Nuclear mRNA export: insights from virology

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Nuclear mRNA export: insights from virology Bryan R. Cullen Howard Hughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA

To maximize the production of progeny virions, several viruses have evolved mechanisms that promote the selective nuclear export of viral mRNA transcripts while, in some cases, inhibiting the export of cellular mRNAs. To achieve this goal, viruses have evolved regulatory proteins and cis-acting RNA elements that selectively interact with key cellular nuclear export factors. Efforts to identify the cellular targets of these viral proteins and RNA elements have led to the identification of Crm1 and Tap as essential human nuclear RNA-export factors and continue to provide insights into how mRNAs are selected for export The segregation of the eukaryotic cell into nuclear and cytoplasmic subcellular compartments requires that pathways must exist to transport specific macromolecules between these compartments [1]. mRNAs represent an essential class of macromolecules that are produced in the nucleus, yet are primarily used in the cytoplasm. Moreover, functional mRNAs require extensive nuclear processing before their use in translation, including capping, splicing and polyadenylation. Therefore, it is important that nuclear export-factor recruitment should be regulated such that it only occurs when an mRNA is fully mature [2]. Efforts to define the mechanisms underlying nuclear mRNA export have proceeded in several distinct but complementary experimental systems. Because nucleocytoplasmic-transport pathways are well conserved among eukaryotes, genetic screens using the yeast Saccharomyces cerevisiae have been valuable in identifying gene products mutation of which perturbs nuclear mRNA export. However, it can be difficult to define the actual function of these gene products in the yeast system. Conversely, microinjection assays in Xenopus oocytes, which can be readily separated into nuclear and cytoplasmic fractions, can be a powerful tool in defining the role of a particular protein in a nucleocytoplasmictransport pathway in metazoan cells, but this system is not genetically tractable. However, the small genome that is a defining characteristic of viruses does enables genetic approaches to be used in metazoan cells. When combined with microinjection and transfection assays, the analysis of mutant viruses that show defects in the nuclear export Corresponding author: Bryan R. Cullen ([email protected]).

of viral mRNAs has therefore provided several key insights into the mechanism of nuclear mRNA export. The HIV-1 Rev protein Although the genome packaged into retroviral virions is a single-stranded mRNA, the retroviral replication cycle proceeds via a double-stranded DNA derivative, termed a provirus, that is integrated into the host genome [3]. The pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1) has a total of nine genes that are expressed by alternative splicing of a single, initial proviral transcript that also forms the RNA genome. Importantly, HIV-1 replication requires the nuclear export and translation of unspliced, singly-spliced and multiply-spliced derivatives of this initial transcript (Fig. 1). Analysis of the coding potential of the various HIV-1 mRNAs reveals that the fully spliced mRNAs encode the viral regulatory proteins Tat, Rev and Nef, whereas incompletely spliced HIV-1 mRNAs primarily encode viral structural proteins (Fig. 1). Mutational inactivation of Rev was found to selectively block expression of these structural gene products, whereas the Tat, Nef and mutant Rev proteins continue to be expressed at normal, or even elevated, levels [4]. Subsequently, it became clear that, in the absence of Rev function, the incompletely spliced HIV-1 mRNAs that encode the viral structural proteins are retained in the cell nucleus [5,6]. By contrast, nuclear export of fully-spliced HIV-1 mRNAs, including the mRNA encoding Rev itself, is independent of Rev function (Fig. 1). Nuclear export of unspliced HIV-1 mRNAs also requires a structured cis-acting RNA target – the Rev response element (RRE) – which serves as a specific binding site for Rev [6]. Together, these data identified Rev as the first sequence-specific nuclear mRNA-export factor. An important question is why the HIV-1 mRNAs that encode the viral structural proteins are retained in the nucleus in the absence of Rev function but the mRNAs encoding the regulatory proteins continue to be exported normally. In fact, nuclear retention of these incompletely spliced viral mRNAs primarily results from the fact that they retain complete introns, including 50 and 30 splice sites. These splice sites are recognized by cellular mRNAprocessing factors, termed splicing commitment factors, that not only participate in intron removal but also normally prevent cellular pre-mRNAs (i.e. incompletely spliced cellular mRNAs) from exiting the nucleus [2,7].

