Linking nuclear mRNP assembly and cytoplasmic destiny

Biol. Cell (2005) 97, 469–478 (Printed in Great Britain) Scientiae forumPrimer Review

Linking nuclear mRNP assemblyand cytoplasmic destinyScott Kuersten and Elizabeth B. Goodwin1

Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, U.S.A.

From the very beginning, mRNAs have a complex existence. They are transcribed, capped, spliced, modified atthe 3′end, exported from the nucleus, translated, and eventually degraded. These many events not only affect theoverall survival and properties of an mRNA, but are also carefully co-ordinated and integrated with quality controlmechanisms that function to ensure that only ‘proper’ mRNAs are translated at the correct developmental timeand place. This does not mean that all mRNAs follow a single or uniform path from synthesis to death. Instead,there are diverse means by which the activities of specific mRNAs are regulated, and these controls often dependupon multiple events in the mRNA’s life. mRNAs are not found naked in the cell, instead they are part of complexRNPs (ribonucleoproteins) that consist of many factors. These RNPs are highly dynamic structures that changeduring the lifetime of a given RNA; linking events such as synthesis and processing to the final fate of the mRNA.Here, we will discuss what is known of the assembly of RNPs in general, with specific reference to the myriad ofconnections between different nuclear events and the cytoplasmic activity of an mRNA. Due to space limitationsthis review is not comprehensive, instead we focus on specific examples to illustrate these emerging themes ingene expression.

IntroductionThe connection of transcriptionand RNA processingMany data link gene transcription with mRNA pro-cessing (for review see Neugebauer, 2002; Proudfoot,2004; Proudfoot et al., 2002; and Figure 1). A keyplayer in linking transcription with processing is theC-terminal domain (CTD) of RNA Polymerase II(PolII). The CTD of PolII is located near the exitsite of the nascent RNA from the polymerase, andconsists of multiple (approx. 26 in yeast and 52in mammals) conserved heptad repeats (consensusYSPTSPS). The repeats vary in phosphorylation statebetween transcription initiation and elongation, andthis regulates the association of different RNA-pro-

1To whom correspondence should be addressed (email [email protected]).Key words: localization, mRNA, ribonucleoprotein, translation, transport.Abbreviations used: CBC, cap binding complex; CRM1, chromosome regionmaintenance protein 1; CTD, C-terminal domain; EJC, exon junction complex;GLD-1, germ-line defective 1; hnRNP, heteronuclear RNP; MBP, myelin basicprotein; NMD, nonsense-mediated mRNA decay; NXF, nuclear export factor;PolII, RNA polymerase II; poly(A)+, polyadenylated; PTC, prematuretermination codon; PTC+, PTC-containing; REF, RNA export factor; RNAi, RNAinterference; RNP, ribonucleoprotein; SR protein, serine/arginine-rich protein;TRE, tra-2 retention element; TREX, transcription and export factor;UPF, up-frameshift; UTR, untranslated region.

cessing factors with the CTD during a transcriptioncycle.

The CTD and mRNA processingEarly in transcription of a gene, the phosphorylatedCTD recruits factors involved in 5′ and 3′ end form-ation. Enzymes involved in the addition of the 5′monomethylguanosine cap structure to the 5′ endof the mRNA are associated with the CTD. Thecap is important for a number of downstream RNAprocessing events (for review see Proudfoot, 2004;Proudfoot et al., 2002). This is in part due to thecap being bound by the nuclear cap binding complex(CBC) consisting of CBP80 and CBP20, which affectssplicing (Izaurralde et al., 1994; Lewis et al., 1996), 3′end formation (Flaherty et al., 1997; Das et al., 2000)and translation (Fortes et al., 2000; McKendricket al., 2001). The CTD also recruits factors requiredfor 3′ end formation (Proudfoot, 2004). 3′ End form-ation requires the recognition of the hexanucleotideAAUAAA and a GU-rich sequence by a complex offactors at the 3′ end of the mRNA. This results incleavage of the mRNA and the subsequent addition ofa poly(A)+ tail by a nuclear poly(A) polymerase. The

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S. Kuersten and E.B. Goodwin

Figure 1 Links between transcription, RNA processing, export and translationAs RNA PolII converts from transcription initiation to elongation, the CTD becomes phosphorylated (light green dots) and helps

to recruit factors involved in RNA processing. These include the capping enzymes, splicing factors and components of the 3′ end

formation machinery. The subsequent recruitment of CBC to the cap structure affects splicing, 3′ end formation and translation.

