PROJECT INFORMATION :
Mammalian gene expression involves the formation of mRNPs, which regulate processing, translation and turnover of mRNAs. The exon junction complex (EJC) is a multifunctional mRNP component, which is assembled on mRNAs during splicing in the nucleus. The interaction of the EJC component eIF4A3 with the spliceosomal protein CWC22 enables the deposition of the EJC approximately 20 nucleotides upstream of the splice junction in a sequence-independent manner. EJCs remain bound to their mRNA during and after transport to the cytoplasm and modulate many post-transcriptional steps of gene expression, including pre-mRNA splicing, mRNP packaging, mRNA transport, translation and degradation.
Significant insights into EJC biology have been obtained since its first description 15 years ago. However, important questions about the assembly of EJCs and their biological functions remain open. To address these questions, we put forward three main aims as part of this proposal.
1. Understanding the mechanism of EJC assembly is key to understand its function. Hence, we aim to identify factors and interactions that mediate EJC formation and dissect different steps and assembly intermediates of the EJC.
2. Binding of the EJC to mRNA involves mainly the four core components, but the function of the EJC during gene expression is tightly linked to its interacting protein partners. Therefore, we aim to characterize the interactions of EJC-binding proteins with the EJC and identify novel EJC-binding proteins.
3. The molecular functions of EJCs have been mainly studied with simplified reporter assays. To avoid the shortcomings of such approaches, we plan to study the cellular functions of the EJC using unbiased high-throughput assays.
A combination of low- and high-throughput assays will leverage our understanding on all possible aspects of EJC function. It is this holistic approach that will lead us to a system-wide, quantitative understanding of EJC function and will deliver hypothesis to be tested in reconstituted systems in vitro and through genomic engineering in vivo.
KEY TECHNOLOGIES :
- Cell culture
Steckelberg AL, Altmueller J, Dieterich C, Gehring NH. (2015)
CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes. Nucleic Acids Res. 43(9):4687-700
Boehm, V, Haberman, N, Ottens, F, Ule, J, Gehring, NH (2014)
3′ UTR Length and Messenger Ribonucleoprotein Composition Determine Endocleavage Efficiencies at Termination Codons. Cell Reports 10.1016/j.celrep.2014.09.012
Steckelberg AL, Boehm V, Gromadzka AM, Gehring NH (2012)
CWC22 connects pre-mRNA splicing and exon junction complex assembly. Cell Reports 2(3):454-61
Gehring NH, Lamprinaki S, Hentze MW, Kulozik AE (2009)
The hierarchy of exon-junction complex assembly by the spliceosome explains key features of mammalian nonsense-mediated mRNA decay. PLoS Biology 26;7(5):e1000120
Gehring NH, Lamprinaki S, Kulozik AE, Hentze MW (2009)
Disassembly of exon junction complexes by PYM. Cell 1;137(3):536-48