TrendsThe EJC is a protein complex thatbinds to mRNA in a splicing-depen-dent, but sequence-independent,manner. Deposition of the EJC requiresthe recruitment of the EJC componentEIF4A3 by the spliceosomal proteinCWC22.

High-throughput mapping of EJC-binding sites revealed many noncano-nical binding sites in addition to thecanonical site 20 nucleotides upstreamof exon–exon junctions.

ReviewExon Junction Complexes:Supervising the GeneExpression Assembly LineVolker Boehm1 and Niels H. Gehring1,*

The exon junction complex (EJC) is an RNA-binding protein complex that isassembled and deposited onto mRNAs during splicing. The EJC comprises fourcore components that bind to not only canonical sites upstream of exon–exonjunctions, but also to noncanonical sites at other positions in exons. EJC-associated proteins are recruited by the EJC at different steps of gene expres-sion to execute the multiple functions of the EJC. Recently, new insights havebeen obtained into how EJCs stimulate pre-mRNA splicing, and mRNA export,translation, and degradation. Furthermore, mutations in EJC core componentswere shown to result in severe disorders in humans, demonstrating the criticalphysiological role of the EJC. Hence, the EJC has been identified as an impor-tant player in post-transcriptional gene regulation in metazoans.

The EJC regulates many post-tran-scriptional steps of gene expression,including mRNA export and translationand nonsense-mediated mRNA decay.It is also involved in the global regula-tion of alternative splicing and exondefinition.

Mutations in the genes encoding theEIF4A3 and RBM8A components ofthe EJC result in human disorders withcomplex phenotypes.

Discovery of the Exon Junction ComplexDuring mammalian gene expression, the EJC carries information about the splicing process tosubsequent steps of mRNA metabolism [1]. EJCs exert different functions depending on thecellular compartment in which they are active and on their position on a given mRNA. In manycases, EJCs positively influence gene expression, for example by enhancing pre-mRNA splicing,mRNA transport or mRNA translation. However, the EJC is best known for its function innonsense-mediated mRNA decay (NMD; see Glossary), where it initiates the rapid turnoverof mRNAs with premature translation termination codons (PTCs). Therefore, the initial discoveryand characterization of the EJC occurred mainly with regard to its function in NMD [2–4]. Forexample, the observation that introns have a central function during NMD had been interpretedoriginally as a nuclear effect of the splicing process [5–7]. However, shortly afterwards, it waspostulated that splicing imprints the mRNA with a molecular mark that defines terminationcodons upstream of the site of intron removal as premature [8]. Components of the EJC weresubsequently identified by their incorporation into mRNPs formed by in vitro splicing of shortsubstrate mRNAs [9–11]. Further research has since led to the identification of the complete EJC[12–16], a multicomponent mRNA-binding protein complex that is assembled by the spliceo-some during splicing.

1Institute for Genetics, University ofCologne, 50674 Cologne, Germany

*Correspondence:ngehring@uni-koeln.de (N.H. Gehring).

Splicing-Dependent Assembly of the EJC at Canonical SitesDuring the first years after its discovery, the exact composition of the EJC was not well defined.However, its biochemical characterization revealed two important features: (i) the EJC isassembled onto the mRNA at a position approximately 20–24 nucleotides (nt) upstream ofthe spliced exon–exon junction [9]; and (ii) recruitment and stable binding of this complex occursin a strictly splicing-dependent manner without a specific RNA-sequence element that the EJCdirectly binds to [9]. This sequence-independent mode of mRNA binding was later explained bythe atomic structure and molecular characteristics of the four EJC core components (Figure 1)

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© 2016 Elsevier Ltd. All rights reserved.

GlossaryCanonical EJC (cEJC): deposited�20 nucleotides upstream of anexon–exon junction.Exon junction complex bindingmotif (EBM): a short peptidesequence that mediates theinteraction of some proteins (UPF3B,SMG6, TDRD3, and GIDRP88) withthe EJC.Messenger ribonucleoprotein(mRNP): an mRNA with all itsassociated RNA-binding proteins andnoncoding RNAs, considered to bethe biologically active form of anmRNA.Noncanonical EJC (ncEJCs):deposited at sites other than thecanonical binding site.Nonsense-mediated mRNA decay(NMD): surveillance mechanism thatdetects and degrades mRNAscontaining premature translationtermination codonsSerine/arginine-rich (SR) proteins:members of the SR protein family arecharacterized by the presence of oneor two RNA-binding domains and anRS domain rich in arginine/serinedipeptides. They are required forconstitutive pre-mRNA splicing andare also regulators of alternativesplicing.