During transcription elongation the association of THO affects elongation efficiency and also helps promote the assembly of

factors involved in later steps, most notably UAP56–Sub2p which links splicing with export. UAP56–Sub2p binds the yeast

REF–Aly homologue Yra1p, which is required for association of the mRNA with the export receptor NXF-1. Splicing results in

a number of factors being deposited on the mRNA including the EJC and some SR proteins. Following export, some mRNP

components remain bound to the mRNA to promote translation, affect degradation and/or localization of mRNAs.

CTD is also important in recruiting factors requiredfor splicing to the nascent mRNA (Proudfoot, 2004).

The THO–TREX (transcription and exportfactor) complexDuring transcriptional elongation, a complex, calledTHO, links transcription with ribonucleoprotein(RNP) assembly (Jimeno et al., 2002; Strasser et al.,

2002). In yeast the THO complex is composed of fivesubunits: THO2, HPR1, MFT1, THP2 and TEX1.The complex differs in humans: homologues ofTHO2, HPR1 and TEX1 are present while homo-logues of MFT1 and THP2 have not been iden-tified (Strasser et al., 2002). The THO complex isimportant for PolII elongation and can recruit somefactors involved in mRNA splicing and export such

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Nuclear history and cytoplasmic fate of mRNAs Scientiae forum

as UAP56 (Sub2p in yeast) and RNA export factor(REF)–Aly (Yra1p in yeast). Both genetic and bio-chemical analyses demonstrate that the yeast spli-cing factor Sub2p, and its mammalian homologueUAP56, bind THO, resulting in a complex calledTHO–TREX that affects mRNA export (Strasseret al., 2002). Sub2p and UAP56 influence mRNAexport, since Sub2p interacts with the nuclear exportfactor Yra1p (Strasser and Hurt, 2001), and in Caen-orhabditis elegans and Drosophila, removal of UAP56by RNAi (RNA interference) results in accumulationof poly(A)+ mRNA in the nucleus (Gatfield et al.,2001; MacMorris et al., 2003). Nuclear export of mostmRNAs requires the heterodimeric export receptorNXF1–p15 (Mex67p–Mtr2p in yeast), and it isthought that REF–Aly can facilitate the associationof a given mRNA with NXF1 (Conti and Izaurralde,2001).

A model for the role of THO–TREX in linkingdifferent aspects of mRNA metabolism suggests thatTHO stimulates transcription elongation and recruitsUAP56–Sub2p, facilitating splicing and the associ-ation of the export factor REF–Aly to the mRNA.Interestingly, Sub2p and Mex67p compete with oneanother for binding to Yra1p (Strasser and Hurt,2001). This suggests that remodelling of a Sub2pRNP into one that contains Mex67p may be im-portant in altering the RNP so that it is compet-ent for export. The fact that THO–TREX influences(either directly or indirectly) elongation, splicingand export emphasizes how these events are highlyintegrated.

At present it is unclear how general the THO–TREX complex is in regulating RNP assembly. Re-cent data indicate that reduction of the THO complexcomponents by RNAi affected export of approx. only20% of the total pool of mRNA in Drosophila cells(Rehwinkel et al., 2004), while reduction of UAP56or NXF1 blocked export of approx. 75% of cellulartranscripts (Herold et al., 2003); moreover, in yeastthe THO components were not essential. Hence itis possible that the THO–TREX complex regulatesonly a subset of mRNAs and that other mRNAs use

different means to recruit splicing and export factors.In support of this, the reduction of THO complexcomponents by RNAi affects the export of intronlessheat shock mRNAs in Drosophila cells (Rehwinkelet al., 2004).

Splicing, the exon junction complex(EJC) and gene expressionSeveral years ago, it was found in Xenopus oocytes thatmRNAs derived by splicing produced higher proteinlevels than intronless mRNAs, indicating that splic-ing affected the cytoplasmic activity of the mRNA(Matsumoto et al., 1998). Genetic and moleculardata have since identified several splicing dependentevents that affect gene expression by altering the sta-bility, 3′ end formation, export and/or translationof an mRNA. A key event is that splicing depositsprotein complexes on the mRNA that persist in thecytoplasm; hence, the RNPs of a spliced versus anunspliced mRNA are distinct.

