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Journal of Virology, March 1999, p. 1868-1877, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III

Shiori Koseki,1,2,3 Tsuyoshi Tanabe,4 Kenzaburo Tani,4 Shigetaka Asano,4 Tatsuo Shioda,5 Yoshiyuki Nagai,5 Takashi Shimada,6 Jun Ohkawa,1,2,3,* and Kazunari Taira1,2,3,*

National Institute for Advanced Interdisciplinary Research, AIST, MITI, Tsukuba Science City 305-8562,1 National Institute of Bioscience and Human Technology, AIST, MITI, Tsukuba Science City 305-8566,2 Department of Hepatology/Oncology4 and Department of Viral Infection,5 Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8539, Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602,6 and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City 305-8572,3 Japan

Received 3 September 1998/Accepted 4 December 1998


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

A hammerhead ribozyme is one of the smallest catalytic RNA molecules (19, 28). Because of its small size and potential as an antiviral agent, numerous mechanistic studies (10, 11, 13, 31, 37, 57-60) and studies directed towards application in vivo have been performed (13, 14, 32, 38, 40, 42, 50). Many successful experiments aimed at the use of ribozymes for suppression of gene expression in different organisms have been reported (12, 15, 16, 24, 27, 30, 41, 53, 55, 56). However, the efficacy of ribozymes in vitro is not necessarily correlated with functional activity in vivo. Some of the reasons for this ineffectiveness in vivo are as follows: (i) cellular proteins may inhibit the binding of the ribozyme to its target RNA or may disrupt the active conformation of the ribozyme, (ii) the intracellular concentrations of the metal ions that are essential for ribozyme-mediated cleavage might not be sufficient for functional activity, and (iii) ribozymes are easily attacked by RNases. However, we are now starting to understand the parameters that determine ribozyme activity in vivo (7, 17, 18). Studies in vivo have suggested that the following factors are important for effective ribozyme-mediated inactivation of genes: a high level of ribozyme expression (55), the intracellular stability of the ribozyme (13, 39), colocalization of the ribozyme and its target RNA in the same cellular compartment (6, 7, 46), and the cleavage activity of the transcribed ribozyme (49). Recently, it was shown that these various features depend on the expression system that is used (7).

The RNA polymerase II (Pol II) system, which is employed for transcription of mRNAs, and the polymerase III (Pol III) system, employed for transcription of small RNAs such as tRNA and snRNA, have been used as ribozyme expression systems (50). Transcripts driven by the Pol II promoter have extra sequences at the 3' and 5' ends (for example, an untranslated region, a cap structure, and a poly(A) tail) in addition to the coding region. These extra sequences are essential for stability in vivo and functional recognition as mRNA. A transcript containing a ribozyme sequence driven by the Pol II promoter includes all of those sequences unless such sequences are trimmed after transcription (32-34, 47). As a result, in some cases the site at which the ribozyme recognizes its target may be masked, for example, by a part of the coding sequence. By contrast, the Pol III system is suitable for expression of short RNAs, and only very short extra sequences are generated. In addition, the level of expression is at least 1 order of magnitude higher than that obtained with the Pol II system (9). Indeed, in our hands, Pol III-driven ribozymes (30), but not Pol II-driven ribozymes (34), were detected by Northern blotting analysis, demonstrating the higher transcription level of the former RNA. Thus, it was suggested that the Pol III system might be very useful for expression of ribozymes (36, 55). However, in many cases, the expected effects of ribozymes could not be achieved in spite of the apparently desirable features of the Pol III system (7, 23, 25).

In this study, in order to investigate the parameters that determine ribozyme activity in vivo, we designed three types of ribozyme with the same ribozyme sequence, driven by the Pol III promoter, and we demonstrated that the entire structure of the transcript determined not only the cleavage activity but also the intracellular half-life of the ribozyme. All three types of chimeric tRNAVal ribozymes that were transcribed in the cell nucleus were exported to the cytoplasm. Thus, the ribozymes and their target were present within a single cellular compartment. Under these conditions, we found that the cytoplasmic localization of tRNAVal ribozymes and the intracellular half-life and steady-state level of each tRNAVal ribozyme were the major determinants of functional activity in cultured cells. Moreover, we demonstrated that cells that expressed the specifically designed ribozyme with the longest half-life in cultured cells were almost completely resistant to challenge by human immunodeficiency virus type 1 (HIV-1).


