Characterization of pre-EJC factors and their role in cell fate determination – spEJCificity

The Exon-Junction Complex (EJC) plays a central role in post-transcriptional gene expression control. This multiprotein complex deposited onto mRNAs, participates to major steps in the mRNA life-cycle. The EJC is organized around a tetrameric core assembled by the splicing machinery. This core constitutes a binding platform for more than a dozen of peripheral factors associated with distinct functions. Deposited on most exonic junctions, EJCs constitute cornerstones for mRNP packaging. Developmental defects and human pathological disorders due to altered expression of EJC proteins show that the EJC dosage is critical for some cell fate specifications, and in particular brain development. However, the underlying mechanisms are not known. EJC assembly requires adaptor proteins, such as CWC22 that escorts the EJC core component eIF4A3 to the spliceosome. EJC assembly on specific junctions is hypothesized to be tightly regulated, and to control gene expression in a cell-specific manner. This project addresses the mechanisms controlling EJC deposition on target mRNAs and aims to dissect how EJC-dependent transcripts are involved in cell fate decisions.
Three axes will be developed. (i) Pre-EJC complexes will be characterized. Partner 1 recently identified complexes containing eIF4A3 core EJC subunit together with the CWC22, CWC27 spliceosome subunits and SR splicing factors. As EJC core components are assembled together by the splicing machinery, these complexes might constitute new intermediates, on which eIF4A3 piggybacking is regulated to control EJC assembly. (ii) The role of pre-EJC components in modulating EJC deposition and mRNA destiny will be determined. Thus, transcriptome-wide deposition of EJCs will be analyzed following pre-EJC factor down-regulations. This analysis will be repeated in the context of recently discovered mutations of eIF4A3, CWC22 and CWC27 that are associated to novel human disorders presenting common neurological phenotypes. (iii) The role of EJC and pre-EJC components in RNA localization in relation to cell-fate decisions will be investigated. We have observed that EJCs concentrate around centrosomes in quiescent neural stem cells (NSC) but not during their differentiation. This argues for a massive post-transcriptional program involving localized untranslated EJC-bound transcripts in NSCs. Given that core EJC subunits are involved in cell division and differentiation, are centrosomal EJC-associated transcripts responsible for these effects? Thus, the role of EJC and pre-EJC factors in mRNA localization and local translation will be investigated in different cell types, including NSCs.
This project will use complementary cutting-edge methodologies such as establishing human cell lines with endogenous EJC components tagged by genome editing for stringent affinity purification, inducible protein degradation and single mRNP molecule imaging. Hence, we will clarify the mechanisms governing splicing-dependent EJC assembly, their functional impacts on specific gene circuits and their intriguing relationship to highly specific developmental defects and human syndromes.