One of the first hints that splicing leaves ‘marks’on the mRNA came from in vitro experiments. RNPscreated through splicing in HeLa cell nuclear extractshave a slower mobility on native gels than complexesformed on the corresponding intronless version of thesame mRNA (Zhang et al., 1998b; Luo and Reed,1999). Also, in mammals, the distinction betweena normal and a premature termination codon (PTC)is determined by its position with respect to the lastexon–exon junction. Premature codons result in rapiddegradation of the mRNA due to a process referredto as nonsense-mediated mRNA decay (NMD; seebelow). If the termination codon is >50–55 nt up-stream of the last exon–exon junction, it is consideredpremature (Zhang et al., 1998b).

A splicing-dependent complex, the EJC is depos-ited 20–24 nt upstream of presumably all exon–exonjunctions (Le Hir et al., 2000a, 2000b). The core com-ponents of the EJC, as well as other EJC-associatedfactors, contribute to multiple aspects of mRNAmetabolism. The core is thought presently to con-sist of the Y14–Magoh heterodimer and eIF4AIII(for review see Tange et al., 2004). Cross-linking

Exon junction complex (EJC): A complex of proteins deposited approx. 20–24 nucleotides upstream of the junctions of adjoining exons. The EJC forms duringthe course of pre-mRNA splicing and is thought to link important aspects of nuclear mRNA processing such as transcription, splicing and perhaps export withcytoplasmic events like translation and mRNA turnover.

Nonsense-mediated mRNA decay (NMD): A phenomenon in which steady state levels of mutant mRNAs containing a premature termination codon (PTC) arereduced relative to wild-type mRNAs. This mechanism is thought to minimize the accumulation of protein fragments synthesized from PTC-containing mRNAsthat could be detrimental to survival. It is currently thought that the EJC helps to recruit some NMD components in the nucleus.



experiments suggest that eIF4AIII probably bindsthe RNA directly and recruits Y14–Magoh (Shibuyaet al., 2004). These factors influence NMD andmRNA cytoplasmic localization (see below), illus-trating the fact that splicing and the EJC can affectsubsequent cytoplasmic events.

Some of the more peripheral EJC-associated factors,such as RNPS1, Pinin, SRM160, REF–Aly, UAP56,NXF1, up-frameshift protein UPF3a and UPF3b,span the spectrum of functions in RNA processing.RNPS1, Pinin, UAP56 and SRM160 have roles insplicing and 3′ end formation. REF–Aly, UAP56,and NXF1 are involved in mRNA nuclear export, andUPF3a, UPF3b, and RNPS1 are required for NMD(Tange et al., 2004).

The fact that several EJC factors affect differentmRNA processing events strongly indicates that theEJC integrates splicing of mRNAs with export andcytoplasmic fate. Indeed, there is evidence that spli-cing enhances the export of mRNAs, particularlymRNAs that are inherently poor substrates for theexport machinery such as short mRNAs (<300 nt)(Luo and Reed, 1999; Rodrigues et al., 2001). Con-sistent with the EJC helping to recruit the REF–Alyexport factors, co-injection of excess REF–Aly pro-teins overcomes the deficiency in export of some shortmRNAs (Rodrigues et al., 2001).

Splicing is not the only way to recruit export factorsto an RNA; the length of an RNA can also affect re-cruitment of export factors. Insertion of approx.300 nt of sequence into a synthetic intronless U1snRNA resulted in the binding of REF–Aly to theRNA, and a switch in the export of the RNA fromchromosome region maintenance protein 1 (CRM1)-dependent (the route normally used by U1 snRNAs)to the NXF1-dependent pathway in Xenopus oocytes;however, insertion of shorter sequences did not re-sult in efficient association of REF–Aly or a switchto the NXF1 mediated pathway (Masuyama et al.,2004). This change in export pathway was not depen-dent upon the position of the inserted sequence. Inaddition, export of an intronless mRNA could beconverted to the CRM1-dependent pathway by pro-gressively shortening the mRNA, further indicatingthe importance of RNA length on choice of exportpathway.

