and Transport of tRNA-Attached Ribozymes to the - American

Biomacromolecules 2001, 2, 1229-1242

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Recognition of Engineered tRNAs with an Extended 3′ End by Exportin-t (Xpo-t) and Transport of tRNA-Attached Ribozymes to the Cytoplasm in Somatic Cells Tomoko Kuwabara,†,‡,§ Masaki Warashina,†,‡,§ Masayuki Sano,‡,| Hengli Tang,⊥ Flossie Wong-Staal,⊥ Eisuke Munekata,| and Kazunari Taira*,†,‡ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan, Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan, Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan, and Department of Medicine and Biology, University of California, San Diego, La Jolla, California 92093 Received June 19, 2001; Revised Manuscript Received August 22, 2001

Our recent analysis indicates that the cytoplasmic localization of tRNA-attached ribozymes (tRNA-Rz) is critical for its high-level intracellular activity, suggesting that mature mRNAs in the cytoplasm are more accessible to ribozymes than pre-mRNAs in the nucleus (Kato et al. J. Biol. Chem. 2001, 276, 1537815385; Kuwabara et al. Nucleic Acids Res. 2001, 29, 2780-2788). Although studies in Xenopus oocytes led to the proposal that only correctly processed mature tRNAs are exported from nuclei in a RanGTPdependent manner (Lund and Dahlberg Science 1998, 282, 2082-2085), our tRNA-Rz with an extended 3′ end can also be exported to the cytoplasm in somatic cells. Xpo-t/RanGTP bound to tRNA-attached ribozymes in vitro and in somatic cells, with recognition basically resembling the recognition of mature tRNAs. In contrast, no binding to tRNA-attached ribozymes occurred in Xenopus oocytes. The injection of a nuclear extract of Xenopus oocytes together with tRNA-attached ribozymes inhibited the export of tRNAattached ribozymes but not mature tRNAs in somatic cells, suggesting the existence of an inhibitor(s) of the Xpo-t-dependent export pathway. Moreover, the inhibitor(s) appears responsible for a proofreading mechanism that operates in oocytes. Introduction The transport of molecules through nuclear pore complexes (NPCs) has been the focus of much recent attention. The exchange of proteins between the nucleus and the cytoplasm has been relatively well-characterized and appears to be mediated by saturable transport receptors that recognize signals on cargo molecules and then interact directly with NPCs, shuttling continuously between the nucleus and the cytoplasm.1-3 In addition to the transport receptors, a small GTPase called Ran appears to be essential for most nucleocytoplasmic transport.4-6 However, the mechanism of transport of RNA molecules, and in particular that of mRNAs, remains relatively unclear. The mechanism for transport of tRNAs has been much better characterized, and rapid progress has recently been made in efforts to understand the * Correspondence should be addressed to Prof. Kazunari Taira, at the following address: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Phone: 81(Japan)-3-5841-8828 or 81(Japan)-298-61-3015. Fax: 81(Japan)-3-5841-8828 or 81(Japan)-298-61-3019. E-mail: taira@ chembio.t.u-tokyo.ac.jp. † The University of Tokyo. ‡ National Institute of Advanced Industrial Science and Technology (AIST). § These authors contributed equally to the work. | Institute of Applied Biochemistry. ⊥ University of California, San Diego.

mechanism involved in the export of tRNAs to the cytoplasm from the nucleus.7-24 Such transport requires a tRNA-binding protein called exportin-tRNA (Xpo-t) and the Ran GTPase, and transport requires the hydrolysis of GTP. Only mature tRNAs, with accurately trimmed 5′ and 3′ ends and an attached 3′ CCA end appear to be recognized by Xpot.7,8,10,11,14 Aminoacylation of each tRNA also appears to be critical for the export of tRNAs from the nucleus to the cytoplasm in Xenopus oocytes11 and in yeast.19 In Xenopus oocytes, immature tRNAs with several extra nucleotides at the 3′ end are not recognized by Xpo-t, and thus, they are not exported to the cytoplasm. This phenomenon suggests the existence of a proofreading mechanism in cells whereby only tRNAs that are usable in the cytoplasm, with mature 5′ and 3′ ends, can be exported to the cytoplasm. However, in our studies of tRNA-attached ribozymes, which might be considered equivalent to a kind of immature tRNAs because of the extra sequences at their 3′ ends, such ribozymes were efficiently exported to the cytoplasm in mammalian cells.25-35 In the RNA polymerase III (pol III) transcription system that we use, the promoter is located within the tRNA sequence that is being transcribed. Thus, it is inevitable that a portion of the tRNA becomes incorporated into the ribozyme. In our expression system, therefore, ribozymes are linked downstream of a human tRNAVal

10.1021/bm0101062 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/08/2001

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Figure 1. Secondary structures of mature tRNAs and tRNA-driven ribozymes. (A) Secondary structures of wild-type tRNAs. (B) Secondary structures of the various tRNA-Rz’s that were efficiently exported to the cytoplasm. Secondary structures of (C) tBR-Rz and (D) U6-Rz, which accumulated in the nucleus. The sequences underlined in purple are the sequences complementary to the sequences of probes used for in situ hybridization.

though a linker (Figure 1). Figure 1 shows predicted secondary structures of ribozymes that we designed that are targeted against various genes and are expressed via the tRNAVal promoter. All of the ribozymes shown in Figure 1B exhibit extremely high activities in mammalian cells, and

all are efficiently exported from the nucleus to the cytoplasm.25-35 Our tRNA promoter-driven ribozymes (tRNA-Rz) can be classified as non-aminoacylated immature tRNAs because each has an unprocessed 5′ end and an extremely extended