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Plasmid constructs and expression vectors. The plasmids (pUCdt-Rz series) that expressed each tRNAVal ribozyme were constructed as follows. The tRNAVal ribozymes were designed to be targeted to the U5 region of HIV-1 RNA. The sense and antisense oligonucleotide linkers encoding the sequence of the promoter region, derived from the human gene for placental tRNAVal (pHtV1 [3]), were annealed and ligated into the EcoRI/SalI site of pUC19. The sequences of the oligonucleotide linkers were as follows: sense, 5'-AAT TCA GGA CTA GTC TTT TAG GTC AAA AAG AAG AAG CTT TGT AAC CGT TGG TTT CCG TAG TGT AGT GGT TAT CAC GTT CGC CTA ACA CGC GAA AGG TCC CCG GTT CGA AG-3'; antisense, 5'-TCG ACT TCG AAC CGG GGA CCT TTC GCG TGT TAG GCG AAC GTG ATA ACC ACT ACA CTA CGG AAA CCA ACG GTT ACA AAG CTT CTT CTT CTT TTT GAC CTA AAA GAC TAG TCC TG-3'. Next, the sense and antisense oligonucleotide linkers that encoded the terminator sequence were also annealed and ligated into the NspV/SalI site of pUC19, which contained the sequence of the promoter region. The sequences of the oligonucleotide linkers were as follows: sense, 5'-CGA AAC CGG GCA CCC GGG GAA TAT AAC CTC GAG CGC TTT TTT TCT ATC GCG TC-3'; antisense, 5'-TCG ACG CGA TAG AAA AAA AGC GCT CGA GGT TAT ATT CCC CGG GTG CCC GGT TTC-3'. The resultant plasmid, which contained the A and B boxes of tRNAVal and a terminator, was designated pUCdt.

DNA fragments encoding the sequence of each ribozyme and the tRNAVal portion were amplified by PCR by using pUCdt as a template with an upper primer (5'-CGC CAG GGT TTC CCA GTC ACG AC-3') and a lower primer that included the sequences of both the ribozyme and the terminator (Rz1, 5'-CTG CAG GTC GAC GCG ATA GAA AAA AAG CGC TCG AGG TGC CCG TTT CGT CCT CAC GGA CTC ATC AGT GTT GTG TGG GTG CCC GGT TTC GAA CCG GGG ACC TTT-3'; Rz2, 5'-CTG CAG GTC GAC GCG ATA GAA AAA AAC CGT TTC CGA CGT GCC CGT TTC GGA CCT TTC GGT CCT CAT CAG TGT TGT GTT TGT AGT GCC CGG TTT CGA ACC GGG GAC CTT T-3'; Rz3, 5'-CTG CAG GTC GAC GCG ATA GAA AAA AAC CGT TTC CGA CGT GCC CGT TTC GGA CCT TTC GGT CCT CAT CAG TGT TGT GTG TTG GTT TGT AGT GCC CGG TTT CGA ACC GGG GAC CTT T-3'). After digestion of products of PCR with EcoRI and SalI, each fragment was ligated into the EcoRI/SalI site of pUC19 to yield pUCdt-Rz. The members of the pUC-Rr series, which each contained a reference gene expression cassette in addition to the gene for the tRNAVal ribozyme (see Fig. 3A), were constructed by inserting the PvuII fragment of pUCdt into the HincII site of each pUCdt-Rz plasmid. The direction of the inserted fragment was confirmed by digestion with restriction enzymes. The pHyg dt-Rz series, which was used for generation of ribozyme-transduced HeLa cells, was constructed by inserting each PvuII-SalI fragment of the pUCdt-Rz series into the EcoRV/SalI site of pHyg (54). All oligonucleotide linkers and primers for PCR were synthesized by a DNA/RNA synthesizer (model 392; Applied Biosystems, Foster City, Calif.).