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Fig. 1. Role of Rev in the HIV-1 life cycle. HIV-1 replication requires the cytoplasmic expression of unspliced, singly spliced and fully spliced viral mRNAs. In the absence of Rev function, or early in the viral life cycle, only fully spliced viral mRNAs, encoding the regulatory proteins Tat, Nef and Rev itself, are exported from the nucleus and expressed. By contrast, incompletely spliced viral mRNAs, encoding primarily viral structural proteins, are retained in the nucleus by cellular proofreading proteins that also prevent the nuclear export of cellular pre-mRNAs [5–7]. However, in the presence of Rev, these incompletely spliced viral mRNAs are exported and expressed due to the recruitment of Rev and its associated cellular cofactors to the cis-acting Rev response element (RRE) RNA target.

Although these commitment factors can prevent incompletely spliced mRNAs from accessing the canonical cellular mRNA-export pathway, they cannot prevent the nuclear export of HIV-1 mRNAs bound by Rev (Fig. 1). Of course, fully spliced HIV-1 mRNAs are not subject to nuclear retention by splicing commitment factors and are therefore competent to exit the nucleus by the same mechanism used by fully spliced cellular mRNAs. Mutational analysis of the HIV-1 Rev protein identified two distinct functional domains, an N-terminal sequence required for RRE binding and Rev multimerization and an , 10-amino-acid leucine-rich domain near the C terminus that serves as the Rev nuclear export signal (NES) [8,9]. The identification of the Rev NES enabled Fischer et al. [9] to investigate whether Rev functioned via the same pathway used by cellular mRNAs or by different nuclear RNA export pathway. These experiments were based on earlier work [10] that showed that different classes of RNA – specifically mRNA, U-rich small nuclear RNA (U snRNA) and tRNA – each used distinct nuclear export factors. This was demonstrated by studying the export of radiolabeled mRNAs, U snRNAs and tRNAs from the nucleus of microinjected Xenopus oocytes in the presence of a large excess of an unlabeled mRNA, U snRNA or tRNA competitor. In each case, the competitor only inhibited the nuclear export of its own RNA class. Microinjection of a large excess of the Rev NES was found to competitively inhibit U snRNA nuclear export, whereas export of mRNA and tRNA was unaffected [9]. These data indicated that http://tibs.trends.com

incompletely spliced HIV-1 mRNAs exit the nucleus via a pathway used by few, if any, cellular mRNAs. Efforts to identify the cellular target for the Rev NES subsequently showed that Rev directly interacts with Crm1, a member of the karyopherin family of nucleocytoplasmic-transport factors [1,11,12]. Crm1, like other karyopherins involved in nuclear export, binds its cargo in the nucleus in the presence of the GTP-bound form of the Ran GTPase (Fig. 2). After nuclear export, hydrolysis of the bound GTP to GDP causes a conformational shift that induces cytoplasmic cargo release, thus providing the directionality of this export pathway [1]. Crm1 also interacts with components of the nuclear pore complex (NPC), the portal used for all nucleocytoplasmic transport, and this interaction is essential for Crm1-mediated nuclear RNA export. It is now clear that Crm1 is also the crucial nuclear export factor for two classes of cellular RNAs, that is, U snRNAs and rRNAs [13] (Fig. 2). Both U snRNA and rRNA nuclear export is mediated by adaptor proteins bearing leucine-rich NESs similar to the prototypic NES first defined in Rev, thus explaining the result of the competition experiment described. As noted, microinjection experiments in Xenopus oocytes suggested that the majority of cellular mRNAs do not use Crm1 as a nuclear export factor. More recently, an extensive analysis of mRNAs expressed in drosophila cells treated with leptomycin B – a selective inhibitor of Crm1 function – failed to identify any mRNAs whose nuclear export is blocked [14]. However, two reports have suggested that a few human mRNAs might be subject to