Recognition of tRNAs and Transport of Ribozymes

3′ end (which includes the ribozyme sequence) without a 3′ CCA end, but each is exported to the cytoplasm in mammalian cells. Therefore, in this study, we investigated the discrepancy between the reported observations that led to the proposal of the existence of a proofreading mechanism and our own observations of the efficient export of tRNARz. All of the previous studies that led to the proposal of a proofreading mechanism involved injection of RNAs into Xenopus oocytes. We investigated the export of various tRNA derivatives in several types of cells. Confirming our previous observations, we observed the efficient export not only of mature tRNAs but also of “immature-like tRNAs”, namely, tRNA-Rz, in somatic cells, and in addition, we detected specific binding of Xpo-t to our tRNA-attached ribozymes. However, in common with the observations by other groups, our observations revealed that the export of tRNA-attached ribozymes was dramatically inhibited in Xenopus oocytes, as predicted by the proofreading hypothesis. Our present analysis strongly suggests the existence of machinery for the transport of the immature-like tRNAs in somatic cells (but not in Xenopus oocytes). Materials and Methods Construction of Plasmids for Expression of tRNA-Rz. The construction of ribozyme-expression vectors using pUCdt (a plasmid that contains the chemically synthesized promoter for a human gene for tRNAVal between the Eco RI and Sal I sites of pUC 19) was described previously.28,29 In all cases, the pUC-dt was double-digested by Csp 45I and Pst I and each ribozyme sequence, with the terminator sequence UUUUU at the 3′ end, was ligated into the plasmid. Northern Blotting Analysis. Cells were grown to approximately 80% confluence (1 × 107 cells) and were transfected with a tRNA-Rz expression vector with the Lipofectin reagent (Gibco-BRL, Gaithersburg, MD). Thirtysix hours after transfection, the cells were harvested. For the preparation of the cytoplasmic fraction, collected cells were washed twice with PBS and then resuspended in digitonin lysis buffer (50 mM HEPES/KOH, pH 7.5, 50 mM potassium acetate, 8 mM MgCl2, 2 mM EGTA and 50 µg/mL digitonin) on ice for 10 min. The lysate was centrifuged at 1000g and the supernatant was collected as the cytoplasmic fraction. The pellets were resuspended in NP-40 lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and held on ice for 10 min, and the resultant lysate was used as the nuclear fraction. Cytoplasmic RNA and nuclear RNA were extracted and purified from the cytoplasmic fraction and the nuclear fraction, respectively, with ISOGEN reagent (Wako, Osaka, Japan). Thirty micrograms of total RNA per lane were loaded on a 3.0% NuSieve (3:1) agarose gel (FMC Inc., Rockland, ME). After electrophoresis, bands of RNA were transferred to a Hybond-N nylon membrane (Amersham Co., Buckinghamshire, U.K.). The membrane was probed with synthetic oligonucleotides that were complementary to the sequences of the ribozymes in question.28 A synthetic probe complementary to the sequence of each respective ribozyme was used, and all probes were labeled with 32P by T4 polynucleotide kinase (Takara Shuzo Co., Kyoto, Japan).

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In Situ Hybridization. HeLa S3 cells on a coverslip, which had been transfected in advance with one of the various plasmids, were washed in fresh PBS and fixed in fix/permeablization buffer (50 mM HEPES/KOH, pH 7.5, 50 mM potassium acetate, 8 mM MgCl2, 2 mM EGTA, 2% paraformaldehyde, 0.1% NP-40, 0.02% SDS) for 15 min at room temperature. Cells were rinsed three times in PBS for 10 min. Seventy micrograms of Cy3-labeled oligodeoxynucleotide probe with a sequence complementary to one of the underlined sequences in Figure 1 (Tm > 90 °C) and 20 µg of tRNA from E. coli MRE 600 (Boehringer Mannheim, Mannheim, Germany), dissolved in 10 µL of deionized formamide, were denatured by heating for 10 min at 70 °C. The mixture was then chilled immediately on ice. Ten microliters of hybridization buffer, containing 20% dextran sulfate and 2% BSA in 4× SSC, were added to the solution of the denatured probe. A 20-µL sample of hybridization solution containing the probe was placed on the coverslip, and the coverslip was inverted on a glass slide, sealed with rubber cement, and incubated for 16 h at 37 °C. Cells were rinsed in 2× SSC/50% formamide and in 2× SSC at room temperature for 20 min each. The coverslip was mounted with Vectashield (Vector Laboratories, Burlingame, CA) on a glass slide, and the cells were analyzed with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany). Pull-down Assay with Biotin-Labeled RNA. Biotinlabeled RNA was synthesized with an AmpliScribe T7 transcription kit (Epicentre Technologies, Madison, WI). The molar ratio of Biotin-21-UTP (Clontech, Palo Alto, CA) to UTP in the reaction was 1:5. A 70-µL sample of streptavidinconjugated agarose beads (Gibco BRL, Gaithersburg, MD) was washed twice with binding buffer (20 mM Tris-HCl, pH 7.5, 60 mM KCl, 2.5 mM EDTA, and 0.1% Triton X-100) and suspended in 100 µL of binding buffer. While the beads were kept on ice, 500 µL of cell extract (from HeLa S3 cells, NIH 3T3 cells, QCl cells, Xenopus oocytes, or A6 cells) was mixed with 70 µg of biotinylated RNA. After incubation on ice for 10 min, the total volume was adjusted to 1 mL with binding buffer. Then the sample was transferred to the tube with agarose beads, and the tube was rotated slowly overnight at 4 °C. The beads were washed five times with washing buffer (20 mM Tris-HCl, pH 7.5, 350 mM KCl, and 0.01% NP-40) and were finally resuspended in binding buffer. Proteins were eluted by boiling the beads and were separated by SDS-PAGE (7% polyacrylamide). For immunodetection of Xpo-t, the proteins were transferred to a PVDF membrane by the standard procedure. Immunoprecipitation and Analysis by RT-PCR. HeLa S3 cells were transfected with a tRNA-Rz expression vector with Lipofectin (Gibco BRL). Thirty-six hours later, the nuclear fraction and the cytoplasmic fraction were separated as described above. Both fractions were incubated overnight at 4 °C with 20 µL of normal rabbit serum that had been conjugated to agarose beads. After removal of the beads, the resulting lysate was divided into two parts. Each part was mixed with 50 µL of protein A-agarose beads (Amersham Pharmacia, Uppsala, Sweden) plus 3 µL of Xpo-tspecific antibodies (RXpo-t) or procaspase-3-specific anti-

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bodies (RCPP32). After incubation at 4 °C overnight, the beads were washed five times with lysis buffer (50 mM Hepes-KOH, pH 7.5, 60 mM KCl, 2.5 mM EDTA, and 0.1% Triton X-100). The bound RNA was extracted from beads by phenol extraction, ethanol precipitation, treatment with DNase I (3-h incubation at 37 °C), phenol extraction, and final precipitation in ethanol. The extracted RNA was subjected to analysis by RT-PCR with the appropriate ribozyme-specific primers, and the products of PCR were visualized on agarose gels under UV light. Recombinant Proteins. Histidine-tagged recombinant Xpo-t and Ran were expressed and purified as described previously.7 Footprinting of tRNA-Rz Bound to Xpo-t/RanGTP. In the RNA protection assays, we used 30 000 cpm of 5′end-labeled tRNA-Rz (tRNA-R32; Figure 1B). The RNA was mixed with a solution of 50 mM HEPES-KOH (pH 7.5), 5 mM magnesium acetate, 200 mM potassium acetate, and 7.5 µM GTP-loaded Ran.8 Recombinant Xpo-t was also added to a final concentration of 0.4-8 µM. Formation of a complex was achieved by incubation at 20 °C for 30 min. Reactions with Fe/EDTA were performed as described elsewhere36,37 for 10 min at 20 °C. Probing with RNase T1 was performed in the abovementioned buffer supplemented with 0.01 units of RNase T1 (Sigma, St. Louis, MO) for 3 min at 20 °C. Probing with RNase A was performed in the same buffer supplemented with 0.0002 units of RNase A (Qiagen, Hilden, Germany) for 3 min at 20 °C. Samples were loaded on a 12% denaturing polyacrylamide gel and subjected to electrophoresis until the region that included the 5′ or 3′ end of tRNA-Rz was well resolved. Microinjection of tRNA-Rz into Xenopus Oocytes. We injected a mixture of 32P-labeled DHFR mRNA, U1∆Sm RNA, U6∆ss RNA, yeast tRNAPhe, and tRNA-Rz into nuclei of Xenopus oocytes, as described in previous studies,7,8,38 and analyzed their distribution in the nucleus and cytoplasm after dissection of the oocytes at various times. In some experiments, 20 ng of recombinant Xpo-t were injected with the RNAs into each oocyte. Microinjection of tRNA-Rz into HeLa S3 Cells. Microinjection analysis using HeLa S3 cells was performed as described previously.39,40 FITC-labeled RNAs [tRNARz (tRNA-HIV Rz2, tRNA-R32), yeast tRNAPhe, U6-Rz, tBR-Rz, and human tRNAVal] were prepared with an AmpliScribe T7 transcription kit in combination with ChromaTide BODIPY FL-14-rUTP (Molecular Probes, Eugene, OR). The molar ratio of FL-14-rUTP to UTP in the reaction was approximately 1:5. Human tRNAVal and yeast tRNAPhe were used in their unmodified forms. For injection of FITC-labeled tRNA-Rz in combination with an excess amount of wildtype tRNAVal, unlabeled tRNAVal was mixed with tRNARz at final concentrations of 3.6 µg/µL and 20 ng/µL. Before injection, HeLa S3 cells were grown in 35-mm glass base dishes (Iwaki, Chiba, Japan). We injected RNA (final concentration in PBS of ∼20 ng/µL) that had been prepared in vitro into nuclei of HeLa S3 cells using a micromanipulation system (micromanipulator 5171 and transjector 5246; Eppendorf, Hamburg, Germany). Simultaneously, FluoroLink

