- Email: [email protected]
mRNA export: Travelling with DEAD box proteins
Dispatch
R961
mRNA export: Travelling with DEAD box proteins Patrick Linder† and Françoise Stutz*
Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene. Addresses: *Institut de Microbiologie, Rue du Bugnon 44, 1011 Lausanne, Switzerland. †Département de Biochimie médicale, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Genève 4, Switzerland. E:mail: [email protected] Current Biology 2001, 11:R961–R963 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Evidence has been accumulating for some time now that the different steps of gene expression in eukaryotic cells — from transcription in the nucleus to translation and degradation in the cytoplasm — are intimately linked to control the fate of RNAs produced by RNA polymerase II. In particular, it has become clear from several studies that splicing facilitates export in metazoan systems, and that mutations that affect splicing in yeast can also inhibit mRNA export [1–3]. Recent studies [4–7] with yeast, Xenopus, Drosophila and mammalian cells have added another example of the way pre-mRNA splicing is coupled to mRNA export from the nucleus. The mammalian protein UAP56, and its yeast homologue Sub2p, have previously been shown to be required for an early ATP-dependent step in pre-mRNA splicing [8–11]. It now appears that, in the absence of functional UAP56/Sub2p, mRNA export is severely affected. Unexpectedly, this export defect involves not only mRNA derived from intron-containing genes, but also mRNA encoded by intron-less genes. UAP56/Sub2p is a member of the DEAD box family of putative RNA helicases [12]. In contrast to other members of this family, the UAP56/Sub2p proteins from different species contain a DECD motif instead of the DEAD motif. The mammalian UAP56 was first described as a U2AF65 interacting protein [8]. U2AF65 is the pyrimidine-richelement binding protein that needs to be removed from the pre-mRNA prior to U2 snRNP binding in spliceosome assembly. Depletion of UAP56 prevents U2 snRNP binding and renders splicing extracts inactive. Interestingly, the growth defect caused by deletion of the SUB2 gene in yeast can be bypassed to some extent by deletion of the MUD2 gene, which encodes the yeast homologue of U2AF65 [9]. In addition, the yeast SUB2 gene was found as a suppressor of the brr1 and ∆nam8/prp40 splicing mutations [10,13]. Thus, Sub2p is, by genetic and biochemical criteria, a bona fide splicing factor.
Once the mRNA is fully processed, it is exported from the nucleus to the cytoplasm through the nuclear pore complexes. Work in yeast and in a mammalian system has shown that the DEAD box protein Dbp5p is required for this process [14–16]. Depletion of Dbp5p leads to a rapid accumulation of poly(A)+ mRNA in the nucleus. Dbp5p is recruited to the cytoplasmic side of the nuclear pore complex through a conserved interaction with Nup159p, and is likely to function in a terminal step of mRNA export [15,16]. The finding that another DEAD box protein is required for mRNA export is rather exciting, and emphasises the importance of ATP-dependent helicaselike proteins in dynamic rearrangements during the export process. Nevertheless, the results of RNA inactivation (RNAi) experiments in Drosophila have indicated that the Dbp5p homologue, DBP80, is not essential for cell growth in this organism [7]. The explanation for the discrepancy between yeast and Drosophila is not yet clear and needs further investigation. Two groups working on yeast [5,6] have recently reported that Sub2p is likewise required for mRNA export. Ed Hurt’s group [5] used a genetic approach to isolate and characterise genes likely to be involved in mRNA export. To do so, they used a mutated YRA1 gene to perform a screen for synthetically lethal mutations. Yra1p, a member of the REF family of RNA and export factor binding proteins, participates in mRNA export by facilitating the recruitment of the essential shuttling export receptor Mex67p to the mRNP [17–19]. Mex67p forms a heterodimeric complex with Mtr2p which mediates the interaction of the mRNP with the nuclear pore complex (Figure 1). Amongst 30 candidates isolated, they found mutations in mex67 (six), mtr2 (two) and sub2 (fourteen). In the second yeast study, Jensen et al. [6] examined the potential role of Sub2p in mRNA export, as SUB2 mutations induce polyadenylation defects. Indeed, it has previously been shown that proper 3′ end formation and polyadenylation is required for efficient mRNA export [2,6,20]. Both groups [5,6] show convincingly that poly(A)+ mRNA accumulates in the nucleus after depletion or inactivation of Sub2p. Most interestingly, the accumulation is not only restricted to mRNAs generated through splicing, but also affects intron-lacking mRNAs. What is true for yeast is also true for higher eukaryotes. Robin Reed’s group [4] found that UAP56 immunoprecipitated with Aly/REF, the homologue of yeast Yra1p in higher eukaryotes. Moreover, injection of UAP56 into Xenopus oocytes interfered with mRNA nuclear export, but not that of U1 snRNA or tRNA. These observations suggest that a limiting export factor, such as Aly/REF, is