Recombinant HIV vector plasmids were constructed as follows. A 2.0-kbp BamHI fragment that encoded the bacterial Neor gene cassette from pMC1 neo (48) was inserted into the SalI site of an HIV-1-derived vector (see Fig. 5A [43]). Then, the tRNAVal ribozyme expression cassette was cloned into the SalI site immediately upstream of TK-Neor, as shown in Fig. 5B.

Chimeric long terminal repeat (LTR) (R-U5)-Luc-expressing plasmids for luciferase assays were constructed as follows. The fragment which encoded the target sequence of the ribozyme was amplified by PCR by using pNL4-3 (1) as a template with an upper primer (5'-TCG ATA TCA AGC TTC ACT GCT TAA GCC TCA ATA TAG CTT GCC TTG AGT GCT CAA AGT AGT GT-3') and a lower primer (5'-AGG CCC GGC GCC TTT CTT GCT CTC CTC TGT CGA GT-3'). After digestion of products of PCR with NarI and HindIII, each fragment was ligated into the NarI/HindIII site of pGV-C1 (Promega, Madison, Wis.). This plasmid was designated pGV-V1.

Cell culture and transfections. HeLa and Cos cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Gaithersburg, Md.) supplemented with 10% (vol/vol) fetal bovine serum (Gibco BRL) and 45 µg of gentamicin (Gibco BRL) per ml. H9 cells were cultured in RPMI (Gibco BRL) supplemented with 10% fetal calf serum (FCS) (Gibco BRL).

Cells were transfected with the Lipofectin reagent (Gibco BRL), according to the manufacturer's protocol. A recombinant HIV vector plasmid (HIVRib.N [see Fig. 5B]) was used to transfect H9 cells by the CaPO4 coprecipitation method.

Preparation of RNA. Total RNA was extracted by the guanidinium thiocyanate phenol-chloroform method. Cytoplasmic RNA and nuclear RNA were separated as described previously (22).

Measurement of steady-state levels and half-lives of ribozymes. The steady-state level of each ribozyme was measured as follows. HeLa cells (106 cells/10-cm plate) were transfected with each pUC-Rr plasmid. Two days after transfection, total RNA was isolated from these cells. The amount of reference RNA, located downstream of the tRNAVal ribozyme in the isolated total RNA, was quantified first by Northern blotting analysis (29) with a probe specific for the reference RNA (5'-AAA TCG CTA TAA AAA GCG CTC GAG GTT ATG CTC CCC GGG T-3'). The amount of reference RNA in each sample was maintained at a constant value, and the level of total RNA in each sample was also kept constant by addition of RNA isolated from untransfected HeLa cells as necessary. Finally, hybridization was repeated with a probe specific for the ribozyme (5'-CTC ATC AGT GTT GTG T-3') or the probe specific for the reference RNA (see Fig. 3B).

The half-life of each ribozyme was determined by Northern blotting analysis after treatment of cells with actinomycin D, as described previously (22). In brief, cells were exposed to actinomycin D at a final concentration of 5 µg/ml for 0, 60, 120, or 180 min and, at each time point, total RNA was isolated (see Fig. 3C). The amount of ribozyme in each preparation of isolated RNA was determined by Northern blotting.

Cleavage assay. Total RNA was isolated from HeLa cells transfected with each pUCdt-Rz or pUCdt plasmid. The amount of ribozyme in each preparation of isolated RNA was determined by Northern blotting with the probe that was specific for the ribozyme. Then, the concentration of each ribozyme was adjusted to the same value by addition of RNA isolated from untransfected HeLa cells. The substrate RNA that encoded the U5 LTR region of HIV-1 (see Fig. 2A) was prepared by T7 transcription in a mixture that contained [alpha -32P]UTP. Cleavage reactions were allowed to proceed in a 50-µl reaction mixture (40 mM Tris-HCl [pH 8.0], 8 mM MgCl2, 5 mM dithiothreitol, 2 mM spermidine, 40 U of placental RNase inhibitor, 30 µg of total RNA, 5 kcpm of radiolabeled substrate RNA) at 37°C for 12 h. Products were identified after electrophoresis on a 6% polyacrylamide-7 M urea gel (see Fig. 2B).

