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
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
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
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 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

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