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Fig. 2. Crm1 is a key nuclear RNA export factor. Crm1 – a member of the karyopherin family of nucleocytoplasmic transport factors – interacts with a wide range of nuclear proteins that contain leucine-rich nuclear export signals (NESs) and then transports these proteins to the cytoplasm via the nuclear pore complex (NPC) [1]. Nuclear cargo binding requires the GTP-bound form of the Ran GTPase and cytoplasmic cargo release is induced by hydrolysis of Ran–GTP to Ran– GDP. Several proteins that bind to Crm1 act as adaptor proteins for different types of RNA cargo, including Rev for Rev response element (RRE)-containing HIV-1 mRNAs, phosphorylated adaptor for RNA export (PHAX) for U-rich small nuclear RNAs (U snRNAs) and Nmd3 for the ribosomal RNAs (rRNAs) present in 60S ribosomal subunits [13]. The adaptor for nuclear export of 40S ribosomal subunits (X) remains unknown, although this process is also Crm1 dependent. It has been proposed that the proteins NXF3, APRIL and pp32 – all of which contain leucine-rich NESs – could act as adapters for the Crm1-mediated export of specific cellular mRNAs, but this hypothesis remains controversial [15,16].

nuclear export by Crm1 (Fig. 2) [15,16]. One line of evidence supporting this proposal comes from study of the AU-rich elements (AREs) present in the 30 -untranslated regions of several genes involved in cell signaling [15]. AREs bind the protein HuR, which in turn interacts with two nucleocytoplasmic shuttle proteins pp32 and APRIL. Both pp32 and APRIL contain leucine-rich NESs and interact with Crm1 (Fig. 2). Importantly, inhibition of Crm1 function using leptomycin B can result in the nuclear accumulation of mRNAs that contain AREs, even though the subcellular distribution of bulk poly(A)þ RNA remains unaltered [15]. These data imply that a specific subset of cellular mRNAs might be substrates for Crm1mediated nuclear mRNA export. Functional analysis of members of the human nuclear export family (NXF) has also suggested that some mRNAs might be exported by Crm1 [16]. As discussed in more detail later, bulk nuclear mRNA export in metazoan cells is mediated by a cellular factor called Tap or NXF1 (Fig. 3). Tap is not a karyopherin, and Tap-mediated nuclear export does not require its interaction with any karyopherin. Instead, Tap contains an essential domain located at the C terminus that directly interacts with components of the NPC. Although Tap is ubiquitously expressed, a closely http://tibs.trends.com

related protein – NXF3 – displays a highly tissue-specific expression pattern [16]. Although NXF3, like Tap, is a nucleocytoplasmic shuttle protein that can export nuclear mRNAs when tethered via a heterologous RNA-binding motif, NXF3 lacks the C-terminal NPC-binding domain that is crucial for Tap function. This conundrum was resolved by the demonstration that NXF3, unlike Tap, contains a leucine-rich NES that binds Crm1 [16]. NXF3 associates with poly(A)þ mRNA in vivo and, therefore, might function as a tissue-specific Crm1-dependent nuclear mRNA-export factor (Fig. 2). The retroviral constitutive transport element HIV-1 is a complex retrovirus; HIV-1 encodes not only the canonical structural proteins (Gag and Env) and enzymes (Pol) required for the retroviral life cycle but also additional auxiliary and regulatory proteins, including Rev (Fig. 1). By contrast, simple retroviruses, such as Mason– Pfizer monkey virus (MPMV), encode only Gag, Pol and Env [3]. Yet MPMV also encodes both an unspliced RNA that serves as the genome and as the Gag and Pol mRNA, as well as a spliced mRNA encoding Env. The same problem of avoiding nuclear retention of the introncontaining genomic MPMV RNA therefore exists.

