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Originally published In Press as doi:10.1074/jbc.M609727200 on April 20, 2007

J. Biol. Chem., Vol. 282, Issue 24, 17507-17516, June 15, 2007
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The Nuclear Export Signal of Splicing Factor Uap56p Interacts with Nuclear Pore-associated Protein Rae1p for mRNA Export in Schizosaccharomyces pombe*

Anjan G. Thakurta{ddagger}12, Saravana P. Selvanathan{ddagger}1, Andrew D. Patterson§, Ganesh Gopal{ddagger}, and Ravi Dhar{ddagger}3

From the {ddagger}Basic Research Laboratory, §Laboratory of Metabolism, Center for Cancer Research, NCI, and Pharmacology Research Associate Program, NIGMS, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 16, 2006 , and in revised form, April 20, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian UAP56 (Saccharomyces cerevisiae Sub2p) and its functional homologs belong to the conserved DECD box class of ATP-dependent RNA helicases that play important functional roles in multiple aspects of DNA and RNA metabolism (1). They are essential in a number of organisms, including yeast, nematode, and fruit fly (2). UAP56/Sub2p is directly involved in pre-messenger RNA splicing and nuclear export of messenger RNAs in S. cerevisiae and in metazoan cells (3). Recently, in Schizosaccharomyces pombe, we found that uap56 is an essential gene for growth and that Uap56p is critical for mRNA export (4). Mammalian UAP56 functions in both ATP-independent and ATP-dependent steps of the spliceosome assembly process (5). Uap56p homologs typically contain a characteristic ATP-binding site, a catalytic DECD box, and an RNA interaction motif. Mutations within the ATP-binding pocket or the catalytic DECD box residues abolished the splicing functions of UAP56 (5). The enzymatic activity of Uap56p homologs has not been directly linked to mRNA export.

In S. cerevisiae, Sub2p, was proposed to function in dissociating an intron branch point-binding protein (BBP)4 Mud2p from pre-mRNAs (6). Sub2p is also critical for the export of mRNAs of both intron-containing and intron-less genes (7, 8). It is known to be recruited to genes by Hpr1p, a component of the THO complex (9). THO is a transcription-elongation complex, and in yeast, in addition to Hpr1p, it contains three other nonessential proteins, Mft1p, Tho2p, and Thp2p (10). Once recruited, the major function of Sub2p in mRNA export is the recruitment of mRNA adapter Yra1p to elongating transcripts (8). Sub2p and Yra1p together with THO form the TREX complex that was suggested to physically link the transcription apparatus to mRNA export steps (7, 11). Although the recruitment of Yra1p to intron-containing messages depended on Sub2p, its recruitment to intron-less genes was found to be unaffected in a sub2-85 ts mutant strain. These results imply a second function of Sub2p unrelated to Yra1p recruitment (8). Both UAP56 and Sub2p directly bind Aly/Yra1p in vitro (7, 12). In a competitive binding experiment, mRNA export carrier Mex67p could displace Sub2p from Yra1p (7). It was suggested that removal of Sub2p from Yra1p in the nucleus allows Mex67p to target mature mRNPs to the nuclear pore complex (NPC). Because Mex67p is not essential in wild type S. pombe cells for mRNA export, these results led us to propose that Uap56p was not removed from mRNPs, rather it played a critical role in NPC targeting and the export of mRNAs.

S. pombe Uap56p was originally identified as a suppressor of a cold-sensitive S. cerevisiae strain ({Delta}nam8 prp40HA) (13). Recently, we found that it is physically and functionally linked to two mRNA export factors as follows: Rae1p, an NPC-associated essential mRNA export factor, and Mlo3p, an S. pombe homolog of Yra1p/Aly (4). Mlo3p and Rae1p do not interact with each other directly, but they could be linked via Uap56p in a ternary protein complex in vitro (4). Another mRNA export factor Dss1p could similarly link Mlo3p and Rae1p in vitro. Based on biochemical and in vivo experiments, we proposed that Uap56p and Dss1p-mediated links are critical for targeting mature mRNPs to the NPC (4).

In this study, we further explored Uap56p functions in mRNA export. Our results indicate that Uap56p does not function as an RNA-dependent helicase in mRNA export. However, its ability to bind RNA is important for exporting mRNA. We found a functionally important NES within Uap56p that mediates direct interactions with Rae1p. Loss of the NES function abrogates Rae1p interaction as well as ability to export mRNA. Taken together, these properties are consistent with Uap56p functioning as an export carrier of mRNA in S. pombe.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Culture—Basic cell culture techniques used in this study were as described previously (14, 15). The construction of the null strain of uap56 was as described (4). The strains used in this study were as follows: wild type cells h– leu1-32 ura4-D18, h– {Delta}uap56/pREP42x-uap56, h– prp2-1 (16), and h–nup184–1 rae1-167/pREP81X-rae1 (17).

