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Previous Article | Next Article 
Molecular and Cellular Biology, April 2002, p. 2057-2067, Vol. 22, No. 7
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.7.2057-2067.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
John E. Donello,2,
and Thomas J. Hope1*
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612,1 Graduate Programs in Molecular Pathology and Biology, University of California, San Diego, La Jolla, California 920372
Received 25 June 2001/ Returned for modification 19 July 2001/ Accepted 21 December 2001
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ABSTRACT |
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INTRODUCTION |
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Much of our present understanding of nuclear export has come from the study of how viruses exploit host cell RNA processing and export pathways. The first viral export system studies were those of complex retroviruses, exemplified by human immunodeficiency virus type 1 (HIV-1). HIV-1 replication requires unspliced and partially spliced RNAs to be exported from the nucleus by the virally encoded Rev protein (9, 13, 44). Rev contains an RNA binding domain, which specifically binds to the Rev response element (RRE), located within the second intron of HIV-1 pre-mRNA, and a nuclear export signal (NES) that interacts with CRM1, a member of the importin ß family of transport receptors (6, 15, 17, 44, 58).
The interaction between Rev and CRM1 is dependent upon CRM1 association with the GTP-bound form of the GTPase Ran protein (RanGTP). Once assembled, the RRE/Rev-CRM1-RanGTP ribonucleoprotein complex interacts with NPs, which trigger its nuclear export. CRM1 has been proposed to mediate this interaction by directly contacting selected nucleoporins (NPs), including CAN/Nup214. Binding of CRM1 to CAN has been mapped to the NP domain located within the extreme carboxy terminus of CAN (16). Overexpression of the isolated NP domain of CAN, termed 
CAN, is able to inhibit Rev-mediated export by competing with the NPs for binding to CRM1 (2). Rev-mediated export is also inhibited by the antibiotic leptomycin B (LMB), which disrupts the interaction between NES and CRM1 (38, 39).
The use of the CRM1 export pathway is common among complex viruses. Other lentiviruses, such as feline immunodeficiency virus and equine infectious anemia virus, encode Rev-like proteins with atypical NESs that can interact with CRM1 (48, 51, 52). Complex oncoretroviruses, for instance, human T-cell leukemia virus and bovine leukemia virus, encode Rex proteins that are believed to use the CRM1 pathway (12, 23, 49). DNA viruses also utilize the CRM1 pathway. Thus, Epstein-Barr and herpes simplex viruses encode the MTA and ICP27 proteins, respectively, which use CRM1 to mediate the export of at least some of the virally encoded messages (45, 54, 56, 57). Additionally, influenza virus A uses the NS2 protein to mediate the export of viral messages via the CRM1 pathway (46, 47). Therefore, the use of virally encoded proteins to couple the export of viral RNA to the CRM1 pathway is a common strategy among the complex retroviruses. In contrast, simple viruses that use cellular RNA binding proteins for nuclear export do not use CRM1. The best characterized are the type D retroviruses. These simple retroviruses, exemplified by the Mason-Pfizer monkey virus, do not encode a Rev-like protein but rather act through a cis-acting RNA element named the constitutive transport element (CTE) (10, 11). Cytoplasmic accumulation of unspliced RNAs from these viruses involves the interaction between their CTEs and the host-encoded RNA binding protein Tap. Tap has been reported to directly bind the CTE and mediate the export of CTE-containing substrates (3, 21, 34). Although CTE and RRE/Rev can functionally substitute for each other in mediating nuclear RNA export, CAN and LMB do not inhibit CTE-mediated nuclear export (2, 48). These data indicate that the Tap export mechanism is distinct from that of Rev and that it is CRM1 independent. Recent data indicated that Tap itself is a nuclear transport factor that directly mediates the nuclear export of its substrate RNA (3, 32, 59). Like CRM1, Tap interacts with CAN/Nup214, albeit through a distinct interaction domain (1, 32, 34). These data suggest that there are at least two independent mRNA export pathways: (i) a CRM1-dependent pathway utilized by virally encoded RNA binding proteins and (ii) a CRM1-independent pathway utilized by cellular factors.
Recently, Tap has also been implicated in the export of spliced cellular mRNAs (3, 34, 50, 55). However, in this case, the interaction between Tap and the cellular spliced messages appears to be mediated via the RNA binding proteins Aly (also known as Ref) and Y14 (60, 62, 65). These proteins are components of the recently identified exon junction complex, which is left associated with the exon-exon junction after the intron is excised (5, 40).
