The ever-increasing complexities of the exon junction complex
Thomas Ø Tange1, Ajit Nott2 and Melissa J Moore1,3
Over the past decade many studies have revealed a complex
web of interconnections between the numerous steps required
for eukaryotic gene expression. One set of interconnections link
nuclear pre-mRNA splicing and the subsequent metabolism of
the spliced mRNAs. It is now apparent that the means of
connection is a set of proteins, collectively called the exon
junction complex, which are deposited as a consequence of
splicing upstream of mRNA exon–exon junctions.
Addresses
1
Howard Hughes Medical Institute, Department of Biochemistry,
MS009, Brandeis University, 415 South Street, Waltham,
Massachusetts, 02454, USA
2
Plant Biology Laboratory, The Salk Institute for Biological Studies, and
the Howard Hughes Medical Institute, La Jolla, California 92037, USA
3
e-mail: [email protected]
Current Opinion in Cell Biology 2004, 16:279–284
This review comes from a themed issue on
Nucleus and gene expression
Edited by Elisa Izaurralde and David Spector
0955-0674/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2004.03.012
Abbreviations
EJC
exon junction complex
mRNP messenger ribonucleoprotein particle
NMD
nonsense-mediated mRNA decay
nt
nucleotide
PTC
premature termination codon
RNAi
RNA interference
RRM
RNA recognition motif
Introduction
The expression of eukaryotic genes is a process with
many individual steps. These include transcription and
pre-mRNA processing in the nucleus, mRNA export
through the nuclear pore complex, translation in the
cytoplasm, and finally decay of the mRNA and protein.
Although these processes were traditionally thought of
and analyzed as entirely separate events, it has recently
become apparent that in vivo they are all highly interconnected [1–3]. One type of interconnection is the
ability of an earlier process in a pathway to influence
one or more later processes. A prime example of this is
pre-mRNA splicing, which can imprint mRNAs with
information that affects their subsequent metabolism.
Early indications of such mRNA imprinting came from
studies of nonsense-mediated mRNA decay (NMD) —
the selective degradation of mRNAs containing premature termination codons (PTCs). The observation that, in
www.sciencedirect.com
mammalian cells, this translation-dependent decay pathway requires the presence of a spliceable intron downstream of a PTC [4–6] suggested that the splicing process
leaves behind some type of ‘mark’ capable of signaling
the positions of exon–exon boundaries to the translation
and decay machineries [7–9]. Another early indication
that splicing can affect subsequent events in the RNA
processing pathway came from experiments in Xenopus
oocytes, wherein spliced mRNAs exhibited altered translational yields compared to mRNAs not generated by
splicing [10,11].
The simplest way in which splicing might ‘mark’ an
mRNA is by altering the complement of associated proteins, which together with the mRNA constitute the
messenger ribonucleoprotein particle (mRNP). The first
direct evidence for this came from gel shift experiments,
where an mRNP generated by splicing in vitro exhibited
reduced mobility on native gels compared with an otherwise identical mRNP not generated by splicing [12].
Subsequently, several proteins that specifically associate
with spliced RNAs were identified by crosslinking and
immunoprecipitation approaches [13,14]. Soon thereafter, it was shown that these splicing-dependent proteins are deposited at a specific position approximately
20–24 nts upstream of exon–exon junctions [15,16]. This
set of proteins is now known as the exon junction complex
(EJC).
In the four years since its discovery, the EJC has become
the focus of intense research by numerous laboratories.
In addition to being a key effector of NMD, the EJC
also appears to play important roles in subcellular
mRNA localization and translational yield. In the following sections, we will review recent progress on EJC
structure and assembly, as well as its proposed functions.
We also speculate on the origin and evolution of the
EJC.
EJC structure, assembly and removal
The EJC is a highly dynamic structure consisting of a few
core proteins plus several more peripherally associated
factors; most of these peripheral proteins join the complex
only transiently, either during EJC assembly or during
subsequent mRNA metabolism. The best-characterized
core component to date is the Y14:Magoh heterodimer.