8 nt

MAGOH

(A) (B) (C)RBM8A

BTZ

EIF4A3

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MAGOH RBM8A

BTZ

EIF4A3

RNA

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BTZ

EIF4A3

Figure 1. Structure and Schematic Representation of the Exon Junction Complex (EJC). The core of the EJCcomprises four proteins: EIF4A3 (orange), MAGOH (blue), RBM8A (green), and Barentsz (BTZ; magenta). The atomicstructure of the EJC core (A) revealed that EIF4A3 binds directly to RNA and occupies approximately eight RNA nucleotides(picture was rendered with PyMOL using PDB ID: 2J0S [18]). This interaction involves mainly the ribose-phosphatebackbone, but not the RNA bases themselves. Within the EJC, MAGOH and RBM8A form a stable heterodimer thatinteracts with the RNA-bound EIF4A3. The MAGOH protein may be synthesized from either of two homologous genes inmammals, referred to as MAGOH and MAGOHB. Direct binding of BTZ to EIF4A3 is independent of MAGOH-RBM8A anddoes not involve RNA. Formation of the EJC also requires ATP (not shown), which is bound by both RecA domains ofEIF4A3. For this review, we use a scheme of the EJC (B) that mirrors the overall structure as well as the interactioncharacteristics of the EJC core. In its RNA-bound form, EIF4A3 adopts a closed conformation with both RecA domainsbeing closely linked to each other (middle panel). By contrast, the RecA domains of EIF4A3 adopt an open conformation inthe absence of ATP and RNA (C). Abbreviation: nt, nucleotide.

[17,18] [EIF4A3 (DDX48), MAGOH, RBM8A (Y14), and Barentsz (BTZ, also called CASC3 orMLN51)] [15]. The DEAD box protein EIF4A3 is the main RNA-binding protein of the EJC. Itcomprises two conserved RecA-like domains that, in the presence of ATP, form a contiguouscleft where the RNA is bound [17,18]. Stable mRNA binding also requires the other three coreproteins of the EJC, of which BTZ directly interacts via its SELOR domain with EIF4A3 [17,18].The main function of the MAGOH-Y14 heterodimer is to lock EIF4A3 in its RNA-boundconformation by inhibiting the release of hydrolyzed ATP by EIF4A3 [19]. This prevents EIF4A3from switching to an open conformation, which would disrupt the RNA-binding site and result inRNA dissociation. The RNA binding of EIF4A3 does not involve the RNA bases, but occursmainly via the ribose-phosphate backbone [17,18]. This explains the sequence-independentmode of RNA binding by the EJC.

Although the EJC can form spontaneously in vitro from its protein components in the presence ofRNA and ATP, the assembly of the EJC in vivo is tightly linked to the process of splicing. Excisionof introns by the spliceosome requires the five spliceosomal small nuclear RNPs (snRNPs) andmore than 100 protein factors [20]. During the two consecutive transesterification steps, whichare the main steps of splicing, the spliceosome undergoes dramatic rearrangements and formsdifferent spliceosomal intermediates [20]. Early during the splicing process, the EJC coreproteins EIF4A3, MAGOH, and RBM8A associate transiently with the spliceosome and forma trimeric pre-EJC at the designated EJC-binding site after the first step of splicing [21]. Afterexon ligation and release from the spliceosome, BTZ joins the pre-EJC to form the mature,tetrameric EJC [21].

The identification of the spliceosomal protein CWC22 as an interaction partner of EIF4A3 hasshed new light on the splicing-dependent assembly of the EJC (Figure 2A) [22–24]. CWC22 is anessential splicing factor [25] and an abundant component of active spliceosomes [26]. CWC22contains a middle of initiation factor 4G (MIF4G) domain, which binds to EIF4A3 and keeps it inan open conformation [27]. This interaction is required for the assembly of the EJC duringsplicing and, therefore, CWC22 is considered the main recruitment factor for EIF4A3 [22–24].Notably, without CWC22, the efficiency of splicing as well as of EJC assembly is impaired

