Nucleocytoplasmic transport of luciferase gene mRNA ... - Springer Link

Download PDF
4 downloads 2855 Views 604KB Size Report
Comment
The constitutive trans- port element (CTE)-mediated nuclear export of luc+ ... corresponding to the major functional classes of RNA; i.e., ribosomal RNA (rRNA) ...
Med Mol Morphol (2009) 42:70–81 DOI 10.1007/s00795-009-0441-3

© The Japanese Society for Clinical Molecular Morphology 2009

ORIGINAL PAPER Tominori Kimura · Iwao Hashimoto · Masao Nishikawa Hisao Yamada

Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase

Received: January 5, 2009 / Accepted: February 17, 2009

Abstract Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency.1,2 We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of ΔCAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors. Key words Nucleocytoplasmic transport · Luciferase gene mRNA · CRM1/Exportin1 · RanGTPase · HIV-1 Rev · tsBN2 cells

T. Kimura (*) · I. Hashimoto Laboratory of Microbiology and Cell Biology, Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan Tel. +81-77-561-2826; Fax +81-77-561-6569 e-mail: [email protected] T. Kimura · I. Hashimoto · M. Nishikawa Department of Microbiology, Kansai Medical University, Osaka, Japan H. Yamada Department of Anatomy and Cell Science, Kansai Medical University, Osaka, Japan

Introduction Correct intracellular localization of RNAs is essential for their function and can be utilized by the cell to regulate gene expression. The cytoplasmic or nuclear localization of RNA results from a balance between two opposing mechanisms, namely, nuclear retention and transport through the nuclear pores.1–3 Nucleocytoplasmic transport occurs through nuclear pore complexes and is mediated by saturable transport receptors that shuttle between the nucleus and the cytoplasm. Cross-competition experiments have revealed that only a few transport pathways exist, roughly corresponding to the major functional classes of RNA; i.e., ribosomal RNA (rRNA), messenger RNA (mRNA), Urich small nuclear RNA (U snRNA), transfer RNA (tRNA), and micro-RNA (miRNA) (see Kohler and Hurt4 for review and references therein). Most RNA export relies on a conserved family of homologous receptor proteins known as karyopherins. Each karyopherin recognizes specific features present within the cargo molecules (nuclear export signals or NESs) either directly or indirectly with the help of adapter molecule (see Rodriguez et al.5 for review). In vitro cross-linking and affinity purification assays have demonstrated that the karyopherin chromosome region maintenance 1 (CRM1)/ Exportin16,7 forms a complex with NES in the presence of the GTP-bound form of RanGTPase (RanGTP),6 the nuclear form of Ras-like GTPase Ran.8 CRM1 mediates the nuclear export of a variety of protein and RNA substrates; these include Rev (regulator of the expression of the virion) response element (RRE)-containing,9,10 incompletely spliced human immunodeficiency virus type 1 (HIV-1) mRNAs (RRE mRNAs) via the virally encoded Rev protein,2,6,11 several U snRNAs,6 and all rRNAs.12,13 While CRM1 is known to be dispensable for the export of most cellular mRNAs,14 the nuclear export of certain early response gene (ERG) transcripts was blocked by leptomycin B (LMB).15–17 LMB is a Streptomyces metabolite that selectively binds to CRM1 and blocks CRM1-mediated nuclear transport.18 To further examine potential CRM1

71

pathways for other cellular mRNAs, we previously studied the nuclear export of human interferon-α1 (IFN-α1) mRNA, another ERG transcript. Rev overexpression caused nuclear retention of IFN-α1 mRNA by titrating CRM1 that was required for the nuclear export of the transcript.19 During this study, we noticed a consistent and significant reduction of luciferase gene (luc+) expression when a plasmid encoding the pRSVluc gene20 was co-transfected into HeLa cells to monitor the transfection efficiency of a Rev-expressing construct (Kimura et al., personal communication; see also Kimura et al.2). This finding is supported by Bond et al.,1 who showed that the observed reduction in expression of the luciferase gene was dependent on coexpression of the Rev protein and was caused by neither competition between two types of transcripts nor a general decrease in expression from pRSVluc caused by the cotransfection of a second expression construct. These results raise the possibility that, similar to IFN-α1 mRNA, luc+ mRNA may also compete with Rev in the nucleocytoplasmic transport pathway. This idea prompted us to examine the nuclear export of luc+ mRNA. In this study, we show that the export of luc+ mRNA is likely to utilize the common nuclear export machinery, including CRM1 and RanGTPase, that the Rev–RRE mRNA complex employs for its export.

Materials and methods Recombinant plasmids Firefly luciferase expression plasmid pRSVluc20 (renamed as pRSVluc-wt in this article) was employed to study the nucleocytoplasmic transport machinery for wild-type (wt) luc+ mRNA. As a negative control, a luciferase reporter plasmid was generated by fusing a rabbit β-globin gene21 fragment that includes the second intron (nt, 718–1290) (pRSVluc-intron) with luciferase cDNA in pRSVluc-wt. pRSVluc-constitutive transport element (CTE)19 was also employed as another control vector in which the rabbit β-globin gene intron was replaced with full-length SRV-1 CTE.22 The effect of coexpressed HIV-1 Rev on the nuclear export of wt luc+ mRNA and luc+ mRNA variants that were transcribed from pRSVlucintron (spliced luc+ mRNA) or from pRSVluc-CTE (CTEplus luc+ mRNA) was examined using co-transfection with pCG-HA-Rev.2 M10, which acts as a trans-dominant repressor of the Rev function,9 was expressed under the control of the Cytomegalovirus (CMV) immediate early region promoter in the pCG-HA vector.2 pCG-HARev27–29A expresses a recessive-negative mutant of Rev that is efficiently transported into the nucleus but lacks RRE mRNA-binding activity.19 An HA-tagged, truncated form of Nup214/CAN (ΔCAN23; amino acids 1864–2090 of human CAN/Nup214) was expressed by using the pCGHA vector.19 pCRRE/ΔRev, a defective provirus that carries a defective rev gene, was derived from its precursor, pCRRE.24

