Talk abstracts

Talk on Saturday 09:45-10:00am submitted by Manu Sanjeev

PYM1 controls of Exon Junction Complex occupancy at canonical and non-canonical positions and can thereby modulate NMD

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 (Molecular Genetics, The Ohio State University)

RNA binding proteins are critical for RNA processes in the cell. The Exon Junction Complex (EJC) is an important RNA binding protein complex present in all vertebrate cells. EJCs are deposited ~24 nucleotides upstream of exon-exon junctions during pre-mRNA splicing. Once deposited, EJCs remain stably bound to mRNA and modulates mRNA fate at multiple post-transcriptional steps. A critical EJC function is to aid the Nonsense Mediated mRNA Decay (NMD) pathway to identify and decay transcripts that undergo premature translation termination. Since there are more exon junctions than EJCs at steady state, EJC subunits are continuously disassembled and recycled. However, the molecular understanding of this process is incomplete. Here, we tested the roles of two activities implicated in EJC disassembly, (i) the elongating ribosome and (ii) a ribosome-associated factor called PYM1, which binds the Y14/MAGOH heterodimer of the EJC core. Using an EJC footprinting assay in HEK293 cells, we find that unlike inhibition of translation elongation, blocking EJC-PYM1 interaction shows no defect in EJC disassembly. Thus, PYM1 does not function as an EJC disassembly factor. Surprisingly, we find that the loss of EJC-PYM1 interaction leads to decreased EJC footprints at the -24 position (canonical EJC binding site) and increased EJC footprints away from exon junctions (non-canonical EJC binding sites). Among transcripts with increased non-canonical EJC binding are intronless mRNAs that do not normally contain the EJC. These observations suggest a role of PYM1 in which it aids proper EJC recycling and suppresses EJC deposition at non-canonical sites. Consistent with this idea, we find that PYM1 depletion upregulates canonical EJC dependent NMD targets and downregulates of transcripts that accumulate non-canonical EJCs. In summary, our work reveals that PYM1 is dispensable for EJC disassembly and suggests a model where PYM1 prevents splicing-independent deposition of EJC onto RNAs.

[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.

Keywords: EJC, PYM1, NMD