Plasmid Constructions—The coding sequence for uap56 was amplified from the genomic DNA and inserted into pREP42X vector (18, 19). To get a genomic construct, the gene for uap56 along with its promoter and 3'-untranslated region were inserted into an S. pombe vector pRL1 that carried the auxotropic marker LEU2 gene of S. cerevisiae. This plasmid was further used to make mutations within the uap56 coding sequences. Site-directed mutagenesis was carried out to introduce GK100–101DA, DECD to DEAD, DECD to EEAD, Q297R F320A, or R382AR385AR388A (RNA binding domain) mutations using the QuikChange site-directed mutagenesis kit from Stratagene. Plasmids for bacterial expression of N-terminal GST fusions of proteins were made using pGEX5X-3 vector.

Spot Assay for Growth—Growth conditions used have been described previously (4). Briefly (OD = 0.5), cells were serially diluted and plated on YEA or EMM media. Growth was monitored after 3 days at 30 °C.

In Situ Hybridization—In situ hybridization method used was described previously (20). Oligo(dT)50 carrying an {alpha}-digoxigenin at its 3'-end was used as the hybridization probe, and rhodamine-anti-digoxigenin was used for detecting the hybridization signal by using fluorescence microscopy. DAPI was used to stain DNA in the nucleus.

Expression and Purification of Recombinant Proteins—All proteins were expressed and purified from Escherichia coli strain BL21 using the standard purification protocols. For GST and GST fusion proteins, GSH-Sepharose beads from Amersham Biosciences were used. Proteins were eluted using 250 mM glutathione. Eluted proteins were dialyzed with the universal binding buffer (20 mM Hepes-KOH, pH 7.0, 100 mM KoAc, 2 mM Mg(OAc)2, 1 mM dithiothreitol, 0.1% Tween 20, and 10% glycerol) (21). For binding reactions, GST and GST fusion proteins were bound to GSH-Sepharose and incubated with 35S-Rae1p protein. The labeled Rae1p protein was synthesized using in vitro coupled transcription translation rabbit reticulocyte kit from Promega. Binding reactions were performed in the universal binding buffer (21). Bound proteins were separated on 4–12% NuPAGE gels (Invitrogen) and transferred to polyvinylidene difluoride membrane, and the membrane was then exposed to x-ray film. To detect the amount of Sepharose-bound proteins used in the binding reactions, polyclonal antibodies raised in rabbits against Uap56p were used. Standard chemiluminescent detection methods (PerkinElmer Life Sciences) were used to detect the proteins.

RT-PCR—Total RNA was extracted from S. pombe wild type and uap56 mutant cells using a Qiagen RNA extraction kit. beta-Tubulin cDNA synthesis was performed using the Titan one-step RT-PCR kit supplied by Roche Applied Science. The products were analyzed on agarose gels.

CHIP—Chromatin immunoprecipitation (CHIP) assays were performed by following published methods using polyclonal antibodies against Uap56p and Rae1p (22, 23). Chromatin DNA was sonicated to 0.5–1-kb size fragments and then immunoprecipitated. Gene-specific PCR primers for different regions of beta-actin gene were used to amplify the precipitated DNA. For control amplification, primer sets for a nontranscribed region was included in each PCR. The sequence of the primer set was 5'-CAACAGGAGCGCTATAATAA-3' and 5'-CAGATAGCTTGGATAGATATG-3'.[{alpha}-32P]dCTP was added to the PCR. The amplified products were separated by electrophoresis on 6% polyacrylamide gel and quantified using the PhosphorImager.

S. pombe NES Assay—The construction of the S. pombe vector, pAG177, for in vivo nuclear export assay has been described previously (24). The details of this assay have been briefly described under "Results."

HeLa Cell Nuclear Export Assay—Nuclear export assay in HeLa cells was performed according to the method described previously (25, 26). DNA sequence coding for specific NES segments of interest was fused to glucocorticoid receptor (Gr) and GFP DNA sequences in Gr-GFP vector. The details of the export assay are described in the figure legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
S. pombe Uap56p Is Not an Essential Splicing Factor—S. pombe uap56 is an essential gene for growth (4). A null strain of uap56 ({Delta}uap56/pREP42X-uap56) was kept viable by expressing Uap56p from a thiamine-repressible nmt1 promoter in pREP42X vector by growing cells in the absence of B1 (henceforth referred to as –B1 condition). Growth in [{alpha}-32P] presence of B1 (referred to as +B1 condition) results in depletion of Uap56p and inhibits growth. As a putative RNA-helicase, Uap56p contains a characteristic ATP-binding motif AKSGMGKT, the conserved catalytic DECD box, and the RNA-binding motif shown in Fig. 1A (5, 27). When the GKT motif was mutated to GNT in the ATP binding domain, both the ATPase activity and splicing were inhibited in S. cerevisiae sub2 mutant cells (5). We made mutations within the ATP binding domain of Uap56p (GK to DA) to determine the role of these mutations in splicing and mRNA export in S. pombe.