Cells and transfections HeLa cells (ATCC CCL2), 293T/17 cells (ATCC CRL11268), and tsBN2 cells (RIKEN RCB1264), maintained in Dulbecco’s minimum essential medium (MEM) containing 10% heat-inactivated fetal calf serum (D10), were transfected with the indicated expression plasmids by the calcium phosphate method as described previously.2 Cells were transfected at either 37°C (HeLa and 293T/17 cells) or 33°C (tsBN2 cells) for 16 h. Transfected HeLa cells were incubated for an additional 16 h and then subjected to the luciferase assay as well as the reverse transcription-polymerase chain reaction (RT-PCR) analysis described next. Transfected 293T/17 cells were collected at 32 h after addition of DNA for nuclear extract preparation, as described below. Transfected tsBN2 cells were incubated for various times up to 7 h at either 33° or 39.5°C. At the end of each incubation period, cells were harvested for following luciferase assay or RT-PCR analysis. The luciferase enzymatic activity per 1 μg total cytoplasmic protein was assayed using the manufacturer’s suggested protocol (Promega, Madison, WI, USA). When HA-tagged, Rev wild-type, or the mutant proteins were expressed to compare their effects on luciferase gene expression, they were separated, blotted, and then visualized with a mouse monoclonal anti-HA epitope antibody (clone SCP-12CA5; BAbCO, Richmond, CA, USA) to confirm an equal level of gene expression among the Rev expression plasmids.19 p55Gag was separated, blotted, and visualized using pooled acquired immunodeficiency syndrome (AIDS) patient sera25 as described previously.2 Relative concentrations of Rev and HIV-1 p55Gag proteins were determined with a Bio-imaging analyzer, MacBAS (Fuji Film, Tokyo, Japan), according to the manufacturer’s instructions.

Nuclear extraction and immunoprecipitation Transfected 293T/17 cells were collected by centrifugation, resuspended in two volumes of cold hypotonic buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol (DTT); pH 7.9] and allowed to swell on ice for 10 min; subsequent steps were all performed at 4°C. The swollen cells were then lysed by a glass Dounce homogenizer (Wheaton, Millville, NJ, USA) using a tight pestle. The homogenate was checked microscopically for cell lysis and centrifuged to pellet nuclei. Nuclei were washed once with the hypotonic buffer and resuspended with a volume of low-salt buffer [20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM PMSF, 0.5 mM DTT; pH 7.9] equal to one-half the volume of the nuclear pellet. The same amount of high-salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT; pH 7.9) was then added to the extract for 30 min with continuous gentle stirring. The mixture was then centrifuged for 30 min at 25 000 g. The resulting supernatant was dialyzed against 50 volumes of dialysis buffer (20 mM HEPES, 20% glycerol, 100 mM

72

KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT; pH 7.9) until the conductivities of extract and buffer were equal. The dialysate was centrifuged again for 20 min at 25 000 g and the resulting precipitate discarded. The protein concentration was then determined. To an aliquot of the nuclear extract containing 50 μg total protein, 2 μg mouse monoclonal anti-HA epitope tag antibody was added. The total volume of the mixture was then adjusted to 250 μl by adding 2× immunoprecipitation buffer [2% Triton X-100, 300 mM NaCl, 20 mM Tris·HCl, 2 mM EDTA, 2 mM ethyleneglycotetraacetic acid (EGTA), 0.4 mM PMSF, 1% NP-40; pH 7.4], and double-distilled water (DDW). The mixture was incubated at 4°C for 1 h, and 50 μl 10 % (w/v) protein GSepharose (Pharmacia, Uppsala, Sweden) suspension added. After roll mixing at 4°C overnight, the protein G-Sepharose pellets were washed four times with immunoprecipitation buffer and twice with 10 mM Tris·HCl, pH 7.4. The immunoprecipitated proteins were then blotted and visualized, as described above, using either the same monoclonal antibody (1 μg/ml) or rabbit anti-CRM1 antiserum (1 : 500 dilution).19

land)-labeled antisense oligonucleotide for luc+ mRNA (complementary to nucleotides 28–201 relative to the translation start site). Following hybridization and visualization of the probe, cells were processed for immunolabeling as previously described.2 The primary antibodies were rabbit anti-CRM1 antiserum (1 : 600 dilution in D10) and mouse monoclonal anti-Ran/TC4 (1 : 25 dilution in D10) (clone 20; Transduction Lab, Lexington, KY, USA) antibody. Tetramethylrhodamine isothiocyanate (TRITC)-conjugated swine antirabbit IgG antiserum (1 : 20 dilution in D10) or TRITC-conjugated rabbit antimouse IgG antiserum (1 : 20 dilution in D10) (both from Dakopatts, Glostrup, Denmark) were used as the secondary antibodies. Visualization was done with an Olympus Fluoview confocal laser scanning microscope (Olympus, Tokyo, Japan) as previously described.2 Individual FITC and TRITC images were obtained through separate channels and were then merged using Adobe Photoshop software. No signal was obtained when the fixed cells were treated with RNase before hybridization or with the secondary antibody alone. Analysis of mRNA produced from transfected cells

Microinjections Nuclear injections into HeLa cells were performed essentially as described previously.19 The injected cells were incubated at 37°C for 30 min (cells expressing luc+ mRNAs) or 45 min (cells expressing HIV-1 gag mRNA). They were then treated for an additional 60 min at 37°C in the presence or absence of 20 nM LMB. The cells were then fixed, permeabilized, and subjected to RNA fluorescence in situ hybridization (FISH) alone or together with immunofluorescence, as described next. RNA–FISH and immunocytochemistry In situ hybridization was performed as described previously2 using digoxigenin (DIG)-11-deoxyuridine 5triphosphate (dUTP) (Roche Diagnostics, Basel, Switzer-