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Current Biology Vol 11 No 23
Figure 1
PolII
DNA Sub2p/ UAP56 Yra1p/ Aly Npl3p CBC
Spliceosome
ATP
AAAAAAAA
AAAAAAAA Mex67p/ TAP Mtr2p/ p15
NPC Nucleus
Cytoplasm Dbp5p
AAAA eIF4
Release
Translation Current Biology
The mRNA ‘relay race’ from the site of transcription in the nucleus to the site of delivery to ribosomes in the cytosol. Sub2p/UAP56, Yra1p/Aly/REF and hnRNP proteins such as Npl3p associate co-transcriptionally with the mRNA (CBC is the cap-binding complex). In the case of intron-containing genes, the spliceosome also assembles on the pre-mRNA. In mammalian cells, Aly/REF and UAP56 are part of the exon-junction complex on the spliced mRNA (not shown). The Sub2p/UAP56 protein is replaced by the Mex67p–Mtr2p/TAP-p15 heterodimers, which mediate the interaction of the mRNP with components of the nuclear pore complex (NPC). The DEAD box protein Dbp5p is required for release of mRNP on the cytoplasmic side of the NPC. DEAD box-mediated ATPase activities important for mRNA export are indicated by stars (see text for details).
titrated by an excess of UAP56. Indeed, Reed’s group were able to show that concomitant injection of Aly/REF alleviated the inhibitory effect of UAP56. Elisa Izaurralde’s group [7] found that depletion by RNAi of HEL, the Drosophila homologue of UAP56, caused growth arrest and a defect in mRNA export. They found that depletion of HEL in Drosophila inhibited the export of mRNAs derived from intron-containing as well as intron-less genes, resulting in a general decrease in translation and methionine incorporation.
In principle one would expect that the splicing factor UAP56 is recruited to the mRNA during the splicing process and remains associated with the mRNA as part of the exon-junction complex. In higher eukaryotes, the exonjunction complex is deposited on the mRNA at a fixed position upstream of the exon–exon junction as a result of splicing. The exon-junction complex, which contains Aly/REF, greatly enhances export of spliced mRNAs by facilitating the recruitment of TAP, the human homologue of Mex67p [3,21]. The observation that spliced mRNA coimmunoprecipates with UAP56 from Xenopus oocyte extracts or HeLa nuclear extracts supports the view that UAP56 is part of the exon-junction complex [4,7]. However, UAP56 also interacts, though to a lesser extent, with mRNA lacking the exon-junction complex. Taken together, the genetic and biochemical data suggest that, in higher eukaryotes as well as in yeast, UAP56/Sub2p is loaded on mRNA irrespective of whether it derives from an intron-containing or intronless primary transcript. These data are consistent with the finding that the export of mRNA derived from intron-less genes is also affected by depletion of Sub2p in yeast [5,6]. Thus UAP56/Sub2p/HEL appears to be a conserved and mRNA-specific nuclear export factor. From the results discussed above, it cannot be completely ruled out that the effect of UAP56 deficiency is an indirect consequence of a splicing defect. There is, however, strong evidence against this possibility. Firstly, the onset of the mRNA export defect in a yeast sub2 mutant is very fast (within 10 minutes) [5,6]. Secondly, an excess of UAP56 efficiently blocks mRNA export in Xenopus oocytes [4]. Thirdly, splicing of HSP83 pre-mRNA was not affected in Drosophila cells that were depleted of UAP56 and exhibited a strong mRNA export defect [7]. Finally, the two yeast proteins Sub2p and Yra1p also directly interact and, moreover, Mex67p competes with Sub2p for binding to Yra1p, indicating a common binding site for Sub2p and Mex67p on Yra1p [5]. These observations support the view that Yra1p/Aly/ REF is recruited to the mRNA ribonucleoprotein particle (mRNP) through an interaction with Sub2p/UAP56 which is subsequently displaced by Mex67p/TAP. Additional genetic evidence for a functional link between mRNA maturation and nuclear export has come from the work of Jensen et al. [6]. They found that the slow growth of a sub2 yeast mutant was synthetically enhanced by deletion of the RRP6 gene. RRP6 encodes a subunit of the ‘exosome’ — a multisubunit complex involved in 3′ end processing of mRNAs — and it has recently been shown that Rrp6 is necessary to retain hypo-adenylated as well hyper-adenylated mRNAs at their transcription sites [20]. Although not completely understood, this synthetic enhancement could result from the combination of a deficit in transcript retention in the absence of Rrp6p and reduced level of export activity in the absence of Sub2p.