In contrast to the above examples, human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) encode intronless messages. HBV and WHV messages contain specific cis-acting elements named posttranscriptional regulatory elements (PREs) (the HBV PRE [HPRE] and the WHV PRE [WPRE], respectively) that are essential for their expression (8, 27, 30). The activity of the PREs is independent of any virally encoded protein, suggesting that the PREs require as yet unidentified cellular trans-acting factors for function. Although the function of the PREs is unclear, their ability to work in a Rev-dependent assay and their requirement for RNA cytoplasmic accumulation have been suggestive of a role in export. Earlier experiments demonstrated that HPRE function is LMB resistant, indicating that, similar to CTE, it does not use CRM1 as an export receptor (48, 64). Mapping studies have demonstrated that HPRE and WPRE contain two homologous cis-acting sequences, or subelements, designated PRE
and PREß (7, 8). However, there are significant functional differences between HPRE and WPRE. In a Rev-dependent assay, WPRE is significantly more active than HPRE (8). WPRE also has the unique ability to posttranscriptionally stimulate the expression of heterologous cDNAs (41, 66). The increased activity correlates with the presence of an additional subelement, PRE
, which is not found in HPRE (8). The data suggest that WPRE, compared to HPRE, has an additional posttranscriptional activity that may shed light upon the mechanism by which intronless mRNAs are processed and exported from the nucleus.
In this study we demonstrated that WPRE posttranscriptional activity is both CRM1 dependent and independent. A potential for cooperation between CRM-dependent and -independent posttranscriptional activities is demonstrated by the observed cooperation between CRM1-dependent Rev and the CRM1-independent HPREß subelement. Hence, WPRE is the first example of an RNA element that is partially dependent upon CRM1 function, does not require virally encoded proteins for its cytoplasmic localization, and has an activity that is mediated by several alternative pathways that may not be mutually exclusive but may instead be cooperative.
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MATERIALS AND METHODS |
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HBVPRE surface expression vector (RV) (30) and its derivatives, RVHPRE and RVWPRE (8). To generate p138HPREß/Bul and p138HPREß(AS)/Bul, a fragment encompassing amino acids 1352 to 1684 of HBV, containing the HPREß subelement (7), was inserted in both orientations into the ClaI site located at the 5' end of the Bul fragment in pDM138Bul. To generate p138HPREß/Bul(AS) and p138HPREß(AS)/Bul(AS), the Bul fragment was inserted into the unique ClaI site located at the 3' ends of the HPREß subelements within p138HPREß and p138HPREß(AS). Rev was expressed from the plasmid pRSV-Rev (24) or ptk-Rev (26). The plasmid pBC12-CAN, expressing amino acids 1864 to 2090 of CAN/Nup214 (2), was a gift from Bryan Cullen (Howard Hughes Medical Institute, Duke University Medical Center). The plasmid pDMCRM1, expressing human CRM1, was kindly provided by David McDonald (Dept. of Microbiology and Immunology, University of Illinois at Chicago).
Cells lines and transient transfections.
The human and monkey kidney fibroblast cell lines, 293 and CV-1, respectively, were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and grown at 37°C. Approximately 24 h prior to transfection, 10-cm-diameter plates of confluent cells were split 1:60 into 6-well plates, for CAT or HBV surface antigen (HSAg) assay, and 1:10 into 15-cm-diameter plates, for Northern blot analysis. Transient transfections were performed using the calcium phosphate method (7). For CAT assays, cells were transfected with 0.25 µg of either pDM138 reporter constructs or 0.25 µg of pDM138RRE plus 0.25 µg of pRSV-Rev. CAN inhibition experiments were performed with 0.5 µg of pBC12-CAN. CAN, CRM1, and Rev dose-response experiments were performed in the presence of increasing levels of the pBC12-CAN (0 to 0.5 µg), pDMCRM1 (0 to 4 µg), or ptk-Rev (0 to 0.4 µg) expression plasmid. For CRM1 dose response, cells were transfected with 0.125 µg of pDM138WPRE. Each transfection included 0.25 µg of pCH110 (a simian virus 40 ß-galactosidase [ß-gal] reporter) and enough pUC118 or pCMVpoly(A) plasmid to bring the total amount of DNA to 2 µg. For the HSAg assay, cells were transfected with 0.25 µg of a RV reporter construct and 0.25 µg of a plasmid coding for a secreted alkaline phosphatase (SEAP). The total amount of DNA was kept constant (2 µg) with the pCMVpoly(A) plasmid. All transfections were performed in triplicate, and data were normalized to the cotransfected internal controls (ß-gal for the CAT assay and SEAP for the HSAg assay). For Northern blot analysis, cells in 15-cm-diameter plates were transfected with 4.5 µg of pDM138 constructs or 4.5 µg of pDM138RRE plus 4.5 µg of pRSV-Rev and 4.5 µg of pCH110. For CRM1 treatment, 2.25 µg of pDM138WPRE was used. Total transfected DNA was kept constant (30 µg) with the pUC118 or pCMVpoly(A) plasmids. LMB treatment was performed as previously described (48). Briefly, 18 h posttransfection, culture medium was replaced with fresh medium with or without 5 nM LMB. Cells were harvested for analysis 24 h later. No cell death was observed at the end of the 24 h LMB treatment. The CAT and HSAg assays have been described elsewhere (7, 8).