Fig. 1. Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) (HIV-1 Rev) inhibits nuclear export of cotransfected luciferase reporter gene (luc+) and luc- constitutive transport element (CTE) mRNAs. A Rev-dependent inhibition of luciferase gene expression. HeLa cells were co-transfected with 100 ng each of either pRSVluc-wild type (wt) (open circles), pRSVluc-CTE (closed circles), or pRSVluc-intron (closed boxes), and the various amounts of pCG-HA-Rev shown on the x-axis for 16 h at 37ºC. pCG-HA, the empty parental vector, was included to normalize the amounts of the vector-based expression plasmids. pUC12 was included to give a total DNA level of 20 μg. At 32 h after DNA addition, cytoplasmic lysates were obtained to examine luciferase activity, as described in Materials and methods. Values are presented as percent luciferase activity relative to luciferase expression plasmids alone samples. The mean ± SEM values for a representative of two independent transfection experiments with triplicate samples are shown. The Western blot indicates the levels of Rev protein. B Coexpression of Rev results in a reduction of the cytoplasmic but an increase of the nuclear wt and CTE-fused luc+ mRNA levels. HeLa cells were co-transfected with pRSVluc-wt (wt), pRSVluc-CTE (+CTE), or pRSVluc-intron (spliced), and either pCG-HA-Rev or pCG-HA vector. Cytoplasmic and nuclear total

Total cytoplasmic and nuclear RNAs were extracted from the transfected HeLa or tsBN2 cells by the modified NP40 method as previously described.19 First-strand synthesis was performed, and the cDNA synthesis reaction mixtures were then appropriately diluted to ensure linear amplification of cDNAs in the following PCR reaction. The amplified fragments were randomly labeled with [α-32P]-deoxycytidine triphosphate (dCTP) (111 TBq/mmol; NEN Life Science, Boston, MA, USA), separated on a 6% denaturing polyacrylamide gel and then visualized by autoradiography as described previously.19 The ratio of normalized luc+ or HIV-1 gag transcript levels in the presence of Rev to those in the absence of Rev was determined with MacBAS as already described. For the detection of luc+ mRNA, the nucleotide sequences and locations of the oligonucleotide primers were 5′-GACGCCAAAAACATAAAG-3′ (Photinus pyralis luciferase cDNA20 nt 7–24), and 5′-CGAACG

RNA were then collected and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis as described in Materials and methods. cDNA synthesis reaction mixtures were appropriately diluted to ensure linear amplification of cDNAs in the PCR reaction. luc+ transcript levels were normalized to the corresponding GAPDH mRNA levels. The ratios of normalized luc+ transcript levels in the presence of Rev to those in the absence of Rev were then calculated and are shown below each lane. C, Mock-transfected control. Sizes of amplified fragments corresponding to luc+ mRNAs and GAPDH mRNA are indicated. The autoradiographs shown were obtained after 70 min exposure at −70°C. Results of a representative of two independent experiments are shown. C Rev NES-dependent inhibition of luciferase gene expression. HeLa cells were co-transfected with the combinations of plasmids shown below the figure. Luciferase activity was examined as described in B and presented as per cent luciferase activity expressed in HeLa cells transfected with pRSVluc-wt and pCG-HA vector (column 1). The Western blot shows the levels of Rev and its mutant proteins. Bars represent mean ± SEM. A representative of two independent transfection experiments with triplicate samples is presented

73

A

Rev

Relative luciferase activity

% 100

50

0

B

C

0

wt

-

Rev:

+

5 10 15 0.5 1 1.5 Concentration of Rev expression plasmid (µg)

+CTE spliced

-

-

+

wt

-

+

luc+ mRNAs

179 bases

GAPDH mRNA

177 bases 1 100

2

20 luc+ mRNAs+Rev/GAPDH mRNA : luc+ mRNAs/GAPDH mRNA

3 4 100 40

5 6 100 98

(%)

+

-

+

-

+

1 2 3 4 5 6 100 100 100 230 193 121 (%)

Cytoplasm

Nucleus

% 125

Relative luciferase activity

C

+CTE spliced C

100 75 50 25 0

0

Rev wt Rev27-29A RevM10 Western

GACATTTCGAAG-3′ (P. pyralis luciferase cDNA nt 185–168). The primer pairs used for the detection of HIV-1 gag mRNA and human GAPDH mRNA were as described previously.2

1

-

2

3

-

+

+

-

4

-

+

Numbering of sequences The sequence numbering presented in this report is based on the following GenBank sequences: M15077 for the firefly

74

P. pyralis luciferase gene complete cds, and NC 001802 for HIV-1NL4-3.

Results Coexpressed Rev inhibits luciferase gene expression in a NES-dependent manner To test whether the nuclear export pathway for luc+ mRNA shared common components with that for Rev, we compared the effect of Rev overexpression on the cytoplasmic expression of wild-type (wt) luc+ mRNA with luc+ mRNA variants that are expected to be transported out of the nucleus by the general mRNA export receptor TAP (Tipassociated protein)/NXF1 (Nuclear export factor 1) and associated components (see Kimura and Hashimoto26 for review and references therein). Overexpression of the rev gene was previously applied to show that the Rev NES and certain cellular mRNAs shared some export pathway components, including CRM1.19,27 As shown in Fig. 1A, the increasing level of Rev expression from 0–15 μg of a Rev expression plasmid, pCG-HA-Rev (top panel, Western blot) caused a progressive reduction in luciferase gene expression. The reduced gene expression was, however, reversed when the luciferase gene was fused with a rabbit β-globin gene fragment including the second intron, but not with the CTE. Subsequent RT-PCR analysis for the subcellular localization of luc+ mRNAs in the Rev-cotransfected cells (Fig. 1B) revealed that the observed reduction of luciferase activity, expressed from both pRSVluc-wt and pRSVluc-CTE, was reflected by the corresponding decrease in the cytoplasmic mRNA level (80% reduction in wt luc+ mRNA and 60% reduction in luc-CTE mRNA levels; cytoplasm, lanes 2 and 4, respectively) together with increases in the nuclear mRNA levels (230% and 193%, respectively; nucleus, lanes 2 and 4). By contrast, Rev failed to inhibit the cytoplasmic expression of spliced luc+ mRNA to the degree observed in cells expressing wt luc+ or luc-CTE mRNAs (cytoplasm and nucleus, lanes 5 and 6, respectively; see also mRNA quantification results shown underneath each lane). These results suggest that the decrease in the amount of cytoplasmic wt luc+ and luc-CTE mRNAs relative to the increase in the nuclear fractions upon Rev overexpression is consistent with inhibition of the nuclear export of these mRNAs, as was observed for heat shock mRNAs27 and human IFNα1 mRNA.19 This finding then raises the possibility that the transport pathway for wt luc+ and luc-CTE mRNAs may share common components with that for the Rev–RRE mRNA complex. To further study the mechanism of the inhibitory Rev effect, we employed an inactive Rev NES mutant19 (RevM10) and a recessive-negative mutant Rev27–29A that is efficiently transported into the nucleus but lacks RRE mRNA-binding activity because of its arginine-rich domain (ARD).19 Under conditions in which Rev and the Rev domain mutant proteins were all expressed at a comparable level (Fig. 1C, Western blot), the Rev inhibitory effect was