When the DECD box of mammalian Uap56 or S. cerevisiae Sub2p was mutated to DEAD or EEAD, its RNA helicase and splicing functions were inhibited (5). The conserved cystine and aspartic acid residues are located at the catalytic core and are thought to stabilize the SAT loop (28). To test the role of these mutations in splicing and mRNA export, we introduced these mutations in uap56.

Previous biochemical work in S. cerevisiae eIF4A showed a single arginine to alanine mutation within the RNA binding domain was sufficient to drastically reduce cross-linking of eIF4A to RNA and its helicase activity (27). Based on these experiments, we generated a triple mutant Uap56p (R382A/R385A/R388A) by replacing all three conserved arginine residues in the RNA-binding motif it shares with the helicase family members (1).

We then set out to test the roles of the ATP-binding mutant, the DECD box mutant, and the RNA-binding mutant in growth, splicing, and mRNA export. Surprisingly, {Delta}uap56 expressing the ATP mutant protein (G100D/K101A) or the DEAD or EECD mutant proteins were able to grow like the wild type strain (Fig. 1B). For comparison, {Delta}uap56 strain expressing uap56 (–B1 condition) grew like the wild type strain. But when Uap56p was depleted (+B1 condition) growth of {Delta}uap56 strain was inhibited (Fig. 1B). {Delta}uap56 cells expressing the RNA-binding triple mutant (R382A/R385A/R388A) were also viable (Fig. 1B) and grew only slightly slower than the wild type cells suggesting that the loss of RNA binding also did not affect the essential function of Uap56p.

To test the efficacy of pre-messenger RNA splicing, total RNA was extracted from {Delta}uap56 cells expressing Uap56p, depleted for Uap56p, or {Delta}uap56 cells expressing putative ATP-binding (G100D/K101A), RNA-helicase (DEAD or EEAD), or RNA-binding (R382A/R385A/R388A) mutant proteins. A pair of primers flanking the ends of the coding sequence of the intron-carrying beta-tubulin gene was used for RT-PCR to amplify cDNA from its transcripts (29). RNA extracted from {Delta}uap56 cells expressing Uap56p produced a single RT-PCR product of ~1400 bp, consistent with a fully spliced mRNA (Fig. 1C, lane 2). Interestingly, RNA made from the cells depleted for Uap56p also gave a single product consistent with fully spliced beta-tubulin transcripts (Fig. 1C, lane 3). For two other intron-containing genes dss1 and rae1, similar results were obtained (data not shown). As a control for spliced and unspliced transcripts, we used prp2-1 ts allele, which is known to inhibit RNA splicing at the restrictive temperature (16). RT-PCR performed on total RNA extracted from prp2-1 mutant cells at the permissive temperature (27 °C) produced a product consistent with normal splicing of the beta-tubulin transcript (Fig. 1C, lane 4). However, RNA isolated from cells grown at the nonpermissive conditions (37 °C, 3 h) produced a higher molecular weight RT-PCR product (~1730 bp) consistent with unspliced mRNA (Fig. 1C, lane 5). The data shown above suggest that loss of Uap56p does not result in the corresponding splicing defect. However, in the depletion experiments we could not rule out the possibility that a small amount of Uap56p was still present under depletion conditions that was sufficient for splicing. Because mutation in ATP-binding and DECD box motifs are known to inhibit splicing (5), we tested the efficiency of splicing by ATP-binding or the DECD box mutants. Total RNA was isolated from the cultures of {Delta}uap56 cells expressing either G100D/K101A, DEAD, or the EEAD mutant proteins. In all cases, RT-PCR-generated products were consistent with fully spliced beta-tubulin messenger RNAs (Fig. 1C, lanes 6–8). Finally, we tested whether pre-mRNA was fully spliced in cells expressing the RNA-binding Uap56p mutant protein. We found that the RT-PCR product obtained from cells expressing this mutant protein did not have any defect in the splicing of beta-tubulin mRNA (Fig. 1C, lane 9). These results strongly suggest that Uap56 is not required for splicing of pre-mRNAs in wild type S. pombe cells. It is not known whether the enzymatic activity of Uap56p/Sub2p is essential for the nuclear export of mRNA in eukaryotic cells. These results allowed us to investigate whether any of these properties affect mRNA export in S. pombe (see below).

Intact RNA-binding Motif by Uap56p Is Required for mRNA Export—S. pombe cells expressing the ATP-binding, the DECD box, or the RNA-binding mutant Uap56p proteins are viable. We wanted to know if these mutant cells could support nuclear export of mRNA. As expected, wild type and {Delta}uap56 cells expressing Uap56p had normal mRNA export (Fig. 1D, panels a and b), and cells depleted for Uap56p accumulated poly(A)+ RNA in the nucleus (Fig. 1D, panel c). Surprisingly, however, {Delta}uap56 cells expressing the ATP-binding mutant protein, G100D/K101A, had no detectable accumulation of poly(A)+ RNA in the nucleus (Fig. 1D, panel d), demonstrating that presumptive loss of ATP binding did not affect the ability of Uap56p to export mRNA.