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MAGOH/RBM8A

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Figure 2. Assembly and Disassemblyof the Exon Junction Complex (EJC).(A) Splicing of pre-mRNAs in the nucleusof metazoan cells assembles an EJC fromits individual components. The RNA-binding core component EIF4A3 isrecruited to the spliceosome by theessential splicing factor CWC22.MAGOH and RBM8A form a tight hetero-dimer that is recruited separately to thespliceosome by an as yet unknownmechanism. After completion of splicing,the processed mRNA is releasedtogether with the deposited EJC, whichis joined by the fourth subunit, Barentsz(BTZ). Three potential outcomes of spli-cing and EJC assembly have beendescribed: (i) binding of a canonicalEJC at a position 20 nucleotides (nt)upstream of the exon–exon junction; (ii)binding of a noncanonical EJC at a posi-tion different from the canonical bindingsite; and (iii) absence of an EJC at thecanonical binding site. (B) Disassembly ofEJCs occurs in the cytoplasm. The pro-cessivity of a translating ribosome is suf-ficient to remove all EJCs from the codingsequence of a given mRNA. After disso-ciation from the mRNA, the EJC is dis-assembled into two subcomplexes(MAGOH-RBM8A and EIF4A3-BTZ) bythe protein PYM1. PYM1 may also acttogether with the ribosome to facilitateEJC disassembly during translation. Inaddition, PYM1 is also important to dis-assemble EJCs that are located inregions of mRNAs that are not accessibleby the ribosomes, such as the 30 untrans-lated region (UTR). Binding of PYM1 toMAGOH-RBM8A is incompatible withEJC formation and, therefore, preventsthe reassembly of EJCs in the cytoplasm.

[24,25]. Therefore, combining both functions within CWC22 ensures that splicing and EJCdeposition are tightly linked (Figure 2A). Recent structural insights shed new light on this tightfunctional linkage, because two central domains of CWC22 (MIF4G and MA3) are located closeto the 50 exon in structures of yeast spliceosomes [28–30]. Although Saccharomyces cerevisiaedoes not have an EJC, the position of CWC22 within the spliceosome is consistent with itsfunction to recruit EIF4A3 to the binding site of the EJC upstream of exon–exon junctions inmetazoan cells. The intron-binding helicase IBP160 (Aquarius) has also been shown to beinvolved in EJC assembly [31], but whether intronic sequences also contribute to the recruitmentof the EJC remains to be determined. In contrast to the well-established recruitment of EIF4A3by CWC22, little is known about the interaction of the MAGOH-RBM8A heterodimer with thespliceosome (Figure 2A). The stable interaction of MAGOH-RBM8A with splicing intermediateshas been shown to involve the formation of a trimeric pre-EJC comprising MAGOH-RBM8A andEIF4A3 [21]. Therefore, it remains to be determined whether a recruitment factor for MAGOH-RBM8A similar to CWC22 for EIF4A3 exists. Furthermore, we are lacking insights into thechoreography that is used by the spliceosome to assemble EJCs from its individual compo-nents. Thus, the exact mechanism of EJC assembly, the functions of IBP160 and othercomponents of the spliceosome, and interconnections between EJC modules remain elusive.

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Deposition of EJCs at Noncanonical SitesHigh-throughput analyses of EJC-binding sites in human cells have revealed that a small portionof exon–exon junctions does not carry EJCs (Figure 2A) [32,33]. Remarkably, transcripts encod-ing ribosomal proteins displayed low EJC binding, whereas alternatively spliced mRNAs showedincreased EJC occupancy [34]. Whether the differential EJC binding is due to different stability ordifferent assembly efficiencies of the EJC at the respective sites is an unresolved question. Inaddition to canonical EJCs (cEJCs), which are associated at a position �20 nt upstream ofexon–exon junctions (Figure 2A), so-called noncanonical EJC (ncEJC)-binding sites wereobserved at several other locations in exons (Figure 2A) [32,33]. Surprisingly, in earlier studies,as many as 40–50% of all EJCs were detected as being bound to these noncanonical sites[32,33]. However, when EJC binding was determined by the combined signal of four different EJCcomponents in a recent study, more than 80% of fully assembled EJCs were detected atcanonical sites [34]. While the cEJCs:ncEJCs ratio may be influenced by cell type-specificmolecular determinants, it could also depend on the experimental strategies to detect EJC-binding sites.

Although it was assumed that canonical EJCs bind to mRNA in a sequence-independentmanner, specific sequence features were found associated with both cEJCs and ncEJCs[32]. Since extensive secondary structures prevent EJC deposition, both cEJCs and ncEJCsbind favorably to unstructured regions of the mRNA. Furthermore, EJCs show a bindingpreference to purine-rich sequences, particularly a GAAGAA motif that resembles the bindingsite of serine/arginine-rich proteins (SR proteins) [32,33]. SR proteins are best known for theirfunction as general activators of splicing, although they also fulfill multiple additional postsplicingroles [35–37]. The mechanism of EJC deposition at noncanonical sites is not fully understood. Itis an interesting hypothesis that both types of EJC are assembled by the spliceosome, but thefinal binding site is determined by the context of the mRNP and factors that stabilize the bindingof the EJC to the mRNA. Whether cEJCs and ncEJCs only differ by their binding sites, or if theyalso vary in composition and function(s) still remains to be determined. Therefore, the compari-son of cEJCs and ncEJCs will be an important subject for future studies.