Northern blot analysis of RNA.
Cells from the 293 cell line were harvested using phosphate-buffered saline containing 5 mM EDTA, followed by a brief centrifugation. Nuclear and cytoplasmic RNAs were prepared as previously described (8), except that the cytoplasmic lysis buffer consisted of 0.01 M Tris (pH 8.00), 0.14 M NaCl, 0.0015 M MgCl2, 0.5% Nonidet P-40, and 20% glycerol. Cytoplasmic and nuclear poly(A)+ RNA selection was accomplished by using the Poly(A) Pure kit from Ambion according to the manufacturer's recommendations. Ten micrograms of cytoplasmic and 5 µg of nuclear poly(A)+ RNA were electrophoresed on 1% agarose gels containing 20 mM guanidine thiocyanate (20) and transferred to a Duralon-UV membrane (Stratagene). The 5' LTR probe was obtained by digesting pDM138 with XbaI and NotI. This probe hybridizes with unspliced pDM138 RNA. The ß-gal probe was obtained by digesting pCH110 with SacI and EcoRV. Radiolabeling of the probes with [-32P]dCTP was performed by using the Prime-It kit from Stratagene. Blots were hybridized with the probes in UltraHyb (Ambion), according to the manufacturer's instructions. Quantitation of the cytoplasmic accumulation of the unspliced and ß-gal mRNAs was performed using a Molecular Dynamics PhosphorImager. To correct for different transfection efficiencies, the data for the unspliced mRNAs were normalized to the ß-gal values.
Numbering of sequences. The sequence numbering presented in this report is based on the following GenBank sequences: D003329 for HBV, J02442 for WHV, and K03455 for HIV-1.
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RESULTS |
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CAN and LMB inhibit WPRE-mediated export.
To test whether the functional difference between HPRE and WPRE was CRM1-dependent, we used the dominant negative form of CAN/Nup214 (CAN), which was shown to specifically inhibit CRM1 (2). We hypothesized that, if WPRE function is mediated by CRM1, then the cytoplasmic accumulation of WPRE-containing RNA should be inhibited by overexpression of CAN. For this study we utilized the well-characterized pDM138 system. We have previously shown that the PREs could substitute for RRE/Rev in facilitating the export of unspliced pDM138 transcripts (7). A schematic representation of the pDM138 reporter and the constructs utilized in this study is presented in Fig. 1A. The reporter is derived from the second half of the HIV-1 sequence, with the CAT gene placed within the intron (24). In the absence of any export element, the CAT coding region is removed by splicing and degraded in the nucleus. Insertion of a functional transport element into the unique ClaI site results in cytoplasmic accumulation of unspliced reporter, allowing the CAT gene to be translated. Thus, CAT assay can be used to quantitate export activity of the element of interest. Human 293 cells were transfected with pDM138WPRE in the presence or absence of 0.5 µg of pBC12CAN, a concentration reported to be efficient in inhibiting Rev-mediated export (2). To demonstrate specificity for the observed effects, we used pDM138RRE/Rev as a positive control. As shown in Fig. 1B, overexpression of CAN disrupted Rev function, without perturbing the function of HPRE. In the absence of CAN, WPRE was 2.7 times more active than HPRE. Interestingly, CAN inhibited WPRE activity by 57%, to approximately the same level of activity as the HPRE. CTE RNA, the negative control, was not affected, which is consistent with previous results demonstrating that the CTE does not depend on a leucine-rich NES for its function (2, 48) (data not shown). The inhibition of WPRE function was specific, as demonstrated by a dose-response experiment, while increased concentrations of CAN had no effect on HPRE activity (Fig. 1C).
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CAN could also inhibit WPRE activity in the more natural context of the intronless surface mRNA, we used the HSAg assay. This assay uses an intronless HSAg expression vector (R5) from which the HPRE has been removed (30). Insertion of any of the PREs into the unique ClaI site contained within the intronless HSAg message results in efficient HBV surface gene expression that can be quantified in the culture medium by radioimmunoassay (8). The WPRE and HPRE surface constructs depicted in Fig. 2A were transiently transfected into 293 cells. As previously reported, WPRE-mediated surface protein expression was slightly higher than that mediated by HPRE (8). Similar to data obtained with the pDM138 reporter assay, overexpression of CAN did not inhibit HPRE-mediated surface antigen expression (Fig. 2B). However, WPRE-mediated surface expression was inhibited about 30%, to approximately the same level as HPRE-mediated expression (Fig. 2B). These results demonstrate that CAN can partially inhibit WPRE activity in a specific, dose-response fashion.
