Poster abstracts
Poster number 127 submitted by Lauren Woodward
Alternate exon junction complexes potentiate branched nonsense-mediated mRNA decay pathways
Justin Mabin (Department of Molecular Genetics, the Ohio State University), Lauren Woodward (Department of Molecular Genetics, the Ohio State University), Robert Patton (Department of Physics), Mengxuan Jia (Department of Chemistry and Biochemistry), Vicki Wysocki (Department of Chemistry and Biochemistry), Guramrit Singh (Department of Molecular Genetics, the Ohio State University)
Abstract:
Pre-mRNA splicing deposits the exon junction complex (EJC) 24 nt upstream of most exon-exon junctions in a sequence-independent manner. The stable EJC core consists of eIF4AIII, Y14 and Magoh, which serves as an interaction platform for more dynamic peripheral EJC proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA decay (NMD). To date, the specificity of the peripheral EJC proteins in target RNA selection and the resulting impact on mRNA fate remains largely unknown. We recently reported that MLN51, a protein widely presumed to be an EJC core factor, and several peripheral EJC proteins are sub-stoichiometric in EJCs purified from human cells. We have now discovered that MLN51 and RNPS1, two EJC proteins previously implicated in NMD, exist in two mutually exclusive EJCs in human and mouse cells. Surprisingly, while some mRNAs are preferentially bound by one of the alternate EJC factors, most endogenous mRNAs including many that are targeted for NMD have similar alternate EJC occupancy. Notably, as compared to MLN51-EJCs, RNPS1-EJCs are more enriched in non-canonical sites, which we previously showed to overlap with SR protein binding sites. Indeed, biochemical and proteomic analysis of purified MLN51 and RNPS1 complexes from HEK293 cells shows that while the two alternate EJCs share the three EJC core proteins, only RNPS1-EJCs associate with SR proteins. Intriguingly, RNPS1 depletion causes >2-fold upregulation of most of the endogenous NMD targets tested while MLN51 depletion leads to modest or no increase in their levels. Overall, our data suggests that endogenous EJCs exist in at least two mutually exclusive complexes defined by either RNPS1 or MLN51. While both complexes are likely to be active in NMD, they may represent two distinct NMD branches in which RNPS1-EJCs, in cooperation with SR proteins, promote more efficient RNA turnover as compared to SR protein devoid MLN51-EJCs. We are currently testing this model to understand how EJC composition and its cooperativity with SR protein impacts mRNA fate.
Keywords: EJC, NMD