Dispatch
Jensen et al. [6] also found that the growth and export defect of a sub2 mutant could be partially restored by mutations in another gene encoding a putative helicase, RAD3, which is involved in transcription-coupled excision repair. This suppression effect might indicate that a mutation in the RAD3 gene slows down the transcription machinery, allowing splicing and export to catch up. Alternatively, Sub2p may be more directly connected to the transcription machinery [6]. Indeed, it is interesting to note that SUB2 was isolated as a suppressor of a HPR1 deletion. HPR1 encodes an RNA polymerase II-associated protein that is required for transcription elongation and genome stability [22]. What then could the role of Sub2p be? It is too early to assign a clear function for this protein in mRNA export, but from what is known on DEAD box proteins in general, and Sub2p in particular, some possibilities come to mind. DEAD box proteins are thought to be ATP-driven motors that are mostly RNA-dependent. It is generally accepted that they separate double-stranded RNA molecules, but recent evidence suggests that they could more broadly be considered as ATP-driven translocating machines or dissociation factors (reviewed in [23]). Thus, Sub2p could dissociate double-stranded RNA, ribonucleoprotein complexes or even protein–protein interactions. The current data favour a model in which Sub2p/UAP56 cotranscriptionally recruits Yra1p/Aly/REF to the mRNP. In yeast, genetic interactions functionally link Sub2p to the transcription machinery [6,22], and Yra1p is recruited to the mRNP while the mRNA is still bound to the DNA template [24], and, finally, mammalian Aly/REF interacts with transcription factors [25,26]. The ATPase activity of Sub2p/UAP56 could be involved in a mechanism that coordinates the release of a fully processed mRNP from the transcription site, with the subsequent binding of the export factor Mex67p/TAP. In this view, Sub2p/UAP56p may contribute to a quality control mechanism [20] which helps to ensure that only correctly processed mRNAs are made available to the export machinery. Mex67p/TAP, as Mex67p–Mtr2p or TAP–p15 heterodimers, then direct the mRNP through the nuclear pore complex. On the cytoplasmic side, ATP hydrolysis by Dbp5p is required to release the mRNP for translation and to recycle the export factors to the nucleus. In conclusion, the dual role of UAP56/Sub2p reflects the need of the cell to interlink different processes to control the fidelity of the expression program. It is very likely that future work will reveal more such interconnections. References 1. Luo M-J, Reed R: Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl Acad Sci USA 1999, 96:14937-14942. 2. Brodsky AS, Silver PA: Pre-mRNA processing factors are required for nuclear export. RNA 2000, 6:1737-1749.