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Cy3-labeled goat antibodies against rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ), which cannot pass through nuclear membranes, were injected into the nuclei as an internal control. Approximately 5 min after injection, the location of the injected RNA in the cells was monitored with a confocal laser scanning microscope. For injection of FITC-labeled RNAs in combination with a nuclear extract of Xenopus oocytes, 70 nuclei from Xenopus oocytes were collected and dissolved in 20 µL of lysis buffer that contained 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.02% NP-40, and 5 mM EDTA. For injection of FITClabeled RNAs in combination with a nuclear extract of Xenopus A6 cells, the nuclear extract was collected from 2 × 106 cells as described above and dissolved in 150 µL of the lysis buffer. FITC-labeled RNAs [tRNA-Rz (tRNAR32) and human tRNAVal] were dissolved in PBS at a final concentration of 20 ng/µL and mixed with the nuclear extract from Xenopus oocytes or from A6 cells. The ratio (v/v) of the solution of FITC-labeled RNA to the solution of nuclear extract from Xenopus oocytes in the mixture was 1-to-1. After incubation of the mixture for approximately 10 min on ice, it was injected into nuclei of HeLa S3 cells, with subsequent analysis as described above. Results Northern Blotting and in Situ Hybridization Reveal the Efficient and Specific Export of tRNA-Rz from Nuclei in HeLa Cells. We first reexamined the localization, under various conditions, of several tRNA-Rz’s in mammalian cells. Figure 2A shows the results of analysis by Northern blotting of the subcellular localization of one of our ribozymes (tRNAVal CPP Rz; Figure 1B). HeLa S3 cells were grown to approximately 80% confluence (1 × 107 cells) and were transfected with the vector that encoded the tRNAValdriven ribozyme. Thirty-six hours after transfection, total RNA was extracted as nuclear and cytoplasmic fractions. The conditions for the separation of cytoplasmic and nuclear fractions (see Materials and Methods) were optimized after several kinds of experiments in which we confirmed that the nuclear and cytoplasmic proteins remained in the respective fractions (Western blotting analysis) and also that the nuclear and cytoplasmic RNAs remained in the respective fractions (Northern blotting analysis; data not shown). Because there was a possibility that the strong treatment with the detergent digitonin, which we used for the separation of the cytoplasmic fractions, might have not only disrupted cell membranes but also damaged nuclear membranes, we carefully set the conditions for digitonin treatment such that only cell membranes were removed, without any damage to nuclear membranes. The successful separation of the cytoplasmic fraction was monitored by mixing the digitonintreated suspension of cells with 148-kDa fluorescent dextran (Figure 2A, bottom). If nuclear membranes had remained intact, this large fluorescent dextran molecule should have been excluded from the nuclei. The confocal image indicated that the nuclear membranes had indeed remained intact (Figure 2A, bottom) and that the separation of the cytoplasmic fraction had succeeded. Therefore, using these condi-



Figure 2. Intracellular localization of pol III transcripts. (A) Northern blotting analysis performed with the RNA from two intracellular fractions (N, nuclear; C, cytoplasmic) at various times after transfection. As a control, intracellular U6 snRNA, which remains in the nucleus, was also analyzed. We confirmed that, under our conditions for treatment with digitonin, only cell membranes were disrupted and nuclear membranes remained intact. (B) Analysis by in situ hybridization of tRNA-Rz in HeLa S3 cells. As controls, U6-Rz (Figure 1D) and tBR-Rz were also examined.

tions, we purified cytoplasmic and nuclear RNAs, 36 h after transfection, from the separated cytoplasmic and nuclear fractions. Transcripts of about 130 nucleotides in length, which corresponded in size to tRNA-Rz, were detected with a ribozyme-specific probe (Figure 2A, upper panels, left), and we confirmed that the tRNAVal-ribozyme transcripts (tRNA-Rz) had been exported to the cytoplasm. The predicted secondary structures of the various tRNA-Rz’s are shown in Figure 1B. We also investigated the kinetics of the export of tRNA-Rz. The total RNA from HeLa S3 cells that had been transfected with various plasmids was extracted 6, 12, 18, 24, 30 and 36 h after transfection. These samples of total RNA were also separated into nuclear and cytoplasmic fractions. Even initially, tRNA-Rz was found in the cytoplasmic fraction, and none was detected in the nuclear fraction (Figure 2A, upper panel, right), confirming the high efficacy, for the 5′- and 3′-extended version of tRNA-attached ribozymes, of the transport system in mammalian cells. In contrast, a tRNAiMet-linked ribozyme is known to accumulate in the nucleus,41 although it is also transcribed under the control of a tRNA promoter. The secondary structure of this specific transcript is shown in Figure 1C [tRNAiMet-BR Rz (tBR-Rz)]. We investigated the subcellular localization of this transcript (tBR-Rz) under the conditions described above. As shown in Figure 2A,

tBR-Rz accumulated in the nucleus without being transported into the cytoplasm for 36 h after transfection. To further strengthen our hypothesis, under more natural conditions, that properly designed tRNA-Rz can be exported to the cytoplasm, we attempted to detect the transcript directly by in situ hybridization. Figure 2B shows the results of such an analysis of the localization of tRNA-Rz in HeLa S3 cells. As controls, we also examined the U6 promoterdriven Rz (U6-Rz; Figure 1D) and tBR-Rz. HeLa S3 cells that had been transfected with the various plasmids were fixed and allowed to hybridize with Cy3-labeled oligodeoxynucleotide probes. Because each probe was specific for each ribozyme sequence, endogenous tRNAs and U6 RNAs were not detected by the probes. As seen in Figure 2B, tRNARz was detected only in the cytoplasm. In contrast, U6-Rz and tBR-Rz were detected in the nucleus. These results were in good agreement with the results of the Northern blotting analysis that are shown in Figure 2A and confirmed unambiguously that tRNA-Rz was exported specifically and effectively to the cytoplasm in HeLa cells.33,35 When we compare the secondary structures of the transcripts that are transported to the cytoplasm (Figure 1B) with those that accumulate in the nucleus (Figure 1C), it is clear that the structures are different. In particular, the first half of the transcripts, corresponding to the tRNA portion, is important: the transcripts that are transported to the cyto-

