Talk abstracts

Talk on Saturday 10:00-10:15am submitted by Lauren Woodward

Alternative exon junction complexes differentially mark mRNAs targeted for Nonsense Mediated mRNA Decay

Lauren Woodward (Molecular Genetics The Ohio State University), Justin Mabin (The Ohio State University), Guramrit Singh (Molecular Genetics The Ohio State University)

The spliceosome deposits the exon-junction complex (EJC) ~24nt upstream of exon-exon junctions during pre-mRNA splicing. The EJC is an integral component of spliced RNPs and regulates gene expression at several steps during the lifecycle of mRNA: pre-mRNA splicing and mRNA export from the nucleus to mRNA localization, translation and nonsense-mediated mRNA decay (NMD) in the cytoplasm. The EJC core—composed of the eIF4AIII, Y14, Magoh and MLN51—serves as a binding platform, recruiting a myriad of factors involved in these processes. Intriguingly, proteomic analysis of EJCs purified from HEK293 cells showed that MLN51 and other peripheral EJC proteins are sub-stoichiometric to the EJC core, indicating that they may not be present in all EJCs. We have now established the existence of at least two distinct EJCs in human and mouse cells: MLN51- and RNPS1-containing EJCs. Interestingly, the two complexes differentially associate with the NMD machinery, which detects and degrades mRNAs containing premature stop codons. RNPS1-EJCs are Upf2-rich, whereas MLN51-EJCs are Upf3b-rich. Our findings are in agreement with previous work suggesting distinct branches of EJC-dependent NMD; RNPS1 is Upf2-dependent but Upf3b-independent to trigger NMD, whereas MLN51 is Upf2-independent. Our transcriptome-wide identification of MLN51- and RNPS1-EJC occupancy sites via RIPiT-Seq has revealed that certain endogenous NMD targets are exclusively bound to MLN51-EJCs. These mRNAs are likely to undergo MLN51 (and Upf3b) - dependent but RNPS1 (and Upf2)-independent NMD, predictions that we are currently testing. We are also determining if NMD targets exist that exclusively undergo RNPS1-dependent NMD. Taken together, our findings suggest an exciting possibility that distinct EJCs sort mRNAs into distinct populations, which may be subject to different spatial and temporal regulation by discrete branches of NMD.

Keywords: EJC, alternate EJC, NMD