reversed (Fig. 1C, RevM10; compare lane 4 with lane 2) when rev had a point mutation that made its NES unable to interact with the CRM1 and RanGTP complex.19 By contrast, coexpression of Rev27–29A resulted in a reduction of luciferase activity to a level comparable with that in wt Rev-transfected cells (Fig. 1C, Rev27–29A; compare lane 3 with lane 2). These results indicate that Rev blocks luciferase gene expression in a NES-dependent manner, further suggesting that a high level of Rev NES expression could titrate the common transport pathway components described above, thereby reducing the export of wt luc+ and luc-CTE mRNAs.

LMB inhibits the nuclear export of wt luc+ mRNA but not of the luc+ mRNA variants The important feature of the Rev-dependent nuclear export pathway for RRE mRNA, but not of the bulk cellular mRNA export pathway, is the involvement of CRM1, the receptor for short leucine-rich Rev-type NESs (see Kimura et al.28 for review). To test whether CRM1 is involved in the nuclear export of wt luc+ mRNA, we performed an LMB sensitivity assay. LMB is a Streptomyces metabolite that selectively binds to and blocks CRM1-mediated nuclear transport,29 including nuclear export of HIV-1 gag mRNA and IFN-α1 mRNA.19 As shown in Fig. 2, pRSVluc-wt was microinjected into the nuclei of HeLa cells, and the cells were incubated for 30 min at 37°C to allow transcription (Fig. 2A, t: 0). After an additional 1 h incubation with (Fig. 2C, LMB: 1h) or without (Fig. 2B, D10: 1h) 20 nM LMB, the nuclear export of wt luc+ mRNA was evaluated using RNA-FISH. LMB treatment caused rapid cessation of wt luc+ mRNA export, resulting in the nuclear retention of the mRNA (Fig. 2C). The same concentration of LMB blocked Rev-dependent nuclear export of HIV-1 gag mRNA, as the positive control, as efficiently as it blocked wt luc+ mRNA (Fig. 2C,L), By contrast, nuclear export of spliced luc+ mRNA and luc-CTE mRNA was not affected by LMB (Fig. 2D–I; see also Kimura et al.19), which is consistent with previous reports that bulk cellular mRNA and the CTE do not depend on CRM1 for their export.23,30 This result confirms that the LMB-dependent inhibition of wt luc+ mRNA export does not result from general toxic effects on cell viability. ΔCAN is the dominant negative form of CAN/Nup214 and specifically inhibits CRM1.23 This effect is based on the formation of a complex between ΔCAN and CRM1 (Fig. 3B), enabling the nucleoporin fragment to compete with authentic nucleoporins for binding to CRM1.31 To substantiate the results of the LMB sensitivity experiments shown in Fig. 2, we performed dose–response experiments that measured the effect of ΔCAN on luciferase gene expression. ΔCAN produced a clear dose-dependent inhibition of wt luciferase gene expression (Fig. 3A) to a degree similar to that of Rev/RRE-dependent gag expression. As expected from the experiments in Fig. 2, the enzyme activity of luciferase that was translated from either the spliced luc+ mRNA

75 Fig. 2. Leptomycin B (LMB) suppresses the nuclear export of wt luc+ mRNA but does not negatively affect that of lucCTE mRNA. HeLa cell nuclei were microinjected with either pRSVluc-wt (wt), pRSVluc-CTE (+CTE), pRSVluc-intron (spliced), or pCRRE (HIV-1 gag mRNA) at 37°C for 30 min. Cells were further incubated at 37°C for 30 min (t: 0, A, D, G; cells expressing luc+ mRNAs) or 45 min (t: 0, J; cells expressing HIV-1 gag mRNA), and were then treated for an additional 60 min at 37°C with LMB (LMB: 1h, C, F, I, L) as described in Materials and methods. D10: 1h shows mocktreated cells (B, E, H, K). Cells were subsequently fixed, permeabilized, and subjected to RNA fluorescent in situ hybridization (FISH) as described in Materials and methods. Bars 10 μm

or luc-CTE mRNA was not affected (Fig. 3; closed boxes and closed circles, respectively).

Wt luc+ mRNA is colocalized with CRM1 in the nucleus The foregoing data strongly implicated CRM1 in wt luc+ mRNA export. To confirm the involvement of CRM1 in mRNA export and to examine the relationship between the two molecules in more detail, we studied the subnuclear localization of wt luc+ mRNA and CRM1. pRSVluc-wt was microinjected into HeLa cell nuclei (Fig. 4A–C). After 90 min at 37°C, to allow transcription, processing, and export of wt luc+ transcripts, the cells were subjected to

analysis by RNA-FISH (Fig. 4A) and immunocytochemistry (Fig. 4B). In Fig. 4C, merging of the two colors resulted in a yellow signal, indicating that the majority of nuclear wt luc+ mRNA was colocalized with CRM1. By contrast, colocalization analysis of HeLa cell nuclei that were microinjected with either pRSVluc-CTE or pRSVluc-intron showed that most CRM1 exhibited localization that was distinct from that of both luc-CTE and spliced luc+ mRNAs (compare Fig. 4E,H with 4D,G). These results, when taken together with the LMB sensitivity assay (Fig. 2) and ΔCAN coexpression assay (Fig. 3), imply that wt luc+ mRNA, but neither spliced luc+ mRNA nor CTEluc mRNA, interacts functionally with a CRM1-dependent transport pathway out of the nuclei.

