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

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

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

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

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)

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

EJC Core Components - Y14: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

EJC Core Components - Y14: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

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

DEAD-box Helicases Regulate essentially all processes involving RNA (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

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

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

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)

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)

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

eIF4aIII – Mago Interactions Shibuya et al. RNA. 2006

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