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50%, to levels similar to those observed for HPRE. These data confirm that WPRE-mediated accumulation of cytoplasmic RNA is partially dependent upon CRM1 function. The data also demonstrate that WPRE is not dedicated to an exclusive posttranscriptional processing pathway, but is instead a potential example of CRM1-dependent and -independent posttranscriptional processing events being mediated by a single RNA element.
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CAN to inhibit WPRE function suggests that CRM1 is required for the maximal activity of this element. To further explore this relationship, we determined the effect of CRM1 overexpression on the function of WPRE (Fig. 5). For this study, constant amounts of DNA encoding the reporters for the WPRE, HPRE, and RRE were cotransfected with increasing amounts of a CRM1 expression vector. To facilitate comparison, half as much of the WPRE reporter was transfected. The results of this study revealed that the activity of the WPRE was stimulated by the exogenous expression of CRM1, while the activity of RRE/Rev and HPRE did not change significantly (Fig. 5A). Cotransfection of larger amounts of the CRM1 expression vector did not have any additional stimulatory effect on any of the reporters. To explore the observation further, cytoplasmic (Fig. 5B) and nuclear (Fig. 5C) poly(A)+ RNAs were isolated from 293 cells transfected with either the WPRE or HPRE in the presence or absence of exogenous CRM1 and analyzed by Northern blot analysis. Again, half as much of pDM138WPRE relative to pDM138HPRE was transfected. Quantitation of the Northern blot analysis results, normalized to the ß-gal internal control, is shown below each blot. The RNA analysis revealed that the CRM1-mediated increase in levels of WPRE-stimulated expression was reflected by a corresponding increase in the cytoplasmic RNA levels. Importantly, the coexpression of CRM1 caused an increase in the ratio of cytoplasmic to nuclear RNA. Cytoplasmic WPRE RNA was increased by more than threefold, while the nuclear fraction increased by 50%. The increase in the amount of cytoplasmic WPRE RNA relative to the increase in the nuclear fraction when CRM1 is overexpressed is consistent with a stimulation of the rate of nuclear export of the WPRE-containing messages. In contrast, the levels of HPRE-containing messages are not altered by the overexpression of CRM1.
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This artificial element is relatively inactive by itself (28). However, duplication of the site generates an element with wild-type activity (28). Thus, the duplication of the high-affinity site mimics the function of the respective native response element (RRE), which contains both a high-affinity and several low-affinity binding sites (6, 37, 63). Our chimera, pDM138HPREß-Bul, consisted of HPREß (CRM1 independent) adjacent to Bul (CRM1 dependent). Three control reporters were also generated: both subelements in the antisense (AS) orientation, pDM138HPREß(AS)-Bul(AS), or only one subelement in the AS orientation, pDM138HPREß(AS)-Bul or pDM138HPREß-Bul(AS). The different reporter derivatives used in this experiment are schematically shown in Fig. 6A. The positive control consisted of pDM138Bul II, which contains two copies of the Bul element (28). These reporter constructs were transfected into CV-1 cells in the presence or absence of saturating amounts of Rev (25). The results are presented in Fig. 6B. The negative control, pDM138HPREß(AS)-Bul(AS), was not activated in the presence of Rev. Rev trans-activated pDM138HPREß(AS)-Bul to 20% of the level of the positive control, consistent with the construct containing only one copy of Bul. Due to the presence of HPREß, the construct pDM138HPREß-Bul(AS) exhibited a low level of activity (20% of the positive control) which was Rev-independent. This result was expected for the following reasons: (i) HPRE does not need Rev to function (48, 64), and (ii) HPRE subelements function cooperatively; therefore, the presence of only one subelement results in minimal activity (7). When Rev was cotransfected with pDM138HPREß-Bul, the level of activation was 86% of that of the positive control with Rev. These results demonstrated that combining HPREß and Bul can stimulate the pDM138 reporter to levels greater than those resulting from the sum of their individual activities.
