Relationships between the Activities in Vitro and in Vivo of Various Kinds of Ribozyme and Their Intracellular Localization in Mammalian Cells
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m010570200
ISSN1083-351X
AutoresYoshio Kato, Tomoko Kuwabara, Masaki Warashina, Hirofumi Toda, Kazunari Taira,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoNineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting stepin vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules. Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting stepin vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules. hepatitis delta virus polymerase external guide sequence chronic myelogenous leukemia nucleotide(s) wild-type ribozyme Since the discovery of the first two ribozymes (1Cech T.R. Zaug A.J. Grabowski P.J. 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Small ribozymes that can be designed to cleave RNA strands intermolecularly include hammerhead, hairpin, and HDV1 ribozymes. Thesetrans-acting ribozymes recognize their RNA substrates via formation of Watson-Crick base pairs, and they cleave these RNAs in a sequence-specific manner. Because of their specificity,trans-acting ribozymes show promise as tools for the dysfunction of target RNAs (9–21). The constitutive expression of a ribozyme in vivo, under the control of a strong promoter, represents an attractive strategy for the application of trans-acting ribozymes to gene therapy. As described in our previous reports (22Koseki S. Tanabe T. Tani K. Asano S. Shioda T. Nagai Y. Shimada T. Ohkawa J. Taira K. J. Virol. 1999; 73: 1868-1877Crossref PubMed Google Scholar, 23Kuwabara T. Warashina M. Nakayama A. Ohkawa J. Taira K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1886-1891Crossref PubMed Scopus (55) Google Scholar), we have succeeded in establishing an effective ribozyme expression system, with subsequent efficient transport of transcripts to the cytoplasm, which is based on a promoter that is recognized by RNA polymerase III (pol III). High levels of expression under the control of the pol III promoter are advantageous for the exploitation of ribozymes in vivo. Therefore, we chose an expression system with the promoter of a human gene for tRNAVal. Many ribozymes, such as hammerheads and hairpins, have been effectively expressed under the control of promoters of gene for tRNAs (9Ojwang J.O. Hampel A. Looney D.J. Wong-Staal F. Rappaport J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10802-10806Crossref PubMed Scopus (207) Google Scholar, 11Yu M. Leavitt M.C. Maruyama M. Yamada O. Young D. Ho A.D. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 699-703Crossref PubMed Scopus (109) Google Scholar, 12Bertrand E. Castanotto D. 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Biol. 1998; 58: 1-39Crossref PubMed Scopus (52) Google Scholar). A major advantage of our tRNAVal-directed expression system is that, with appropriate modification of the tRNAValportion, it is possible to colocalize the expressed ribozyme in the cytoplasm with its target mRNA (14Kuwabara T. Warashina M. Orita M. Koseki S. Ohkawa J. Taira K. Nat. Biotechnol. 1998; 16: 961-965Crossref PubMed Scopus (61) Google Scholar, 15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 22Koseki S. Tanabe T. Tani K. Asano S. Shioda T. Nagai Y. Shimada T. Ohkawa J. Taira K. J. Virol. 1999; 73: 1868-1877Crossref PubMed Google Scholar, 23Kuwabara T. Warashina M. Nakayama A. Ohkawa J. Taira K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1886-1891Crossref PubMed Scopus (55) Google Scholar, 25Kuwabara T. Warashina M. Taira K. Trends Biotechnol. 2000; 18: 462-468Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Ribozymes expressed under the control of the tRNAVal promoter are exported to the cytoplasm as effectively as natural tRNAs via the action of Xpo(t), 2T. Kuwabara and K. Taira, unpublished data. a tRNA-binding protein (26Arts G.J. Kuersten S. Romby P. Ehresmann B. Mattaj I.W. EMBO J. 1998; 17: 7430-7441Crossref PubMed Scopus (183) Google Scholar, 27Arts G.J. Fornerod M. Mattaj I.W. Curr. Biol. 1998; 8: 305-314Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) that functions with Ran GTPase, which regulates the transport by catalyzing the hydrolysis of GTP. Mature mRNAs are exported to the cytoplasm for translation. Thus, both ribozymes and their target mRNAs can be co-localized in the same cellular compartment. By contrast, an external guide sequence (EGS), which is added intrans and is able to bind to its target RNA, appears to function in the nucleus because its effect depends on the activity of ribonuclease P (RNase P) (17Plehn-Dujowich D. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7327-7332Crossref PubMed Scopus (89) Google Scholar, 28Forster A.C. Altman S. Science. 1990; 249: 783-786Crossref PubMed Scopus (259) Google Scholar, 29Yuan Y. Altman S. Science. 1994; 263: 1269-1273Crossref PubMed Scopus (83) Google Scholar, 30Guerrier-Takada C. Salavati R. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8468-8472Crossref PubMed Scopus (73) Google Scholar). The EGS RNA binds to the target RNA, yielding a structure that resembles the pre-tRNA that is recognized as a substrate by RNase P. RNase P normally cleaves precursors to tRNAs to generate the 5′ termini of mature tRNAs. Because RNase P is expressed constitutively in cells and accumulates, in particular in the nucleus, the use of an EGS as a gene-inactivating agent does not require expression of additional RNase P from exogenously introduced genes. Although an EGS does not have intrinsic cleavage activity, when it acts in cooperation with endogenous RNase P, it can effectively inactivate its target mRNA. Although there have been many studies both in vitro andin vivo of the activities of the ribozymes mentioned above, further detailed information on the parameters that determine their activities as gene-inactivating agents in vivo is necessary so that we will be able to optimize their effects by optimizing the requisite parameters. In addition, although it has been claimed for each individual ribozyme that it has potential utility as an effective gene-inactivating agent, there has been no systematic analysis in which the activities of various ribozymes have been compared under similar conditions in vivo. In this study, we designed several types of functional RNA targeted to the junction site of theBCR-ABL chimeric mRNA that causes chronic myelogenous leukemia (CML). Using this system, we have accumulated data that might allow correlations to be made between ribozyme activities in cultured cells and the efficacies of the same ribozymes in vivo, namely, in mice (15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Tanabe T. Kuwabara T. Warashina M. Tani K. Taira K. Asano S. Nature. 2000; 406: 473-474Crossref PubMed Scopus (78) Google Scholar). CML occurs as a result of reciprocal chromosomal translocations that result in the formation of the BCR-ABL fusion gene. One of the chimeric mRNAs transcribed from an abnormal BCR-ABL(B2A2) gene (consisting of exon 2 of BCR and exon 2 of ABL; Refs. 31Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar and 32Nowell P.C. Hungerford D.A. Science. 1960; 132: 1497-1499Google Scholar) provides a suitable substrate for comparisons of ribozymes. We used six kinds of functional RNA, including hammerhead, hairpin, and HDV ribozymes; our maxizyme andin vitro selected minizymes; and EGSs to examine parameters that determined activities in vitro and in vivo. Our goal was to determine whether activity in vitro might reflect activity in mammalian cells. Moreover, since we have evidence that suggests that tRNAVal-driven ribozymes with high level of activities are efficiently exported to the cytoplasm, whereas similarly expressed tRNA ribozymes with poor activities are accumulated in the nucleus (22Koseki S. Tanabe T. Tani K. Asano S. Shioda T. Nagai Y. Shimada T. Ohkawa J. Taira K. J. Virol. 