The EJC also plays roles in other steps of gene ex-pression. It contributes to the overall transcriptionof a gene and enhances the translational efficiency of

an mRNA in the cytoplasm (Lu and Cullen, 2003;Nott et al., 2003; Wiegand et al., 2003). The effecton translation may be direct, since artificial tetheringof RNPS1, Y14, or Magoh to mRNAs stimulatestranslation in vitro (Nott et al., 2003).

Despite all of these potential important roles for theEJC, none of the EJC components, except UAP56,is essential for mRNA export in Drosophila cells(Gatfield and Izaurralde, 2002). Thus other mech-anisms may couple splicing with mRNA activity be-sides the EJC. For example, the SR (serine/arginine-rich) splicing factor SF2–ASF shuttles between thenucleus and cytoplasm and is associated with trans-lating ribosomes. In addition, SF2–ASF stimulatestranslation when tethered to a reporter mRNA(Sanford et al., 2004). Moreover, SF2–ASF, and otherSR proteins such as Srp20, 9G8, and the yeast Npl3pbind to NXF-1–Mex67p to influence mRNA export(Huang and Steitz, 2001; Huang et al., 2003; Laiet al., 2003; Gilbert and Guthrie, 2004). Phos-phorylation of these SR proteins regulates their inter-actions with NXF1–Mex67p, suggesting post-trans-lational control may affect export of mRNAs.

The EJC and NMDNuclear events play an important role in helping to‘proofread’ transcripts, to ensure that only properlyprocessed mRNAs that are capable of encoding func-tional proteins are translated. Transcription errors, in-appropriate splicing and mutation can generate defec-tive mRNAs that contain PTCs. As described above,NMD is a mechanism that rapidly degrades PTC-containing (PTC+) mRNAs (for review see Maquat,2004). NMD appears to occur generally when themRNA is associated with the nucleus (Maquat,2004), although some mRNAs are clearly degradedin the cytoplasm (Moriarty et al., 1998). Presently,it is unclear if the nuclear-associated NMD is occur-ring before the mRNA is ‘released’ from the nucleusinto the cytoplasm, or whether it happens inside thenucleus itself.

There is a strong connection between nuclear eventsand NMD. As discussed above, the distinction bet-ween normal and PTC+ mRNAs depends upon theposition of the PTC with respect to the last exon–exonjunction; a PTC that is located more than 50–55 ntupstream of the last exon–exon junction is targetedfor rapid decay (Zhang et al., 1998a). This ‘mark’involves the EJC which is thought to be removed



during the first or ‘pioneering’ round of translation,which occurs on mRNAs that are still associated withCBC (Fortes et al., 2000; Lejeune et al., 2002), be-fore the exchange of the CBC with eIF4E. The ideathat NMD is occurring on CBC mRNA is supportedby immunoprecipitation experiments that find thatPTC+ CBP80-bound mRNA is reduced to similarlevels to PTC+ eIF4E mRNAs relative to controlmRNAs without a PTC (Ishigaki et al., 2001;Lejeune et al., 2002). This indicates that NMD pri-marily affects the levels of CBC-bound mRNA beforethe exchange of CBC with eIF4E, which is thoughtto occur after the first round of translation (Forteset al., 2000). If PTC was occurring mainly on eIF4E-bound mRNA, one would anticipate no decrease inthe PTC+ CBP80-bound mRNA. In addition, inhi-bition of translation results in higher levels of PTC+CBP80-bound mRNA (Ishigaki et al., 2001).