Disassembly of the EJCThe particular ability of the EJC to regulate many different steps of mRNA metabolism requires itstight interaction with the mRNA. Consequently, assembled EJCs do not dissociate spontaneouslyfrom their bound mRNA, but need to be removed from the mRNA by an active dissociationprocess. This may be done by translating ribosomes, whose processivity is sufficient to remove, inprincipal, all RNA-binding proteins from the coding sequence of any given mRNA. Indeed, there isevidence that EJCs are removed from their bound mRNA during the initial round of translation(Figure 2B) [38]. An alternative pathway of EJC disassembly involves the protein PYM1, whichinteracts with MAGOH-RBM8A, but not the other proteins of the EJC [39]. PYM1 and EIF4A3 bindto overlapping sites on the surface of MAGOH-RBM8A. Hence, when PYM1 binds to MAGOH-RBM8A within the EJC, it releases EIF4A3 from the closed conformation and initiates itsdissociation from the mRNA (Figure 2B) [40]. The C-terminal part of human PYM1 has beenshown to mediate its association with ribosomes [41], which could be important to couple EJCdisassembly with translation (Figure 2B) [40]. In contrast to mammals, PYM1 does not interactwith ribosomes in Drosophila melanogaster and, therefore, acts as a translation-independentdisassembly factor [42]. While translation would suffice to dislodge most EJCs, the activity ofPYM1 could be required to disassemble EJCs located outside the coding sequences of mRNA orEJCs assembled on noncoding mRNAs (Figure 2B). Alternatively, PYM1 may prevent thereassembly of EJCs in the cytoplasm or enhance recycling of EJC components that need tobe transported back to the nucleus for the next round of EJC assembly [40]. In conclusion, theEJC is a component of the mRNP that does not integrate spontaneously into mRNPs, butrequires specific processes for its association with, and the dissociation from, its target mRNA.

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The EJC Promotes Efficient and Faithful Gene ExpressionThe deposition of an EJC positively influences almost all subsequent steps of gene expressionleading to the production of transcript-encoded protein (Figure 3, Key Figure). Therefore, thesplicing-dependent assembly of EJCs serves as a quality control mechanism to ensure thatproperly processed mRNAs are loaded with components that enhance gene expression. Tofulfill this demanding role, the four EJC core components use a large number of dynamicallyassociated auxiliary proteins. These factors act at certain steps of the mRNP life cycle andmediate specific functions, such as splicing regulation, export of mature mRNA to the cyto-plasm, and translation stimulation.

One set of peripheral EJC components acting in the nucleus is the so-called Apoptosis andSplicing Associated Protein (ASAP) complex that comprises SAP18, RNPS1, and Acinus[15,43]. Of note, an alternative PSAP complex exists in which the splicing activator Pinin isincluded instead of Acinus [44,45]. To which extent ASAP and PSAP complexes or componentsoverlap in function and how these complexes are recruited to the EJC is currently unknown. Dueto the cellular localization and the copurification with both spliceosome and EJC, it is speculatedthat the ASAP complex joins during the deposition of EJCs on the mRNA [15,43,46]. Ampleevidence for the function of SAP18, RNPS1, and Acinus as splicing regulators supports the viewthat recruited ASAP components modulate adjacent splicing events (Figure 3) [47–52]. Inter-estingly, the core EJC is not essential for most constitutive splice events in human and fly cells[25,53,54]. Nevertheless, several studies in D. melanogaster showed abnormal splicing patternsof certain transcripts upon depletion of EJC or ASAP factors [54–57]. The piwi mRNA, forexample, contains a weak fourth intron that is spliced inefficiently due to its abnormal poly-pyrimidine tract [54,57]. However, the assembly of EJCs on flanking exon junctions facilitates theexcision of intron 4. This function is dependent on the integrity of the trimeric pre-EJC (EIF4A3,MAGOH, and RBM8A) and requires the ASAP components RNPS1 and Acinus. Other con-stituents, such as BTZ and SAP18, were dispensable for the correct expression of the Piwiprotein. This implies that the minimal core of the EJC is sufficient to recruit ASAP factors, whichmay interact with the EJC either individually or as a dimer. Interestingly, the trimeric pre-EJC isrequired not only for certain intron excision events, but also for the definition of exons in specifictranscripts harboring long introns [55,56]. Whereas depletion of the minimal core EJC factors ledto exon skipping of the mapk mRNA, knockdown of RNPS1 had only minor effects and Acinuswas entirely dispensable for exon definition. This points to a differential requirement of peripheralEJC components for specific and functionally distinct splice events.