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To confirm that the observed synergistic activation of pDM138 was the consequence of an increased accumulation of unspliced CAT-containing RNA, we performed a Northern blot analysis. Cytoplasmic (Fig. 7A) and nuclear (Fig. 7B) poly(A)+ RNAs were isolated from 293 cells transfected with pDM138, pDM138HPREß-Bul, pDM138HPREß, and pDM138Bul. Blots were probed for CAT (upper panels) and luciferase (lower panels). PhosphorImager data are shown under each blot. In the absence of Rev, the amount of exported pDM138Bul was the same as that of pDM138, as expected. In the presence of Rev, the amount of cytoplasmic pDM138Bul was increased slightly. In the absence of Rev, the chimeric element accumulated unspliced RNA in the cytoplasm slightly more than pDM138. This is consistent with the low level of activity previously reported for the HPREß subelement in CAT assays (7). In the presence of Rev, the export activity of the chimeric element was higher than those of the other constructs.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CAN overexpression can inhibit WPRE activity, but not HPRE activity, in both CAT and HSAg assays (Fig. 1 and 2). The inhibition of WPRE by CAN suggests that part of the WPRE activity requires functional CRM1. However, recent data have implied that CAN is not a selective inhibitor of CRM1 function (22). A fusion between Tap and an export-incompetent Rev mutant protein (RevM10) was LMB insensitive, indicating that it does not use the CRM1-mediated export pathway. However, CAN was able to inhibit RevM10-Tap-mediated export. It is not clear why CAN affects RevM10-Tap. Even though the C terminus of Tap has been shown to bind the FG repeat domain of CAN/Nup214 in vitro, effective binding by Tap requires a higher number of repeats (residues 1690 to 2090) than the number of repeats present in CAN (1, 34). Additionally, in vivo experiments demonstrated that Tap-mediated CTE and cellular mRNA export is not sensitive to CAN (2). A conformational change in RevM10-Tap could render this fusion protein sensitive to CAN, while the native Tap protein is insensitive. This could explain the ability of CAN to act as a selective inhibitor of CRM1 function. Moreover, if export function is LMB sensitive, it would be expected that function would also be sensitive to the overexpression of CAN, which is what we observe for WPRE function.
Numerous studies have demonstrated that LMB specifically inhibits NES-CRM1 interactions and directly blocks Rev-mediated export. In this study, LMB inhibited the nuclear and cytoplasmic RNA accumulation mediated by both Rev and WPRE but not that mediated by HPRE (lanes 6 in Fig. 4A and B). Prior studies (48) have also demonstrated that LMB treatment reduced the amounts of nuclear RRE RNAs. This suggests that the disruption of CRM1 function causes the unspliced nuclear RRE RNA and WPRE RNA to become unstable in the nucleus and probably to degrade. Alternatively, LMB may lower the amount of nuclear WPRE RNA available for export, which would result in a reduced amount of cytoplasmic WPRE RNA. However, the similar sensitivity and RNA accumulation profiles for WPRE- and RRE-containing RNAs suggests that part of the WPRE function is CRM1-dependent export. Support for this model comes from the observation that the overexpression of CRM1 stimulates WPRE function while having only a minimal effect on the activity of RRE/Rev and HPRE. Importantly, excess CRM1 causes a relative increase in the amount of cytoplasmic WPRE-containing messages relative to the nuclear pool of these messages. This shift is consistent with the increased amounts of CRM1 improving the efficiency of WPRE-mediated mRNA export.
The exact nature of the relationship between WPRE and CRM1 is presently unclear. CRM1 has not been shown to directly interact with RNA; hence, it is unlikely that the WPRE and CRM1 interact directly. One possibility is that CRM1 is involved in the shuttling of a factor essential for WPRE activity. The disruption of CRM1 function would prevent the shuttling of this hypothetical factor, disrupting WPRE function while not having a direct effect on RNA export. However, the similarity between the RRE and WPRE RNA accumulation profiles suggests that partial CRM1 dependence is more direct. Further support for a direct role of CRM1 in WPRE-mediated export is suggested, because CRM1 overexpression increases WPRE activity and the relative abundance of cytoplasmic WPRE-containing RNA. Finally, our studies with an artificial element containing Bul acting in concert with HPREß support this idea. If HIV Rev can act synergistically with the CRM1-independent PREß subelement, it is possible that a cellular equivalent of Rev is doing the same in mediating the action of the WPRE. A more likely scenario is that NES-containing cellular bridging factor(s), functionally similar to Rev, binds to the WPRE and mediates the export of WPRE-containing RNA via the CRM1 pathway. Hence, it will be interesting to identify the cellular adapter protein(s).
The observation that the efficient function of the WPRE is CRM1 dependent has interesting potential implications. The partial CRM1 dependence of the WPRE suggests, as discussed above, that WPRE may be a bona fide RNA export element.
Of potentially greater interest is the observation that WPRE function is both CRM1 dependent and independent. This result is rather surprising, since HPRE is CRM1 independent and HPRE and WPRE share a high degree of sequence similarity. However, the two elements have consistently shown significant functional differences, including the fact that WPRE exhibits a much greater posttranscriptional activity. This study demonstrates that the increased WPRE activity is due to a CRM1-dependent function for WPRE. When WPRE-transfected cells are treated with LMB or CAN is overexpressed, WPRE activity is inhibited to the exact levels observed with HPRE. These data suggest that WPRE can utilize multiple cellular processing pathways that may function cooperatively. The potential for cooperativity between different processing pathways was addressed by testing whether CRM1-dependent Rev-mediated export can act cooperatively with the CRM1-independent HPREß subelement. The observed synergistic activity suggests that it is possible that a cellular equivalent of Rev is doing the same in mediating the action of WPRE. There are interesting parallels between the activity of WPRE and the export of c-fos. Both are partially dependent on CRM1 function and appear to have the ability to utilize CRM1-dependent and CRM1-independent posttranscriptional pathways. These similarities suggest that the WPRE may be mimicking certain aspects of highly regulated cellular messages such as c-fos.