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Fig. 3. Export of viral mRNAs via the cellular mRNA export pathway. Nuclear export of cellular mRNAs initiates with the regulated recruitment of the protein UAP56, which can occur co-transcriptionally or during mRNA processing [28,32]. In turn, UAP56 binds to Aly, which then recruits Tap and its partner Nxt [26,28]. The Tap–Nxt heterodimer interacts with components of the NPC and serves as the actual export factor for bound mRNAs. Although UAP56, Tap and Nxt are all essential for poly(A)þ RNA export in metazoan cells [23,24,35], Aly appears to be dispensable [34], thus suggesting the existence of other intermediate factor(s), indicated here by ‘X’. Viruses can circumvent the proofreading steps thought to be involved in UAP56 recruitment by direct recruitment of either Aly or the Tap/Nxt heterodimer to viral mRNA transcripts, as exemplified here for the ICP27 protein encoded by herpes simplex virus (HSV) and the constitutive transport element (CTE) RNA target found in the retrovirus Mason–Pfizer monkey virus (MPMV), respectively.

Mutational analysis of the MPMV genome identified the constitutive transport element (CTE), a structured RNA element that is required for Gag and Pol, but not Env, expression [17]. Importantly, the CTE was found to rescue structural gene expression when introduced into a Revdefective HIV-1 genome and to do so in the absence of any MPMV gene products. The CTE was, therefore, proposed to act as the target for a cellular nuclear RNA-export factor. Interest in identifying this factor was further increased by experiments in Xenopus oocytes showing that an excess of a CTE competitor inhibited nuclear export of mRNA, but not tRNA or U snRNA [18,19]. These data showed that the target for the CTE was likely to be an essential participant in nuclear mRNA export in human cells. Using biochemical approaches, Gru¨ter et al. [20] were able to identify Tap as the cellular target of the MPMV CTE (Fig. 3). The likely importance of this factor was immediately apparent because the yeast homolog of Tap, Mex67p, had been shown to be essential for poly(A)þ RNA nuclear export in yeast cells [21]. Importantly, Tap differs from Crm1 in not being a member of the karyopherin family of nucleocytoplasmic transport factors and does not require the Ran GTPase as a cofactor. Thus, these findings explained the previous observation that, in contrast to all other nuclear RNA-export pathways, nuclear mRNA export was independent of Ran function [22]. It is now known that Tap forms a heterodimer with a small cofactor termed p15 or Nxt (Fig. 3), and this interaction is essential for the high-affinity interaction of http://tibs.trends.com

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Tap with components of the NPC and, hence, for Tapmediated nuclear mRNA export [23,24]. Genetic screens in yeast cells subsequently identified the protein Yra1p (Ref or Aly in metazoan cells) as a Mex67p (or Tap)binding protein that, at least in yeast cells, is also crucial for nuclear mRNA export [25,26]. In turn, Yra1p interacts with Sub2p (UAP56 in metazoans) a protein that is also essential for nuclear mRNA export [27,28] (Fig. 3). The observation that Sub2p and UAP56 also participate in mRNA splicing and are preferentially recruited to introncontaining mRNAs [28,29] might explain the observation that spliced mRNAs are exported from the nucleus more efficiently than intronless versions of the same mRNA [30,31]. However, Sub2p and UAP56 also appear to be recruited to mRNAs by other, co-transcriptional mechanisms [32,33]. Nevertheless, current models suggest that nuclear export of metazoan mRNAs initiates with the recruitment of UAP56. UAP56, in turn, interacts with Aly leading finally to the recruitment of the Tap –Nxt heterodimer, which directly interacts with components of the NPC and thereby delivers the mRNA to the cytoplasm (Fig. 3). RNA interference experiments have demonstrated that UAP56, Tap and Nxt are all essential for nuclear poly(A)þ RNA export in metazoan cells, although Aly seems to be dispensable [23,24,34,35]. This has led to the proposal that there could be one or more additional factors that can serve as adapters between UAP56 and the Tap– Nxt heterodimer in human cells [34] (Fig. 3). One possible candidate for this role are several serine – arginine-rich (SR) proteins that have recently been shown to shuttle in and out of the nucleus and to specifically interact with Tap [36]. Nuclear export of herpesvirus mRNAs The fact that the retroviral life cycle requires the nuclear export of intron-containing viral RNAs, in the face of cellular proofreading mechanisms that seek to prevent the expression of cellular pre-mRNAs, means that retroviruses have been the most informative viral system in which to study nuclear mRNA export. However, other viruses have also evolved mechanisms to selectively promote the nuclear export of their mRNAs. One interesting example of this occurs in herpes simplex virus (HSV). ICP27 is a multifunctional protein expressed very early in the HSV life cycle and is essential for virus replication. Among other activities, ICP27 inhibits host-cell gene expression by blocking the splicing and, hence, the nuclear export of cellular mRNAs [37]. ICP27 is also a nucleocytoplasmic shuttle protein that has been reported to selectively bind HSV mRNAs, almost all of which are intronless, and to activate their nuclear export [37]. However, it remains unclear how ICP27 is able to specifically recognize the , 80 different HSV mRNAs expressed in infected cells. ICP27 function is not perturbed by inhibition of Crm1 function, suggesting that it must access another nuclear RNA-export pathway. It has now been demonstrated that ICP27 directly interacts with Aly, and hence indirectly with the Tap– Nxt heterodimer, to activate HSV mRNA nuclear export [38,39] (Fig. 3). In this way, ICP27 acts as a