We also found that {Delta}uap56 cells expressing Uap56p with DEAD or the EEAD mutations did not inhibit nuclear export of mRNA (Fig. 1D, panels e and f). Because helicase activity presumably requires intact catalytic residues as well as an ability to bind and hydrolyze ATP, these results indicate that putative enzymatic functions of Uap56p are not essential for driving mRNA export in S. pombe. In contrast, we found extensive nuclear poly(A)+ RNA accumulation in the cells expressing the RNA-binding mutant protein (Fig. 1D, panel g). Taken together, these results indicate that Uap56 functions in mRNA export presumably as a component of the nuclear export complex rather than as an enzyme. The functional requirement of RNA binding further suggests that Uap56p may directly interact with mRNAs for mediating their export.

Uap56p May Shuttle between the Nucleus and the Cytoplasm—In human and yeast cell, Uap56p/Sub2p is stably located in the nucleus. Uap56p/Sub2p is recruited to the transcripts during elongation and is thought to be released from the mRNP complex before the complex is targeted to the NPC (2). Our previous results suggest that Uap56p is likely targeted to the NPC (4). We wanted to know if Uap56 can exit the nucleus. GFP fused to full-length Uap56p at the C terminus localized to the nucleus (4) (reproduced here in Fig. 2B, panel a). First, we determined the region of Uap56p that contains the nuclear import signal. Different segments of Uap56p were fused to GFP at the C terminus, and their cellular localization was determined (Fig. 2, A and B). We found that GFP fusion of the N-terminal half (1–250 aa) of Uap56p localized to the nucleus (Fig. 2B, panel c). In contrast, the C-terminal half (251–434 aa) showed a diffused localization (Fig. 2B, panel b). Within the N-terminal half of Uap56p, we found that a GFP fusion of residues 1–216 aa, 1–100 aa, and 101–250 aa were diffused, whereas that of 50–250 aa was nuclear but less than that observed for the 1–250-aa fragment (N) (Fig. 2B, panels d–g)). Based on these analyses, the N terminus of Uap56 appears to contain a bipartite nuclear import signal, located between 1–100 and 216–250 aa.

We next explored if Uap56p contains an NES by using a previously described nuclear export assay in S. pombe. The construction of the S. pombe vector, pAG177, for in vivo nuclear export assay has been described previously (24). pAG177 contains RNA-binding motifs (RRM) of crp79 fused to GFP at the C terminus. To identify an NES within Uap56, DNA fragments for various portions of the coding sequences were inserted between the RRM and the GFP encoding sequences creating RRM-NES-GFP fusions. Plasmids expressing the RRM-NES-GFP fusions were transformed into rae1-167 nup184-1/pREP81X-rae1 SL27 double mutant. The RRM-GFP (pAG177) control fusion protein is cytoplasmic when Rae1p is expressed (–B1) in the double mutant (Fig. 2D, panel a). Under synthetic lethal conditions when Rae1p is depleted, mRNA accumulates in the nucleus and retains the fusion protein (Fig. 2D, panel b). However, in the presence of NES the fusion protein can exit the nucleus showing a diffused localization of the fusion protein throughout the cell, indicative of a functional NES (25).


Figure 1
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  		FIGURE 1.
Splicing and poly(A)+ RNA localization in uap56 mutant cells. A, schematic diagram of Uap56p and the location of different mutation sites used in this study. B, growth comparisons of different {Delta}uap56 cells expressing Uap56p mutants with wild type cells are as indicated. C, total RNA was extracted from {Delta}uap56/pREP42X-uap56 strain in the presence and absence of B1 (+B1 and –B1), prp2-1 mutant cells, and {Delta}uap56 cells expressing the mutant Uap56p proteins, Uap56p (G100D/K101A (GK100–101DA)), Uap56p (DEAD), Uap56p (EEAD), or Uap56p RNA-binding mutant. Lanes 1 and 10 are the marker lanes. The beta-tubulin cDNA is as indicated for {Delta}uap56/pREP42X-uap56 cells in the absence and presence of B1 (lanes 2 and 3), prp2-1 cells at 27 and 37 °C (lanes 4 and 5), uap56 (G100D/K101A), uap56 (DEAD), uap56(EEAD), and uap56 RNA-binding mutant cells (lanes 6–9). D, poly(A)+ RNA localization in wild type cells (panel a), {Delta}uap56/pREP42X-uap56 cells in the absence and presence of B1 (panels b and c), uap56 (G100D/K101A) GK100–101DA)(panel d), uap56 (DEAD) (panel e), uap56 (EEAD) (panel f), and uap56 (RNA-binding) (panel g) mutant cells. The corresponding lower panels (panels h–n) show DAPI staining of the DNA.