It is interesting to point out the WPRE can posttranscriptionally stimulate the expression of heterologous messages, which do not contain introns or other inhibitory sequences. This stimulatory ability is vector and transgene independent, making the WPRE a potentially important tool in situations, such as gene therapy or large-scale protein production, where optimal gene expression is advantageous. This ability is unique. Neither CTE nor HPRE can stimulate transgene expression. The unique combination of CRM1-dependent with CRM1-independent elements within WPRE may be responsible for its potent activity. For instance, one element could influence CRM1-dependent export, while another could increase the efficiency of 3'-end processing. This would be consistent with recent data demonstrating that intronless mRNA export elements could be involved in other steps of pre-mRNA processing beside nuclear export, such as splicing and polyadenylation (29). Many other observations suggest the existence of intimate links between the various steps in the posttranscriptional regulation of gene expression, particularly between splicing and downstream events, including mRNA export, translation, stability, and cytoplasmic localization (35, 36, 40, 42, 65). Therefore, the ability of the WPRE to facilitate multiple levels of RNA processing and export may lead to efficient handling of the transgene message in the nucleus and result in optimal gene expression. It seems probable that analysis of the WPRE function will lead to major insight into the steps governing the nuclear export and processing of cellular mRNA.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Health grant AI35477 (T.J.H.) and Arthur Kramer. Ileana Popa was supported by the Sarswatiben M. Upadhyaya Scholarship.
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FOOTNOTES |
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Present address: Walter Reed Army Institute of Research, Division of Retrovirology, Rockville, MD 20850.
Present address: Allergan, Inc., Irvine, CA 92623.
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REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2.
Bogerd, H. P., A. Echarri, T. M. Ross, and B. R. Cullen. 1998. Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J. Virol. 72:8627-8635.
3. Braun, I. C., E. Rohrbach, C. Schmitt, and E. Izaurralde. 1999. TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus. EMBO J. 18:1953-1965.[CrossRef][Medline]
4.
Brennan, C. M., I. E. Gallouzi, and J. A. Steitz. 2000. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J. Cell Biol. 151:1-14.
5.
Cullen, B. R. 2000. Connections between the processing and nuclear export of mRNA: evidence for an export license? Proc. Natl. Acad. Sci. USA 97:4-6.
6. Daly, T. J., K. S. Cook, G. S. Gray, T. E. Malone, and J. R. Rusche. 1989. Specific binding of HIV-1 recombinant Rev protein to the Rev-response element in vitro. Nature 342:816-819.[CrossRef][Medline]
7. Donello, J. E., A. A. Beeche, G. J. Smith III, G. R. Lucero, and T. J. Hope. 1996. The hepatitis B virus posttranscriptional regulatory element is composed of two subelements. J. Virol. 70:4345-4351.[Abstract]
8.
Donello, J. E., J. E. Loeb, and T. J. Hope. 1998. Woodchuck hepatitis virus contains a tripartite postranscriptional regulatory element. J. Virol. 72:5085-5092.
9. Emmermman, M., R. Vazeux, and K. Pedeu. 1989. The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell 57:1155-1165.[CrossRef][Medline]
10. Ernst, R. K., M. Bray, D. Rekosh, and M.-L. Hammarskjold. 1997. Secondary structure and mutational analysis of the Mason-Pfizer monkey virus constitutive transport element. RNA 3:219-222.
11. Ernst, R. K., M. Bray, D. Rekosh, and M.-L. Hammarskjold. 1997. A structural retroviral element that mediates nucleocytoplasmic export of intron-containing RNA. Mol. Cell. Biol. 17:135-144.[Abstract]
12. Felber, B. K., D. Derse, A. Athanassopoulos, M. Campbell, and G. N. Pavlakis. 1989. Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements. New Biol. 1:318-328.[Medline]
13. Felber, B. K., M. Hadzopoulou-Cladaras, C. Cladara, T. Copeland, and G. Pavlakis. 1989. Rev protein of human immunodeficiency virus affects the stability and transport of viral mRNA. Proc. Natl. Acad. Sci. USA 86:1496-1499.