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3. Le Hir H, Gatfield D, Izaurralde E, Moore MJ: The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J 2001, 20:4987-4997. 4. Luo M-J, Zhou Z, Magni K, Christoforides C, Rappslibere J, Mann M, Reed R: Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 2001, 413:644-647. 5. Strässer K, Hurt E: The splicing factor Sub2p interacts directly with the transport factor Yra1p and is required for nuclear mRNA export. Nature 2001, 413:648-652. 6. Jensen TH, Boulay J, Rosbash M, Libri D: The DECD-box putative ATPase Sub2p is an early mRNA export factor. Curr Biol 2001, 11:1711-1715. 7. Gatfield D, Le Hir H, Schmitt C, Braun IC, Köcher T, Wilm M, Izaurralde E: The DExH/D protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr Biol 2001, 11:1716-1721. 8. Fleckner J, Zhang M, Valcarcel J, Green MR: U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev 1997, 11:1864-1872. 9. Kistler AL, Guthrie C: Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for Sub2, an essential spliceosomal ATPase. Genes Dev 2001, 15:42-49. 10. Libri D, Graziani N, Saguez C, Boulay J: Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev 2001, 15:36-41. 11. Zhang M, Green MR: Identification and characterization of yUAP/Sub2p. Genes Dev 2001, 15:30-35. 12. de la Cruz J, Kressler D, Linder P: Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci 1999, 24:192-198. 13. Staley JP, Guthrie C: Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 1998, 92:315-326. 14. Tseng SS-I, Weaver PL, Liu Y, Hitomi M, Tartakoff AM, Chang T-H: Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export. EMBO J 1998, 17:2651-2662. 15. Snay-Hodge CA, Colot HV, Goldstein AL, Cole CN: Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export. EMBO J 1998, 17:2663-2676. 16. Schmitt C, von Kobbe C, Bach IA, Pante N, Rodrigues JP, Boscheron C, Rigaut G, Wilm M, Seraphin B, Carmo-Fonseca M, Izaurralde E: Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p. EMBO J 1999, 18:4332-4347. 17. Strässer K, Hurt E: Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export. EMBO J 2000, 19:410-420. 18. Stutz F, Bachi A, Doerks T, Braun IC, Seraphin B, Wilm M, Bork P, Izaurralde E: REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 2000, 6:638-650. 19. Zenklusen D, Vinciguerra P, Strahm Y, Stutz F: The yeast hnRNP-like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p. Mol Cell Biol 2001, 21:4219-4232. 20. Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH: Quality control of mRNA 3′′ end processing requires the nuclear exosome. Nature 2001, 413:538-542. 21. Zhou Z, Luo MJ, Straesser K, Katahira J, Hurt E, Reed R: The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 2000, 407:401-405. 22. Fan HY, Merker RJ, Klein HL: High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability. Mol Cell Biol 2001, 21:5459-5470. 23. Tanner NK, Linder P: DExD/H box RNA helicases: From generic motors to specific dissociation functions. Mol Cell 2001, 8:251-262. 24. Lei EP, Krebber H, Silver PA: Messenger RNAs are recruited for nuclear export during transcription. Genes Dev 2001, 15:1771-1782. 25. Bruhn L, Munnerlyn A, Grosschedl R: ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function. Genes Dev 1997, 11:640-653. 26. Virbasius C, Wagner S, Green M: A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins. Mol Cell 1999, 4:219-228.
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mRNA export: Travelling with DEAD box proteins Patrick Linder† and Françoise Stutz*
Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene. Addresses: *Institut de Microbiologie, Rue du Bugnon 44, 1011 Lausanne, Switzerland. †Département de Biochimie médicale, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Genève 4, Switzerland. E:mail: [email protected] Current Biology 2001, 11:R961–R963 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Evidence has been accumulating for some time now that the different steps of gene expression in eukaryotic cells — from transcription in the nucleus to translation and degradation in the cytoplasm — are intimately linked to control the fate of RNAs produced by RNA polymerase II. In particular, it has become clear from several studies that splicing facilitates export in metazoan systems, and that mutations that affect splicing in yeast can also inhibit mRNA export [1–3]. Recent studies [4–7] with yeast, Xenopus, Drosophila and mammalian cells have added another example of the way pre-mRNA splicing is coupled to mRNA export from the nucleus. The mammalian protein UAP56, and its yeast homologue Sub2p, have previously been shown to be required for an early ATP-dependent step in pre-mRNA splicing [8–11]. It now appears that, in the absence of functional UAP56/Sub2p, mRNA export is severely affected. Unexpectedly, this export defect involves not only mRNA derived from intron-containing genes, but also mRNA encoded by intron-less genes. UAP56/Sub2p is a member of the DEAD box family of putative RNA helicases [12]. In contrast to other members of this family, the UAP56/Sub2p proteins from different species contain a DECD motif instead of the DEAD motif. The mammalian UAP56 was first described as a U2AF65 interacting protein [8]. U2AF65 is the pyrimidine-richelement binding protein that needs to be removed from the pre-mRNA prior to U2 snRNP binding in spliceosome assembly. Depletion of UAP56 prevents U2 snRNP binding and renders splicing extracts inactive. Interestingly, the growth defect caused by deletion of the SUB2 gene in yeast can be bypassed to some extent by deletion of the MUD2 gene, which encodes the yeast homologue of U2AF65 [9]. In addition, the yeast SUB2 gene was found as a suppressor of the brr1 and ∆nam8/prp40 splicing mutations [10,13]. Thus, Sub2p is, by genetic and biochemical criteria, a bona fide splicing factor.