 
Fig. 2C shows a schematic diagram of the fragments used in the nuclear export assay. By using this assay, we found that a region between 250 and 434 aa (Fig. 2D, panels c and d) of Uap56p but not 1–250 aa (data not shown) was capable of exporting the RRM-NES-GFP fusion in SL27 cells under synthetic lethal conditions. But the fusion protein relocated to the nucleus in the double mutant following inactivation of Rae1-167p at 35 °C for 30 min (Fig. 2D, compare panels c, d, and e). These data suggest that under synthetic lethal conditions the fusion protein was not immobilized in the cytoplasm by some retention mechanism. By deletion analyses, the NES activity was mapped between 250 and 350 aa (Fig. 2D, panel f). A further deletion of 250–280 aa or 282–350 aa substantially damaged the export activity (Fig. 2D, panels g and h). Thus, we conclude that the full NES is contained within the 250–350-aa region. We next made several mutations within the NES (250–350 aa) to identify residues that are critical for mediating nuclear export of the fusion protein. Among these residues we identified two single mutations Q297R and F320A within the 100-aa region (Fig. 2C) that caused partial loss of the nuclear export activity (data not shown). When these two mutations were combined, there was a significant loss of nuclear export of the fusion protein as judged by the nuclear localization of the RRM-NES (Q297R/F320A)-GFP fusion (Fig. 2D, panel i).

To further test whether the NES property is evolutionarily conserved, we used a HeLa cell-based nuclear export assay. For this assay, as described previously (24, 25), the test export sequence was inserted between DNA fragments encoding Gr and GFP, and its ability to export the fusion was tested. As a positive control, nuclear export activity from Rev-NES was used, whereas Gr-GFP was used as a negative control (Fig. 2E). By using this assay we confirmed that the 250–350 aa of Uap56p was able to export a Gr-GFP fusion from the HeLa cell nucleus (Fig. 2E). The same sequence carrying the Q297R/F320A double mutations was unable to export Gr-GFP under the conditions of the export assay (Fig. 2E). Thus, Uap56p contains a conserved nuclear export signal within the C-terminal half between the 250- and 350-aa region. Taken together, Uap56p carries a nuclear import and a conserved nuclear export signal raising the possibility that it shuttles between the two cellular compartments.

Mutations within the NES Region Inhibit Growth and mRNA Export—We next tested the role of the NES in mRNA export and growth. Cells expressing the NES mutant Uap56p grew slower than {Delta}uap56 cells expressing the wild type Uap56p (Fig. 3A). Therefore, mutations within the NES affected cellular growth.

In parallel we tested the level of mRNA export in the strains expressing mutant Uap56p (Fig. 3B, panels c–e). The presence of Uap56p Q297R led to some loss of function as {Delta}uap56 cells expressing the mutant protein accumulated poly(A)+ RNA in the nucleus (Fig. 3B, panel c). In the case of Uap56p F320A, there was a distinct pattern of poly(A)+ RNA at the nuclear pore suggesting a loss of Uap56p function affecting movement of mRNA at the NPC (Fig. 3B, panel d and inset). When Q297R and F320A mutations were combined in Uap56p, the double mutant accumulated mRNA in the nucleus (Fig. 3A, panel e). These results suggest that the functional integrity of NES is important for Uap56p to export mRNA.

NES Region of Uap56p Binds Rae1p—We previously showed that Rae1p and Uap56p interact with each other (4). To analyze which region of Uap56p interacts with Rae1p, we made GST fusions of full-length, N-terminal (1–252 aa) and C-terminal halves (252–434 aa) of Uap56p. We then tested their ability to interact with 35S-Rae1p made in rabbit reticulocyte extract. As expected, GST-Uap56p was able to interact with Rae1p, whereas GST alone could not (Fig. 3C, top, compare lanes 3 and 2). Furthermore, the C-terminal half, but not the N-terminal half, was able to bind 35S-Rae1p under the same experimental condition (Fig. 3C, top, compare lanes 5 and 4). Because the C-terminal region contains the NES, we tested GST fusion of 250–350 aa (GST-NES) of Uap56p for its ability to bind Rae1p. We found that GST-NES fusion was able to retain Rae1p efficiently (Fig, 3C, lane 6). We then tested the effect of the NES mutations on Rae1p interaction. A GST-NES fusion carrying the double mutation Q297R/F320A did not interact with 35S-Rae1p (Fig. 3C, lane 7). GST fusion of a full-length Uap56p with these two mutations was also unable to bind 35S-Rae1p (Fig. 3C, top, lane 8). These results suggest that the NES is the only region of Uap56p that interacts with Rae1p. It is likely that a loss of this biochemical interaction is the basis for loss of mRNA export function of Uap56p.