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viral homolog of the cellular UAP56 protein (Fig. 3) and it appears possible that ICP27 might, in fact, compete with UAP56 for Aly binding. Well-conserved homologs of ICP27 are present in all herpesviruses that have been sequenced to date, including Epstein – Barr virus (EBV) in which the ICP27 homolog is termed EB2 or SM. Recent data show that EB2 also participates in EBV mRNA nuclear export and have demonstrated a specific interaction between EB2 and Aly that is crucial for this activity [40,41]. Therefore, it seems probable that the selective recruitment of the cellular Aly mRNA-export factor might be key to the nuclear export and expression of mRNAs encoded by many members of the herpesvirus family. Other viruses, other export pathways Although the viral nuclear mRNA-export systems discussed here are the most fully developed, study of other viruses has also shed light on nuclear mRNA export or has, at least, raised unresolved issues that have the potential to lead the mRNA-export field in new directions. Among retroviruses, it is clear that the majority of complex retroviruses, including all lentiviruses and members of the T-cell leukemia retrovirus family, encode Rev homologs [3]. An interesting Rev homolog was found in the human endogenous retrovirus K (HERV-K) family. The HERV-Ks appear to be extinct in nature and instead exist solely in the form of , 100 endogenous proviral ‘fossils’ that are integrated into the genomes of higher primates, including humans. Although these HERV-K proviruses entered the human genome up to 30 million years ago, many still encode, and indeed express, functional forms of the Rev protein [42]. Whether this viral protein serves any purpose in the human host is, however, unknown. Although the MPMV CTE provides one well-understood solution to the question of how simple retroviruses activate the nuclear export of their intron-containing genomic RNAs, this issue remains unresolved for most other simple retroviruses, including the intensely studied murine leukemia viruses. However, in the case of retroviruses belonging to the avian leukemia/sarcoma virus family, it has been possible to identify a CTE-like element that mediates the nuclear export of unspliced viral mRNAs [43]. However, the avian retroviral CTE does not bind to Tap, and the identity of the cellular factor that mediates its function is unknown. Two other virus families also merit a brief mention. In the case of adenoviruses, viral mutants that are unable to express functional forms of the E1B 55-kDa (E1B 55K) or E4 34-kDa (E4 34K) proteins are defective both for nuclear export of viral mRNAs and for inhibition of cellular mRNA export [44]. The E1B 55K – E4 34K heterodimer undergoes nucleocytoplasmic shuttling, and evidence has been presented showing that E4 34K directly interacts with Crm1 via a leucine-rich NES. Although this issue remains to be fully resolved, it appears that the E1B 55K –E4 34K heterodimer is likely to function as a Crm1dependent nuclear export factor that is specific for adenoviral mRNAs [44]. A final, and potentially very interesting, experimental system is presented by hepatitis B virus (HBV). HBV replicates as a nuclear episomal DNA and encodes several http://tibs.trends.com

