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1 Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan
2 Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan
3 Department of Physics, School of Science and Engineering, Waseda University, Okubo, Tokyo 169-8555, Japan
4 Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
5 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan
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Abstract |
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-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells. |
Introduction |
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Genetic studies in yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe have been an effective approach for identifying components involved in mRNA export (for a review see Sträßer & Hurt 1999). Temperature-sensitive mutants defective in mRNA export were isolated by screening with fluorescence in situ hybridization (FISH) using an oligo dT as a probe. Characterization of those mutants has implicated a variety of proteins in mRNA export. A synthetic lethal screen in yeast also worked effectively to identify factors required for mRNA export. mex67, the wild-type gene of which encodes an export receptor, was identified in a synthetic lethal screen with the nup85
allele (Segref et al. 1997). Yra1p, a conserved nuclear RNA binding protein, which acts as a bridge between mRNA and Mex67p, was also identified as an adapter protein for mRNA export in a synthetic lethal screen with a temperature-sensitive allele of MEX67 (Sträßer & Hurt 2000). The conserved spliceosomal component Sub2p was proposed to recruit Yra1p to the mRNA during pre-mRNA splicing, thereby performing its role in mRNA export in yeast (Luo et al. 2001; Sträßer & Hurt 2001). Yra1p and Sub2p are members of the TREX (transcription and mRNA export) complex, which is thought to link transcription and mRNA export in yeast (Sträßer et al. 2002).
Fischer et al. (2002) reported a genetic interaction between Yra1p and Sac3p. Sac3p was shown to interact with Sub2p in vivo and form a stable complex with Thp1p involved in transcriptional elongation (Fischer et al. 2002). Sac3p was also shown to associate with Mex67p and nucleoporin Nup1p. Recently, it was found that the Sac3p-Thp1p complex interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, via a novel protein Sus1p (Rodríguez-Navarro et al. 2004). These findings also support a connection between transcription and mRNA export in yeast. In addition to yeast genetics, microinjection of RNAs or proteins into Xenopus laevis oocytes has been taken as a complementary approach to analyze the mechanisms of mRNA export. Early studies using this technique showed that the cap structure and polyA tail can facilitate mRNA export from the nucleus. A 32P-labeled mRNA synthesized with the m7G cap structure was exported from the nucleus to the cytoplasm in X. laevis oocytes more rapidly than an mRNA synthesized with an adenosine (A) or tri-methylguanosine (m3G) cap (Hamm & Mattaj 1990; Jarmolowski et al. 1994). In addition, co-injection of excess m7GpppG dinucleotide into the oocyte nucleus effectively inhibited mRNA export (Hamm & Mattaj 1990; Dargemont & Kühn 1992). The export of injected mRNA lacking the polyA tail was also slower than that of mRNA with the polyA tail in X. laevis oocytes (Jarmolowski et al. 1994).
Using the X. laevis oocytes system, Reed and coworkers (Luo & Reed 1999; Zhou et al. 2000) showed that mRNAs generated through splicing are exported at high rates, whereas the corresponding mRNAs transcribed from the intron-less cDNAs are poor substrates for export. They also found that Aly, the metazoan homolog of the yeast Yra1p, is recruited to mRNP complexes generated by splicing and injection of excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo (Zhou et al. 2000). These results suggest that pre-mRNA splicing is coupled to mRNA export. Drawing upon data from genetic screens in yeast and microinjection studies in Xenopus oocytes, it is clear that there exists a complex relationship among the steps in eukaryotic gene expression including transcription, splicing and mRNA export.
In the present study, we analyzed nucleocytoplasmic transport of fluorescently labeled pre-mRNA microinjected into the nuclei of mammalian cells (HeLa cells). The injected pre-mRNAs were exported to the cytoplasm from the nucleus via an energy-dependent process after splicing. Interestingly, we found that inhibition of gene transcription impairs export of the injected pre-mRNA and causes the accumulation of the fluorescent mRNA in nuclear domains named TIDR (transcriptional-inactivation dependent RNA domain), which are localized adjacent to the SC35 speckles. Our results suggest a linkage between nucleocytoplasmic transport of mRNA and ongoing gene transcription in mammalian cells.