76

Relative luciferase activity

%

%

125

125

100

100

75

75

50

50

25

25

0

Relative p55Gag concentration

A

0 0

0.25

0.5

0.75

1

Concentration of ΔCAN expression plasmid (µg)

B

C HAΔCAN

IP: α-HA-ΔCAN WB: α-CRM1

IP: α-HA-ΔCAN WB: α-HA-ΔCAN Tfx

C

Input

Tfx

Input

CRM1

Fig. 3. ΔCAN inhibits wt luciferase gene expression. A HeLa cells were co-transfected with 100 μg each of either pRSVluc-wt (open circles), pRSVluc-CTE (closed circles), pRSVluc-intron (closed boxes), or pCRRE (open triangles), and the various amounts of pCG-HAΔCAN shown on the x-axis. At 32 h after DNA addition, cytoplasmic lysates were obtained to examine luciferase activity or were employed for the analysis of p55Gag expression by Western blotting (see legend to Fig. 1 and Materials and methods). Values are presented as luciferase activity or p55Gag concentration relative to luciferase expression plasmids or pCRRE alone samples. The mean ± SEM values for a representative of two independent transfection experiments with triplicate samples are shown. B ΔCAN interacts with chromosome region maintenance 1 (CRM1) in 293T/17 cell nuclear extract. 293T/17 cells were transfected with pCG-HA-ΔCAN (Tfx). Nuclear extracts were then prepared from the transfected cells as described in Materials and methods. Input lanes show 5% of the total nuclear extract (from 5 × 105 cells) used in adjacent lanes. Immunoprecipitations were performed and analyzed as described in Materials and methods with the antibodies shown above each panel. C, Mock-transfected control

The nuclear export of wt luc+ mRNA requires RanGTP It was shown that RanGTPase regulates the interaction between CRM1 and NES, as required for Rev-dependent RRE mRNA export.6 We, therefore, examined whether RanGTPase also has a role in the nucleocytoplasmic transport of wt luc+ mRNA. For this, we employed tsBN2 cells,32 which have a temperature-sensitive allele of RCC1 (regulator of chromosome condensation 1), the nuclear GTP–GDP exchange factor for Ran.33 RCC1 has been reported to be almost undetectable in cells after a 3-h incubation at the restrictive temperature of 39.5°C, resulting in the depletion of nuclear RanGTP in a later stage of incubation.34,35 Indeed, the cytoplasmic expression of HIV-1 Rev was substantially reduced at the end of a 7-h incubation period at 39.5°C when compared to cells incubated at the permissive temperature of 33°C (Fig. 5B).

tsBN2 cells were then transfected with pRSVluc-wt at 33°C. After 16 h transfection, cells were kept at 39.5°C for various time periods before measuring luciferase activity. Incubation at 39.5°C approximately doubled the wt enzyme activity by the end of the 7-h incubation period (Fig. 5A, top/bottom panels, closed circles). By contrast, incubation at 33°C (top/bottom panels, open circles) increased the luciferase activity by more than 8 fold at the end of the period. However, when pRSVluc-intron was alternatively used for transfection, incubation at 39.5°C for 7 h did not negatively affect the luciferase activity but showed a similar 5.6-fold increase when compared to cells incubated at 33°C (Fig. 5A, bottom panel, closed and open triangles, respectively). These results thus suggested that wt luciferase gene expression may require the GTP-bound form of RanGTPase in the nucleus. Comparative increases of the luciferase activity expressed from spliced luc+ transcript at both temperatures further indicate that reduction of wt luciferase activity at 39.5°C did not result from an increase of the rate of turnover of mRNA or protein at the elevated temperature. Interestingly, tsBN2 cells that were transfected with pRSVluc-CTE showed an approximately threefold increase of enzyme activity at 33°C when compared with cells at 39.5°C (Fig. 5A, top panel; compare open and closed boxes at 7 h). This result raises the possibility that expression of the luciferase gene fused with the CTE may also require RanGTP. To study the mechanisms of inhibitory effect on luciferase gene expression at the restrictive temperature in more detail, we used RT-PCR to analyze the subcellular localization of wt luc+ mRNA and the luc+ mRNA variants in the transfected cells described in Fig. 5A. As shown in Fig. 5C, the observed reduction of luciferase activity at 39.5°C, expressed from both pRSVluc-wt and pRSVlucCTE, were reflected by corresponding decreases in cytoplasmic mRNA levels (Fig. 5C: 62% reduction in wt luc+ mRNA and 65% reduction in luc-CTE mRNA levels; cytoplasm, lanes 2 and 4) together with increases in nuclear mRNA levels (187% and 240%, respectively; nucleus, lanes 2 and 4). Similar to these cells, the RT-PCR analysis of tsBN2 cells described in Fig. 5B showed that incubation at 39.5°C caused a 75% reduction of cytoplasmic HIV-1 gag mRNA (Fig. 5C, cytoplasm, lane 8) with a concomitant increase in nuclear gag mRNA (189 %; nucleus, lane 8). The temperature shift, by contrast, failed to reduce the cytoplasmic expression or to increase the nuclear level of spliced luc+ mRNA (103%, cytoplasm, lane 6; 105%, Nucleus, lane 6, respectively), indicating that the restrictive temperature-dependent reduction of the cytoplasmic mRNA level does not result from an increase of the mRNA turnover rate. These results therefore suggest that the decrease in the amount of cytoplasmic wt luc+ and luc-CTE mRNAs relative to the increase in the nuclear fraction upon the temperature shift is consistent with inhibition of the nuclear export of these mRNAs, as was observed for Rev-dependent gag mRNA (Fig. 5C, cytoplasm/nucleus, lanes 7 and 8; see also Fornerod et al.6). These findings thus imply that expression of the wt luciferase and lucif-