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plasm can assume a cloverleaf structure similar (but not identical) to that of a native tRNA, whereas the transcripts that accumulate in the nucleus have structures that are quite different from that of a tRNA (one example is shown in Figure 1C). Thus, it appears that, in the case of the export of tRNA-attached ribozymes with extended 5′ and 3′ ends (tRNA-Rz), receptors that are involved in the recognition of native tRNA might also be responsible for the export of tRNA-Rz in mammalian cells. Evidence that tRNA-Rz Exported to the Cytoplasm Has Not Been Processed at Its 5′ and 3′ Ends. Xpo-t has been identified as a specific receptor that is responsible for the export to the cytoplasm of native tRNAs, and only mature tRNAs are recognized as the correct cargo by Xpo-t in Xenopus oocytes. Therefore, we examined tRNA-Rz that had been exported to the cytoplasm in mammalian cells in terms of processing at the 5′ and 3′ ends to confirm that the various tRNA-Rz’s were exported to the cytoplasm in their immature forms rather than as trimmed and matured forms in mammalian cells. The size, enzymatic activity, and sequencing of the cytoplasmic tRNA-Rz unambiguously demonstrated that each exported tRNA-Rz in mammalian cells did indeed have unprocessed 5′ and 3′ ends with the specific sequences depicted in Figure 1B (see details in ref 35). Specific Binding of tRNA-Rz to the Xpo-t/RanGTP Complex. Our conclusion that all of the exported tRNARz had unprocessed 5′ ends was confirmed not only in HeLa cells but also in all other cells tested, which included other lines of human cells, such as 293 cells and H9 cells, and several lines of mouse cells, such as NIH 3T3 cells and BaF3 cells (data not shown). Because the tRNA-Rz that were transported to the cytoplasm (Figure 1B) apparently retained the cloverleaf motif, we postulated that the transcripts might be transported preferentially to the cytoplasm via the same transport pathway as followed by endogenous native tRNA. In the export of native tRNA, the receptor protein Xpo-t, acting in cooperation with Ran, has been proposed as the major pathway-specific transport receptor.7,10 To examine potential interactions between our tRNA-Rz and Xpo-t, we incubated an extract of HeLa cells with biotin-labeled tRNA-Rz. Proteins that had bound to the biotin-labeled tRNA-Rz were “pulled down” with streptavidin beads. The beads were washed extensively, and then the proteins were eluted. The eluted proteins were analyzed by SDS-PAGE with subsequent staining with Coomassie Blue (Figure 3A, middle). Then, the proteins were probed with antibodies against Xpo-t (Figure 3A, right). The immunoblot revealed that the tRNAVal transcript without a ribozyme sequence (tRNA linker), whose secondary structure is shown in Figure 1B, bound Xpo-t under these conditions. Furthermore, the tRNA-Rz designated tRNA CPP Rz also bound intracellular Xpo-t (Figure 3A, right), demonstrating the interaction between tRNA-Rz and Xpo-t. To examine the specificity in the interaction between other types of tRNA-Rz and Xpo-t and, in addition, to compare the binding affinity with that of various negative and positive controls, we repeated the pull-down assay with each biotin-

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labeled RNA. As negative controls, we prepared biotinlabeled RNAs with the sequence of the multicloning site (MCS) in pBlueScript II SK+ (Stratagene, La Jolla, CA), the U6 promoter-driven Rz (U6-Rz), and the CTE (constitutive transport element). The CTE is known as a cis-acting viral RNA that is involved in the export of unspliced genomic RNA to the cytoplasm for the expression of viral structural proteins and packaging. The CTE RNA is localized in the cytoplasm. Transcription of U6-Rz is driven by the U6 promoter, and U6-driven transcripts are found exclusively in the nucleus (Figure 2B). These three control RNAs, with different intracellular locations, were not expected to interact with Xpo-t, and so we used them as negative controls. As positive controls, we prepared biotin-labeled yeast tRNAPhe and biotin-labeled human tRNAVal (Figure 1A). The wildtype tRNAPhe from yeast binds specifically to Xpo-t.8 In addition to these controls, we prepared three tRNAVal-driven transcripts (tRNAVal HIV Rz2, tRNAVal TAR Rz, and tRNAVal-R32; Figure 1B) that were all efficiently exported to the cytoplasm (data not shown), as well as the tRNAiMetdriven transcript (tBR-Rz; Figure 1C) that stayed in the nucleus (Figure 2), to examine their ability to interact with Xpo-t. As we had expected, the negative control RNAs did not interact with Xpo-t (Figure 3B; lanes 1, 2, and 9). In contrast, both wild-type tRNAPhe from yeast (lane 3) and wild-type human tRNAVal (lane 4) clearly interacted with Xpo-t. Moreover, as anticipated, all of our tRNA-Rz’s interacted with Xpo-t, and to our surprise, the affinity and specificity were the same as those for wild-type tRNAPhe and tRNAVal. In the case of tBR-Rz, which was not transported to the cytoplasm (Figure 2), the binding of tBR-Rz to Xpo-t was very limited. To quantify the affinity of the binding between these RNAs and Xpo-t, we used fluorescein isothiocyanateconjugated (FITC-conjugated) antibodies against IgG as second antibodies during the same Western blotting analysis, and then we quantified the intensities of the bands with a Fluoro-imager (Figure 3B, right). Relative intensities are indicated in Figure 3B below the images of the blot, with the intensity for human tRNAVal taken as 100%. Clearly, the extent of the interaction between tBR-Rz and Xpo-t was significantly lower than that between tRNA-Rz and Xpo-t. The levels of interaction appeared, moreover, to reflect the efficiency of export of each respective RNA (note that a small amount of tBR-Rz was exported to the cytoplasm, as indicated in Figure 2A). Because the above pull-down assay strongly suggested the involvement of Xpo-t in the export of tRNA-Rz, we attempted to confirm the interaction between tRNA-Rz and Xpo-t in vivo. We used Xpo-t-specific antibodies (RXpo-t) to immunoprecipitate RNAs that interacted with Xpo-t from HeLa cells that had been transfected with the vectors that encoded the tRNA-Rz. Antibodies raised against procaspase-3 (RCPP32) were used as negative controls in these experiments. The extracted RNA was subjected to analysis by RT-PCR with ribozyme-specific primers. Figure 3C shows that the Xpo-t-specific antibodies brought down tRNA-Rz transcripts. In contrast, the procaspase-3-specific antibodies did not bring down tRNA-Rz transcripts (Figure