Once the mRNA is fully processed, it is exported from the nucleus to the cytoplasm through the nuclear pore complexes. Work in yeast and in a mammalian system has shown that the DEAD box protein Dbp5p is required for this process [14–16]. Depletion of Dbp5p leads to a rapid accumulation of poly(A)+ mRNA in the nucleus. Dbp5p is recruited to the cytoplasmic side of the nuclear pore complex through a conserved interaction with Nup159p, and is likely to function in a terminal step of mRNA export [15,16]. The finding that another DEAD box protein is required for mRNA export is rather exciting, and emphasises the importance of ATP-dependent helicaselike proteins in dynamic rearrangements during the export process. Nevertheless, the results of RNA inactivation (RNAi) experiments in Drosophila have indicated that the Dbp5p homologue, DBP80, is not essential for cell growth in this organism [7]. The explanation for the discrepancy between yeast and Drosophila is not yet clear and needs further investigation. Two groups working on yeast [5,6] have recently reported that Sub2p is likewise required for mRNA export. Ed Hurt’s group [5] used a genetic approach to isolate and characterise genes likely to be involved in mRNA export. To do so, they used a mutated YRA1 gene to perform a screen for synthetically lethal mutations. Yra1p, a member of the REF family of RNA and export factor binding proteins, participates in mRNA export by facilitating the recruitment of the essential shuttling export receptor Mex67p to the mRNP [17–19]. Mex67p forms a heterodimeric complex with Mtr2p which mediates the interaction of the mRNP with the nuclear pore complex (Figure 1). Amongst 30 candidates isolated, they found mutations in mex67 (six), mtr2 (two) and sub2 (fourteen). In the second yeast study, Jensen et al. [6] examined the potential role of Sub2p in mRNA export, as SUB2 mutations induce polyadenylation defects. Indeed, it has previously been shown that proper 3′ end formation and polyadenylation is required for efficient mRNA export [2,6,20]. Both groups [5,6] show convincingly that poly(A)+ mRNA accumulates in the nucleus after depletion or inactivation of Sub2p. Most interestingly, the accumulation is not only restricted to mRNAs generated through splicing, but also affects intron-lacking mRNAs. What is true for yeast is also true for higher eukaryotes. Robin Reed’s group [4] found that UAP56 immunoprecipitated with Aly/REF, the homologue of yeast Yra1p in higher eukaryotes. Moreover, injection of UAP56 into Xenopus oocytes interfered with mRNA nuclear export, but not that of U1 snRNA or tRNA. These observations suggest that a limiting export factor, such as Aly/REF, is
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Current Biology Vol 11 No 23
Figure 1
PolII
DNA Sub2p/ UAP56 Yra1p/ Aly Npl3p CBC
Spliceosome
ATP
AAAAAAAA
AAAAAAAA Mex67p/ TAP Mtr2p/ p15
NPC Nucleus
Cytoplasm Dbp5p
AAAA eIF4
Release
Translation Current Biology
The mRNA ‘relay race’ from the site of transcription in the nucleus to the site of delivery to ribosomes in the cytosol. Sub2p/UAP56, Yra1p/Aly/REF and hnRNP proteins such as Npl3p associate co-transcriptionally with the mRNA (CBC is the cap-binding complex). In the case of intron-containing genes, the spliceosome also assembles on the pre-mRNA. In mammalian cells, Aly/REF and UAP56 are part of the exon-junction complex on the spliced mRNA (not shown). The Sub2p/UAP56 protein is replaced by the Mex67p–Mtr2p/TAP-p15 heterodimers, which mediate the interaction of the mRNP with components of the nuclear pore complex (NPC). The DEAD box protein Dbp5p is required for release of mRNP on the cytoplasmic side of the NPC. DEAD box-mediated ATPase activities important for mRNA export are indicated by stars (see text for details).
titrated by an excess of UAP56. Indeed, Reed’s group were able to show that concomitant injection of Aly/REF alleviated the inhibitory effect of UAP56. Elisa Izaurralde’s group [7] found that depletion by RNAi of HEL, the Drosophila homologue of UAP56, caused growth arrest and a defect in mRNA export. They found that depletion of HEL in Drosophila inhibited the export of mRNAs derived from intron-containing as well as intron-less genes, resulting in a general decrease in translation and methionine incorporation.