Uap56p Is Recruited Early in mRNA Export—We used immunoprecipitation of chromatin DNA by anti-Uap56p antibody in the wild type strain to test whether Uap56p is recruited to genes. PCR amplification of different regions of beta-actin gene (promoter, 5'-end, middle region, and 3'-end) was used to determine Uap56p recruitment (Fig. 4A). For control, a nontranscribed region from S. pombe genome was co-amplified after immunoprecipitation (see figure legends and "Experimental Procedures" for details). Using [32P]dCTP, quantitative PCR was performed to amplify different regions of the beta-actin gene as well as the nontranscribed control region from the immunoprecipitated extract by the anti-Uap56p antibody or pre-bleed sera (–IP). For quantitative measurement, the amount of PCR product obtained from the actin gene was normalized against the control region and the pre-bleed samples (Fig. 4C). Uap56p appeared to be enriched progressively from the promoter to the 3'-end region of beta-actin gene. Average enrichment was 1.4-fold at the promoter, 2.1-fold at the 5'-end, 3.2-fold at the middle portion, and 5.9-fold at the 3'-end, respectively (Fig. 4, B, panel a, and D, bars 1–4). These results together suggest that Uap56p associates with the beta-actin gene, and its recruitment is biased toward the 3'-end of the gene, consistent with its association with elongating transcripts. We conclude that Uap56p, like Sub2p, is recruited during early mRNA export steps.


Figure 2
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  		FIGURE 2.
Characterization of the import and export signals of Uap56p. A, schematic diagram of Uap56p and its different coding fragments fused to GFP. The expression of the fusion proteins was driven by uap56 genomic promoter. The GFP localizations of the fusion proteins are shown as N for nuclear and D for diffused. B, localization of Uap56 and its fragments fused to GFP in S. pombe cells. FL stands for full-length Uap56-GFP (panel a), C-terminal half (251–434 aa-GFP) (panel b), N-terminal half (1–250 aa-GFP) (panel c), 1–216 aa-GFP (panel d), 50–250 aa-GFP (panel e), 1–100 aa-GFP (panel f) and 101–250 aa GFP (panel g). Their corresponding DAPI panels are shown (panels h–n). C, schematic diagram showing the C-terminal fragments of Uap56 fused to GFP used in D and E. Asterisk denotes the site of mutations. D, localization of RRM-GFP fusion proteins pAG177-GFP and various RRM-NES-GFP fusions expressed in nup184-1 rae1-167/pREP81X-rae1 double mutant cells in the presence (+B1) or absence (–B1) of thiamine. RRM-GFP localization in –B1 and +B1 (panels a and b) and various RRM-NES-GFP fusions in the absence (panel c) or presence of +B1 for 20 h at permissive temperature (panel d) and at the restrictive temperature of 35 °C for 30 min after the cells were grown in presence of B1 for 20 h (panel e) are shown. Panels f–i correspond to different fragments used to map the NES region within the C terminus of Uap56. Their corresponding DAPI panels are shown below (panels j–r). E, regions of NES or their mutants as indicated in the figure were expressed in HeLa cells as Gr-GFP chimeric proteins. The localization of the fusion proteins under different treatment conditions is indicated. No treatment, the fusion protein is largely cytoplasmic. Import, cells treated with 2 µM corticosteroid for 30 min at 37 °C, and the fusion protein accumulates in the nucleus. Export, cells were washed following treatment with the hormone with PBS to remove the hormone; 30 min of incubation was performed at 37 °C in the presence of cycloheximide to inhibit new protein synthesis. Gr-GFP was used as negative control and Gr-Rev-NES-GFP as positive control.

 
We next wanted to test whether the RNA-binding mutant could be recruited to genes. We used {Delta}uap56 cells expressing the triple mutant Uap56p (R382A/R385A/R388A) in similar immunoprecipitation experiments as above and found that the recruitment pattern of the mutant Uap56p was similar to the wild type protein, i.e. less at the promoter (1.9-fold over the control) and more toward the 5'-end (2.9-fold), middle (3.9-fold), and 3'-end (4.2-fold) of beta-actin gene (Fig. 4, B, panel b, and D). These results indicate that RNA binding was not a critical factor in the initial recruitment of Uap56p. This is not surprising, however, becauseUap56p, like Sub2p, may be recruited by multiple mechanisms, including protein-mediated interactions with THO components such as Hpr1p (8). In addition, our results suggest that interaction between Uap56p and RNA takes place after the initial recruitment step.