77

luc+ mRNA

Merged

CRM1

A

B

C

D

E

F

G

H

I

wt

+CTE

Spliced

Fig. 4. wt, but not CTE+ or luc+ mRNA, is colocalized with CRM1 in cell nuclei. HeLa cell nuclei were microinjected with either pRSVlucwt (wt: A–C), pRSVluc-CTE (+CTE: D–F), or pRSVluc-intron (spliced: G–I) at 37°C for 30 min as described in the legend to Fig. 2. The cells

erase genes fused with CTE requires generation by RCC1of the GTP-bound form of RanGTPase. Wt luc+ mRNA and CTE-luc mRNA are colocalized with RanGTPase in the nucleus To confirm the involvement of RanGTPase in the nuclear export of wt luc+ and CTE-luc mRNAs and to examine the functional relationship between these molecules in more detail, we studied the subnuclear localization of RanGTPase and the mRNAs. Either pRSVluc-wt or pRSVlucCTE was microinjected into HeLa cell nuclei (Fig. 6A–F). After 90 min at 37°C, cells were subjected to analysis by RNA-FISH (Fig. 6A,D) and immunocytochemistry (Fig. 6B,E). Merging of the mRNA signal into the RanGTPase signal resulted in a yellow signal (Fig. 6C,F), indicating that the majority of nuclear wt luc+ and CTE-luc mRNAs colocalized with RanGTPase. By contrast, as was expected from the experiments presented in Fig. 5, most RanGTPase exhibited localization that was distinct from that of spliced luc+ mRNA (Fig. 6G–I). These results, when taken together with the temperature shift assay using tsBN2 cells in Fig. 5, support the foregoing conclusion that both wt luc+ and luc-CTE mRNAs employ RanGTP for their nuclear export.

were further incubated for 90 min at 37°C and subsequently fixed, permeabilized, and subjected to RNA FISH and immunocytochemistry as described in the legend to Fig. 2. Bars 10 μm

Discussion The experiments presented here demonstrated that inhibition of luciferase gene expression by coexpressed Rev resulted from nuclear retention of the reporter gene transcript (see Fig. 1A,B). Subsequent analysis of Rev mutants for luciferase gene expression revealed that the inhibitory effect relied on Rev NES, but not its ARD (Fig. 1C). It is reported that NES interacts directly or indirectly with multiple cellular components that primarily constitute nuclear export machinery for noncoding RNAs and proteins (see Kimura and Hashimoto26 for review and references therein). Indeed, NES conjugated to BSA was able to inhibit the export of coinjected U snRNA and 5S rRNA in Xenopus oocytes.11 However, it is also reported that a fraction of cellular mRNAs such as ERG mRNAs15–17,19 are also transported by CRM1, one of the components that acts as the receptor for leucine-rich Rev type NES. The Rev NESdependent nuclear retention could, therefore, result from interference with wt luc+ mRNA export by limiting the availability of common transport factors for use in a similar transport process by the non-RRE-containing reporter gene transcripts (Fig. 1B,C; and see Bond et al.1). The following experiments demonstrated that this was indeed the case. First, we examined the effect of LMB, a

78

A

Relative luciferase activity

1000

B

%

C

7

(hr)

33°C 39°C Cytoplasmic HA-Rev

500

C wt

C 0 0h

1h

3h

5h

7h

% 1000 Relative luciferase activity

0

HIV-1 gag

HIV-1 +CTE spliced gag

wt

33 39 33 39 33 39 33 39°C

luc+ mRNAs

179 bases

HIV-1 gag mRNA GAPDH mRNA

167 bases

C

33 39 33 39 33 39 33 39°C

172 bases

luc+ mRNAs at 39°C/ GAPDH mRNA luc+ mRNAs at 33°C/ GAPDH mRNA

500

+CTE spliced

1 2

: 100

3

4

100 38

35

5

6

7

8

100 100 103 25 (%)

Cytoplasm

1

2

100

3

4

100 187

5

6

100 240

7

8

100 105

189 (%)

Nucleus

0 0h

1h

3h

5h

7h

Time after temperature shift (hrs) Fig. 5. pRSVluc-wt or pRSVluc-CTE-transfected tsBN2 cells reduce luciferase gene expression at the restrictive temperature of 39.5°C. A tsBN2 cells were transfected with either pRSVluc-wt (circles), pRSVluc-CTE (boxes), or pRSVluc-intron (triangles). Following transfection at 33°C, cells were incubated at either 33°C (open symbols) or 39.5°C (closed symbols) for various time periods up to 7 h. At the end of each time period, luciferase activity was examined as described in the legend to Fig. 1. Values are presented as luciferase activities relative to those obtained at time 0 for the temperature shift. Mean relative luciferase activity ± SEM values for a representative of two independent experiments with duplicate samples are shown. Error bars are not visible under the scale employed. B Incubation at 39.5°C reduces the cytoplasmic expression of Rev in tsBN2 cells. tsBN2 cells were cotransfected with pCRRE/ΔRev and pCG-HA-Rev at 33°C. Transfected cells were further incubated for 7 h at either 33°C or 39.5°C. At time 0 and 7 h after the temperature shift, cells were collected for the analy-

sis of Rev expression as described in Materials and methods. C Incubation at 39.5°C causes a reduction of cytoplasmic but an increase of nuclear wt and CTE-fused luc+ mRNA levels. tsBN2 cells were transfected with pRSVluc-wt (wt), pRSVluc-CTE (+CTE), pRSVluc-intron (spliced), or pCRRE (HIV-1 gag). Following transfection at 33°C, cells were incubated for 7 h at either 33°C or 39.5°C. Cytoplasmic and nuclear total RNA were then collected and subjected to RT-PCR analysis as described in the legend to Fig. 1. cDNA synthesis reaction mixtures were appropriately diluted to ensure linear amplification of cDNAs in the PCR reaction. The ratios of normalized luc+ or HIV-1 gag transcript levels at 39.5°C to those at 33°C are shown below each lane. C, Mock-transfected control. Sizes of amplified fragments corresponding to luc+ mRNAs, HIV-1 gag mRNA, and GAPDH mRNA are indicated. The autoradiographs shown were obtained after 70 min exposure at −70°C. Results of a representative of two independent experiments are shown