Luciferase assay. Luciferase activity was measured with the Dual-Luciferase Reporter assay system (Promega) according to the manufacturer's protocol. The target gene-expressing plasmid (pGV-V1) was used to transfect, separately, HeLa cells and stable transformants that express ribozymes. HeLa cells transfected with pUCdt-Rz and the target-expressing plasmid (see Fig. 4A) or stable transformants that had been transduced with the ribozyme-expressing plasmid followed by transfection with the target-expressing plasmid (see Fig. 4B) were lysed in 150 µl of 1× passive lysis buffer for 15 min and scraped off the plate. The cell debris was removed by centrifugation. After addition of 20 µl of the centrifuged lysate to 100 µl of Luciferase Assay Reagent II, the luminescent signal was immediately quantitated with a luminometer (Lumant LB 9501; Berthold, Bad Wildbad, Germany). Furthermore, for normalization of the activity of firefly luciferase, we measured the luminescent signal generated by Renilla luciferase by adding 100 µl of Stop & Glo reagent to the sample tube immediately after quantitation of the reaction catalyzed by firefly luciferase. The recorded value of firefly luciferase activity was normalized by reference to the activity of Renilla luciferase (see Fig. 4).

Each normalized value of firefly luciferase activity was further normalized by reference to the concentration of protein in the lysate. The protein was quantitated with a Protein Assay Kit (Bio-Rad, Richmond, Calif.) which was based on Bradford's method.

Ribozyme-stable transformants. Ribozyme-stable transformants were obtained by transfecting HeLa cells with pHyg dt or a member of the pHyg dt-Rz series and selection in DMEM that contained 300 µg of hygromycin B (Wako Chemicals, Osaka, Japan) per ml. Twelve hours after transfection, the medium was replaced by growth medium and the cells were cultured for another 48 h. The cells were subcultured at a dilution of 1:5 in DMEM that contained 300 µg of hygromycin B (selection medium) per ml. The medium was replaced by fresh medium every 3 days. Cells resistant to hygromycin B were expanded in DMEM that contained 250 µg of hygromycin B per ml.

Production of virus and transduction of the ribozyme by an HIV vector. A supernatant containing recombinant virus was produced as described previously (43). COS cells (2 × 106 cells/10-cm dish) were cultured and transfected with 10 µg of the packaging vector plasmid and 10 µg of the recombinant HIV vector plasmid (HIVRib.N [shown in Fig. 5B]). The supernatant, which contained recombinant virus, was collected after 48 h and filtered through a 0.22-µm-pore-size filter. Then, 2 × 106 H9 cells were incubated with 5 ml of the filtered supernatant that contained 6 µg of Polybrene (Abbott Laboratories) per ml. After 24 h, the medium was replaced with RPMI supplemented with 10% FCS and 1 mg of G418 per ml. The cells were cultured for 48 h further, and then G418-resistant clones were isolated. Transduction of the ribozyme gene was confirmed by reverse transcriptase (RT) PCR analysis.

Quantitation of tRNAVal ribozyme produced in H9 cells. Quantitative RT-PCR was carried out as follows (20, 35). Total RNA was extracted from H9 cells that had been stably transduced with a ribozyme. cDNA was synthesized in a 20-µl reaction mixture (1 µg of total RNA, 20 mM Tris-HCl [pH 8.3], 50 mM KCl, 5 mM MgCl2, 1 mM deoxynucleoside triphosphate, 1 pmol of primer [for beta -actin, 5'-GTG GCC ATC TCT TGC TCG AA-3'; for ribozyme, 5'-GAC CTT TCG GTC CTC ATC-3'], and 0.25 U of Moloney murine leukemia virus RT [Takara Shuzo, Kyoto, Japan] per ml) at 42°C for 30 min.

cDNA for beta -actin was amplified by PCR with two oligonucleotide primers (upper, 5'-GAC TAC CTC ATG AAG ATC CT-3'; lower, 5'-GTG GCC ATC TCT TGC TCG AA-3') and 13, 15, or 17 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. Ribozyme cDNA was amplified by PCR with two oligonucleotide primers (upper, 5'-GTT ATC ACG TTC GCC TAA-3'; lower, 5'-GAC CTT TCG GTC CTC ATC-3') and 13, 15, or 17 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min.