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intronless viral mRNAs. Efficient expression of these mRNAs is dependent on a structured RNA element termed the post-transcriptional regulatory element (PRE) [45]. Although the PRE, like the MPMV CTE, functions independently of any viral proteins, it is distinct from the CTE in that it is not able to effectively support the nuclear export of intron-containing retroviral mRNAs. However, unlike the CTE, the PRE is able to functionally substitute for introns in supporting the efficient expression of cDNA copies of cellular genes [46] and, therefore, might function analogously to the exon-junction complex – a large protein complex assembled during mRNA splicing that marks the site where introns have been removed [47,48]. Thus, identification of the cellular target for the HBV PRE has the potential to shed significant light on the mechanism(s) by which splicing facilitates eukaryotic gene expression. Concluding remarks Virology has made several key contributions to our current understanding of how mRNAs are exported from the nucleus. Study of the HIV-1 replication cycle led to the identification of Rev as the first nuclear mRNA-export factor, enabled the definition of the most common form of NES and identified Crm1 as the cellular target for Rev NES function (Fig. 2). Analysis of MPMV CTE function has led to the identification of Tap as a crucial factor in the nuclear export of cellular mRNAs and, in conjunction with genetic analyses in yeast, represented a key step towards our current understanding of how cellular mRNAs are recruited for nuclear export (Fig. 3). By directly recruiting cellular nuclear RNA export factors to cis-acting viral RNA target sequences, these retroviruses are able to circumvent the proofreading mechanisms that regulate the nuclear export of cellular mRNAs. The ability of viruses to thereby uncouple viral mRNA export from mRNA processing has proven valuable in revealing the importance of this coupling in regulating the nuclear export and expression of host mRNA transcripts [49]. Although these are clearly important contributions, I note that the nuclear export and expression of mRNAs encoded by avian leukemia virus and by HBV is subject to regulation by cis-acting RNA elements whose cellular binding partners remain to be identified. Therefore, virology will probably continue to provide unexpected insights into the processes that regulate the post-transcriptional fate of both viral and cellular mRNAs. References 1 Nakielny, S. and Dreyfuss, G. (1999) Transport of protein and RNAs in and out of the nucleus. Cell 99, 677– 690 2 Stutz, F. and Izaurralde, E. (2003) The interplay of nuclear mRNP assembly, mRNA surveillance and export. Trends Cell Biol. 13, 319 – 327 3 Cullen, B.R. (1998) Retroviruses as model systems for the study of nuclear RNA export pathways. Virology 249, 203 – 210 4 Sodroski, J. et al. (1986) A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature 321, 412 – 417 5 Emerman, M. et al. (1989) The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell 57, 1155– 1165 6 Malim, M.H. et al. (1989) The HIV-1 Rev transactivator acts through a