In human cells, transcriptome-wide analysis of alternative splicing revealed that the depletion ofEJC core components primarily resulted in skipping of cassette exons [58]. In agreement withthe findings in Drosophila, Acinus was not involved in the exon definition function conferred bythe EJC. Therefore, it remains unknown how the trimeric pre-EJC core mechanistically regulatesand influences individual splice events in metazoan cells.

Transport of mature mRNAs from the nucleus to the cytoplasm is required for the final steps ofgene expression and to make the mRNA available for protein biosynthesis. Supporting the ideaof coupling between nuclear export and successful pre-mRNA processing, splicing was foundto increase the kinetics and abundance of transcripts reaching the cytoplasm [59]. One commondenominator of this phenomenon is the EJC-dependent recruitment of export factors, such asALYREF and UAP56, to the spliced mRNA [15,60–62]. These components of the transcription-export (TREX) complex serve as binding sites of the general export receptor NXF1/NXT1, whichmediates translocation of the mRNP through the nuclear pore (Figure 3) [63–66]. Once on thecytoplasmic side of the nuclear envelope, the mRNA can, in principle, be engaged by thetranslational machinery. However, specific transcripts require transport to a certain subcellularlocalization to drive spatially confined protein synthesis [67]. For instance, the localization of the

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Key Figure

The Exon Junction Complex (EJC) as Multifaceted Modulator of GeneExpression

Cap Pol II

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ppp

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CBC

Capping enzymes

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Cap

AAATerAUG

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AA

A

NMD factorrecruitment

EIF4F

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ASAPPinin

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?

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mRNPlocaliza�on

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Export

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mRNA decay

ASAP/PSAPcomplexes

SR proteins

EIF4A3/CWC22

MAGOH/RBM8A

BTZ

Tetrameric EJC core forma�on

Intron excision/Exon defini�on

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Higher-orderEJC/SR

complexes

TREX complexNXF1/NXT1 recruitment

BTZEIF3recruitment

UPF3NMD complexassembly

Peripheral EJCCore EJCKey:

Nuclear pore

PYM1

CWC22EIF4A3

Trimeric pre-EJC

Figure 3.

(Figure legend continued on the bottom of the next page.)

This schematic overview is intended to illustrate how the EJC influences the fate of a spliced mRNA during its lifecycle (left side). Important processes of gene expression and the key contribution of core or peripheral EJC components to

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oskar mRNA determines the site of Oskar protein synthesis and thereby regulates where germcells are formed during embryogenesis in D. melanogaster [68,69]. Splicing and concomitantEJC deposition at the first exon–exon junction are necessary for oskar mRNA localization to theposterior pole in oocytes [70]. In combination with the splicing-dependent formation of aspecialized mRNA structure called the spliced oskar localization element (SOLE), the assembledEJC promotes kinesin-mediated intracellular transport via the cytoskeleton. This particular set-up might explain why no other transcripts have yet been reported to utilize a similar EJC-dependent localization mechanism.

After benefitting from enhanced splicing, export, and potentially intracellular localization, theEJC-loaded mRNP is ready to recruit ribosomal subunits for translation initiation. Althoughincreased protein production as a consequence of pre-mRNA splicing and EJC assembly wasdescribed more than a decade ago [71–73], the mechanism for this effect remained elusive untilrecently. Interestingly, not only peripheral, but also core EJC factors participate in the upregu-lation of translation. In contrast to the other core factors, BTZ was reported to be a general EJC-independent activator of translation. However, this activity of BTZ was more pronounced whenspliced and EJC-loaded mRNAs were analyzed [74]. Notably, BTZ directly interacts with thetranslation initiation factor 3 (EIF3), which is required for several early steps of translation.Therefore, an attractive mechanistic link between the EJC and enhanced translational outputis conceivable (Figure 3). According to this model, initiation factors are efficiently recruited tospliced mRNAs via the combined interaction with 50 cap-binding complexes and EJCs. Impor-tantly, the recruitment of EIF3 is compatible with BTZ being a constituent of the EJC core,because the central SELOR domain of BTZ is interacting separately with EIF4A3 and EIF3. Giventhat BTZ and EIF4A3 also form stable complexes, this heterodimer might influence translation inan EJC-independent manner [21]. Along this line, it was shown recently that EIF4A3 is able tounwind 50 untranslated region (UTR) secondary structures of mRNAs bound by the nuclear cap-binding complex to allow ribosome scanning and translation initiation [75]. BTZ could have a rolein this mode of translation enhancement by simultaneously recruiting EIF3 as well as promotingthe helicase activity of EIF4A3 [76]. As a peripheral EJC component, the protein SKAR (S6K1Aly/REF-like substrate) was also implicated to stimulate translation [77]. It was shown that, via its