Molecular analysis is elucidating the role of theEJC in NMD. In mammalian cells, the UPF proteinsco-operate with the EJC to identify PTC+ mRNAs.The up-frameshift (UPF) proteins, UPF3a, UPF3b,and UPF2, co-immunoprecipitate with EJC compon-ents in both nuclear and cytoplasmic fractions, in-dicating that these factors could associate with theEJC in the nucleus (Kim et al., 2001; Le Hir et al.,2001; Lykke-Andersen et al., 2001). Consistent withthis, is the finding that UPF3a and UPF3b shuttleand co-purify with Y14 (Maquat, 2004). There arealso reasonable, but not definitive, data to suggestshuttling of UPF2 and UPF1 (Mendell et al., 2002;Ohnishi et al., 2003). Components of the EJC, suchas Y14, Magoh, eIF4IIIA and RNPS1 are also re-quired for NMD (see above). Other UPF factors,such as the cytoplasmic UPF1, are not associatedwith the EJC, indicating that although recruitmentof some of the NMD factors on the mRNA occursin the nucleus, other critical factors do not bind tothe mRNA until a later step(s). Barentsz is anotherfactor required for NMD in mammalian cells and isalso thought to bind the mRNA in the cytoplasm.Barentsz binds eIF4AIII, suggesting that the EJCmay help this factor associate with the mRNA(Palacios et al., 2004).

A current model for how nuclear events influenceNMD in mammalian cells is as follows: the form-ation of a functional complex for NMD is initiatedby the deposition of the EJC upstream of the exon–exon junction after splicing (Figure 2). Subsequently,

Figure 2 The role of splicing and the EJC in NMDDuring RNA splicing the EJC is positioned 20–24 nt upstream

of exon–exon junctions. RNPS1 interacts with the core EJC

factors via a direct interaction with Y14 and in turn recruits

UPF3. Immediately following or during export, UPF2 binds

to UPF3 and promotes the association of a key NMD factor

UPF1. The Barentsz (Btz) protein also associates with the core

EJC factors and is involved in NMD. During the first round of

translation, if a PTC exists (*) > 50–55 nt upstream of the ter-

minal exon junction, UPF1 is activated and triggers the de-

struction of the PTC-containing mRNA.

UPF3a and UPF3b, and then UPF2 are recruited toform an EJC competent for NMD. Probably in thecytoplasm, both Barentsz and UPF1 are recruitedto the mRNA, and during the first round of trans-lation the ribosome displaces the EJCs. However, thepresence of a PTC upstream of the last exon–exonjunction results in premature termination of trans-lation and perhaps one EJC remaining bound to the



mRNA. Activation of UPF1 then triggers NMD,although how this occurs is not known.

The role of the EJC in NMD may not be uni-versal, since although many of the components areconserved, the EJC is not essential for NMD in yeast,Drosophila, and C. elegans (Maquat, 2004). These or-ganisms must have alternative methods to recruit theUPF factors to the mRNA.

Nuclear events and mRNA localizationSplicing, the EJC and oskar mRNA localizationRegulated mRNA localization and translation is crit-ical for early embryo development in many organ-isms, and is also important for the correct expressionof genes in neurons (Bashirullah et al., 1998; Palaciosand St Johnston, 2001). Recent evidence from Droso-phila indicates that splicing and the resulting nuc-lear RNP is essential for proper mRNA cytoplasmiclocalization of at least one mRNA. In Drosophila,establishment of the anterior-posterior and dorsal-ventral axes depends upon the correct localization andtranslational control of a number of maternal mRNAs.oskar mRNA, which encodes a posterior determin-ant, is localized to the posterior pole, and its localizedtranslation is critical for posterior patterning.

Localization of the oskar mRNA to the posteriorpole requires the splicing of the first exon–exon junc-tion (Hachet and Ephrussi, 2004). Interestingly, thereare three introns in the gene, and only the removalof the first intron is necessary for correct localization.This is unlikely to be due to specific cis-acting ele-ments in the intron, since substitution of a differ-ent intronic sequence does not affect the splicing-dependent localization. Consistent with a molecularlink between splicing and localization are the findingsthat several components of the EJC, such as the Y14–Mago heterodimer (Hachet and Ephrussi, 2001) andeIFIIIA (Palacios et al., 2004), are required for local-ization of oskar mRNA, suggesting that localizationis mechanistically coupled by the splicing-dependentdeposition of the EJC (Figure 3).