1999; 73: 1868-1877Crossref PubMed Google Scholar), we decided to examine the correlation between nuclear localization and/or transport of functional RNAs and the activity in vivo. For this purpose, we used two kinds of promoter. One promoter was the promoter of the gene for tRNAVal described above, and the other was a U6 promoter (33Das G. Henning D. Wright D. Reddy R. EMBO J. 1988; 7: 503-512Crossref PubMed Scopus (154) Google Scholar, 34Ohkawa J. Taira K. Hum. Gene Ther. 2000; 11: 577-585Crossref PubMed Scopus (73) Google Scholar). Transcripts expressed under the control of these promoters are located in the cytoplasm and the nucleus, respectively. We found that the intrinsic cleavage activity of a ribozyme is not the sole determinant of activity in cultured cells and that it is the cytoplasmic localization and the association of the ribozyme with its substrate that regulate activity. The construction of vectors for expression of ribozymes from the tRNAVal promoter using pUC-dt (a plasmid that contains the chemically synthesized promoter for a human gene for tRNAVal between the EcoRI and SalI sites of pUC 19) was described previously (22Koseki S. Tanabe T. Tani K. Asano S. Shioda T. Nagai Y. Shimada T. Ohkawa J. Taira K. J. Virol. 1999; 73: 1868-1877Crossref PubMed Google Scholar, 23Kuwabara T. Warashina M. Nakayama A. Ohkawa J. Taira K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1886-1891Crossref PubMed Scopus (55) Google Scholar). pUC-dt was double-digested with Csp45I and SalI, and a fragment having a linker sequence with 5′ Csp45I site and the restriction sites for KpnI and EcoRV and the terminator sequence TTTTT at the 3′ end with 3′ SalI site was cloned into the double-digested plasmid to yield pUC-tRNA/KE. TheKpnI and EcoRV sites were used for subsequent insertion of the each ribozyme sequence. The construction of vectors for ribozyme expression from the U6 promoter has been described elsewhere (17Plehn-Dujowich D. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7327-7332Crossref PubMed Scopus (89) Google Scholar). The EcoRI and XhoI sites were used for insertion of each ribozyme sequence. Each ribozyme and two substrates, namelyBCR-ABL and ABL RNAs, were preparedin vitro using T7 RNA polymerase. Assays of ribozyme activity in vitro were performed, in 25 mmMgCl2 and 50 mm Tris-HCl (pH 8.0) at 37 °C, under enzyme-saturating (single-turnover) conditions, as described elsewhere (14Kuwabara T. Warashina M. Orita M. Koseki S. Ohkawa J. Taira K. Nat. Biotechnol. 1998; 16: 961-965Crossref PubMed Scopus (61) Google Scholar). Each ribozyme (50 μm) was incubated with 2 nm 5′-32P-labeled substrate. The substrate and the products of each reaction were separated by electrophoresis on an 8% polyacrylamide, 7 m urea denaturing gel and detected by autoradiography. HeLa S3 cells on a coverslip, which had been transfected in advance with an appropriate plasmid, were washed in fresh phosphate-buffered saline and fixed in fix/permeabilization buffer (50 mm HEPES/KOH, pH 7.5, 50 mm potassium acetate, 8 mm MgCl2, 2 mm EGTA, 2% paraformaldehyde, 0.1% Nonidet P-40, 0.02% SDS) for 15 min at room temperature. Cells were rinsed three times in phosphate-buffered saline for 10 min each. Seventy micrograms of Cy3-labeled oligodeoxynucleotide probe with a sequence complementary to the ribozyme and 20 μg of tRNA from Escherichia coli MRE 600 (Roche Molecular Biochemicals, 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, and 10 μl of hybridization buffer, containing 20% dextran sulfate and 2% BSA in 4× SSC, were added. Twenty microliters of the hybridization solution containing the probe were 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 cells were analyzed with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany). Cells were grown to ∼80% confluence (1 × 107 cells) and were transfected with a tRNAVal-Rz expression vector with the Lipofectin™ reagent (Life Technologies, Inc.). Thirty-six hours after transfection, cells were harvested. For the preparation of the cytoplasmic fraction, collected cells were washed twice with phosphate-buffered saline and then resuspended in digitonin lysis buffer (50 mmHEPES/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 1,000 × g, and the supernatant was collected as the cytoplasmic fraction. The pellet was resuspended in Nonidet P-40 lysis buffer (20 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-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 Pharmacia Biotech, Buckinghamshire, United Kingdom). The membrane was probed with a synthetic oligonucleotide that was complementary to the sequence of the relevant ribozyme. Each probe was labeled with32P by T4 polynucleotide kinase (Takara Shuzo Co., Kyoto, Japan). Luciferase activity was measured with a PicaGene® kit (Toyo-inki, Tokyo, Japan) as