Figure 3. Specific binding of tRNA-Rz to endogenous Xpo-t. (A) Pull-down assay with biotin-labeled tRNA transcripts. Total proteins in an extract of HeLa cells (left), the proteins bound to biotin-labeled tRNA transcripts (center), and the protein (Xpo-t) that reacted with Xpo-t-specific antibodies (right) are shown. (B) Comparison of the binding affinities for Xpo-t of the various transcripts. The pull-down assay was performed with each biotin-labeled RNA. The relative affinity of the binding between each RNA and Xpo-t is shown under the Western blot obtained with RXpo-t/FITC-IgG (Xpo-t; bottom right). Bands of an unidentified protein of about 200 kDa that appeared upon staining with Coomassie Brilliant Blue after transfer to the membrane were used as internal controls. (C) Immunoprecipitation of Xpo-t that interacted with RNAs from HeLa cells. Xpo-t-specific antibodies (RXpo-t) were used to immunoprecipitate Xpo-t-interacting RNAs. Antibodies specific for procaspase-3 (RCPP32) were used as negative controls. Extracted RNA, after DNase I treatment, was subjected to analysis by RT-PCR with ribozyme-specific primers. In the case of (-)RT-PCR, PCR was performed without prior reverse transcription, and it revealed that no DNA was amplified from the plasmid DNA. Lanes M9 and M4 were loaded with DNA size markers (Toyobo, Osaka, Japan).

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Figure 4. Sites within tRNA-Rz involved in formation of the complex with Xpo-t/RanGTP, as determined by footprinting. (A) Protection analysis of the binding of Xpo-t/RanGTP to tRNA-Rz using a variety of probing reagents. (B) Summary of results of footprinting analysis of binding of tRNA-Rz to Xpo-t/RanGTP. Pink underlining and circles indicate identified sites of interaction. The yellow ribbon indicates part of the target substrate for tRNA-Rz (blue ribbon). However, the footprinting analysis was performed in the absence of the substrate.

3C, right). These results further strengthened our hypothesis that Xpo-t did indeed interact with tRNA-Rz and was involved in the transport of tRNA-Rz to the cytoplasm in mammalian cells. However, we could not exclude the possibility that the interaction might be mediated by some other cellular protein(s) or nucleic acid(s) in vivo.

Sites within tRNA-Rz of Interaction with Xpo-t/ RanGTP as Revealed by Footprinting. Because our analysis strongly suggested that tRNA-Rz interacted with Xpo-t in vivo, we attempted to identify the specific sites within tRNA-Rz that interact with Xpo-t. Figure 4A shows the results of protection analysis of the binding of Xpo-t/


RanGTP to tRNA-Rz for a variety of probing reagents. Treatment with Fe/EDTA results in cleavage of the ribose sugar rings through the local production of hydroxyl radicals. Extensive protection of tRNA-Rz against cleavage by Fe/ EDTA was evident in the presence of Xpo-t and RanGTP. We determined the positions of protected riboses using 5′end-labeled tRNA-Rz (tRNA-R32, Figure 1B). We also used, ribonuclease T1 and ribonuclease A as probing reagents. RNase T1 specifically cleaves internucleotide bonds at the 3′ phosphate of G residues in RNA. Under mild conditions, RNase A cleaves RNAs at the 3′ phosphates of pyrimidine (C and U) residues. Using these nucleases, we identified several protected regions in tRNA-Rz RNA (Figure 4). The data from our experiments with Fe/EDTA, RNase T1, and RNase A are summarized in Figure 4B, in which the identified sites of interaction are indicated by pink lines (left) and pink circles (right). The binding between Xpo-t/RanGTP and yeast tRNAPhe has been examined by footprinting and phosphate ethylation analysis.8 Arts et al. found that the region of the acceptor stem (in particular, the 3′ acceptor arm) and the TΨC arm were protected during binding with Xpo-t/RanGTP. This region is located in the upper half of the tertiary structure of tRNAPhe, and the nucleotide sequence of this region differs among various tRNAs. Therefore, the transport of tRNAs to the cytoplasm by Xpo-t seemed to require some aspects of the structure of tRNAs, rather than a specific sequence. We also identified the TΨC arm and the 3′ acceptor region as sites of interaction with Xpo-t. However, in addition, protection that was dependent on the concentration of Xpo-t was also apparent in the 5′ acceptor region and the D loop, as indicated by the pink lines and circles in Figure 4B. In fact, the extended 5′ and the unnatural 3′ acceptor (linker) sequences were also protected by Xpo-t/RanGTP. In particular, we detected evidence for the binding of Xpo-t/ RanGTP in the 3′ acceptor region, which had been partially replaced by the artificial linker sequence. In previous analyses, an extended 3′ acceptor stem on tRNA strongly inhibited export to the cytoplasm in Xenopus oocytes.7,8,10,11,14 In our analysis, the ribozyme portion, which we can regard as a region extended from the tRNA structure, did not bind Xpo-t/RanGTP. However, the extended ribozyme portion did not inhibit the binding of Xpo-t/RanGTP to the tRNA portion nor did it inhibit export to the cytoplasm in mammalian cells. The D loop and the TΨC arm are essential for the tertiary folding of tRNAs. In our tRNA-Rz, these regions were extensively protected by Xpo-t/RanGTP. Therefore, it is possible not only that the secondary structure of exported tRNA-Rz resembles a cloverleaf structure but also that the higher-order structure resembles the L-shaped structure of wild-type tRNA. It seems likely that Xpo-t can recognize and bind tRNA transcripts as long as their tertiary structures resemble those of a parental tRNA, as indicated in Figure 1B. Moreover, the 3′ extended portion does not appear to inhibit export into the cytoplasm at all in mammalian cells. Comparison of the Fate of tRNA-Rz (Immature-like tRNA) Transcripts in Xenopus Oocytes with That in Somatic Cells. The various tRNA-Rz’s can be classified