Rae1p Is Not Recruited to Genes—Rae1p interacts with Dss1p and Uap56p, both of which appear to be recruited to genes (see Ref. 4 and the results above). We wanted to test whether Rae1p was also recruited to genes. We performed immunoprecipitation experiments with wild type cells by anti-Rae1p antibody and tested for its enrichment on beta-actin gene by using similar experimental conditions as before. Rae1p did not associate with any region of beta-actin gene significantly. (Fig. 4, B, panel c, and D). These results therefore suggest that Rae1p may not be associated with the genes. Uap56p-Rae1p interaction is therefore likely a later step in mRNA export.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that Uap56p is not essential for splicing in S. pombe, and its mRNA export functions do not require a functional ATP-binding pocket or catalytic residues of the DECD box. In contrast, RNA binding is critical for Uap56p to function in mRNA export. We provide evidence that Uap56p has import as well as NES. Thus, Uap56p may be able to shuttle between the nucleus and the cytoplasm. Uap56p physically interacts with Rae1p via the NES. This interaction is critical for both its nuclear export activity as well as mRNA export function. These results suggest Uap56p functions nonenzymatically in mRNA export presumably as part of the mRNP-export complex.

S. pombe Uap56p May Function as an mRNA Carrier—Based on the Rev paradigm, an ability to bind RNA and the possession of a NES are considered critical attributes in a protein to potentially function as an RNA carrier (2). Export carriers are also known to shuttle between the two cellular compartments. Our results suggest that Uap56p possesses all of these attributes. Mutational analyses of Uap56p revealed that the RNA-binding motif was functionally important for Uap56p in mRNA export. In RNA-helicase E1F4A, these mutations were shown to abolish RNA binding (30). We found that RNA binding was not important for the recruitment of Uap56p to genes. At this time it is not clear at what step of mRNA export RNA binding becomes critical for function of Uap56p. If Uap56p functions as a carrier, functional interaction with RNA can take place during or after splicing of the messages.

We found a 100-amino acid sequence at the C-terminal half of Uap56p that contains a functional nuclear export signal that is also conserved in human cells. Nonclassical import sequences were located in the N-terminal half of Uap56p. These results are consistent with Uap56p shuttling between the two cellular compartments. Notably the Chironomus tentans homolog of Uap56p, HEL, was shown to co-migrate with exporting mRNPs and was seen to be released within the nuclear pores prior to the release of Aly (31). Taken together, our results suggest that Uap56p likely functions as an mRNA export carrier in S. pombe.

How does S. pombe Uap56p function in mRNA export? We found that in S. pombe Uap56p was recruited to genes early. Based on biochemical interaction studies, we previously demonstrated the formation of an Mlo3p-Uap56p-Rae1p complex. These results suggested that Uap56p may link Mlo3p to Rae1p (4). In this study we show that Uap56p interacts with Rae1p via the NES, and this interaction is vital for the function of Uap56p in mRNA export. Unlike human RAE1, S. pombe Rae1p is mostly localized at the NPC and is thought to function at the nuclear pore (4). We also found that Rae1p is not actively recruited to genes. It is reasonable to hypothesize that Uap56p-Rae1p interaction takes place at the nuclear pore where Rae1p is bound to Nup98p of the NPC. Uap56p could be involved with targeting and export of mature mRNP to the NPC via its interaction with Rae1p.


Figure 3
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  		FIGURE 3.
Growth and poly(A) +RNA localization of NES uap56 mutant cells and interaction with Rae1p. A, growth of {Delta}uap56 cells expressing only the Uap56 NES mutant proteins are shown as indicated (see also "Experimental Procedures"). Growth of {Delta}uap56 cells expressing wild type Uap56p from a thiamine-repressible promoter (–B1) or {Delta}uap56 depleted for Uap56p (+B1) as shown. B, poly(A)+ RNA localization in different strains used in A (panels a–e). Lower panels show their corresponding DAPI localization (panels f–j). C, 35S-labeled Rae1p was made in rabbit reticulocytes using the Promega in vitro rabbit transcription and translation kit. Uap56p and different Uap56p fragments were fused to the C terminus of GST. The fusion proteins as indicated were expressed and purified from E. coli. The purified proteins were bound to beads and incubated with 35S-labeled Rae1p protein. Bound 35S-Rae1p was identified by transferring the gel to nitrocellulose membrane and exposing the membranes to an x-ray film (upper panel). Lower panel shows corresponding GST fusion proteins (positions indicated by arrows) by Western blot analysis of the same membrane treated with {alpha}-Uap56 antibody. GST-Uap56p (Q297R, F320A) fusion protein showed a significant amount of degradation following purification.