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structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, 254 – 257 Chang, D.D. and Sharp, P.A. (1989) Regulation by HIV depends upon recognition of splice sites. Cell 59, 789– 795 Malim, M.H. et al. (1989) Functional dissection of the HIV-1 Rev transactivator – derivation of a trans-dominant repressor of Rev function. Cell 58, 205 – 214 Fischer, U. et al. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475– 483 Jarmolowski, A. et al. (1994) Nuclear export of different classes of RNA is mediated by specific factors. J. Cell Biol. 124, 627 – 635 Fornerod, M. et al. (1997) Crm1 is an export receptor for leucine rich nuclear export signals. Cell 90, 1051– 1060 Neville, M. et al. (1997) The importin-b family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr. Biol. 7, 767– 775 Cullen, B.R. (2003) Nuclear RNA export. J. Cell Sci. 116, 587 – 597 Herold, A. et al. (2003) Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J. 22, 2472– 2483 Brennan, C.M. et al. (2000) Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J. Cell Biol. 151, 1 – 13 Yang, J. et al. (2001) Two closely related human nuclear export factors utilize entirely distinct export pathways. Mol. Cell 8, 397 – 406 Bray, M. et al. (1994) A small element from the Mason– Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc. Natl. Acad. Sci. U. S. A. 91, 1256 – 1260 Pasquinelli, A.E. et al. (1997) The constitutive transport element (CTE) of Mason – Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16, 7500– 7510 Saavedra, C. et al. (1997) The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export. Curr. Biol. 7, 619 – 628 Gru¨ter, P. et al. (1998) TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, 649– 659 Segref, A. et al. (1997) Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)þ RNA and nuclear pores. EMBO J. 16, 3256 – 3271 Clouse, K.N. et al. (2001) A Ran-independent pathway for export of spliced mRNA. Nat. Cell Biol. 3, 97 – 99 Wiegand, H.L. et al. (2002) Formation of Tap-NXT1 heterodimers activates Tap-dependent nuclear mRNA export by enhancing recruitment to nuclear pore complexes. Mol. Cell. Biol. 22, 245– 256 Herold, A. et al. (2001) NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. RNA 7, 1768 – 1780 Stra¨sser, K. and Hurt, E. (2000) Yra1p, a conserved nuclear RNAbinding protein, interacts directly with Mex67p and is required for mRNA export. EMBO J. 19, 410 – 420 Stutz, F. et al. (2000) REF, an evolutionarily conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6, 638 – 650 Stra¨sser, K. and Hurt, E. (2001) Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p. Nature 413, 648 – 652 Luo, M-J. et al. (2001) Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 413, 644 – 647 Libri, D. et al. (2001) Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev. 15, 36– 41

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30 Rodrigues, J.P. et al. (2001) REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc. Natl. Acad. Sci. U. S. A. 98, 1030 – 1035 31 Luo, M-J. and Reed, R. (1999) Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. U. S. A. 96, 14937 – 14942 32 Kiesler, E. et al. (2002) HEL/UAP56 binds cotranscriptionally to the Balbiani ring pre-mRNA in an intron-independent manner and accompanies the BR mRNP to the nuclear pore. Curr. Biol. 12, 859– 862 33 Zenklusen, D. et al. (2002) Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol. 22, 8241 – 8253 34 Gatfield, D. and Izaurralde, E. (2002) REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export. J. Cell Biol. 159, 579 – 588 35 Gatfield, D. et al. (2001) The DexH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr. Biol. 11, 1716– 1721 36 Huang, Y. et al. (2003) SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol. Cell 11, 837 – 843 37 Sandri-Goldin, R.M. (1998) ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 12, 868 – 879 38 Koffa, M.D. et al. (2001) Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway. EMBO J. 20, 5769– 5778 39 Chen, I-H.B. et al. (2002) ICP27 interacts with the RNA export factor Aly/REF to direct herpes simplex virus type 1 intronless mRNAs to the TAP export pathway. J. Virol. 76, 12877 – 12889 40 Hiriart, E. et al. (2003) A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein. J. Biol. Chem. 278, 335– 342 41 Boyer, J.L. et al. (2002) The Epstein – Barr virus SM protein is functionally similar to ICP27 from herpes simplex virus in viral infections. J. Virol. 76, 9420– 9433 42 Yang, J. et al. (1999) An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein. Proc. Natl. Acad. Sci. U. S. A. 96, 13404 – 13408 43 Paca, R.E. et al. (2000) Rous Sarcoma virus DR posttranscriptional elements use a novel RNA export pathway. J. Virol. 74, 9507– 9514 44 Dobbelstein, M. et al. (1997) Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence. EMBO J. 16, 4276 – 4284 45 Huang, J. and Liang, T.J. (1993) A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products. Mol. Cell. Biol. 13, 7476– 7486 46 Lu, S. and Cullen, B.R. (2003) Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 9, 618 – 630 47 Kataoka, N. et al. (2000) Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell 6, 673 – 682 48 Le Hir, H. et al. (2000) The spliceosome deposits multiple proteins 2024 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19, 6860– 6869 49 Maniatis, T. and Reed, R. (2002) An extensive network of coupling among gene expression machines. Nature 416, 499 – 506