Y14 and Magoh are both found in spliceosomes that have
completed the first chemical step of splicing (lariat formation) [17–19]. After the second chemical step (exon
ligation), they remain associated with the mRNA, traveling with it to the cytoplasm [14,20–22]. Because Y14
contains a well-defined RNA recognition motif (RRM),
Current Opinion in Cell Biology 2004, 16:279–284
280 Nucleus and gene expression
Exon 2
Exon 1
Exon 2
20–24 nts
mR
loca NA
lizatio
n
Nonsense-mediated mRNA decay
Upf2
oh
Upf1
Upf3
ag
Pi
ni
M
14
Y
TAP
p15
F/
RE
n
S1
eIF4AIII
y
Al
6
P5
A
U
RNP
g
Current Opinion in Cell Biology 2004, 16:279–284
Splicing
Exon 1
licin
Once in the cytoplasm, the majority of EJCs are removed
during the first or ‘pioneering’ round of translation
[37,38]. Immunoprecipitation studies of nuclear and cytoplasmic fractions from mammalian cells have indicated
EJC
Sp
All of the other known EJC proteins associate more
peripherally than Y14, Magoh and eIF4AIII. In the nucleus and/or nuclear extracts, complexed proteins include
the splicing co-activators SRm160 and RNPS1, the alternative splicing factor Pinin, the mRNA export factors
UAP56, REF/Aly and TAP/NFX1:p15 and the NMD
factor Upf3b [14,15,20,21,32,33,34,35,36]. Although
DEK, a nuclear oncoprotein, was originally thought to
be EJC-associated, more recent data suggest that DEK is
not a genuine EJC component [18]. Immunoprecipitation
experiments with the nuclear and cytoplasmic fractions of
Xenopus oocytes have indicated that SRm160, RNPS1,
REF/Aly and TAP/NFX1:p15 all dissociate from spliced
mRNPs during or shortly after mRNA export to the
cytoplasm [20–22]. Upf3b might also dissociate, but it
is more likely that it is made inaccessible to antibodies by
its interaction with Upf2, an NMD factor that joins the
EJC in the cytoplasm [20] (see Figure 1).
Intron
0
16
m
SR
Recently, a better candidate for an EJC anchoring factor
has emerged. This protein, eIF4AIII, is a member of the
eIF4A family of RNA helicases and is a nuclear shuttling
protein [26,27] that forms a stable complex with Y14
and Magoh when co-expressed in E. coli [28]. Furthermore, eIF4AIII has been shown to be an integral component of EJCs assembled in vitro [26,27,29].
eIF4AIII readily forms crosslinks to spliced mRNA in
the area of the EJC, and anti-eIF4AIII antibodies interfere with EJC formation in vitro [26]. Intriguingly,
crystal structures of related helicases indicate that their
nucleic acid binding site spans about 8–10 nts [30,31], a
length remarkably similar to the size of the EJC footprint
on spliced mRNA [15]. Furthermore, eIF4A-type helicases generally interact with RNA in a sequenceindependent fashion [31], which correlates with the
apparent sequence-independence of EJC deposition
[15]. If eIF4AIII does prove to be the EJC anchor, an
important question yet to be addressed is how it remains
bound so stably to mRNA in contrast to related RNA
helicases, which cycle through RNA-bound and RNAfree states coupled to NTP hydrolysis [31].
Figure 1
mRNA export
an early hypothesis was that it might constitute part of the
RNA-binding platform upon which the rest of the EJC
assembles. However, crystal structures of the Drosophila
and human Y14:Magoh heterodimers [23–25] revealed
an unexpected mode of interaction between Y14 and
Magoh that completely masks Y14’s RRM. Thus Y14
is unlikely to bind the spliced mRNA directly, a conclusion supported by Y14’s inability to form crosslinks to
highly reactive photoactivable groups incorporated at the
site of EJC deposition [18].
?
?
?
Current Opinion in Cell Biology
Schematic representation of EJC deposition and associated proteins.
Proteins are grouped and color-coded according to their known
functions in vivo. The gray section indicates that there are likely to
be other, as-yet-unknown proteins that also make a contribution
to the function of the EJC.
that this first round of translation occurs while mRNAs are
still associated with the nuclear cap-binding CBP80:
CBP20 heterodimer. In subsequent rounds of translation,
CBP80:CBP20 is replaced by the cytoplasmic capbinding protein, eIF4E, and EJC factors are not detectably associated with eIF4E-bound mRNAs.
EJC and subcellular mRNA localization
After mRNAs are generated in the nucleus, they are
exported through nuclear pores to the cytoplasm, a process that in metazoans is mediated primarily by the TAP/
NXF1:p15 heterodimer [36]. Early experiments in Xenopus oocytes indicated that the presence of an EJC could
enhance the efficiency of mRNA export [12,20,39]. As
mentioned above, three export factors, UAP56, REF/Aly
and TAP/NXF1:p15, have been reported to associate
with spliced mRNAs. UAP56 (also known as Sub2 in
S. cerevisiae), initially identified as a splicing factor, was
recently demonstrated to be essential for mRNA export
and is thought to be responsible for recruiting REF/Aly
to mRNAs [35,40,41]. REF/Aly can then in turn interact
directly with NXF1:p15, a protein which facilitates bulk
mRNA export through its interactions with components
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The ever-increasing complexities of the exon junction complex Tange, Nott and Moore 281
of the nuclear pore complex [20,33,39,42,43]. Another
link between the EJC and mRNA export was recently
suggested to involve the novel EJC factor, Pinin.