the next step are summarized on the right side. As a first step of gene expression, pre-mRNA is transcribed by RNApolymerase II (Pol II). Cotranscriptional loading of pre-mRNA processing factors, such as capping enzymes, is mediated bythe C-terminal domain (CTD) of Pol II. After the 7-methylguanosine cap is attached to the 50 end of the transcript, it is boundby the nuclear cap-binding complex (CBC). During transcription, intronic sequences emerge that lead to the assembly of thespliceosome. Mediated by the interaction with the spliceosomal factor CWC22, EIF4A3 is brought in physical proximity tothe pre-mRNA. Assembly of the EJC during splicing requires MAGOH/RBM8A, whereas Barentsz (BTZ) joins the EJC aftersplicing. These core proteins constitute the tetrameric EJC core. During these early steps or shortly thereafter, additionalauxiliary EJC components are recruited. These include the ASAP or PSAP complex, multiple serine/arginine-rich (SR)proteins, and finally the TREX components UAP56 and ALYREF, which require also the interaction with the CBC for efficientpre-mRNA binding. The EJC may regulate via the ASAP/PSAP components (either as full complex or as subcomplexes) thesplicing of other introns in the same transcript and can aid in exon definition. Multiple EJCs deposited on a single mRNAmolecule cooperate with SR proteins to form higher-molecular-weight structures, thereby potentially compacting themRNP. After 30 end processing, polyadenylation, and binding of the nuclear poly(A) binding protein (PABPN), the maturemRNA is exported to the cytoplasm. CBC/EJC/SR protein-mediated recruitment of TREX components facilitates this stepby interactions with the general export factors NXF1/NXT1. During and after the translocation of the mRNP to the cytoplasm,several proteins are retained in the nucleus (e.g., Acinus/Pinin and TREX components), while others are exchanged [PABPNfor the cytoplasmic poly(A) binding protein, PABPC]. For specific transcripts, the EJC mediates the subcellular localizationvia the cytoskeleton. During the first loading of ribosomal subunits onto the mRNA, the core EJC component BTZ stimulatestranslation initiation by recruiting the initiation factor 3 (EIF3). Displacement of the EJC during translation is facilitated by theaction of the disassembly factor PYM1. In case of premature translation termination, indicated by the presence of an EJCdownstream of the ribosome, the assembly of the nonsense-mediated mRNA decay (NMD) complex is initiated. This occursnormally during the first round of translation (indicated by the presence of CBC), but the replacement of CBC with thecytoplasmic cap-binding complex EIF4F also supports NMD. The NMD factor and peripheral EJC component UPF3 arealready attached to the EJC in the nucleus and support NMD assembly during translation termination. This leads to therecruitment of decay-inducing proteins, which degrade the transcript using endo- and exonucleolytic activities.

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interaction with SKAR, the EJC recruits activated S6K1 kinase, which in turn phosphorylatesand, therefore, stimulates factors required for translation initiation.