CRM1-specific inhibitor, on wt luc+ mRNA export. LMB is an antifungal antibiotic that specifically binds covalently to a cysteine residue in CRM1,18 leading to stabilization of the Ran-free conformation of the NES receptor.36 The compound thereby prevents the binding of both the GTP-bound form of Ran and the export cargo to CRM1: hence, export was arrested.6,37 LMB-dependent inhibition of wt luc+ mRNA export, but not that for spliced luc+ mRNA or that mediated by CTE (Fig. 2), thus suggests that CRM1 is also involved in the nuclear export machinery for wt luc+ mRNA. This conclusion was also supported by employing ΔCAN, another selective inhibitor of the function of CRM1.23

Overexpression of ΔCAN inhibited luciferase gene expression to a degree that was comparable to that of Rev-dependent HIV-1 gag expression; whereas luciferase activity, which was expressed from spliced luc+ mRNA or luc-CTE mRNA, was not affected (Fig. 3). The temperature shift assay of luciferase gene expression using tsBN2 cells and the following RT-PCR analysis of the cells revealed that the GTP-bound form of RanGTPase was also required for the nucleocytoplasmic translocation of wt luc+ mRNA (Fig. 5). Based on these data, together with the colocalization assay shown in Figs. 4 and 6, we concluded that the nuclear

79

luc+ mRNA

RanGTPase

Merged

wt

A

B

C

D

E

F

G

H

I

+CTE

spliced

Fig. 6. RanGTPase is colocalized with both wt and CTE+ luc+ mRNAs in the cell nuclei. HeLa cell nuclei were microinjected with either pRSVluc-wt (wt: A–C), pRSVluc-CTE (+CTE: D–F), or pRSVlucintron (spliced: G–I) at 37°C for 30 min as described in the legend to

Fig. 2. The cells were further incubated for 90 min at 37°C and subsequently fixed, permeabilized, and subjected to RNA FISH and immunocytochemistry as described in the legend to Fig. 2. Bars 10 μm

export of wt luc+ mRNA is mediated by CRM1 and RanGTPase. Zolotukhin and Felber, however, reported that increasing amounts of HIV-1 Rev did not affect luciferase gene expression.38 To overexpress Rev in their experiments, cells were transfected with a rev expression plasmid in various amounts ranging from 0 to 2 μg,38 whereas we examined the effect of Rev overexpression in the range of 0 to 15 μg (Fig. 1A; see also Kimura et al.19). The difference in the levels of Rev protein examined in each study could therefore account for the contradictory conclusions reached by the two groups. We believe that the effect of Rev on luciferase gene expression depends on titration of nuclear export factors, which becomes more prominent when higher levels of Rev are expressed. It is, however, of interest to note that, even in the range of rev expression plasmid that Zolotukhin and Felber examined, increasing amounts resulted in some reduction of luciferase activity, although this was not very clear when the change of enzyme activity was plotted along a semilogarithmic scale.38 As we have reported,19 the nuclear export of luc+ mRNA fused with CTE was not affected by LMB or ΔCAN (see Figs. 2, 3). This finding is consistent with other reports that CTE does not depend on a leucine-rich NES or the receptor CRM1 for its function23,30 but does depend on TAP.39 Contradictory to these findings and reports, Rev overexpression inhibited the nuclear export of luc-CTE mRNA, suggesting that the luc+ mRNA transport pathway directed by CTE

may share common components other than CRM1 with that for the Rev–RRE mRNA complex. Indeed, experiments using tsBN2 cells revealed that luc-CTE mRNA may require RanGTP for its export (Fig. 5). Subsequent colocalization analyses supported this finding (Fig. 6). In light of these results, we should like to note that RanQ69L, a dominant negative form of RanGTPase, blocked CTE-mediated nuclear export of an intron-containing viral mRNA, suggesting that CTE-mediated export was dependent on Ranmediated GTP hydrolysis.40 CRM1 acts as the export receptor for Rev-like leucinerich NES in a Ran-regulated manner.6,7 Moreover, RNA export requires RNA-binding proteins that recognize specific RNAs and mediate their interaction with the actual export receptors.41 Thus, an implication of CRM1 involvement in luc+ mRNA export is that an RNA-binding protein containing Rev-like leucine-rich NES acts as an adapter that mediates formation of the luc+ mRNA–CRM1– RanGTP complex to be exported. Intron-less luc+ mRNA20 may therefore contain a binding site for the adapter, which has been identified in the intron-less RNA of hepatitis B viruses42 and the intron-less thymidine kinase gene mRNA of the human herpes simplex virus.43 The finding of such an adapter protein will facilitate identification of the cellular export cargo(s) of the adapter, leading to a better characterization of the cellular export pathway that luc+ mRNA and HIV-1 Rev utilize.

80 Acknowledgments This work was supported by grants to T.K. from the Ministry of Education, Science, Sports and Culture of Japan.