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Results |
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To examine nuclear export of mRNA in mammalian cells, we synthesized a m7G capped Cy3-labeled fushi tarazu (ftz) pre-mRNA containing one intron and a 30 nt polyA tail (497 nt, see Experimental procedures) and microinjected it into the nuclei of HeLa cells. After incubation for 15, 30, 60 or 120 min at 37 °C, the distribution of Cy3-labeled mRNA was examined using fluorescence microscopy. FITC-Dextran (70 kDa), which cannot move to the cytoplasm through nuclear pores by passive diffusion, was co-injected into the nuclei to confirm the nuclear injection of the fluorescent mRNA. Figure 1A shows results of a typical experiment. The injected ftz pre-mRNA was initially distributed throughout the nucleus, except for the nucleolus (Fig. 1A, 0 min), and then foci enriched with injected mRNA were observed in the nucleus at 15 min (15 min). The fluorescence in the cytoplasm increased with incubation time, suggesting that the injected mRNA was transported to the cytoplasm (60 min). In some cells, the intensity of the mRNA fluorescence decreased 120 min after injection, suggesting that degradation of the injected mRNA had occurred.
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The injected ftz pre-mRNA is co-localized with the splicing factor SC35 and spliced
It was previously shown that the splicing factor SC35 localizes to nuclear speckles (Fu & Maniatis 1990). Here, we examined whether the nuclear foci, which were detected in the nucleus of the injected cells 15 min after injection, are nuclear SC35 speckles. The injected cells were stained with anti-SC35 antibody as shown in Fig. 2. Most of the ftz foci co-localized with the SC35 nuclear speckles, suggesting that the Cy3-labeled pre-mRNA associates with splicing factors.
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Since the above result indicated that a portion of the injected fluorescently labeled pre-mRNA can be spliced, we next wondered whether the cytoplasmic fluorescence resulted from export of the spliced mRNA as opposed to the unspliced intron-containing pre-mRNA. To this end, unlabeled ftz pre-mRNA was injected into the nucleus and in situ hybridization was performed using two different oligonucleotide probes. These probes distinguish between unspliced (P-probe) and spliced (M-probe) products (Fig. 3B). RT-PCR analysis revealed that the injected unlabeled ftz pre-mRNA was spliced more efficiently than the fluorescently labeled ftz pre-mRNA (Fig. 3C). As shown in Figure 3D, P-probe and M-probe gave nucleoplasmic staining with a speckled pattern at 15 min after injection. After 60 min incubation, significant cytoplasmic staining was observed with the M-probe, whereas no hybridization signal was detected in cells with the P-probe. These results suggest that the majority of the injected unlabeled ftz pre-mRNA is spliced completely after 60 min incubation and the spliced ftz mRNA is transported to the cytoplasm.
The intron-less ftz mRNA is diffusely distributed in the nucleus and is unstable
To investigate whether splicing has an effect on the export of the injected mRNA, we injected a Cy3-labeled intron-less ftz mRNA with a 30nt polyA tail (346 nt) into the nuclei of the HeLa cells. In contrast with intron containing ftz pre-mRNA, the intron-less ftz mRNA was diffusely distributed throughout the nucleus with the exception of the nucleolus and no speckle-like distribution was observed (Fig. 4A). The intensity of the Cy3 fluorescence was found to decrease gradually. We could not detect significant quantities of amplified product corresponding to the intron-less ftz mRNA by RT-PCR at 2 h after injection (Fig. 4B). These data suggest that injected intron-less ftz mRNA is degraded and is less stable than its intron-containing counterpart. Some cells showed a faint cytoplasmic fluorescent signal at 60 min, suggesting that a small amount of intron-less ftz mRNA could be exported to the cytoplasm (Fig. 4A). However, we could not exclude the possibility that the increase in cytoplasmic fluorescence in these cells was due to simple diffusion of the degraded mRNA fragments to the cytoplasm.
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To determine whether the nuclear export of fluorescently labeled mRNA is general or unique to the ftz mRNA derived transcript, we carried out similar experiments using another substrate derived from the human ß-globin sequence. When a m7G capped Cy3-labeled ß-globin pre-mRNA containing one intron and a 30nt polyA tail (543 nt, see Experimental procedures) was injected into the nuclei of HeLa cells, the cells showed a non-nucleolar nucleoplasmic distribution at 15 min (Fig. 5A). Unexpectedly, the nuclear speckled pattern of the fluorescent mRNA was not detected at that time. However, a weak speckled distribution began to appear and an increase in cytoplasmic signal was observed after 60 min (Fig. 5A). These observations suggest that the kinetics of the splicing reaction of the ß-globin pre-mRNA is slower than that of the ftz pre-mRNA. RT-PCR analysis of the ß-globin pre-mRNA showed that a certain amount of pre-mRNA remains even after 120 min incubation, suggesting that splicing of the injected ß-globin pre-mRNA is inefficient (data not shown).
