Poster abstracts
Poster number 59 submitted by Lauren Woodward
A subpopulation of Exon Junction Complexes (EJC) containing CASC3 represents a late stage of EJC composition and is displaced during translation to the 3’UTR
Lauren Woodward (Department of Molecular Genetics), Justin Mabin (Department of Molecular Genetics), Robert Patton (Department of Physics), Mengxuan Jia (Department of Chemistry and Biochemistry), Vicki Wysocki (Department of Chemistry and Biochemistry), Ralf Bundschuh (Department of Physics)
Abstract:
Proteins bind nascent mRNAs cotranscriptionally to form ribonucleoprotein complexes (mRNPs). An integral component of mRNPs is the exon junction complex (EJC), which assembles 24nt upstream of exon junctions during splicing and remains bound until its co-translational removal. The EJC is composed of a trimeric core (consisting of EIF4AIII, MAGOH, and RBM8A) and serves as a binding platform for other proteins that influence mRNP fate. Our proteomic analyses in mammalian cells depict two mutually exclusive EJC compositions, which we characterize as either RNPS1- or CASC3-containing EJCs. Both RNPS1 and CASC3 shuttle between the nucleus and cytoplasm; however, RNPS1 is primarily nuclear, while CASC3 localizes to the cytoplasm and cytoplasmic granules. Sequencing of the footprints of these distinct EJCs shows that RNPS1- and CASC3- containing EJCs bind the same site on mRNAs, suggesting EJCs undergo a constitutive compositional switch from RNPS1 to CASC3. Change in EJC protein composition is accompanied by transition from the previously described multimeric structure to a monomeric conformation. The CASC3-containing EJC occupancy is highly sensitive to translating ribosomes resulting in a dramatic shift away from the -24nt position. Additionally, we observe that CASC3-EJCs are more enriched on inefficiently translated mRNAs. This demonstrates a pre-translational switch in mRNP composition where the monomeric CASC3-EJCs bind mRNPs that interact directly with the translation machinery. Surprisingly, concomitant with displacement of the CASC3-EJCs away from the -24nt position, we see an accumulation of CASC3-EJC footprints at the 3’end of actively translating mRNAs. We show EJCs are moved to the 3’ end of mRNAs by ribosomes before disassembly, and these 3’ localized EJCs influence the stability of some mRNAs. This study reveals new insights into the changes that EJCs undergo in the cytosol prior to and during translation and how these changes can influence mRNA fate.
Keywords: EJC, translation, mRNP