Products of PCR after 13, 15, and 17 cycles were analyzed by Southern blotting with a radiolabeled probe specific for the ribozyme (5'-ACG CGA AAG GTC CCC GGT-3') or for beta -actin (5'-GCG GGA AAA TCG TGC GTG A-3'). The radioactivity of each band (see Fig. 6A and 6B) was measured with the BAS2000 system (Fuji Film, Tokyo, Japan).

HIV-1 challenge assay. H9 cells transduced with the ribozyme by the HIV vector (HIVRib.N) and mock-transduced control cells were incubated with HIV-1NL432 at a multiplicity of infection of 0.01 for 4 h. After two washes with phosphate-buffered saline, these cells were cultured at 105 cells/ml in RPMI 1640 medium supplemented with 10% FCS. The supernatant was collected on days 3, 7, and 11 after viral infection. The level of the p24 antigen of HIV-1 in each supernatant was determined with an HIV-1 antigen capture enzyme-linked immunosorbent assay (ELISA) kit (DAINABOT, Tokyo, Japan) according to the manufacturer's protocol.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Secondary structures of tRNAVal ribozymes and their cleavage activities in vitro. To construct a Pol III-driven ribozyme expression cassette, we cloned a ribozyme sequence targeted to the 5' leader sequence of HIV-1 RNA (1, 55) adjacent to the sequence of a tRNAVal promoter, with three kinds of short linker between them (see Fig. 1), to yield a set of pUCdt-Rz plasmids. In this analysis, we focused on three types of ribozyme with the identical ribozyme sequence (Rz1 to Rz3). The insertion of the short linkers changed the overall structure of the transcripts and thus affected the accessibility of the recognition arms of the ribozyme. Naturally, it is important that both the 5' and 3' substrate recognition arms of the ribozyme be available to the substrate so that the ribozyme can form stem structures with the substrate RNA that ensure subsequent cleavage of the substrate. In order to clarify the relationship between structure and functional activity, we chose linkers that altered the extent of availability of the recognition arms. Figure 1 shows the secondary structures of the tRNAVal ribozymes as predicted by computer modeling (Mulfold Biocomputing Office, Biology Department, Indiana University, Bloomington). In one case (Fig. 1A), the linker was inserted before the terminator sequence and restricted the flexibility of the 3' substrate recognition arm of the ribozyme. In addition, the 5' substrate recognition arm was unavailable. Therefore, in the case of tRNAVal ribozyme 1 (Rz1 [Fig. 1A]), both the 5' and 3' substrate recognition arms were mostly embedded in a helical structure. tRNAVal ribozyme 2 (Rz2) has one restricted substrate recognition arm on the 5' side. By contrast, tRNAVal ribozyme 3 (Rz3) had no restricted substrate recognition arms, and both arms were available for binding to the substrate. Judging from the flexibility of the substrate recognition arms, we might expect that the cleavage activity of Rz3 would be the highest, followed by Rz2 and Rz1 in that order.


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  		FIG. 1.   Secondary structures of tRNAVal ribozymes predicted by computer folding. The sequence of the hammerhead ribozyme (boldface capital letters; also shown in top right panel) was ligated downstream of that of tRNAVal (capital letters) with various linker sequences (lowercase letters). The sequences that correspond to the internal promoter of seven-base-deleted tRNAVal, namely the A and B boxes, are indicated by shading. (A to D) Secondary structures of tRNAVal ribozymes 1 (Rz1), 2 (Rz2), and 3 (Rz3) and Rz-N, respectively. Inactive tRNAVal ribozyme was made from Rz2 by changing the G in the core region to A (B). (E) Secondary structure of Bertrand's tRNAiMet ribozyme (Rz-BR). The recognition arms of ribozymes are indicated by underlining. The predicted secondary structures of human placental tRNAVal and tRNAiMet are shown in the bottom right panel; the tRNA is processed at three sites (arrowheads) to yield the mature tRNAVal (capital letters).

