The Exon Junction Complex

Charlotte Leblang '16 and Daniel Jurgens '16

Contents:

I. Introduction

The Exon Junction Complex (EJC) is a eukaryotic molecular machine that interacts with spliced mRNA upstream of exon-exon junctions, providing a binding platform for other trans-acting proteins that determine the fate of the mRNA.  The spliceosome deposits the ~335kD EJC in a non-sequence specific manner 20-24 nucleotides upstream of an exon-junction.  Functionally, the EJC aids in nuclear export of spliced mRNAs, assists in nonsense-mediated decay of incorrectly spliced mRNAs containing premature stop codons, and enhances translation efficiency.

The heterotetramer core of the EJC is composed of the subunits eIF4AIII, Y14 (RBM8A), Magoh, and Barentsz (BTZ, CASC3, MLN51).  The two subunits Y14 and Magoh form a heterodimer, which stabilizes the interaction between ATP-bound eIF4AIII and ssRNA.  The Barentsz subunit then completes the core by binding to eIF4AIII.  Furthermore, both ATP and ssRNA are necessary for the stable, closed conformation of the EJC.

Other transient factors associated with the EJC include heterogenous nuclear ribonuclear proteins (hnRNPs) that bind in complexes (known as nuclear mRNPs) to the EJC core.  Such hnRNPs include CWC22, REF, SMG, UPF2, and UPF3.

II. Structural Characteristics and Functions

Subunits:

A DEAD-box ATP-dependent RNA helicase that functions as a clamp to the bound mRNA. It consists of two domains: and . Both of these domains bind to the

Forms a heterodimer with Magoh, but has various functions separate from the entire EJC complex. Interestingly, the heterodimer remains on the mRNA into the cytoplasm and requires translation in order to be removed.

A post-splicing processing factor that aids in cytoplasmic mRNA localization. The Magoh-Y14 heterodimer stabilizes the EJC when bound to ssRNA.

Overexpression increases translation of mRNAs with the EJC, and silencing decreases translation. Barentsz also associates with ribosomal subunits and translation-initiating factors. Thus, it links the EJC to translation regulation and machinery.

III. Nuclear Export and Regulation

Pre-mRNA bound by a spliceosome is usually not exported from the nucleus, so as to make sure that only fully-processed mRNA travels to the cytoplasm to be translated.  A protein called the mRNP exporter binds to the EJC, both through RNA interactions and interactions with the EJC-associated protein REF (RNA export factor) to help pre-mRNA exit the nuclear pore complex. Interestingly, the efficiency of unspliced mRNA export is dependent on the length; longer mRNAs are exported more efficiently than shorter mRNAs. In spliced mRNAs, however, once the 5' exon is long enough to bind the EJC, the length of the spliced mRNA does not affect the export efficiency. 

The splicing factor, Complex with Cef1 (CWC22), binds to the eIF4AIII (to W263, D266, D270, and D273) in such a way that the ATPase activity of eIF4AIII is diminished and thus causes the EJC to be in an inactive state. This interaction between eIF4AIII and CWC22 blocks the binding of the Magoh-Y14 heterodimer, which is necessary for the EJC to function. Thus, when CWC22 binds to eIF4AIII, Magoh-Y14 cannot, and the EJC is in an inactive state. In this way CWC22 negatively regulates the EJC.
Click Here for CWC22 and eIF4AII interaction.

There are a certain number of EJCs in a cell, and they must be recycled in order to continue tagging mature mRNAs. Once in the cytoplasm, the ribosome-associated regulator protein (PYM) acts as a dissociation factor. PYM binds the Magoh-Y14 heterodimer and breaks up the EJC. Thus, the EJC can be recycled to make sure that new mRNAs can be bound. This interaction also enhances translation of mRNAs with bound EJCs by recruiting them directly to the ribosomal 48A preinitiation complex.

IV. Nonsense-Mediated Decay

Correctly-spliced mRNAs contain a stop codon in the final exon of the exon-junction, but incorrectly-spliced mRNAs contain premature termination codons (PTCs) located before (3') this proper exon location.  Unless degraded via nonsense-mediated decay (NMD), these incorrectly-spliced mRNAs contain altered reading frames that lead to abnormal protein production.  
 
Click Here for mRNA decay diagram.

Nonsense-mediated decay involves mRNA with a tightly bound EJC in the cytoplasm.  The EJC is bound to hnRNPs UPF2 and UPF3 when attached to the mRNA. Triggering of nonsense-mediated decay is caused by the trans phosphorylation of UPF1 by SMG-1. SMG-1 is part of the SURF complex (composed of UFP1, SMG1, SMG8, and SMG9) that recognizes the PTCs. SMG1 then binds to the EJC through the C-terminal domain of UPF3 to create the DECID complex that is the bridge between the SURF and EJC. This triggers the recognition of premature termination codons and thus nonsense-mediated decay.

This interaction of the SURF and EJC also causes the defective mRNA to interact with P-body deadenylases, leading to the de-capping of the poly(A) tail and the subsequent decay of the defective mRNA.

V. References

(1) Andersen C, Ballut L, Johansen J, Chamieh H, Nielsen K, Oliveria C, Pedersen J.S., Seraphin B, Le Hir H, Andersen G. 2006. Structure of the Exon Junction Core Complex with a Trapped DEAD-Box ATPase Bound to RNA. Science: 1968-1972.

(2) Chazal P, Daquenet E, Wendling C, Ulryck N, Tomasetto C, Sarqueil B, Le Hir H. 2013. EJC core component MLN51 interacts with eIF3 and activates translation. Proc Natl Acad Sci: 5903-5908.

(3) Gehring N, Lamprinaki S, Kulozik A, Hentze M. 2009. Disassembly of Exon Junction Complexes by PYM. Cell: 536-548.

(4) Kashima I, Yamashita A, Izumi N, Kataoka N, Morishita R, Hoshino S, Ohno M, Dreyfuss G, Ohno S. 2006. Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay.  Genes and Development: 355-367.

(5) Le Hir H, Gatfield D, Izaurralde E, Moore M. 2001. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.  The EMBO Journal: 4987-4997.

(6) Lodish et al.  Molecular Cell Biology. 7th ed. New York: W.H. Freeman, 2012. Print.

(7) Rasooly R, Hamosh A, Lopez A. "Magoh Nashi, Drosophila, Homolog Of; Magoh." OMIM Entry - 602603. N.p., 6 May 1998. Web. 09 Dec. 2014.

(8) Steckelberg A, Boehm V, Gromadzka A, Gehrig N. 2012. CWC22 Connects Pre-mRNA Splicing and Exon Junction Complex Assembly. Cell Reports: 454-461.


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