Download 053107_EJC

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 premRNA 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
EJC
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
and C. elegans (Longman. RNA. 2003)
suggest mRNA export may be
enhanced by EJCs.
Bio. 2002)
Drosophila oskar mRNA Localization
EJC components (homologues of
human Mago and Y14) required for
proper localization during oogenesis
Hatchet et al. Nature. 2004
(Palacios. Nature. 2004, Hatchet. 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
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
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
(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 VI :
(HRIGRGGR)
Motif II:
Walker B
(DEAD)
GG
Motif Ia :
(PTRELA)
Motif V:
(RGID)
Motif IV :
(VIFCNT)
Conserved
“R” Motif
Motif I :
Walker A
(SGTGKT)
Motif III :
(SAT)
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