as immature tRNAs that have unprocessed 5′ ends, extremely extended 3′ ends (including a ribozyme sequence), no 3′ CCA end, and no aminoacylation. In previous studies of tRNA export, the specific export of correctly processed and aminoacylated mature tRNA was demonstrated.7,8,10,11,14 The proposal of a proofreading mechanism that would allow such tRNA export was based on studies in Xenopus oocytes with an injection system. In fact, almost all previous tRNA-export studies, with some recent exceptions,12,13,15,18,19,21-24 have been based on Xenopus oocytes and an injection system. Because all of our data indicated that tRNA-Rz was recognized by Xpo-t and efficiently exported to the cytoplasm in mammalian cells, we decided to examine the fate of tRNA-Rz in Xenopus oocytes. We injected a mixture of 32P-labeled DHFR mRNA for dihydrofolate reductase (DHFR), U1∆Sm RNA, U6∆ss RNA, yeast tRNAPhe, and our tRNA-Rz into nuclei of Xenopus oocytes in the same way as reported in previous studies,7,8,38 and we analyzed the distribution of each RNA in the nucleus and cytoplasm after dissecting the oocytes at various times after injection. The results are shown in Figure 5A. As expected, U6∆ss RNA stayed in the nucleus, and thus, it can be regarded as an internal control for successful injection into nuclei. In agreement with previous findings, within 45 min after injection, a significant proportion of yeast tRNAPhe (30%) had reached the cytoplasm. Moreover, when human Xpo-t was injected with yeast tRNAPhe into the nuclei of Xenopus oocytes, export of the tRNAPhe was greatly accelerated (100% export within 45 min). Export of DHFR mRNA and U1∆Sm RNA was unaffected by simultaneous injection of human Xpo-t, clearly demonstrating that Xpo-t specifically enhanced the export of tRNA only. In marked contrast, tRNA-Rz was not exported even 6 h after injection, and the addition of Xpo-t did not stimulate the export of tRNA-Rz from nuclei in Xenopus oocytes. Our observation that tRNA-Rz was not exported to the cytoplasm in Xenopus oocytes is in full agreement with the conclusion reached in earlier studies.8,11,14 However, this observation completely contradicts the results shown in Figure 2. As a possible reason for this discrepancy, because we could not exclude the possibility that export might be coupled with transcription, we had to consider the effects of an RNA that was transcribed on one hand and injected on the other. Figure 5A shows results for RNAs, prepared in vitro, that had been microinjected into oocytes. Figure 2 shows the export of RNAs that had been expressed intracellularly in HeLa cells. To determine whether RNAs prepared in vitro could also be exported from the nucleus to the cytoplasm in mammalian cells, we prepared internally FITC-labeled tRNA-Rz, yeast tRNAPhe, human tRNAVal, U6-Rz, and tBR-Rz and then injected each RNA into the nuclei of HeLa S3 cells. Simultaneously, Cy3-labeled IgG, which should not pass through the nuclear membrane, was injected into the nuclei as an internal control. Figure 5B shows the export of RNAs from the nuclei of HeLa cells 5 min after injection. In contrast to export in Xenopus oocytes, tRNA-Rz was efficiently exported into the cytoplasm in HeLa cells within just 5 min. The rate of transport of tRNA-Rz was nearly identical to that of the

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wild-type tRNAVal (Figure 5B) and of wild-type tRNAPhe (data not shown). As expected, U6-Rz and tBR-Rz remained within nuclei. As described above, our various tRNA-Rz’s can be regarded as a kind of immature tRNAs with an unprocessed 5′ end, an extremely extended 3′ end (more than 60 nucleotides), no 3′ CCA end, and no aminoacylation. Nonetheless, the efficient export of the tRNA-Rz from the nucleus into the cytoplasm of HeLa cells was confirmed even after microinjection, and moreover, the results are in good agreement with the results in Figure 2. Furthermore, when an excess amount of the unlabeled wildtype tRNAVal was simultaneously injected into HeLa cells, the wild-type tRNAVal apparently competed with the FITC-

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labeled tRNA-Rz for nucleocytoplasmic transport and significantly inhibited the export of the FITC-labeled tRNARz, indicating that the tRNA-Rz indeed shares the same transport pathway as wild-type tRNAVal (Figure 5B, lower panel). Each tRNA-Rz expression cassette did not contain an internal intron sequence in its cDNA sequence. The expressed tRNA-Rz transcripts were not processed at their 5′ and 3′ ends, and no 3′ CCA end was added. Therefore, conventional maturation of tRNA in the nucleus was not required for the export of tRNA-Rz. Because the injected tRNA-Rz that had been prepared in vitro was effectively exported to the cytoplasm in somatic cells, the coupling of any step in tRNA processing to export does not seem to be



Figure 5. Comparison of the fates of tRNA-Rz in Xenopus oocytes and in somatic cells. (A) Microinjection analysis of various RNAs in Xenopus oocytes. (B) Microinjection analysis of tRNA-Rz in HeLa S3 cells. (C) Pull-down assay with tRNA-Rz from various cell extracts. XO, Xenopus oocytes; Q, QCl-3 cells (quail cell line); M, NIH 3T3 cells (mouse cell line); H, human HeLa cells. (D) Pull-down analysis using differentiated A6 cells derived from Xenopus laevis. The red # highlights A6 cells derived from Xenopus laevis (XA6). (E) Northern blotting analysis and microinjection analysis of the tRNA-Rz. Northern blotting analysis of intracellular RNAs (N, nuclear; C, cytoplasmic) from various cells (left). XA6, A6 cells (Xenopus cell line); Q, QCl-3 cells (quail cell line); M, NIH 3T3 cells (mouse cell line); H; human HeLa cells. FITC-labeled tRNA-Rz was injected into nuclei of A6 cells with (bottom) and without (top) an excess amount of wild-type tRNAVal (right). (F) Microinjection analysis in HeLa S3 cells of tRNA-Rz in the presence and absence of a simultaneously injected extract of Xenopus oocytes. (G) Microinjection analysis in HeLa S3 cells of tRNA-Rz in the presence of simultaneously injected nuclear extracts from Xenopus oocytes (top) and from Xenopus A6 cells (bottom). Figure 5G shows the export of RNAs from the nuclei of HeLa cells 25 min after injection.

important during the export not only of tRNA-Rz but also of standard tRNAs. We next focused on the differences between Xenopus oocytes and somatic cells. As shown in Figure 4, the human Xpo-t/RanGTP complex apparently bound to tRNA-Rz in vitro. Therefore, it is likely that human recombinant Xpo-t

protein (in Figure 5A) could potentially bind to tRNA-Rz in Xenopus oocytes. However, such interaction was not sufficient and appeared not to be important for the export of tRNA-Rz in Xenopus oocytes. Alternatively, the interaction might have been strongly inhibited by some factors in Xenopus oocytes. To examine this possibility, we repeated

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the pull-down assay for which the results are shown in Figure 5C using biotin-labeled tRNA-Rz and extracts of various cells. We prepared four kinds of cell extract using Xenopus oocytes, QCl-3 cells (quail cells), NIH 3T3 cells (mouse cells) and human HeLa cells. First we confirmed that the antibodies against human Xpo-t (RXpo-t) reacted with Xpo-t from each extract (Figure 5C, left). The positive results allowed us to use RXpo-t in this set of experiments. The pulled-down proteins were analyzed by SDS-PAGE with subsequent staining with Coomassie blue (Figure 5C, right). The patterns of bound proteins were almost identical for the extracts of quail (lane Q), mouse (lane M), and human (lane H) cells (Figure 5C, right). However, in the case of Xenopus oocytes, the pattern was quite different (lane XO). The pulleddown proteins were then probed with RXpo-t. To our surprise, in the case of Xenopus oocytes, tRNA-Rz bound barely any intracellular Xpo-t. This result might imply the absence of some adaptor(s) that supports strong binding between tRNA-Rz and intracellular Xpo-t/RanGTP. However, because tRNA-Rz bound to Xpo-t/RanGTP in vitro without any adaptor molecule, we postulated the existence of a strong inhibitor(s) of the binding of tRNA-Rz and Xpot/RanGTP in Xenopus oocytes. These differences in transport of tRNA-attached ribozymes from the nucleus to the cytoplasm might have originated from the obvious differences between unfertilized Xenopus oocytes and the somatic (quail, mouse, and human) cells. We wondered whether the difference might have originated from a difference in the developmental stage of cells or from a difference in species. Therefore, we repeated our pull-down analysis using differentiated A6 cells derived from Xenopus laeVis. Figure 5D shows that tRNA-Rz bound to intracellular Xpo-t in A6 cells and that the patterns of bound proteins were very similar among differentiated cells from Xenopus, quail, mouse, and human. Importantly, tRNA-Rz was exported efficiently into the cytoplasm in Xenopus A6 cells, just as was the case in the quail QCl cells, mouse NIH 3T3 cells, and human HeLa cells (Figure 5E, left). Furthermore, when an excess amount of the unlabeled wild-type tRNAVal was injected simultaneously into A6 cells, wild-type tRNAVal significantly inhibited the export of the FITC-labeled tRNARz in a competitive fashion (Figure 5E, right). We also confirmed that each exported tRNA-Rz had ribozyme activity.35 Thus, the various tRNA-Rz’s had not been processed, in agreement with our previous findings.35 It should be emphasized that the binding interaction between tRNA-Rz and Xpo-t was completely different in unfertilized Xenopus oocytes and in somatic A6 cells derived from Xenopus laeVis. The difference might be related to differences in properties of nuclear membranes at different stages of the cell cycle, namely, whether cells are arrested prior to meiosis or not. Our results suggested the possible existence of an inhibitor(s) that is specific to the stage of development of the Xenopus oocyte. To examine this possibility, we investigated the effect of a nuclear extract from Xenopus oocytes on the export of tRNA-attached ribozymes in somatic cells. A nuclear fraction was prepared from Xenopus oocytes. After incubation of this nuclear extract with FITC-labeled tRNA-