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Taken together with the results of the ftz mRNA microinjection, the rate and the efficiency of mRNA export seem to be dependent on the sequence. Splicing may also affect the mRNA export and stability of RNA transcripts. These observations are consistent with the data obtained from previous Xenopus oocyte microinjection assays.
Treatment with transcriptional inhibitors affects export of the injected mRNA
Next, we investigated effects of several drugs on the export of the fluorescently labeled mRNA in injected HeLa cells (Fig. 6A). HeLa cells injected with Cy3-labeled ftz pre-mRNA were incubated in medium containing either leptomycin B (LMB; an inhibitor for nuclear export of proteins containing a leucine rich nuclear export signal (NES)) or cycloheximide (a translational inhibitor). As shown in Figure 6A-b,d, neither LMB nor cycloheximide significantly inhibited mRNA export. In contrast, when the injected cells were treated with transcriptional inhibitors, such as actinomycin D, -amanitin, or 5,6-dichloro-1-ß-D-ribofuranosylbenzimadizole (DRB), respectively, no increase in cytoplasmic fluorescence was observed (Fig. 6A-f,h,j). The number of the nuclear ftz speckles decreased and the size of each speckle increased relative to those in untreated cells. These observations suggest that transcriptional inactivation causes inhibition of fluorescently labeled mRNA export and alters the subnuclear structure.
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To examine if the transcriptional inhibition also causes defects in protein import and export, we transfected HeLa cells with pcDNA-R1GLFPA6 that expresses a GST-NES-NLS-GFP fusion protein, and then intracellular distribution of the expressed fusion protein was observed. As shown in Fig. 6C, the fusion protein distributed predominantly in the cytoplasm in the absence of LMB, whereas addition of LMB led to the accumulation of the fusion protein in the nucleus. These observations suggest that the expressed fusion protein is shuttling between the nucleus and the cytoplasm in HeLa cells. The same phenomenon was observed in the presence of actinomycin D (Fig. 6C-e–g), suggesting that protein import and export are not affected by transcriptional inactivation, in contrast to the export of the injected mRNA.
Injected ftz mRNA is enriched in novel nuclear domains, TIDRs, in the transcriptionally inactivated cells
To identify the precise localization of the reorganized nuclear ftz speckles, actinomycin D treated cells were stained with several antibodies against components of known nuclear bodies, including splicing SC35 speckles, Cajal bodies and PML (promyelocytic leukemia protein) bodies. Immunostaining with antibodies against p80 coilin (a marker for Cajal bodies, Andrade et al. 1991) and PML (Santa Cruz, PG-M3) showed no significant stained signals in actinomycin D treated HeLa cells, although those nuclear bodies were clearly stained by the antibodies in non-treated cells (data not shown). These results suggest that Cajal and PML bodies disassemble in transcriptionally inactivated HeLa cells. On the other hand, reorganized ftz speckles mostly co-localize with the SC35 speckles, but are also found to be more intense in areas adjacent to the SC35 speckles after actinomycin D treatment (Fig. 7A, upper panels). The same phenomenon was observed in actinomycin D treated HeLa cells injected with ß-globin pre-mRNA (Fig. 7A, lower panels).
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Endogenous poly(A)+ mRNA is also retained in the nucleus after treatment with a transcriptional inhibitor
We wondered whether transcriptional inactivation inhibits the nuclear export of endogenous poly(A)+ mRNAs leading to their accumulation in TIDRs, as was observed for injected Cy3 labeled ftz pre-mRNA. To this end, Cy3-labeled ftz pre-mRNA without a polyA tail [poly(A)–ftz pre-mRNA] was injected into the nuclei of the HeLa cells and in situ hybridization was performed with an oligo dT probe to detect endogenous poly(A)+ mRNA, after acitinomycin D treatment (Fig. 8A). This assay allowed us to label specifically both the endogenous poly(A)+ mRNA and injected ftz pre-mRNA. Similar to the poly(A)+ transcript, poly(A)–ftz pre-mRNA was found to accumulate in TIDRs, adjacent to SC35 speckles, after 2 h treatment with actinomycin D (Fig. 8A). However, we detected no drastic enrichment of endogenous poly(A)+ mRNA in TIDRs. Due to the sensitivity of in situ hybridization, we cannot exclude the possibility that a small amount of poly(A)+ mRNA accumulated in TIDRs after the transcriptional inactivation.