Genetic analysis has also identified other factorsthat are necessary for localization of the oskar mRNA

Figure 3 Nuclear oskar RNP assembly is required forproper cytoplasmic localization and translational controlProper posterior localization of oskar mRNA in Drosophila re-

quires splicing of the first intron and the functions of the core

EJC factors eIF4A3 and the Mago–Y14 heterodimer. It is pro-

posed that the EJC helps recruits other factors, such as hrp48

and Barentsz, to form an RNP competent for localization. Ad-

ditional factors involved in either localization or translational

repression of oskar mRNA include Bruno, which binds to the

Bruno response elements (BRE) in the oskar 3′ UTR, Staufen,

Orb and p68. Once in the cytoplasm oskar mRNP localization

complexes assemble into large granules in which the mRNA

is translationally repressed. These particles are then transpor-

ted to the posterior pole where the mRNA is subsequently

translated.

(Figure 3). Hrp48, a heteronuclear RNP (hnRNP)A/B homologue, is required for oskar mRNA localiz-ation/translational control, and binds elements near

mRNA localization: A process by which some specialized mRNAs are prevented from immediate translation once they reach the cytoplasm, and are instead‘transported’ to specific cellular destinations and translated locally. Many of these mRNAs encode for proteins that have important functions in developmentaldecisions or cell signalling processes.

Translational control: A diverse array of mechanisms designed to prevent specific mRNAs from being translated until the proper time or place in the cell isachieved. Most of the currently known mechanisms are associated with preventing translation initiation.



the 5′end and in the 3′ untranslated region (UTR)(Huynh et al., 2004; Yano et al., 2004). Since hrp48can homodimerize, it is proposed that binding of theprotein to the ends of the mRNA results in circular-ization, which may be necessary for both localizationand translational regulation (Yano et al., 2004). Thefact that hrp48 is both nuclear and cytoplasmic sug-gests that it may bind the mRNA in the nucleus(Huynh et al., 2004; Yano et al., 2004). Staufen andBarentsz are also required for posterior localizationof the mRNA but are thought to associate with themRNA in the cytoplasm (Palacios et al., 2004).

Although it is thought that an EJC is depositedapprox. 20–24 nt upstream of every exon–exon junc-tion, the results with oskar mRNA suggests thatEJCs and associated complexes are not always equal.For oskar, it is possible the EJC at the first exon–exon junction mediates interactions between factorsbound to other regions of the mRNA, such as the 5′cap, the 3′ UTR, or even the coding region, resultingin the formation of an RNP destined for posteriorlocalization. This may help to explain why localiz-ation of other mRNAs, such as bicoid and gurken(both of which are spliced transcripts), are not depen-dent upon splicing and why all EJC positions do notpromote mRNA localization.

Nuclear events may affect localizationof other mRNAsThe cytoplasmic localization of other mRNAs mayalso depend upon the binding of specific factors in thenucleus (Farina and Singer, 2002). Drosophila hrp48not only controls oskar but also gurken mRNA local-ization and translation. In addition, the hnRNPA/B-like factor, Squid, governs the localization and trans-lation of gurken mRNA (Goodrich et al., 2004).In Xenopus oocytes, the homologue of hnRNP I,VgRBP60, may influence the localization of the Vg1mRNA at the vegetal pole (Cote et al., 1999). In ratoligodendrocytes, hnRNPA2 regulates localizationand translation of the myelin basic proteins (MBP)mRNAs (Hoek et al., 1998). Export and localizationof MBP mRNAs also depends upon the activity of theQuaking proteins (Larocque et al., 2002). In addition,other shuttling proteins such as the zipcode-bindingprotein-1 (ZBP1), required for localization of β-actinmRNA in chick fibroblasts, may bind their mRNAsin the nucleus (Farina and Singer, 2002).

Nuclear export and translational controlChoice of nuclear export pathway and tra-2translational regulationAs discussed above, splicing and deposition of theEJC affects the stability or cytoplasmic localizationof an mRNA. The EJC also influences the translat-ability of an mRNA, although how it does so is notunderstood. Recent work from C. elegans indicatesthat other nuclear events such as the choice of exportpathway can also influence translation of an mRNA(Figure 4).

C. elegans has two sexes, males and hermaphrodites.Hermaphrodites are essentially female animals thatfirst produce sperm (a male cell fate) and then switchand produce oocytes (a female cell fate) (for reviewsee Goodwin and Ellis, 2002). The choice of the malecell fate, at least in part, depends upon the trans-lational regulation of the sex determining gene tra-2.Translational repression is mediated by the bindingof GLD-1 (germ-line defective 1), a member of theSTAR family (Jones et al., 1996), to elements locatedin the 3′ UTR (Jan et al., 1997, 1999).

