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The Exon Junction Complex

The Exon Junction Complex. May 31, 2007. Introduction. mRNA Processing The Exon Junction Complex – Components and Roles The Core: Y14, Mago, Barentsz and eIF4AIII Previous Biochemical Studies. Fates of mRNA.

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The Exon Junction Complex

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  1. The Exon Junction Complex May 31, 2007

  2. Introduction • mRNA Processing • The Exon Junction Complex – Components and Roles • The Core: Y14, Mago, Barentsz and eIF4AIII • Previous Biochemical Studies

  3. Fates of mRNA A – 5’ end export B – non 5’ end export C – translationally silent D - transport Moore. Science. 2005

  4. What is the Exon Junction Complex (EJC)? • Macromolecular complex deposited on mRNA due to pre-mRNA splicing • Deposited ~20 nucleotides upstream of exon-exon junctions • Position-dependent • Sequence-independent • ATP-dependent complex • Four core proteins: • eIF4AIII • Y14 • Magoh (Mago) • Barentsz (MLN51) • Peripheral proteins Tange et al. RNA. 2005 Stroupe et al. JMB. 2006

  5. EJC as a ‘Mediator’ of mRNA Functions EJC • Localization • Translation • Decay (Quality Control) EJC Le Hir et al. TIBS. 2003

  6. EJC and mRNA Localization • Nuclear Export • Early studies - EJC stimulates mRNA export in Xenopus oocytes by acting as a binding platform. (Le Hir. EMBO J. 2001) • Knockdowns in Drosophila(Gatfield. J Cell Bio. 2002)and C. elegans(Longman. RNA. 2003)suggest mRNA export may be enhanced by EJCs. • Drosophila oskar mRNA Localization • EJC components (homologues of human Mago and Y14) required for proper localization during oogenesis (Palacios. Nature. 2004, Hatchet. Nature 2004) Hatchet et al. Nature. 2004

  7. EJC and mRNA Translation • Splicing influences mRNA translational yield in Xenopus oocytes (Braddock. Nucleic Acids Res. 1994) • Also found to be true in mammalian cells: • Spliced mRNA leads to more protein than unspliced (Lu. RNA. 2003, Nott. RNA. 2003) • Expression profiles of mRNAs with exons too short or just long enough to accept EJC (Wiegand. PNAS. 2003, Nott. Genes Dev. 2004) • Y14 and Mago can enhance translational yield when tethered to reporter mRNA (Nott. Genes Dev. 2004) • Polysome analysis (Nott. Genes Dev. 2004)

  8. EJC and Nonsense-Mediated Decay (NMD) • a mechanism of surveillance/ “QC” whereby aberrant mRNA’s with Premature Translation-Stop Codons (PTCs) are degraded Rehwinkel et al. TIBS. 2006

  9. EJC Core Components - Y14:Mago Mago • Associate to form a tight heterodimer • Associate with the spliceosome • Y14 has RNA Recognition Motif (RRM) • Does not bind RNA • RRM is buried at dimer interface with Mago Y14 Lau et al. Curr Bio. 2003

  10. EJC Core Components - Y14:Mago Mago • Associate to form a tight heterodimer • Associate with the spliceosome • Y14 has RNA Recognition Motif (RRM) • Does not bind RNA • RRM is buried at dimer interface with Mago Y14 Lau et al. Curr Bio. 2003 • Main role of Y14:Mago - inhibition of ATP hydrolysis to lock eIF4AIII into a conformation that cannot release RNA

  11. EJC Core Components - Barentsz and eIF4AIII • Barentsz and eIF4AIII directly interact in EJC • Both contribute to RNA binding • Barentsz contains SeLoR motif at N-terminus • Speckle Localizer and RNA binding • Non-specific RNA binding • Directs to sub-nuclear speckle domains enriched in splicing factors • eIF4AIII is a DEAD-box Helicase

  12. DEAD-box Helicases (asp-glu-ala-asp) • Regulate essentially all processes involving RNA • ATP-dependent • Several conserved motifs • Grouped into superfamilies based on these • ATPase and helicase activity, RNA binding Cordin et al. Gene. 2006

  13. eIF4A – A DEAD-box Helicase Motif Ib: (TPGRVFD) Motif II: Walker B (DEAD) Motif VI : (HRIGRGGR) GG Motif Ia : (PTRELA) Motif V: (RGID) Motif I : Walker A (SGTGKT) Motif III : (SAT) Conserved “R” Motif Motif IV : (VIFCNT) Caruthers et al. PNAS. 2000

  14. Ded1: A “Mischievous” DEAD-box Helicase EJC Ded1 Cordin et al. Gene. 2006

  15. RNA Binding of eIF4AIII • Stimulates ATPase activity • Cooperativity between RNA and ATP binding • Reduced binding in the presence of ADP • Not known how distinguishes RNA from DNA • Helicase activity of eIF4AIII not used in EJC • Nucleotides protected (6 v. 8)

  16. Previous Biochemical Studies • Co-precipitations show interactions between 4 core components (Ballut et al. Nature Struct Mol Bio. 2005) • Mutational analysis of eIF4AIII identifies regions required for EJC formation (Shibuya et al. RNA. 2006)

  17. Co-precipitation of Core Components * * * Ballut et al. Nature Struct Mol Bio. 2005

  18. eIF4aIII – Mago Interactions Shibuya et al. RNA. 2006

  19. EJC - Leading to the paper… • Core Complex – Y14/Mago/Barentsz/eIF4AIII • Formed during mRNA splicing • Binds RNA in an ATP-dependent manner • Involved in mRNA localization, translation, and decay • Chris: Crystal Structure of the Core Complex

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