described elsewhere (15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In order to normalize the efficiency of transfection by reference to β-galactosidase activity, cells were cotransfected with the pSV-β-galactosidase control vector (Promega, Madison, WI), and then the chemiluminescent signal due to β-galactosidase was quantitated with a luminescent β-galactosidase genetic reporter system (CLONTECH, Palo Alto, CA) as described previously (15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In order to express ribozymesin vivo, we used two kinds of pol III promoter (Fig.1 A). Transcripts with the promoter of the gene for tRNAVal can be efficiently transported to the cytoplasm when the appropriate choice of and combination of linker and ribozyme sequence is made (14Kuwabara T. Warashina M. Orita M. Koseki S. Ohkawa J. Taira K. Nat. Biotechnol. 1998; 16: 961-965Crossref PubMed Scopus (61) Google Scholar, 15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 22Koseki S. Tanabe T. Tani K. Asano S. Shioda T. Nagai Y. Shimada T. Ohkawa J. Taira K. J. Virol. 1999; 73: 1868-1877Crossref PubMed Google Scholar, 23Kuwabara T. Warashina M. Nakayama A. Ohkawa J. Taira K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1886-1891Crossref PubMed Scopus (55) Google Scholar,25Kuwabara T. Warashina M. Taira K. Trends Biotechnol. 2000; 18: 462-468Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We used the mouse U6 promoter, which controls expression of U6 RNA that is localized in the nucleus, for expression and accumulation of transcripts in the nucleus. Ten functional RNAs (Fig. 1 B) directed against sites within a limited region (<100 nt) ofB2A2 and ABL mRNAs (Fig. 1 C; target sites are underlined; the identical cleavage site could not be chosen because of different cleavable sequence for each different ribozyme) and expression vectors that encoded each respective functional RNA were designed such that each functional RNA was produced under the control both of the tRNAVal promoter and of the U6 promoter. The product translated from B2A2 chimeric mRNA causes CML. We demonstrated previously that the RNA maxizyme that functions as a dimer cleaves B2A2 chimeric mRNAin vitro and in vivo with extremely high specificity without any damage to normal ABL mRNA (15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar,21Tanabe T. Kuwabara T. Warashina M. Tani K. Taira K. Asano S. 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Biol. 2000; 33: 359-365Google Scholar, 47Warashina M. Kuwabara T. Taira K. Structure. 2000; 8: R207-R212Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The minizymes shown in Fig. 1 B are minimized hammerhead ribozymes with stem II deletions and relatively high activity, and each of them was identified recently by in vitro selection (43Conaty J. Hendry P. Lockett T. Nucleic Acids Res. 1999; 27: 2400-2407Crossref PubMed Scopus (48) Google Scholar, 44McCall M.J. Hendry P. Mir A.A. Conaty J. Brown G. Lockett T.J. Mol. Biotechnol. 2000; 14: 5-17Crossref PubMed Google Scholar). These minizymes function in vitro even at low concentrations of Mg2+ ions. Therefore, they may have advantage at low concentrations of Mg2+ ions in vivo; thus, we included them in our study. The maxizyme is an effector-inducibletrans-activated ribozyme, and the maxizyme shown in Fig.1 B recognizes the junction region of B2A2; it cleaves B2A2 mRNA but not normal ABL mRNA and, therefore, we used this well characterized tRNAVal-driven maxizyme as a positive control in studies in cultured cells (15Kuwabara T. Warashina M. Tanabe T. Tani K. Asano S. Taira K. Mol. Cell. 1998; 2: 617-627Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Tanabe T. Kuwabara T. Warashina M. Tani K. Taira K. Asano S. Nature. 2000; 406: 473-474Crossref PubMed Scopus (78) Google Scholar, 25Kuwabara T. Warashina M. Taira K. Trends Biotechnol. 2000; 18: 462-468Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 45Kuwabara T. Warashina M. Taira K. Curr. Opin. Chem. Biol. 2000; 4: 669-677Crossref PubMed Scopus (38) Google Scholar, 46Kurata H. Miyagishi M. Kuwabara T. Warashina M. Taira K. J. Biochem. Mol. Biol. 2000; 33: 359-365Google Scholar, 47Warashina M. Kuwabara T. 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