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Rz, the mixture was injected into the nuclei of HeLa S3 cells, as in the experiments for which the results are shown in Figure 5B. As a control, we also examined the export of FITC-labeled wild-type tRNAVal. To our surprise, the export of tRNA-Rz in HeLa cells was significantly inhibited by the addition of the extract of oocyte nuclei (Figure 5F, top). In contrast and again to our surprise, the extract of oocyte nuclei had no effect on the export of mature tRNA (Figure 5F, bottom). Furthermore, when a nuclear extract from A6 somatic cells was used instead of the nuclear extract from Xenopus oocytes, the extract did not inhibit the export of the tRNA-Rz in HeLa cells (Figure 5G, bottom). These observations strongly suggest that an inhibitor(s), present only in the unfertilized oocyte and not in the somatic cells, interacts specifically with tRNA-attached ribozymes (immature tRNA in Figure 6). Discussion A Proofreading Mechanism that Involves the Putative Inhibitor(s) in Xenopus Oocytes and the Absence of Such a Mechanism in Somatic Cells. Xpo-t was first identified as a homologue of Los1p, a yeast member of the importin β superfamily, and as a protein that binds to RanGTP.7,10 Simultaneous injection of Xpo-t increased the rate of export of tRNA (as shown in Figure 5A), and injection of antibodies against Xpo-t into the nuclei of Xenopus oocytes significantly inhibited the export of tRNA from the nuclei.8 Although both unspliced pre-tRNAs and mature tRNAs bound to Xpo-t/ RanGTP,8 the export of such unspliced tRNAs into the cytoplasm was inefficient without maturation of the 5′ and 3′ ends and attachment of the CCA 3′-terminal sequence (the formation of a functional amino acid acceptor helix) or without simultaneous injection of an excess amount of Xpo-t into the oocyte nucleus.8,11,14 In every case, direct evidence that Xpo-t functions in the export of tRNAs to the cytoplasm was provided by experiments in which Xpo-t is injected into Xenopus oocytes together with tRNA substrates. Moreover, the results in Xenopus oocytes led to the elegant proposal of a mechanism for proofreading tRNAs before their export from the nucleus. Such a proofreading system was considered to be general and to be conserved in all kinds of eukaryotic cell. In yeast, Los1p, which is the homologue in Saccharomyces cereVisiae of Xpo-t, is not required for cell viability.42 Because tRNA export can be assumed to be essential for cell survival, the existence of an additional pathway for tRNA export was suggested by this finding in yeast, and indeed, recently, a protein, eEF-1A, that was essential for translation was found as an aminoacylation-dependent tRNA transporter in yeast.19 Our initial finding that tRNA-attached ribozymes (tRNA-Rz’s) could be exported to the cytoplasm in mammalian cells led us to consider the possible existence of such an alternative pathway for the export of tRNAs.25-29 However, contrary to our expectations, we found that Xpo-t was likely to be involved in the transport of tRNA-attached ribozymes in somatic cells and, moreover, that a mechanism similar to that for recognition of the tertiary structure of tRNAs was involved in the interaction of Xpo-t with tRNA-



Figure 6. Schematic representation of transport to the cytoplasm of mature tRNAs and of tRNA-attached ribozymes. The original proofreading mechanism11 has been modified to include the proposed inhibitor-mediated proofreading of tRNA transcripts in Xenopus oocytes (upper panel). A more general pathway for the export of mature and tRNA-attached ribozymes in somatic cells, based on the present study, is presented in the lower panel.

attached ribozymes (Figures 3-5). Nevertheless, in Xenopus oocytes, tRNA-attached ribozymes were not exported to the cytoplasm (Figure 5A), as predicted by the proofreading hypothesis. Further investigation revealed that, despite the presence of Xpo-t in Xenopus oocytes, the Xpo-t/RanGTP complex did not interact with tRNA-attached ribozymes in oocytes (Figure 5C), even though such an interaction could be observed in vitro (Figure 4) and, more importantly, in several lines of somatic cells (Figure 5D). These findings hinted at the presence of an inhibitor(s) in Xenopus oocytes

rather than the involvement of an alternative pathway in somatic cells. In fact, an extract of nuclei from Xenopus oocytes strongly inhibited the export of tRNA-attached ribozymes in somatic cells (Figure 5F and G), suggesting the existence of a strong inhibitor(s) in oocytes exclusively (Figure 6). A Rigorous Proofreading at a Very Specific Stage. Our present analysis demonstrates clearly that a rigorous proofreading mechanism appears to operate in cells at a very specific stage or of a very specific type. It seems likely that