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-amanitin that inhibits transcription by RNA polymerase II. These results suggest that export of endogenous mRNAs is also impaired by transcriptional inactivation. |
Discussion |
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We found that the intron-less ftz and ß-globin mRNAs transcribed from the cDNAs are not distributed to the SC35 speckle domains in contrast to the cases of pre-mRNAs (Figs 4 and 5B). They degraded more rapidly than pre-mRNA after injection into the nucleus. Formation of the exon junction complex (EJC) on the spliced mRNAs during pre-mRNA splicing (Le Hir et al. 2001) might enhance the stability of the injected pre-mRNAs in cells. It was not apparent that degradation of the injected intron-less mRNA occurred in the nucleus or in the cytoplasm. Thus, we could not determine if splicing reaction promotes the export of the injected mRNA in mammalian cells, as reported in the Xenopus oocyte system by Reed and coworkers (Luo & Reed 1999; Luo et al. 2001).
It was previously reported that fluorescently labeled ß-globin or adenovirus pre-mRNA microinjected into the nuclei of cultured mammalian cells localizes in 30–60 discrete nuclear sites (Wang et al. 1991; Melcák et al. 2001). In those experiments, however, neither splicing of the injected pre-mRNA nor its export from the nucleus was analyzed.
Watanabe et al. (1999) also examined nuclear export of digoxigenin-labeled, capped mature TAK1 mRNA (1.7 kb) injected into the nuclei of rat fibroblastic 3Y1 cells. They found that it takes 5 h until the majority of the injected mRNA becomes cytoplasmic. The slow export of the injected mRNAs in their system is probably due to their use of the intron-less mRNA as a substrate. In their assay system, export of the injected mRNA was inhibited by treatment with leptomycin B. In contrast to their results, leptomycin B had no significant effect on the export of injected fluorescent ftz pre-mRNA, while at the same concentration NES-mediated protein export was completely inhibited (Fig. 6C). Previous experiments in S. cerevisiae, X. laevis oocytes and HeLa cells suggested that CRM1-mediated export pathway is not a major mRNA export route (Fornerod et al. 1997; Neville & Rosbash 1999; Ossareh-Nazari et al. 2000). Our results also support the notion that CRM1 is not involved in the major mRNA export pathway in mammalian cells.
Unexpectedly, we found that treatment of cells with a transcriptional inhibitor, such as actinomycin D, DRB or -amanitin, leads to a drastic inhibition of nucleocytoplasmic transport of the injected fluorescent mRNA (Fig. 6A). Those transcriptional inhibitors block the transcription by totally different mechanisms. Actinomycin D exerts its effects by binding to DNA templates, thereby interfering with the elongation of growing RNA chains, and inhibits transcription by the RNA polymerases I to III (Kersten et al. 1960). DRB inhibits RNA polymerase II transcription specifically via inhibition of RNA polymerase II phosphorylation (Sehgal et al. 1976). -amanitin blocks transcription by binding stoichiometrically to RNA polymerases forming a tight complex (Lindell et al. 1970), and inhibited the export of the injected mRNA at the concentration required for specific inhibition of the RNA polymerase II transcription. Thus, inhibition of mRNA export by the transcriptional inhibitors seems to be a result of RNA polymerase II inactivation.
In situ hybridization of HeLa cells with the oligo dT probe showed that the amount of poly(A)+ mRNAs localized in speckled regions does not drastically decrease after treatment with actinomycin D (Fig. 8B,C). If there are no defects in mRNA export in cells treated with the transcriptional inhibitors, then poly(A)+ mRNAs present in the speckled regions would decrease rapidly by virtue of their transport to the cytoplasm. It is likely that the nuclear export of endogenous poly(A)+ mRNAs is also blocked by inactivation of gene transcription. Shopland et al. (2002) showed that, by using in situ hybridization, mRNAs do not disperse from SC35 domains in the nucleus when transcription is arrested. Thus, inhibition of mRNA export by transcriptional inactivation seems to be a general phenomenon that is not specific for injected mRNA.
As a high concentration of the translational inhibitor, cycloheximide, had no effects on mRNA export, blockage of mRNA export by the inhibition of transcription is not a secondary effect of decreased translation of factors required for mRNA export. Inactivation of gene transcription itself seems to affect the mRNA export process, raising the possibility of a functional cross-talk between gene transcription and mRNA export in mammalian cells.
