Poster abstracts
Poster number 85 submitted by Justin Mabin
Biochemical and proteomic characterization of alternate exon junction complexes in mammalian cells
Justin Mabin (Molecular Genetics, The Ohio State University), Mengxuan Jia (Chemistry and Biochemistry, The Ohio State University), Lauren Woodward (Molecular Genetics, The Ohio State University), Kate Ehrensberger (Molecular Genetics, The Ohio State University), Vicki Wysocki (Chemistry and Biochemistry, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)
Abstract:
In mammalian cells, the exon junction complex (EJC) is deposited during pre-mRNA splicing ~24 nt upstream of most exon-exon junctions in a sequence-independent manner. The EJC core is comprised of a stable tetrameric protein complex consisting of eIF4AIII, Y14:Magoh heterodimer, and MLN51. This stable core travels with mRNA to the cytoplasm, and serves as a platform for recruitment of more dynamic peripheral/adapter proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA degradation (NMD). To date, it is unknown if peripheral proteins interact with all EJC cores. In EJCs purified from cultured human cells, we found levels of the so-called core protein, MLN51, and several peripheral proteins to be sub-stoichiometric to those of the core suggesting that they are part of only a subset of EJCs. Spurred by these findings, we have now discovered at least two stable alternate EJCs that are distinguished by the presence of either MLN51 or RNPS1. Biochemical analysis of FLAG-affinity purified MLN51 and RNPS1 protein complexes from HEK293 cells showed that they are unique in composition and size. A comprehensive proteomic analysis confirmed their mutually exclusive nature, and further revealed their distinct compositional differences. We find that RNPS1 is associated with the EJC core (except MLN51), it's previously known partners (ACIN1, SAP18 and PNN), and several SR proteins and splicing factors. On the other hand, MLN51 is mainly associated with the EJC core proteins and is missing almost all of the RNPS1 associated factors. Instead, it purifies the EJC disassembly protein PYM, and some components of the translation and NMD machinery. Our results suggest that EJCs undergo a dynamic remodeling process before or upon MLN51 incorporation. We are now investigating underlying mechanisms that lead to a sequential exchange of peripheral EJC factors and overall EJC maturation.
Keywords: Exon Junction Complex, post-transcriptional gene regulation, Alternate EJCs