In principle one would expect that the splicing factor UAP56 is recruited to the mRNA during the splicing process and remains associated with the mRNA as part of the exon-junction complex. In higher eukaryotes, the exonjunction complex is deposited on the mRNA at a fixed position upstream of the exon–exon junction as a result of splicing. The exon-junction complex, which contains Aly/REF, greatly enhances export of spliced mRNAs by facilitating the recruitment of TAP, the human homologue of Mex67p [3,21]. The observation that spliced mRNA coimmunoprecipates with UAP56 from Xenopus oocyte extracts or HeLa nuclear extracts supports the view that UAP56 is part of the exon-junction complex [4,7]. However, UAP56 also interacts, though to a lesser extent, with mRNA lacking the exon-junction complex. Taken together, the genetic and biochemical data suggest that, in higher eukaryotes as well as in yeast, UAP56/Sub2p is loaded on mRNA irrespective of whether it derives from an intron-containing or intronless primary transcript. These data are consistent with the finding that the export of mRNA derived from intron-less genes is also affected by depletion of Sub2p in yeast [5,6]. Thus UAP56/Sub2p/HEL appears to be a conserved and mRNA-specific nuclear export factor. From the results discussed above, it cannot be completely ruled out that the effect of UAP56 deficiency is an indirect consequence of a splicing defect. There is, however, strong evidence against this possibility. Firstly, the onset of the mRNA export defect in a yeast sub2 mutant is very fast (within 10 minutes) [5,6]. Secondly, an excess of UAP56 efficiently blocks mRNA export in Xenopus oocytes [4]. Thirdly, splicing of HSP83 pre-mRNA was not affected in Drosophila cells that were depleted of UAP56 and exhibited a strong mRNA export defect [7]. Finally, the two yeast proteins Sub2p and Yra1p also directly interact and, moreover, Mex67p competes with Sub2p for binding to Yra1p, indicating a common binding site for Sub2p and Mex67p on Yra1p [5]. These observations support the view that Yra1p/Aly/ REF is recruited to the mRNA ribonucleoprotein particle (mRNP) through an interaction with Sub2p/UAP56 which is subsequently displaced by Mex67p/TAP. Additional genetic evidence for a functional link between mRNA maturation and nuclear export has come from the work of Jensen et al. [6]. They found that the slow growth of a sub2 yeast mutant was synthetically enhanced by deletion of the RRP6 gene. RRP6 encodes a subunit of the ‘exosome’ — a multisubunit complex involved in 3′ end processing of mRNAs — and it has recently been shown that Rrp6 is necessary to retain hypo-adenylated as well hyper-adenylated mRNAs at their transcription sites [20]. Although not completely understood, this synthetic enhancement could result from the combination of a deficit in transcript retention in the absence of Rrp6p and reduced level of export activity in the absence of Sub2p.
Dispatch
Jensen et al. [6] also found that the growth and export defect of a sub2 mutant could be partially restored by mutations in another gene encoding a putative helicase, RAD3, which is involved in transcription-coupled excision repair. This suppression effect might indicate that a mutation in the RAD3 gene slows down the transcription machinery, allowing splicing and export to catch up. Alternatively, Sub2p may be more directly connected to the transcription machinery [6]. Indeed, it is interesting to note that SUB2 was isolated as a suppressor of a HPR1 deletion. HPR1 encodes an RNA polymerase II-associated protein that is required for transcription elongation and genome stability [22]. What then could the role of Sub2p be? It is too early to assign a clear function for this protein in mRNA export, but from what is known on DEAD box proteins in general, and Sub2p in particular, some possibilities come to mind. DEAD box proteins are thought to be ATP-driven motors that are mostly RNA-dependent. It is generally accepted that they separate double-stranded RNA molecules, but recent evidence suggests that they could more broadly be considered as ATP-driven translocating machines or dissociation factors (reviewed in [23]). Thus, Sub2p could dissociate double-stranded RNA, ribonucleoprotein complexes or even protein–protein interactions. The current data favour a model in which Sub2p/UAP56 cotranscriptionally recruits Yra1p/Aly/REF to the mRNP. In yeast, genetic interactions functionally link Sub2p to the transcription machinery [6,22], and Yra1p is recruited to the mRNP while the mRNA is still bound to the DNA template [24], and, finally, mammalian Aly/REF interacts with transcription factors [25,26]. The ATPase activity of Sub2p/UAP56 could be involved in a mechanism that coordinates the release of a fully processed mRNP from the transcription site, with the subsequent binding of the export factor Mex67p/TAP. In this view, Sub2p/UAP56p may contribute to a quality control mechanism [20] which helps to ensure that only correctly processed mRNAs are made available to the export machinery. Mex67p/TAP, as Mex67p–Mtr2p or TAP–p15 heterodimers, then direct the mRNP through the nuclear pore complex. On the cytoplasmic side, ATP hydrolysis by Dbp5p is required to release the mRNP for translation and to recycle the export factors to the nucleus. In conclusion, the dual role of UAP56/Sub2p reflects the need of the cell to interlink different processes to control the fidelity of the expression program. It is very likely that future work will reveal more such interconnections. References 1. Luo M-J, Reed R: Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl Acad Sci USA 1999, 96:14937-14942. 2. Brodsky AS, Silver PA: Pre-mRNA processing factors are required for nuclear export. RNA 2000, 6:1737-1749.