References 1. Bond VC, Huang MB, Person B, Hairston R, Ye XY, Saltarelli M (1997) Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression. Cell Mol Biol (Noisy-Le-Grand) 43:995–1005 2. Kimura T, Hashimoto I, Yamamoto A, Nishikawa M, Fujisawa JI (2000) Rev-dependent association of the intron-containing HIV-1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin-B. Genes Cells 5: 289–307 3. Custodio N, Carmo-Fonseca M, Geraghty F, Pereira HS, Grosveld F, Antoniou M (1999) Inefficient processing impairs release of RNA from the site of transcription. EMBO J 18:2855–2866 4. Kohler A, Hurt E (2007) Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 8:761–773 5. Rodriguez MS, Dargemont C, Stutz F (2004) Nuclear export of RNA. Biol Cell 96:639–655 6. Fornerod M, Ohno M, Yoshida M, Mattaj IW (1997) CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051–1060 7. Stade K, Ford CS, Guthrie C, Weis K (1997) Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041–1050 8. Izaurralde E, Kutay U, von Kobbe C, Mattaj IW, Gorlich D (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J 16:6535–6547 9. Malim MH, Bohnlein S, Hauber J, Cullen BR (1989) Functional dissection of the HIV-1 Rev trans-activator: derivation of a transdominant repressor of Rev function. Cell 58:205–214 10. Mann DA, Mikaelian I, Zemmel RW, Green SM, Lowe AD, Kimura T, Singh M, Butler PJ, Gait MJ, Karn J (1994) A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression. J Mol Biol 241:193–207 11. Fischer U, Huber J, Boelens WC, Mattaj IW, Luhrmann R (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 12. Ho JH, Kallstrom G, Johnson AW (2000) Nmd3p is a Crm1pdependent adapter protein for nuclear export of the large ribosomal subunit. J Cell Biol 151:1057–1066 13. Moy TI, Silver PA (2002) Requirements for the nuclear export of the small ribosomal subunit. J Cell Sci 115:2985–2995 14. Herold A, Teixeira L, Izaurralde E (2003) Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J 22:2472–2483 15. Brennan CM, Gallouzi IE, Steitz JA (2000) Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol 151:1–14 16. Jang BC, Munoz-Najar U, Paik JH, Claffey K, Yoshida M, Hla T (2003) Leptomycin B, an inhibitor of the nuclear export receptor CRM1, inhibits COX-2 expression. J Biol Chem 278:2773–2776 17. Higashino F, Aoyagi M, Takahashi A, Ishino M, Taoka M, Isobe T, Kobayashi M, Totsuka Y, Kohgo T, Shindoh M (2005) Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism. J Cell Biol 170:15–20 18. Kudo N, Matsumori N, Taoka H, Fujiwara D, Schreiner EP, Wolff B, Yoshida M, Horinouchi S (1999) Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc Natl Acad Sci U S A 96:9112–9117 19. Kimura T, Hashimoto I, Nagase T, Fujisawa J (2004) CRM1dependent, but not ARE-mediated, nuclear export of IFN-alpha1 mRNA. J Cell Sci 117:2259–2270 20. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737

21. van Ooyen A, van den Berg J, Mantei N, Weissmann C (1979) Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence. Science 206:337–344 22. Tang H, Gaietta GM, Fischer WH, Ellisman MH, Wong-Staal F (1997) A cellular cofactor for the constitutive transport element of type D retrovirus. Science 276:1412–1415 23. Bogerd HP, Echarri A, Ross TM, Cullen BR (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 24. Kimura T, Hashimoto I, Nishikawa M, Fujisawa JI (1996) A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal beta-actin and viral protein expression. Biochimie (Paris) 78: 1075–1080 25. Kimura T, Ohyama A (1994) Interaction with the Rev response element along an extended stem I duplex structure is required to complete human immunodeficiency virus type 1 rev-mediated trans-activation in vivo. J Biochem 115:945–952 26. Kimura T, Hashimoto I (2004) Functional links between the steps in mRNA biogenesis and nuclear export. In: Pandalai SG (ed) Recent research developments in molecular and cellular biology. Research Signpost, Kerala, pp 197–208 27. Saavedra CA, Hammell CM, Heath CV, Cole CN (1997) Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p. Genes Dev 11:2845–2856 28. Kimura T, Hashimoto I, Nishikawa M (2003) HIV-1 Rev protein: an RNA adapter between the cis-acting viral target site and the cellular CRM1/XPO1 nuclear export factor. In: Pandalai SG (ed) Recent research developments in biochemistry. Research Signpost, Kerala, pp 519–548 29. Wolff B, Sanglier JJ, Wang Y (1997) Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem Biol 4:139–147 30. Otero GC, Harris ME, Donello JE, Hope TJ (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 31. Fornerod M, van Deursen J, van Baal S, Reynolds A, Davis D, Murti KG, Fransen J, Grosveld G (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 32. Nishimoto T, Eilen E, Basilico C (1978) Premature of chromosome condensation in a ts DNA- mutant of BHK cells. Cell 15:475–483 33. Kadowaki T, Goldfarb D, Spitz LM, Tartakoff AM, Ohno M (1993) Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. EMBO J 12:2929–2937 34. Nishitani H, Ohtsubo M, Yamashita K, Iida H, Pines J, Yasudo H, Shibata Y, Hunter T, Nishimoto T (1991) Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis. EMBO J 10:1555–1564 35. Richards SA, Carey KL, Macara IG (1997) Requirement of guanosine triphosphate-bound ran for signal-mediated nuclear protein export. Science 276:1842–1844 36. Gorlich D, Kutay U (1999) Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 15:607–660 37. Kudo N, Wolff B, Sekimoto T, Schreiner EP, Yoneda Y, Yanagida M, Horinouchi S, Yoshida M (1998) Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res 242:540–547 38. Zolotukhin AS, Felber BK (1999) Nucleoporins nup98 and nup214 participate in nuclear export of human immunodeficiency virus type 1 Rev. J Virol 73:120–127 39. Gruter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi A, Wilm M, Felber BK, Izaurralde E (1998) TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell 1:649–659 40. Saavedra C, Felber B, Izaurralde E (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

81 41. Mattaj IW, Englmeier L (1998) Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 67:265–306 42. Popa I, Harris ME, Donello JE, Hope TJ (2002) CRM1-dependent function of a cis-acting RNA export element. Mol Cell Biol 22:2057–2067

43. Otero GC, Hope TJ (1998) Splicing-independent expression of the herpes simplex virus type 1 thymidine kinase gene is mediated by three cis-acting RNA subelements. J Virol 72:9889–9896