DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity

International audienceThe U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common. However, how U6 2’-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2’-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2′-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator

Non-canonical role for the BAF complex subunit DPF3 in mitosis and ciliogenesis.

peer reviewedDPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex that plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and in centrosome, spindle midzone/bridging fiber area and midbodies during mitosis. Loss of DPF3 causes K-fiber instability, unstable kinetochore-microtubules attachment and defects in chromosome alignment, thus resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes in centriolar satellites at the basis of primary cilia and is required for ciliogenesis by regulating axoneme extension. Together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis

Phospho-Dependant Regulation Of TFIP11 Splicing Factor

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.Phospho-dependent regulation of TFIP11 splicing factor activit