Expression of antisense Pinin RNA was shown to lead
to some accumulation of bulk polyA RNA in the nucleus,
indicating a possible role for Pinin in nuclear mRNA
export [34].
While a model in which the EJC licenses export of spliced
mRNAs is appealing, several recent results suggest that
REF/Aly and the EJC are not essential for this process
[36]. RNAi-mediated knockdown of several EJC proteins
(including REF/Aly), individually or in combination, only
partially affected bulk polyA mRNA export in Drosophila
cells [44]. Furthermore, two studies reported no defect
in mRNA export upon simultaneous knockdown of all
three REF/Aly genes in C. elegans [45,46]. These results
suggest that although EJC deposition may enhance the
export of some spliced mRNAs, it is not the only means
by which mRNAs can become associated with export
adaptor proteins.
Regardless of its role in mRNA export from the nucleus, it
is becoming increasingly clear that the EJC plays an
important function in the proper localization of at least
one mRNA within the cytoplasm. Although it has not yet
been formally proven that splicing results in EJCs being
deposited on Drosophila mRNAs, the Drosophila Y14:
Magoh and eIF4AIII homologs are essential for proper
localization of oskar mRNA during oogenesis [28,47,48].
Localization of oskar to the posterior pole of the developing oocyte is essential for subsequent specification of
the germline [49]. A currently open question is whether
this apparent role for the EJC in mediating oskar mRNA
localization is an exception, or whether it reflects a more
general function of the EJC. It might therefore be of
interest to investigate whether the EJC has any role in
mRNA localization in highly asymmetric somatic cells
such as neurons, which are highly dependent on localized
translation for synaptic function [50].
A clue to the mechanism by which splicing leads to
greater protein yields was provided by cytoplasmic polysome analysis [55]. In this study, significantly greater
proportions of spliced mRNAs were found associated
with polysomes compared with otherwise identical
mRNAs transcribed from cDNAs. Moreover, this difference in polysome association could be reproduced by
tethering the same EJC proteins that enhanced translational yield above. Although the mechanism is currently
unknown, having an EJC appears to enhance the uptake
of spliced mRNAs into the translationally active pool.
Whether this occurs via EJC-dependent recruitment of
translation initiation factors or EJC-dependent targeting
of mRNPs to subcellular locations highly active in translation (e.g. the cytoskeleton) is currently unknown.
Surprisingly, another reason why some spliced mRNAs
yield greater levels of protein is because they lend
enhanced stability to the protein product. Dihydrofolate
reductase protein expressed from stably transfected minigenes was found to have a 2.7-fold longer half-life when
expressed from an intron-containing construct than from
a cDNA construct [53]. The mechanism by which this
occurs, however, is a complete mystery at present.
EJC and nonsense-mediated decay
EJC and translation
In mammalian cells, EJC deposition is pivotal for the
process of NMD. It is now well-established that EJCs
provide one means of distinguishing premature stop
codons, which are upstream of the last EJC, from natural
stop codons, which are downstream of the last EJC
[9,56,57]. According to prevailing models, the NMD
factor Upf3a/b joins the EJC in the nucleus [33,58,59].
Before or immediately after mRNA export, Upf2 is
recruited to the cytoplasmic EJC via interactions with
Upf3a/b [20,37,58]. If during the pioneering round of
translation a stop codon is encountered before removal
of one or more EJCs, eRF1/eRF3-bound Upf1 bridges the
terminating ribosome to Upf3 and Upf2 in the downstream EJC(s). The formation of such an activated NMD
complex then leads to rapid decay of the PTC-containing
mRNA [20,37,58,60–62].
As mentioned above, early experiments in Xenopus
oocytes showed that splicing can influence mRNA translational yield [10,11]. More recently this has been shown
to be true in mammalian cells as well. Quantitative
analysis of intron effects on various steps in the process
of gene expression revealed that greater amounts of
protein are produced per molecule of spliced mRNA
than from otherwise identical mRNA molecules not produced by splicing [51,52,53]. Expression profiles of
mRNAs with first exons either too short or just long
enough to accept an EJC directly implicated the EJC
in this phenomenon [54,55]. Further, the EJC proteins RNPS1, Y14 and Magoh could all enhance mRNA
translational yield when artificially tethered to a reporter
mRNA [55].
Although NMD and the NMD-specific Upf proteins are
conserved in all eukaryotes examined thus far [57], discrimination between PTCs and normal termination
codons seems to involve different mechanisms in yeast,
flies and mammals. In budding yeast, PTC recognition
does not require either introns or splicing [63], and the
S. cerevisiae genome contains no apparent orthologs for
any of the known EJC core factors (i.e. Y14, Magoh and
eIF4AIII). Instead, somewhat loosely defined DSEs
(downstream sequence elements), which can destabilize
an mRNA when located 30 to a termination codon, apparently serve as the EJC equivalent for NMD in budding
yeast [63]. A recent surprise, however, came from RNAi
knockdown experiments in flies. Although all of the
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Current Opinion in Cell Biology 2004, 16:279–284
282 Nucleus and gene expression
known EJC core factors are conserved in Drosophila, none
of them appear to be essential for NMD in that organism
[64]. Rather, flies seem to be more yeast-like in their
mechanism of PTC recognition.