Recently, functional cross-talk between transcription and mRNA export, as well as cross-talk among splicing, poly(A)+ addition and mRNA export, have been reported (reviewed in Reed 2003). Sub2p and Yra1p, factors involved in nuclear export of mRNA, were shown to associate with multisubunit THO complex required for transcription elongation (Jimeno et al. 2002). Yeast null mutants of THO components accumulate poly(A)+ RNA and heat shock SSA1 mRNA in the nuclei at the non-permissive temperature (Sträßer et al. 2002). The THO complex with mRNA export factors was designated TREX, which is thought to link between transcription and nuclear mRNA export. Sus1p that functions in mRNA export was shown to interact with a SAGA histone acetylase complex involved in transcription initiation (Rodríguez-Navarro et al. 2004). In addition, ptr6, one of mRNA transport mutants in S. pombe, was found to have a mutation in the gene encoding a transcriptional coactivator TAFII 55 (Shibuya et al. 1999). These findings suggest that there is a mechanism for co-transcriptional loading of export factors on to mRNAs. Such a co-transcriptional loading system, however, is not necessarily a prerequisite for formation of export competent complexes, as biologically active transport factors can be loaded on to mRNA post-transcriptionaly in the Xenopus oocyte system and in our export assay system. Our experiments also suggested that transcription and mRNA export is linked even when mRNAs have been exogenously synthesized and microinjected.
Nup98p, a GLFG (glycine-leucine-phenylalanine-glycine) repeat containing nucleoporin, is thought to aid in directing RNAs to the nuclear pore (Griffis et al. 2002). Photobleaching of green fluorescent protein-Nup98p in living cells revealed that Nup98p is mobile within the nucleoplasm and dynamically associates with both the nuclear bodies (Nup98 bodies) and nuclear pore complexes (Griffis et al. 2002). The mobility of Nup98p within the nucleus and at the pores was highly dependent on ongoing transcription by RNA polymerases I and II. Similarly, the activity of gene transcription might also affect dynamics of mRNA export factors in the nucleus.
Interestingly, some populations of the injected fluorescent mRNAs accumulated to form foci, which we designated TIDR, in the nucleus upon arrest of gene transcription (Fig. 7A). Those TIDRs localize side by side with several SC35 speckles. Such nuclear localization of TIDRs resembles that of paraspeckles (Fox et al. 2002). Paraspeckles contain at least three proteins, namely, PSP1, PSP2 and p54/nrb, which re-localize to the nucleolar periphery when transcription is inhibited. Immunostaining analysis revealed that PSP1 does not reside in TIDRs (Fig. 7B). Thus, it is likely that paraspeckles and TIDRs are distinct nuclear structures. Paraspeckles were suggested to function in RNA metabolism based on the fact that the paraspeckle proteins are RNA binding proteins (Fox et al. 2002). Since nuclear localization patterns of TIDRs and paraspeckles are quite similar, those domains might have a functional relationship. Further experiments are underway to clarify the function of TIDRs and its relationships to paraspeckles and SC35 speckles.
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Experimental procedures |
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HeLa cells were plated on to CELLocate coverslips (Eppendorf Inc.) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in 5% CO2.
Preparation of fluorescently labeled RNAs
For preparation of ftz pre- and intron-less mRNAs, pGEM-pre ftz and pGEM-mature ftz (Rio 1988) were digested with HindIII and SacI, and fragments containing a part of the Drosophila fushi tarazu (ftz) genomic gene or cDNA were inserted between HindIII and SacI sites of pSP64-polyA (Promega). ftz mRNAs (497 nt) with a polyA sequence (30 nt) was prepared from plasmids digested with EcoRI. The linearized plasmids were transcribed in a 50 µL solution containing 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 10 mM DTT, 1 mM of ATP, CTP and UTP, 0.2 mM GTP, 0.8 mM m7GpppG (Promega), 40 U SP6 RNA polymerase and 20 U RNase inhibitor for 4 h at 40 °C. After the reaction, the template DNA was digested with DNase I.
For preparation of ß-globin mRNAs, pSP64-Hß6 that contains a part of the human ß-globin gene or pSP64-Hß6-IVS1,2 that contains a part of the ß-globin cDNA (Krainer et al. 1984) was digested with HindIII and BamHI, and subcloned between HindIII and BamHI sites of pSP64-PolyA.
