Poster abstracts
Poster number 7 submitted by Justin Mabin
Alternate exon junction complexes specify parallel branches of nonsense-mediated mRNA decay
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), Vicki Wysocki (Chemistry and Biochemistry, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)
Abstract:
In mammalian cells, pre-mRNA splicing deposits the exon junction complex (EJC) ~24 nt upstream of most exon-exon junctions in a sequence-independent manner. The minimal EJC core is a trimeric protein complex consisting of eIF4AIII, Y14 and Magoh. This stable core travels with mRNA to the cytoplasm, and serves as a recruitment platform for more dynamic peripheral proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA decay (NMD). To date, the EJC core and peripheral protein factors RNA target specificity, along with their resulting impact on mRNA fate, remains unknown. We recently reported that MLN51, a protein widely presumed to be the fourth EJC core factor, and several peripheral proteins are sub-stoichiometric in EJCs purified from human cells. 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, two EJC proteins previously implicated in NMD. Biochemical and proteomic analysis of FLAG-affinity purified MLN51 and RNPS1 complexes from HEK293 cells shows that the two alternate complexes are unique in composition and size. Importantly, the alternate EJCs associate differentially with NMD proteins Upf2 and Upf3b, and therefore could lie at the root of previously documented Upf2- or Upf3-independent alternate NMD branches. Our identification of in vivo RNA binding sites of these mutually exclusive EJCs shows their differential occupancy on many mRNAs including on some known NMD targets. Furthermore, endogenous NMD-targeted transcripts are differentially sensitive to MLN51 or RNPS1 depletion in HEK293 cells. We are now working to identify transcriptome-wide NMD targets regulated by MLN51 or RNPS1 EJCs. We also aim to delineate the underlying protein-protein interactions that lead to alternate EJC-Upf complex assembly within the NMD pathway. Our results for the first time uncover how the variable composition of endogenous EJCs impacts NMD.
Keywords: Exon-Junction Complex, Nonsense-mediated mRNA decay, Post-transcriptional gene regulation