Pre-mRNA splicing and concomitant EJC deposition were described as processes thatpositively influence multiple gene expression steps, leading ultimately to more gene products.In light of this, it is remarkable that the EJC can act under certain circumstances as a potentand efficient activator of mRNA decay, more specifically, NMD. The molecular principleunderlying this process is the persistent presence of an EJC on a transcript after translationtermination. Normally, all EJCs are removed during the first round of translation, because thestop codon of protein-coding genes is located primarily in the last exon [38,78,79]. However,there are several possibilities why ribosomes can terminate translation at a position upstreamof an EJC. These include, but are not limited to, the usage of upstream open readingframes (uORFs), alternative splicing events, or mutations leading to premature terminationcodons (PTC) [80]. Due to steric restrictions, the distance between the stop codon anddownstream EJC has to be at least 30 nt for NMD activation to occur. The specific function ofthe EJC is to initiate a cascade of protein–protein interactions culminating in the assembly ofa functional NMD complex (Figure 3) [81,82]. To this end, the EJC directly interacts with thecore NMD factor UPF3, which is recruited via its EJC-binding motif (EBM) to a compositebinding site formed by EIF4A3, RBM8A, and MAGOH [83,84]. UPF3 aids in the formation ofthe NMD complex, which ultimately leads to the phosphorylation of the central NMD factorUPF1 [85]. This in turn provides binding sites for decay-inducing factors that either promoteexonucleolytic degradation or catalyze the endonucleolytic cleavage of the NMD substrate[86,87]. Remarkably, the EJC-induced decay of mRNAs is in most cases robust, fast,and efficient, which is in contrast to an additional type of NMD that is activated by long30 UTRs [88].

In addition to these clearly defined functions, it has also been shown that the presence of multipleEJCs on human mRNPs leads to the formation of stable, high-molecular-weight mRNP com-plexes [33]. Hence, the cooperation of cEJCs and ncEJCs with other components of the mRNP,such as SR proteins, is thought to result in the compaction of mRNPs (Figure 3). Although theprecise molecular role of such compaction is not well understood, it may contribute to theefficient execution of post-transcriptional steps of gene expression, such as mRNA export andtranslation in the cytoplasm. Hence, some of the reported functions of EJCs may be related to itsrole as ‘nucleosome-like’ component of mRNPs.

Physiological Role of the EJCThe biological importance of the EJC is further underlined by the observation that mutations inRBM8A and EIF4A3 are associated with human disorders. Thrombocytopenia with absentradius (TAR) syndrome is a rare genetic disease that mainly presents as a disorder of the bloodand the upper extremities [89]. Patients with TAR syndrome not only have low numbers ofmegakaryocytes and a dramatically reduced platelet count (hypomegakaryocytic thrombocy-topenia), but also show skeletal abnormalities, specifically the absence of the radius bones [89].Genetically, patients with TAR usually have one allele with a reduced expression of the RBM8Agene, which is caused by noncoding SNPs in its 50 UTR or in the first intron [90]. The other allelecontains either a small chromosomal deletion including the RBM8A gene or a frameshift insertionin the fourth exon of the RBM8A gene [90]. Hence, TAR syndrome is caused by the hypomorphicexpression of RBM8A due to the compound inheritance of an RBM8A null allele (deletion orframeshift mutation) and an allele with reduced RBM8A expression [90]. Deletions in thechromosomal region containing RBM8A have also been identified in patients with brain sizeabnormalities as well as mental disorders [91]. It remains to be determined what the tissue-specific functions of RBM8A are and why some tissues are specifically affected by its reducedexpression.

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Another core component of the EJC, EIF4A3, has also been recently linked to a genetic disease.Richieri-Costa-Pereira Syndrome (RCPS) is an autosomal-recessive syndrome described in acohort of Brazilian families with craniofacial and limb defects [92]. RCPS is caused by thereduced expression of EIF4A3 due to expansions of noncoding repeats in the 50UTR of its mRNA[93]. The EIF4A3 transcripts containing repeat expansions are reduced in abundance by30–40% and, therefore, lead to a haploinsufficiency of EIF4A3 [93]. However, the mechanismby which the partial loss of EIF4A3 leads to the phenotype of RCPS is not understood. In general,it is notable that the TAR and RCP syndromes are caused by the reduced expression of RBM8Aand EIF4A3, respectively. This suggests that the complete absence of core EJC components isnot compatible with normal embryonic development and/or cell survival in mammals. Also, in D.melanogaster, no homozygous oocytes lacking EIF4A3 could be obtained [13], supporting thenotion that the knockout of EIF4A3 is lethal in metazoans.

The physiological importance of EJC components, particularly during neural development,has also been demonstrated with mouse models [94]. In recent studies, it was shown thatMagoh and Rbm8a are involved in brain development and neurogenesis [95–97]. A mousemutant with a haploinsufficiency of Magoh due to a germline mutation in the Magoh gene hasreduced body size and microcephaly [95]. Likewise, haploinsufficiency of Rbm8a in a condi-tional mouse model resulted in a microcephaly phenotype [96]. Overall, the brains of Magoh-or Rbm8a-haploinsufficient mice had fewer neural progenitor cells and showed increasedapoptosis [95,96]. These observations support the hypothesis that EJC components haveimportant functions during embryonic neurogenesis and the maintenance of neurons in thenervous system.

