Poster abstracts
Poster number 23 submitted by Manu Sanjeev
PYM controls Exon Junction Complex occupancy at non-canonical positions
Manu Sanjeev (Molecular Cellular and Developmental Biology, OSU), Lauren Woodward (Department of Molecular Genetics, OSU), Robert Patton (Department of Physics, OSU), Ralf Bundschuh (Department of Physics,OSU), Guramrit Singh (Department of Molecular Genetics, OSU)
Abstract:
RNA processes such as transcription, splicing, export, translation, and decay are mediated by proteins that bind the RNA. The Exon Junction Complex (EJC) is a major RNA binding protein complex deposited ~24 nucleotides upstream of mRNA exon-exon junctions during the splicing reaction. The EJC remains stably bound to mRNA after splicing, modulates mRNA fate at multiple post-transcriptional steps, and is essential for viability in vertebrates [1]. A ribosome-associated factor called PYM, which binds 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 viability of Drosophila PYM null mutants [3] and sub-stoichiometric levels of PYM to both EJC and ribosomes. Here, we investigated the role of PYM in human cells by using a MAGOH mutant that assembles into EJC but is impaired in PYM interaction. Using a high-throughput footprint sequencing assay (RIPiT-Seq) in human embryonic kidney (HEK293) cells, we find that EJCs lacking PYM interaction show no defect in translation dependent disassembly but have increased footprints away from the -24 position (non-canonical EJC binding sites). Surprisingly, PYM interaction deficient EJCs are also enriched on unspliced transcripts from many single-exon genes that do not normally contain the EJC. PYM also has a dose dependent effect on the Nonsense Mediated mRNA decay (NMD) pathway. 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, May 2017, doi: 10.1002/wrna.1411.
[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, doi: 10.1016/j.cell.2009.02.042.
[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, doi: 10.1371/journal.pgen.1004455.
[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, doi: 10.1038/s41564-019-0375-z.
Keywords: Exon Junction Complex, RNA decay