48H 96H HeLa E F T U D 2 si R N A #1 G L 3 si R N A N o si R N A E F T U D 2 si R N A #3 EFTUD2 NoSi -48H SiEF TUD2 #1-4 8H SiEF TUD2 #3-4 8H SiGL 3-48H NoSi -96H SiEF TUD2 #1-9 6H SiEF TUD2 #3-9 6H SiGL 3-96H 0 20 40 60 80 G1 S G2 NoSi-4 8H SiEF TUD2 #1-48 H SiEF TUD2 #3-48 H SiGL3 -48H NoSi-9 6H SiEF TUD2 #1-96 H SiEF TUD2 #3-96 H SiGL3 -96H 0 20 40 60 80 G1 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 0.00
ALYREF intron retention
Nosi siEFTUD2 #1 siEFTUD2 #3 gl3
DAPI TFIP11 SC35 Merged Cytoplasm Nucleoplasm Chromatin
CK2-∝*
Lamin A+C MEK2