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the export of tRNAs in oocytes is subject to a special kind of regulation (Figure 6). Moreover, the proofreading mechanism that is operative in Xenopus oocytes seems to involve a specific inhibitor(s) that appears to recognize immature tRNAs (tRNA-Rz) specifically. It remains to be determined why the rigorous selection of exclusively mature tRNAs for export into the cytoplasm is necessary at the oocyte stage. The nature of the inhibitor(s) also remains to be determined. Efforts to resolve these issues are underway in our laboratory. Acknowledgment. The authors thank Professors Iain W. Mattaj and Mutsuhito Ohno for letting one of the authors (M.S.) carry out the Xenopus oocyte experiments whose results are shown in Figure 5A in the Mattaj lab at the European Molecular Biology Laboratory (EMBL), for helpful comments,43 and for the gifts of Xpo-t and Ran expression vectors and Xpo-t-specific antibodies. The authors thank Professor David Engelke of the Department of Biological Chemistry, University of Michigan, for providing the protocol for in situ hybridization. They also thank Professors Haruhiko Siomi and Mikiko Siomi of the Division of Gene Function Analysis, Institute for Genome Research, University of Tokushima, for providing the A6 cells44 and Professor Bryan R. Cullen of the Howard Hughes Medical Institute and Department of Genetics, Duke University, for providing the QCl cells. This research was supported by various grants from the Ministry of Economy, Trade and Industry (METI) of Japan and also by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.K. and M.W. are recipients of research fellowships for young scientists from the Japan Society for the Promotion of Science. References and Notes (1) Go¨rlich, D.; Mattaj, I. W. Science 1996, 271, 1513-1518. (2) Ullman, K. S.; Powers, M. A.; Forbes, D. J. Cell 1997, 90, 967970. (3) Ohno, M.; Fornerod, M.; Mattaj, I. W. Cell 1998, 92, 327-336. (4) Drivas, G. T.; Shih, A.; Coutavas, E.; Rush, M. G.; D’Eustachio, P. Mol. Cell. Biol. 1990, 10, 1793-1798. (5) Izaurralde, E.; Kutay, U.; von Kobbe, C.; Mattaj, I. W.; Go¨rlich, D. EMBO J. 1997, 16, 6535-6547. (6) Richards, S. A.; Carey, K. L.; Macara, I. G. Science 1997, 276, 18421844. (7) Arts, G.-J.; Fornerod, M.; Mattaj, I. W. Curr. Biol. 1996, 6, 305314. (8) Arts, G.-J.; Kuersten, S.; Romby, P.; Ehresmann, B.; Mattaj, I. W. EMBO J. 1998, 17, 7430-7441. (9) Hopper, A. K. Science 1998, 282, 2003-2004. (10) Kutay, U.; Lipowsky, G.; Izaurrald, E.; Bischoff, F. R.; Schwarzmaier, P.; Hartmann, E.; Go¨rlich, D. Mol. Cell 1998, 1, 359-369. (11) Lund, E.; Dahlberg, J. E. Science 1998, 282, 2082-2085.

Kuwabara et al. (12) Hellmuth, K.; Lau, D. M.; Bischoff, F. R.; Kunzler, M.; Hurt, E.; Simos, G. Mol. Cell. Biol. 1998, 18, 6374-6386. (13) Sarkar, S.; Hopper, A. K. Mol. Biol. Cell 1998, 9, 3041-3055. (14) Lipowsky, G.; Bischoff, F. R.; Izaurrald, E.; Kutay, U.; Scharfer, S.; Gross, H. J.; Beier, H.; Go¨rlich, D. RNA 1999, 5, 539-549. (15) Sarkar, S.; Azad, A. K.; Hopper, A. K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14366-14371. (16) Simos, G.; Hurt, E. Curr. Biol. 1999, 9, 238-241. (17) Wolin, S. L.; Matera, A. G. Gene DeV. 1999, 13, 1-10. (18) Cleary, J. D.; Mangroo, D. Biochem. J. 2000, 347, 115-122. (19) Grosshans, H.; Hurt, E.; Simos, G. Genes DeV. 2000, 14, 830-840. (20) Grosshans, H.; Simos, G.; Hurt, E. J. Struct. Biol. 2000, 129, 288294. (21) Hellmuth, K.; Grosjean, H.; Motorin, Y.; Deinert, K.; Hurt, E.; Simos, G. Nucleic Acids Res. 2000, 28, 1045-1052. (22) Kruse, C.; Willkomm, D. K.; Gru¨nweller, A.; Vollbrandt, T.; Sommer, S.; Busch, S.; Pfeiffer, T.; Brinkmann, J.; Hartmann, R. K.; Mu¨ller, P. Biochem. J. 2000, 346, 107-115. (23) Qiu, H.; Hu, C.; Anderson, J.; Bjo¨rk, G. R.; Sarkar, S.; Hopper, A. K.; Hinnebusch, A. G. Mol. Cell. Biol. 2000, 20, 2505-2516. (24) Azad, A. K.; Stanford, D. R.; Sarkar, S.; Hopper, A. K. Mol. Biol. Cell 2001, 12, 1381-1392. (25) Kawasaki, H.; Eckner, R.; Yao, T.-P.; Taira, K.; Chiu, R.; Livingston, D. M.; Yokoyama, K. K. Nature 1998, 393, 284-289. (26) Kuwabara, T.; Warashina, M.; Tanabe, T.; Tani, K.; Asano, S.; Taira, K. Mol. Cell 1998, 2, 617-627. (27) Kuwabara, T.; Warashina, M.; Orita, M.; Koseki, S.; Ohkawa, J.; Taira, K. Nature Biotechnol. 1998, 16, 961-965. (28) Koseki, S.; Tanabe, T.; Tani, K.; Asano, S.; Shioda, T.; Nagai, Y.; Shimada, T.; Ohkawa, J.; Taira, K. J. Virol. 1999, 73, 1868-1877. (29) Kuwabara, T.; Warashina, M.; Nakayama, A.; Ohkawa, J.; Taira, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1886-1891. (30) Kuwabara, T.; Warashina, M.; Taira, K. Curr. Opin. Chem. Biol. 2000, 4, 669-677. (31) Kuwabara, T.; Warashina, M.; Taira, K. Trends Biotechnol. 2000, 18, 462-468. (32) Warashina, M.; Kuwabara, T.; Taira, K. Structure 2000, 8, 207212. (33) Kato, Y.; Kuwabara, T.; Warashina, M.; Toda, H.; Taira, K. J. Biol. Chem. 2001, 276, 15378-15385. (34) Warashina, M.; Kuwabara, T.; Kato, Y.; Sano, M.; Taira, K. Proc. Natl. Acad Sci. U.S.A. 2001, 98, 5572-5577. (35) Kuwabara, T.; Warashina, M.; Koseki, S.; Sano, M.; Ohkawa, J.; Nakayama, K.; Taira, K. Nucleic Acids Res. 2001, 29, 2780-2788. (36) Hu¨ttenhofer, A.; Noller, H. F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7851-7855. (37) Lentzen, G.; Moine, H.; Ehresmann, C.; Ehresmann, B.; Wintermeyer, W. RNA 1996, 2, 244-253. (38) Jamolowski, A.; Boelens, W. C.; Izaurralde, E.; Mattaj, I. W.; Dreyfuss, G.; Fischer, U. J. Cell. Biol. 1994, 137, 27-35. (39) Li, J.; Tang, H.; Mullen, T.-M.; Westberg, S.; Reddy, T.; Rose, D.; Wong-Staal, F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 709-714. (40) Kang, Y.; Cullen, B. R. Genes DeV. 1999, 13, 1126-1139. (41) Bertrand, E.; Castanotto, D.; Zhou, C.; Carbonnelle, C.; Lee, G. P.; Chatterjee, S.; Grange, T.; Pictet, R.; Kohn, D.; Engelke, D.; Rossi, J. J. RNA 1997, 3, 75-88. (42) Hurt, D. J.; Wang, S. S.; Lin, Y. H.; Hopper, A. K. Mol. Cell. Biol. 1987, 7, 1208-1216. (43) Ohno, M.; Segref, A.; Bachi, A.; Wilm, M.; Mattaj, I. W. Cell 2000, 101, 187-198. (44) Siomi, M. C. Cell Struct. Funct. 2000, 25, 227-235.

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