14. Fisher, U. J., J. Huber, W. C. Boelens, I. W. Mattaj, and R. Luhrmann. 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.[CrossRef][Medline]
15. Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051-1060.[CrossRef][Medline]
16. Fornerod, M., J. Vandeursen, S. Vanbaal, A. Reynolds, D. Davis, K. Gopal Murti, J. Fransen, and G. Grosveld. 1997. The human homologue of yeast CRM1 is in a dynamic subcomplex with Can/Nup214 and a novel nuclear pore component nup88. EMBO J. 16:807-816.[CrossRef][Medline]
17. Fukuda, M., S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, and E. Nishida. 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390:308-311.[CrossRef][Medline]
18. Gallouzi, I. E., C. M. Brennan, and J. A. Steitz. 2001. Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock. RNA 7:1348-1361.[Abstract]
19.
Gallouzi, I. E., and J. A. Steitz. 2001. Delineation of mRNA export pathways by the use of cell-permeable peptides. Science 294:1895-1901.
20.
Goda, S. K., and N. P. Minton. 1995. A simple procedure for gel electrophoresis and Northern blotting of RNA. Nucleic Acids Res. 23:3357-3358.
21. Gruter, P., C. Tabernero, C. von Kobbe, C. Schmitt, C. Saavedra, A. Bachi, M. Wilm, B. K. Felber, and E. Izaurralde. 1998. TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1:649-659.[CrossRef][Medline]
22.
Guzyk, B. W., L. Levesque, S. Prasad, Y.-C. Bor, B. E. Black, B. M. Pashal, D. Rekosh, and M.-L. Hammarskjold. 2001. NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells. Mol. Cell. Biol. 21:2545-2554.
23.
Hope, T. J., B. L. Bond, D. McDonald, N. P. Klein, and T. G. Parslow. 1991. Effector domains of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex are functionally interchangeable and share an essential peptide motif. J. Virol. 65:6001-6007.
24.
Hope, T. J., X. J. Huang, D. McDonald, and T. G. Parslow. 1990. Steroid-receptor fusion of the human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions of the arginine-rich motif. Proc. Natl. Acad. Sci. USA 87:7787-7791.
25.
Hope, T. J., N. P. Klein, M. E. Elder, and T. G. Parslow. 1992. Trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes. J. Virol. 66:1849-1855.
26.
Hope, T. J., D. McDonald, X. J. Huang, J. Low, and T. G. Parslow. 1990. Mutational analysis of the human immunodeficiency virus type 1 Rev transactivator: essential residues near the amino terminus. J. Virol. 64:5360-5366.
27.
Huang, J., and T. J. Liang. 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.
28. Huang, X. J., T. J. Hope, B. L. Bond, D. McDonald, K. Grahl, and T. G. Parslow. 1991. Minimal Rev-response element for type 1 human immunodeficiency virus. J. Virol. 65:2131-2134.
29. Huang, Y., K. M. Wimler, and G. G. Carmichael. 1999. Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. EMBO J. 18:1642-1652.[CrossRef][Medline]
30. Huang, Z.-M., and T. S. B. Yen. 1995. Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Mol. Cell. Biol. 15:3864-3869.[Abstract]
31. Huang, Z. M., W. Q. Zang, and T. S. Yen. 1996. Cellular proteins that bind to the hepatitis B virus posttranscriptional regulatory element. Virology 217:573-581. (Erratum, 240:382.)
32.
Kang, Y., H. P. Bogerd, and B. R. Cullen. 2000. Analysis of cellular factors that mediate nuclear export of RNAs bearing the Mason-Pfizer monkey virus constitutive transport element. J. Virol. 74:5863-5871.
33.
Kang, Y., and B. R. Cullen. 1999. The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. Genes Dev. 13:1126-1139.
34. Katahira, J., K. Strasser, A. Podtelejnikov, M. Mann, J. U. Jung, and E. Hurt. 1999. The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human. EMBO J. 18:2593-2609.[CrossRef][Medline]
35. Kataoka, N., J. Yong, V. N. Kim, F. Velazquez, R. A. Perkinson, F. Wang, and G. Dreyfuss. 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.[CrossRef][Medline]
36. Kim, V. N., J. Yong, N. Kataoka, L. Abel, M. D. Diem, and G. Dreyfuss. 2001. The Y14 protein communicates to the cytoplasm the position of exon-exon junctions. EMBO J. 20:2062-2068.[CrossRef][Medline]
37. Kjems, J., A. D. Frankel, and P. A. Sharp. 1991. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell 67:169-178.[CrossRef][Medline]
38.
Kudo, N., N. Matsumori, H. Taoka, D. Fujiwara, E. P. Schreiner, B. Wolff, M. Yoshida, and S. Horinouchi. 1999. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96:9112-9117.