Labeling of the transcribed mRNAs with a fluorescent dye (Cy3) was carried out using a Label IT Cy3 nucleic acid labeling kit (Pan Vera Corp) according to a manufacturer's instruction. After the reaction, the labeled RNAs were purified using spin columns (Sephadex G-50) and ethanol-precipitated. The concentration of each RNA was measured using a spectrophotometer with absorbance at 260 nm. The amount of Cy3 coupled with mRNA was measured spectrofluorometrically at an excitation wavelength of 550 nm. On the average, each RNA was labeled with 
18 molecules of Cy3 per molecule.
Nuclear microinjection of RNA
All RNA samples were dissolved in microinjection buffer (10 mM NaH2PO4 (pH 7.2) and 70 mM KCl) and injected at a concentration of 1.5 µM with 1.5 mg/mL of lysine fixable FITC-conjugated 70 kDa dextrans (Molecular Probes Inc.). For microinjection, we used cells cultured for 2 days after plating, which were 80% confluent. Microinjection was done using an Eppendorf 5170 microinjector (injection pressure 50 hPa, compensation pressure 45 hPa and injection duration 0.5 s).
Detection of fluorescent signals
After injecting fluorescent mRNA into the nuclei, cells were incubated at 37 °C in 5% CO2. To detect the fluorescent signal, cells were washed with PBS and fixed for 15 min at room temperature in 3% formaldehyde in PBS. After washing, fluorescence images of the cells were obtained using an Olympus AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera.
Analysis by RT-PCR
Approximately 50 cells in a culture dish were injected with the fluorescent mRNAs and incubated at 37 °C for the indicated time. TRIZOL reagent (Invitrogen) was added to the culture dish to lyze the cells and to isolate total RNA. Reverse transcription of the injected mRNA was carried out using Ready-to-Go You-Prime First-Strand Beads (Amersham Biosciences) and primers corresponding to 3' end of the injected ftz or ß-globin mRNA (for the ftz mRNA, ftz4: 5'-TGGTAGCGGGTGTACGTCTG-3', and for the ß-globin mRNA, 5'-GGGGATCCACGTGCAGCTTG-3'). The PCR reaction was done using the resultant cDNA sample and appropriate primers (for the ftz mRNA, ftz4 and 5'-GCAGAAGCTGAAGAATGGCG-3', and for the ß-globin mRNA, 5'-ACACAACTGTGTTCACTAGC-3' and 5'-CACAGTGCAGCTCACTCAGT-3'). The amplified products were electrophoresed on an 8% polyacrylamide gel.
In situ hybridization
Cells were prepared for fluorescence in situ hybridization by fixing with 3% formaldehyde for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% triton X-100 for 5 min on ice, rinsed with PBS and 2x SSC. Hybridization was done at 42 °C in a solution containing 2x SSC, 20% formamide, 1 mg/mL of tRNA, 10% dextran sulfate and an oligo dT probe labeled with digoxigenin at the 3' end. After hybridization, cells were washed 3 x 10 min each, in 2x SSC, and incubated with anti-digoxigenin antibody followed by treatment with anti-mouse IgG antibody conjugated with FITC.
Analysis of protein import and export
To examine the defects in protein import and export in HeLa cells, the NdeI/SmaI fragment of pR1GLFPA6 encoding a GST-NES (Pap1)-NLS (SV40)-GFP (Kudo et al. 1999) was subcloned into pcDNA3.1 (Invitrogen). After transfection with the resultant plasmid, HeLa cells were cultured for 20 h and then treated with leptomycin B (LMB; 50 ng/mL), actinomycin D (5 µg/mL) or both. When cells were treated with both LMB and actinomycin D, actinomycin D was added to the culture 60 min prior to LMB addition. After incubation for 60 min, localization of the GFP fusion protein was examined using an OLYMPUS AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera.
Immunofluorescence analysis
For indirect immunofluorescence analysis, cells injected with the Cy3 labeled mRNAs were fixed with 2% formaldehyde for 15 min (5 min for detection of PSP1), treated with a solution containing 0.5% BSA and 0.2% Triton X-100 for 5 min on ice, and incubated with the mouse anti-SC35 antibody (PharMingen) or the rabbit anti-PSP1 antibody (kindly provided by Dr Fox) for 1 h. Subsequently, cells were incubated with FITC-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Cappel) for 2 h and analyzed using a Nikon Digital Eclipse confocal microscopy system. For inhibition of transcription by actinomycin D, a concentration of 5 µg/mL was used.
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Acknowledgements |
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Footnotes |
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aPresent address: Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
These authors contributed equally to this work.
* Correspondence: E-mail: ttani{at}sci.kumamoto-u.ac.jp |
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Received: 8 September 2005
Accepted: 24 November 2005
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