Box 1. Experimental Tools Used for the Analysis of the EJC

Previously, three different experimental systems were mainly used to study EJC assembly, composition, and function (Figure I). Each experimental approach offersadvantages for the analysis of certain aspects of the EJC, whereas the combination of two or three approaches will provide a more complete picture. EJC analysisusing recombinant proteins (Figure IA) is used to characterize mutants and to study EJC assembly in a splicing-independent manner. EJC assembly requires ATP andRNA, omitting either or both of them controls for the specificity of the assembly reaction. In vitro splicing reactions (Figure IB) are used for the analysis of splicing-dependent EJC assembly. In vitro splicing enables the characterization of mutant proteins and to dissect separate steps of EJC deposition. No EJC will be formedwithout splicing; hence, intronless substrate mRNAs are used as controls. Cultured cells (Figure IC) are used to identify binding sites of EJCs in a transcriptome-widemanner by CLIP-based approaches. In addition, the biological functions of EJCs are analyzed in cell culture. In living cells, the reduction of EJC levels can be achievedby siRNA transfection or by the overexpression of a mutant of the EJC assembly factor CWC22. Genomic knockouts of EJC core factors using CRISPR-Cas9 are likelynot feasible, due to their essential cellular functions.

Cultured cellsNuclear extractRecombinant proteins

Splicing independent Splicing dependent

Pull-down In vitro splicing Immunoprecipita�on, CLIP

EJC- binding sitesin vivo EJC assembly and binding

EJC assemblycharacteriza�on of mutants

Splicing dependent

EJC assemblyassembly intermediates

Reagent

(A) (B) (C)

EJC characteris�c

Methodology

Aims

EJC manipula�on +/– ATP+/– RNA

recombinant PYMintronless substrate

siRNA knock-downCWC22 mutant

Figure I. Comparison of Different Methods to Analyse the EJC.

732 Trends in Genetics, November 2016, Vol. 32, No. 11

Outstanding QuestionsWhat are the mechanism and factorsinvolved in MAGOH-RBM8A recruit-ment to the spliceosome?

What is the molecular trigger that ini-tiates the assembly of the EJC at itsdesignated binding site? In addition tothe EJC, which factors are involved?

What is the exact function of CWC22 inthe spliceosome and why is EJCassembly coupled to splicing?

What proteins are directly interactingwith the core of the EJC and, therefore,are authentic peripheral NMD compo-nents? What is their mode ofinteraction?

How does the EJC coordinate its mul-tiple functions at the molecular level?Can these functions be disentangled?

What is the mechanism of ncEJCassembly? Are ncEJCs assembled attheir ultimate binding sites or are theyrepositioned?

Are cEJCs and ncEJCs composition-ally and functionally equivalent?

How are the functions of EJC compo-nents outside the EJC regulated?

Concluding Remarks and Future PerspectivesSignificant progress has been made in recent years, leading to a greatly improved understandingof the molecular characteristics and the biological functions of the EJC. It is important to notethat, although high-throughput techniques have already contributed important details to acomprehensive model of the EJC, biochemical methods are still widely used and importantfor studying EJCs (Box 1). Nevertheless, the rapid development of high-throughput technologieswill likely provide novel insights into the functions of the EJC. This will be an important task forfuture studies, since many results concerning the EJC have so far only been obtained with fewmRNA substrates.

Furthermore, many peripheral EJC components or EJC-associated factors have been describedin recent years. However, we only have a detailed (structural) view of the CWC22-EIF4A3complex and EJC interactions that involve an EBM, whereas little is known about othercomplexes. Since CWC22 and EBM bind to an overlapping region of EIF4A3, it will be interestingto elucidate whether additional proteins bind to the same surface area, despite lacking con-served EBM sequences (see Outstanding Questions).

The diverse roles of the EJC represent a major challenge for the functional analysis of the EJC.Isolated functions of the EJC are either not amenable to analysis or only in artificial settings, suchas tethering systems. It remains to be elucidated whether the different functions mutuallyinfluence each other and whether methods can be developed to disentangle them.

Finally, the mechanism and determinants of EJC assembly are another unexplored area. In thisregard we will need a better insight into these processes to understand the decision by thespliceosome whether an EJC is assembled and whether it is deposited on a canonical ornoncanonical site.

AcknowledgmentsWe would like to thank members of the Gehring lab for helpful discussions. This research was funded by a grant from the

Deutsche Forschungsgemeinschaft (GE2014/6-1) to N.H.G.

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