The Exon Junction Complex

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The Exon Junction Complex
May 31, 2007
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Introduction

• mRNA Processing
• The Exon Junction Complex Components and Roles
• The Core Y14, Mago, Barentsz and eIF4AIII
• Previous Biochemical Studies

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Fates of mRNA
A 5 end export
B non 5 end export C
translationally silent D - transport
Moore. Science. 2005
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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
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EJC as a Mediator of mRNA Functions
EJC

• Localization
• Translation
• Decay (Quality Control)

EJC
Le Hir et al. TIBS. 2003
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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
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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)

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• EJC and Nonsense-Mediated Decay
  (NMD)
• a mechanism of surveillance/ QC whereby
  aberrant mRNAs with Premature Translation-Stop
  Codons (PTCs) are degraded

Rehwinkel et al. TIBS. 2006
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EJC Core Components - Y14Mago
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
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EJC Core Components - Y14Mago
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 Y14Mago - inhibition of ATP
  hydrolysis to lock eIF4AIII into a conformation
  that cannot release RNA

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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

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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
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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
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Ded1 A Mischievous DEAD-box Helicase
EJC
Ded1
Cordin et al. Gene. 2006
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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)

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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)

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Co-precipitation of Core Components

Ballut et al. Nature Struct Mol Bio. 2005
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eIF4aIII Mago Interactions
Shibuya et al. RNA. 2006
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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