Unlike most mRNAs, tra-2 mRNA nuclear ex-port is not dependent upon NXF-1 but instead re-quires CRM1 activity (Graves et al., 1999). ThisCRM1-dependent export is regulated by a 3′ UTRelement, called the TRE (for tra-2 retention element),and at least four factors: the C. elegans REF–ALY-1,REF–ALY-2, NXF-2, and the sex-determining factorTRA-1 (Kuersten et al., 2004). NXF-2 is in the samefamily as NXF-1, but it lacks a domain required fornuclear pore binding and export. TRA-1 is a zinc-finger transcription factor that is necessary for femalecell fate.

The data suggest that the association of the REF–ALY and NXF-2 proteins with the TRE results in anRNP that blocks NXF-1-dependent export. The sub-sequent binding of TRA-1 to the 3′ UTR remodelsthe RNP resulting in the export of a TRA-1–tra-2mRNA complex (and possibly other TRE-bindingfactors) into the cytoplasm. The association of at leastTRA-1 with the mRNA may help GLD-1 represstra-2 translation since loss of the TRE control notonly results in export of the mRNA by NXF-1,but also increased tra-2 translation and inappropriatefemale development (Kuersten et al., 2004).

This work indicates that the REF–Aly factors havemore complex roles in mRNA metabolism than ori-ginally thought. It is possible that REF–Aly may



Figure 4 Choice of nuclear export pathway for tra-2 mRNA in C. elegans affects translational control in the cytoplasmIn the nucleus NXF-2, REF–ALY-1 and REF–ALY-2 bind to the TRE within the 3′ UTR of tra-2 mRNA. This complex functions

to prevent export of tra-2 via the NXF-1 pathway. When present, TRA-1 binds to the 3′ UTR altering the RNP such that a

TRA-1–tra-2 mRNA complex exits the nucleus via a CRM1-dependent process. This specific export pathway is required for

proper translational control in the cytoplasm. It is possible that TRA-1 (and perhaps other TRE binding factors) acts with GLD-1,

which binds the translational control elements (black arrows), to repress translation.

bind a subset of mRNAs to regulate their exportactivities. This idea is consistent with data that indi-cate removal of REF–Aly activity in Drosophila tis-sue culture cells by RNAi had only a minor effecton the export of poly(A)+ mRNA in a populationof cells (Gatfield and Izaurralde, 2002). Hence, theremay be other adaptor molecules that recruit NXF-1to the mRNA besides REF–Aly. Alternatively, simi-lar to the organism-specific roles for eIF4AIII andBarentsz (in mammals they regulate NMD, but inflies they control mRNA localization), it is possiblethat REF–Aly in mammals affect many mRNAs viathe EJC, but in flies and worms REF–Aly has morespecific regulatory functions.

Concluding remarksIn summary, the different processing, export andcytoplasmic events that affect an mRNA are tightlylinked, and RNP formation is crucial for the finalactivity of a given mRNA. RNPs are dynamic withproteins entering and exiting at every step along theway. How an RNP is remodelled and how this affectsthe activity of an mRNA is not known. Also it is

not well understood how the different events, such astranscription and processing, recruit different factorson the mRNA. Work from several labs has indicatedthat there may be translation in the nucleus (Iborraet al., 2001; Buhler et al., 2002; Wang et al.,2002). Although nuclear translation is controversial(Hentze, 2001; Dahlberg et al., 2003), it is conceiv-able that expression and processing of mRNAs couldalso affect this process. It is apparent also that aspecific protein can play different roles in the livesof an mRNA depending upon the structure ofthe mRNA, other factors that are associated with thegiven mRNA and what organism is being investi-gated. Presumably, the remodelling of RNPs playsa critical role in moving the mRNA from one pro-cessing event to another. It will not be too surpris-ing if many other examples of nuclear events linkingcytoplasmic destiny are discovered in the near future.

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Received 19 August 2004; accepted 4 November 2004

Published on the Internet 24 May 2005, DOI 10.1042/BC20040106


Linking nuclear mRNP assembly and cytoplasmic destiny

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