Abstract :
[en] Pre-mRNA splicing is a fundamental process in mammalian gene expression contributing to protein diversity. Eukaryotic genes are alternatively spliced by the spliceosome, a large conserved machinery complex comprising RNA, five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and around 200 proteins and splicing factors. These splicing factors are frequently phosphorylated by kinases. Such phosphorylation regulates their subcellular localization and interactions with target transcripts and 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 (see A.Duchemin abstract). We and others have demonstrated that TFIP11 is highly phosphorylated. This phosphorylation might be critical for its nuclear localization, transport and/or its interaction with RNA or proteins such as DHX15. So, the present project is divided into three main goals: 1) Identification of kinases regulating phosphorylation of TFIP11 2) Identification of phospho-residues of TFIP11 and 3) Impact of phosphorylation on TFIP11 activity.
Phosphoproteomic analyses have shown that at least 4 serine residues (S59, S96, S98, S210) and 2 tyrosine residues (Y162, Y722) on TFIP11 undergo phosphorylation. Interestingly, some of these potential phospho-residues are within functional TFIP11 domains including G-Patch domain, nuclear localization signal (NLS) and nuclear speckle targeting sites (NSTS) and belong to consensus sites for CK2 and Prp4K, two kinases known to regulate the activity of splicing factors.
Immunofluorescence experiments demonstrated that CK2 and Prp4K colocalize with TFIP11 in the nuclear speckles. Co-immunoprecipitation experiments and/or proximity ligation assay (PLA) demonstrate that TFIP11, CK2 and Prp4K were associated in the same complex. Interestingly, we identified EFTUD2 as an interacting partner of TFIP11 and it seems that EFTUD2 coordinates the same splicing program as TFIP11. Finally, preliminary PLA data show that CK2 inhibition increases the interaction between TFIP11 and EFTUD2, suggesting that a CK2-dependent phosphorylation of TFP11 might be important to regulate its interaction with partner protein EFTUD2. Further investigations are ongoing to determine the functional role of the TFIP11-EFTUD2-CK2 complex in the regulation of the splicing program.
