Poster abstracts
Poster number 86 submitted by Manu Sanjeev
PYM controls Exon Junction Complex occupancy at non-canonical positions
Manu Sanjeev (Molecular Genetics, The Ohio State University), Lauren Woodward (Molecular Genetics, The Ohio State University), Robert Patton (Physics, The Ohio State University), Ralf Bundschuh (Physics, The Ohio State University), Guramrit Singh (Physics, The Ohio State University)
Abstract:
As mRNAs undergo the splicing reaction in the nucleus, the spliceosome deposits the Exon Junction Complex (EJC) ~24 nucleotides upstream of mRNA exon-exon junctions. EJCs thus deposited remains stably bound to mRNA and modulates mRNA fate at multiple post-transcriptional steps until its disassembly during translation and is essential for viability in vertebrates[1]. A ribosome-associated factor called PYM, which directly binds to the Y14/MAGOH heterodimer of the EJC core, is reported to aid in EJC disassembly during translation[2]. However, such a role for PYM is contradicted by the observation that Drosophila PYM null mutants are fully viable[3]. Furthermore, PYM levels are sub-stoichiometric to both EJC and ribosomes. Here, we have investigated the role of PYM in human cells by using a previously reported MAGOH mutant that assembles into EJC but is impaired in PYM interaction. Using RNA immuno-precipitation in tandem and high-throughput sequencing (RIPiT-Seq) to identify footprints of EJC from human embryonic kidney (HEK293) cells, we find that EJCs lacking PYM interaction show no defect in translation dependent disassembly. We find that the loss of EJC-PYM interaction leads to increased EJC footprints away from the -24 position (non-canonical EJC binding sites). Surprisingly, PYM interaction deficient EJC is also enriched on unspliced transcripts from many single-exon genes that do not normally contain the EJC. Our findings suggest a model for PYM function where PYM prevents spontaneous, splicing-independent deposition of EJC at non-canonical sites across the transcriptome. We are currently testing this model using biochemical and transcriptomic analyses. Our work reveals a potential mechanism for EJC binding at non-canonical sites and has important implications for viral pathogenesis as PYM is targeted by Kaposi Sarcoma-associated Herpes Virus to enhance viral RNA translation and by flaviviruses to promote viral replication via NMD inhibition[4].
References:
1] L. A. Woodward, J. W. Mabin, P. Gangras, and G. Singh, “The exon junction complex: a lifelong guardian of mRNA fate: EJC: assembly, structure, and function,” WIREs RNA, vol. 8, no. 3, p. e1411.
[2] N. H. Gehring, S. Lamprinaki, A. E. Kulozik, and M. W. Hentze, “Disassembly of Exon Junction Complexes by PYM,” Cell, vol. 137, no. 3, pp. 536–548, May 2009.
[3] S. Ghosh, A. Obrdlik, V. Marchand, and A. Ephrussi, “The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila,” PLoS Genet, vol. 10, no. 6, p. e1004455, Jun. 2014.
[4] M. Li et al., “Identification of antiviral roles for the exon–junction complex and nonsense-mediated decay in flaviviral infection,” Nat Microbiol, vol. 4, no. 6, pp. 985–995, Jun. 2019.
Keywords: EJC