What is the evolutionary origin of the EJC?
The finding that EJC core proteins are present in flies but
not are required for NMD provides new fodder for speculation regarding the evolutionary origin of the EJC. A
model we favor is that the EJC originally arose as a means
to promote selective translation of spliced mRNAs. If, in
early eukaryotes, pre-mRNA splicing was a relatively
inefficient process, there would have been a strong selective pressure to tag RNAs as spliced so that only these
RNAs would be efficiently translated. Additionally, the
EJC may have helped to retain these spliced mRNAs on a
solid support (e.g. the cytoskeleton) where they could be
readily shunted into the cytoskeleton-bound translation
machinery. Thus, the present functions of the EJC in
mRNA localization and regulated translation during
Drosophila oogenesis may reflect these primordial roles.
In the mammalian lineage, the abundance of introns in
pre-mRNAs, coupled with poor conservation of splice site
consensus sequences, has led to an explosion of alternative splicing. Hand-in-hand with productive alternative
splicing, however, comes nonproductive mis-splicing,
defined here as alternative splicing events leading to
mRNAs containing prematurely truncated open reading
frames. The prevalence of mis-spliced mRNAs in the
mammalian lineage may therefore have provided an
impetus to also employ the EJC as a signal for premature
termination codon recognition. This could have been
accomplished by simply adding to one of the EJC core
factors a domain that recruits the Upf proteins. In this
scenario, the core EJC factors may be important for NMD
in mammalian cells not because they are directly involved
in NMD, but rather because they are essential for the
integrity of the EJC structure which, in turn, serves as a
platform for recruitment of the Upf factors.
Conclusions
The discovery and characterization of the EJC over the
past few years have added significantly to our understanding of the mechanisms by which pre-mRNA splicing can influence subsequent steps in gene expression.
It has also opened an important new window into the
complexities and dynamics of mRNP formation and
metabolism. However, much remains to be done. Even
though numerous EJC proteins have been identified, the
current list is almost certainly incomplete. Determination of the 3D structure of the EJC will undoubtedly lend
insight into its means of assembly, its mechanism of RNA
binding and its evolutionary origin. Further functional
analysis, particularly with regard to the roles of the EJC in
mRNA localization and translational efficiency, are sure
not only to provide additional surprises, but also to
increase our understanding of the advantages of allowing
Current Opinion in Cell Biology 2004, 16:279–284
the synthetic histories of mRNAs to dictate their subsequent metabolism.
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whereas NXF1/Tap is essential.
46. MacMorris M, Brocker C, Blumenthal T: UAP56 levels affect
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47. Mohr SE, Dillon ST, Boswell RE: The RNA-binding protein
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48. Hachet O, Ephrussi A: Drosophila Y14 shuttles to the posterior of
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49. Palacios IM: RNA processing: splicing and the cytoplasmic
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51. Lu S, Cullen BR: Analysis of the stimulatory effect of splicing
on mRNA production and utilization in mammalian cells.
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52. Nott A, Meislin SH, Moore MJ: A quantitative analysis of
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9:607-617.
This study and [51] report quantitative analyses of the effects of splicing
on various steps in gene expression and suggest that, in addition to
stimulating mRNA production, splicing enhances the amount of protein
produced per mRNA molecule.
53. Noe V, MacKenzie S, Ciudad CJ: An intron is required for
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54. Wiegand HL, Lu S, Cullen BR: Exon junction complexes mediate
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enhanced translation of spliced mRNA in mammalian cells using short
first-exon b-globin constructs. It additionally shows that tethering RNPS1
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of the mRNA. Other experiments clearly demonstrate that the EJCdependent enhancement in mRNA translation is an effect distinct from
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55. Nott A, Le Hir H, Moore MJ: Splicing enhances translation in
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translation is due to EJC deposition and, in mammalian cells, correlates
with a greater percentage of spliced mRNAs being associated with
polysomes. Tethering of the EJC proteins Y14, Magoh and RNPS1 or
of the Upf proteins Upf1, Upf2 and Upf3b leads to enhanced translation
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56. Schell T, Kulozik AE, Hentze MW: Integration of splicing,
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Current Opinion in Cell Biology 2004, 16:279–284
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In this study, it is found that NMD occurs independently of splicing in
Drosophila and that RNAi knockdown of several EJC proteins fails to
abrogate NMD. Thus, Drosophila NMD seems to be more yeast-like than
mammal-like in its mode of PTC recognition.
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