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3. Le Hir H, Gatfield D, Izaurralde E, Moore MJ: The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J 2001, 20:4987-4997. 4. Luo M-J, Zhou Z, Magni K, Christoforides C, Rappslibere J, Mann M, Reed R: Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 2001, 413:644-647. 5. Strässer K, Hurt E: The splicing factor Sub2p interacts directly with the transport factor Yra1p and is required for nuclear mRNA export. Nature 2001, 413:648-652. 6. Jensen TH, Boulay J, Rosbash M, Libri D: The DECD-box putative ATPase Sub2p is an early mRNA export factor. Curr Biol 2001, 11:1711-1715. 7. Gatfield D, Le Hir H, Schmitt C, Braun IC, Köcher T, Wilm M, Izaurralde E: The DExH/D protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr Biol 2001, 11:1716-1721. 8. Fleckner J, Zhang M, Valcarcel J, Green MR: U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev 1997, 11:1864-1872. 9. Kistler AL, Guthrie C: Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for Sub2, an essential spliceosomal ATPase. Genes Dev 2001, 15:42-49. 10. Libri D, Graziani N, Saguez C, Boulay J: Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev 2001, 15:36-41. 11. Zhang M, Green MR: Identification and characterization of yUAP/Sub2p. Genes Dev 2001, 15:30-35. 12. de la Cruz J, Kressler D, Linder P: Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci 1999, 24:192-198. 13. Staley JP, Guthrie C: Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 1998, 92:315-326. 14. Tseng SS-I, Weaver PL, Liu Y, Hitomi M, Tartakoff AM, Chang T-H: Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export. EMBO J 1998, 17:2651-2662. 15. Snay-Hodge CA, Colot HV, Goldstein AL, Cole CN: Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export. EMBO J 1998, 17:2663-2676. 16. Schmitt C, von Kobbe C, Bach IA, Pante N, Rodrigues JP, Boscheron C, Rigaut G, Wilm M, Seraphin B, Carmo-Fonseca M, Izaurralde E: Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p. EMBO J 1999, 18:4332-4347. 17. Strässer K, Hurt E: Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export. EMBO J 2000, 19:410-420. 18. Stutz F, Bachi A, Doerks T, Braun IC, Seraphin B, Wilm M, Bork P, Izaurralde E: REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 2000, 6:638-650. 19. Zenklusen D, Vinciguerra P, Strahm Y, Stutz F: The yeast hnRNP-like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p. Mol Cell Biol 2001, 21:4219-4232. 20. Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH: Quality control of mRNA 3′′ end processing requires the nuclear exosome. Nature 2001, 413:538-542. 21. Zhou Z, Luo MJ, Straesser K, Katahira J, Hurt E, Reed R: The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 2000, 407:401-405. 22. Fan HY, Merker RJ, Klein HL: High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability. Mol Cell Biol 2001, 21:5459-5470. 23. Tanner NK, Linder P: DExD/H box RNA helicases: From generic motors to specific dissociation functions. Mol Cell 2001, 8:251-262. 24. Lei EP, Krebber H, Silver PA: Messenger RNAs are recruited for nuclear export during transcription. Genes Dev 2001, 15:1771-1782. 25. Bruhn L, Munnerlyn A, Grosschedl R: ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function. Genes Dev 1997, 11:640-653. 26. Virbasius C, Wagner S, Green M: A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins. Mol Cell 1999, 4:219-228.