 
Role of Rae1p in mRNA Export—Mex67p/TAP is a major mRNA export carrier in most eukaryotic cells. S. pombe {Delta}mex67 strain is viable and exhibits no mRNA export defect indicating that it is functionally redundant in wild type cells. But a combination of rae1-167 ts mutation with {Delta}mex67 resulted in synthetic lethality, suggesting a functional relationship between Rae1p and Mex67p (32). Our working hypothesis is that Rae1p functions at the nuclear pore for the assembly and disassembly of mRNP complexes. The physical interactions of Rae1p with Dss1p shown previously and its interactions with the Uap56p NES region described in this study provide new clues about how it may function during mRNA export. Based on whether Rae1p is static or dynamic at the NPC, we can envision two scenarios. First, the role of Rae1p could be limited to the targeting step itself where its interaction with Uap56p and Dss1p may help concentrate mRNP complexes. Subsequently, the mRNP complexes may interact with other components of the NPC for negotiating passage through the pore channels. Second and more interestingly, Rae1p could act dynamically as a receptor for the Uap56p NES and help the export of Uap56p (and thereby the mRNP complex) through the nuclear pore. Although S. pombe Rae1p was seen stably associated at the NPC, human RAE1 has been shown to shuttle between the nucleus and the cytoplasm. S. cerevisiae Gle2p was shown to be asymmetrically disposed within the NPC in electron microscopic studies (33). Irrespective of the mode of action of Rae1p, it appears to play a direct and critical role in mRNP targeting and export in S. pombe.


Figure 4
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  		FIGURE 4.
Recruitment of export factors Uap56p and Rae1p to genes. A, schematic diagram of the beta-actin gene and locations of PCR-amplified regions. Primer set 1 spans the promoter; set 2 spans the 5'-region; set 3 spans the middle; and set 4 spans the 3'-region of beta-actin gene, respectively. A nontranscribed region from S. pombe genome (see "Experimental Procedures") was used as control for immunoprecipitation. B, CHIP experiments were performed using extracts from wild type S. pombe strain with anti-Uap56p antibody (panel a) or anti-Rae1p antibody (panel c). Extracts made from {Delta}uap56 strain expressing R382A/R385A/R388A mutant Uap56p was used to immunoprecipitate chromatin by anti-Uap56p (panel b). WCE, whole cell extract; –IP, pre-bleed sera; +IP, anti-Uap56p or anti-Rae1p antibody. Control, co-amplified PCR fragment from nontranscribed region between SPAC4G8.03c and SPAC4G8.15c.of the S. pombe genome. PCR products were labeled using [32P]deoxycytidine triphosphate. C, relative precipitated enrichment was calculated based on formula as shown (23). D, quantification of CHIP experiments in B (panels a–c). Average band intensities corresponding to PCR-amplified fragments from three independent immunoprecipitation experiments were normalized according to the formula in C for quantification by a PhosphorImager. 1, 2, 3, and 4 represent fold amplification of different beta-actin region with respect to the double controls (pre-bleed and nontranscribed region). Amplification of beta-actin gene fragments in each immunoprecipitation experiment was quantified with respect to the nontranscribed control set to 1.

 
By combining functions of Uap56p and Dss1p in S. pombe, a picture emerges regarding mRNP targeting in S. pombe. Interactions between Rae1p and Mlo3p are mediated by Uap56p and Dss1p (4). In vivo and in vitro, Dss1p and Uap56p are both able to link the two proteins. Based on growth and the level of mRNA accumulation in the nucleus in the {Delta}dss1 and uap56 NES mutant strains, it appears that individually each interaction is sufficient for growth but not for efficient mRNA export. It is likely that simultaneous interactions with both proteins are required for productive association between mRNPs and NPC leading to efficient export of mRNAs.

Uap56p and Splicing in S. pombe—In S. cerevisiae, Sub2p is involved in an ATP-dependent early spliceosome assembly step where, together with DEX(H/D) protein Prp5p, it promotes an exchange of binding partners at the pre-mRNA branch site (5, 6). Branch point-binding protein initially binds the branch site. Mud2p is thought to stabilize the binding of BBP to the branch point. Sub2p may displace Mud2p, thereby destabilizing binding of BBP to the branch point (6). The removal of BBP allows the formation of a short duplex between pre-mRNA and U2 small nuclear RNP. This proposed function is deemed essential because Sub2p was no longer essential in a {Delta}mud2 background. The unexpected nonessentiality of Uap56p in splicing leads us to suspect the presence of another protein that may work with U2AF59p. The S. pombe data base includes at least 9 DEXD box helicases by comparison with the splicing machineries of S. cerevisiae and mammalian cells (16). It is possible that one of these proteins substitutes for Uap56p in splicing reactions.


   FOOTNOTES
 
* This work was supported by the Center for Cancer Research, the intramural program of the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Present address: AstraZeneca Pharmaceuticals, 1700 Auburn Ave., Rockville, MD 20850. Back

3 To whom correspondence should be addressed: Basic Research Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Bldg. 37, Rm. 5016, 37 Convent Dr., 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-0990; Fax: 301-480-5088; E-mail: dharr{at}mail.nih.gov.

4 The abbreviations used are: BBP, branch point-binding protein; aa, amino acid; NES, a nuclear export signal; GST, glutathione S-transferase; RT, reverse transcription; CHIP, chromatin immunoprecipitation; mRNP, messenger ribonucleoprotein; Gr, glucocorticoid receptor; RRM, RNA-binding motifs; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; NPC, nuclear pore complex. Back



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