39. Kudo, N., B. Wolff, T. Sekimoto, E. P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, and M. Yoshida. 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242:540-547.[CrossRef][Medline]
40. Le Hir, H., E. Izauralde, L. E. Maquat, and M. J. Moore. 2000. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19:6860-6869.[CrossRef][Medline]
41. Loeb, J. E., W. S. Cordier, M. E. Harris, M. D. Weitzman, and T. J. Hope. 1999. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum. Gene Ther. 10:2295-2305.[CrossRef][Medline]
42.
Luo, M. J., and R. Reed. 1999. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. USA 96:14937-14942.
43.
Malim, M. H., and B. R. Cullen. 1993. Rev and the fate of pre-mRNA in the nucleus: implication for the regulation of RNA processing in eukaryotes. Mol. Cell. Biol. 13:6180-6189.
44. Malim, M. H., J. Hauber, S. E. Le, J. V. Maizel, and B. R. Cullen. 1989. The HIV-1 Rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNAs. Nature 338:254-257.[CrossRef][Medline]
45. Murata, T., F. Goshima, T. Koshizuka, H. Takakuwa, and Y. Nishiyama. 2001. A single amino acid substitution in the ICP27 protein of the herpex simplex virus type 1 is responsible for its resistance to leptomycin B. J. Virol. 75:1039-1043.
46. Neumann, G., M. T. Hughes, and Y. Kawaoka. 2000. Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J. 19:6751-6758.[CrossRef][Medline]
47. O'Neill, R. E., J. Talon, and P. Palese. 1998. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J. 17:288-296.[CrossRef][Medline]
48.
Otero, G. C., M. E. Harris, J. E. Donello, and T. J. Hope. 1998. Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus Rev function but not the function of the hepatitis B virus posttranscriptional regulatory element. J. Virol. 72:7593-7597.
49. Palmeri, D., and M. H. Malim. 1996. The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a nuclear export signal. J. Virol. 70:6442-6445.[Abstract]
50. Pasquinelli, A. E., R. K. Ernst, E. Lund, C. Grimm, M. L. Zapp, D. Rekosh, M. L. Hammarskjold, and J. E. Dahlberg. 1997. The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16:7500-7510.[CrossRef][Medline]
51.
Phillips, T. R., C. Varmont, D. Konings, B. Shacklett, C. Hamson, P. Luciw, and J. H. Elder. 1992. Identification of the Rev transactivation and Rev-responsive elements of feline immunodeficiency virus. J. Virol. 66:5464-5471.
52.
Rosin-Arbesfeld, R., M. Rivlin, S. Noiman, P. Mashiah, A. Yaniv, T. Miki, S. R. Tronick, and A. Gazit. 1993. Structural and functional characterization of rev-like transcripts of equine anemia virus. J. Virol. 67:5640-5646.
53. Saavedra, C., B. Felber, and E. Izauralde. 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.[CrossRef][Medline]
54.
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.
55. Segref, A., K. Sharma, V. Doye, A. Hellwig, J. Huber, R. Luhrmann, and E. Hurt. 1997. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J. 16:3256-3271.[CrossRef][Medline]
56.
Semmes, O. J., L. Chen, R. T. Sarisky, Z. Gao, L. Zhong, and S. D. Hayward. 1998. Mta has properties of an RNA export protein and increases cytoplasmic accumulation of Epstein-Barr virus replication gene mRNA. J. Virol. 72:9526-9534.
57.
Soliman, T. M., and S. J. Silverstein. 2000. Herpesvirus mRNAs are sorted for export via CRM1-dependent and -independent pathways. J. Virol. 74:2814-2825.
58. Stade, K., C. S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041-1050.[CrossRef][Medline]
59.
Strasser, K., J. Bassler, and E. Hurt. 2000. Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export. J. Cell Biol. 150:695-706.
60. Strasser, K., and E. Hurt. 2000. Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export. EMBO J. 19:410-420.[CrossRef][Medline]
61.
Stutz, F., and M. Rosbash. 1998. Nuclear RNA export. Genes Dev. 12:3303-3319.
62. Stutz, F., A. Bachi, T. Doerks, I. C. Braun, B. Seraphin, M. Wilm, P. Bork, and E. Izaurralde. 2000. REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6:638-650.[Abstract]
63.
Tiley, L. S., M. H. Malim, H. K. Tewary, P. G. Stockley, and B. R. Cullen. 1992. Identification of a high affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein. Proc. Natl. Acad. Sci. USA 89:758-762.
64. Zang, W. Q., and B. Yen. 1999. Distinct export pathway utilized by the hepatitis B virus posttranscriptional regulatory element. Virology 259:299-304.[CrossRef][Medline]
65. Zhou, Z., M. J. Luo, K. Straesser, J. Katahira, E. Hurt, and R. Reed. 2000. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407:401-405.[CrossRef][Medline]
66.
Zufferey, R., J. E. Donello, D. Trono, and T. J. Hope. 1999. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73:2886-2892.
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