To examine whether the ribozymes had the cleavage activity that we predicted from their secondary structures (Fig. 1), we first compared activities in vitro. Total RNA was isolated from HeLa cells that had been transfected with the various pUCdt-Rz plasmids that encoded the ribozymes (tRNAVal ribozymes). We mixed a fixed amount (based on Northern blotting data) of each ribozyme within the isolated RNA and radiolabeled substrate RNA to initiate the cleavage reaction, and we monitored the progress of each reaction, after a 12-h incubation, on a 6% polyacrylamide-7 M urea gel (Fig. 2). As expected, the cleavage activity of Rz3, with both recognition arms available, was the highest, followed by that of Rz2, while that of Rz1, with both recognition arms unavailable, was very low. It was clear, therefore, that the cleavage activity of tRNAVal ribozymes in vitro could be deduced from their computer-generated secondary structures.


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  		FIG. 2.   Cleavages mediated by tRNAVal ribozymes in vitro. (A) Schematic representation of substrate RNA (the substrate RNA corresponds to nucleotides 498 to 711 of the pNL432 infectious molecular clone of HIV-1 [the U5 region of HIV-1 RNA]). The substrate RNA was cleaved into two fragments (5' fragment, 70-mer; 3' fragment, 156-mer) by the tRNAVal ribozymes. (B) Autoradiogram showing results of cleavage reactions. Lanes: M, markers; vector, tRNAVal vector alone without a ribozyme; Rz1, ribozyme 1; Rz2, ribozyme 2; Rz3, ribozyme 3.

Steady-state levels and half-lives of tRNAVal ribozymes. We expected that minor structural changes would occur in the entire structure as a result of the intervening (linker) sequence. Thus, the linker should exert considerable influence on the stability of each ribozyme in vivo. We compared the intracellular stability of each tRNAVal ribozyme by using two different approaches, as follows. We compared the steady-state levels of each transcript from HeLa cells that had been transiently transfected with pUC-Rr (a sequence of reference gene was added to each ribozyme-coding pUCdt-Rz plasmid to yield pUC-Rr [Fig. 3A]) by Northern blotting analysis (transient expression assay). In this system, the reference gene (unrelated, meaningless sequence), which had been connected in tandem, was expressed under the control of the identical tRNAVal promoter used for the expression of each ribozyme so that the level of expression of each tRNAVal ribozyme could be normalized by adjustment of the amount of the transcript of the reference gene, which was a reflection of the efficiency of transfection. Transcripts of about 150 nucleotides, which corresponded to the size of the chimeric tRNAVal ribozyme, were detected in all samples of RNA that we isolated from HeLa cells that had been transfected with each plasmid that encoded a tRNAVal ribozyme. The steady-state levels of the tRNAVal ribozymes differed over a 30-fold range of concentrations. The level of Rz2, which was the highest, was about 26 times that of Rz1, which was the lowest, and the level of Rz3 was about 5 times that of Rz1. Since no modifications had been made in the promoter region of each ribozyme expression cassette and thus the efficiency of transcription was assumed to be the same in each case, we postulated that these differences among steady-state levels of transcripts were a consequence of the stability in cultured cells of each respective transcript.


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  		FIG. 3.   Stability of tRNAVal ribozymes in cultured cells. (A) Schematic representation of pUC-Rr, which allowed normalization of the efficiency of transfection by the use of a reference gene. The reference gene was expressed downstream of the ribozyme expression cassette. The sequences of the promoter and terminator were the same in the two expression cassettes. (B) Steady-state levels of expression of tRNAVal ribozymes. The figure shows Northern blotting analysis with the probes specific for the ribozyme and for the reference gene. (C) Half-lives of tRNAVal ribozymes in stable transformants that express ribozymes. Circles indicate relative amounts of tRNAVal ribozyme 1 (Rz1). Squares and diamonds indicate relative amounts of ribozymes 2 (Rz2) and 3 (Rz3), respectively. Bars show standard errors of results from three assays.

As a second approach and to test the above hypothesis, we attempted to compare the stability of each transcript under more natural, intracellular conditions. We established stable transformants that produced each tRNAVal ribozyme and measured the intracellular half-life of each transcript directly by interrupting cellular transcription with actinomycin D. As shown in Fig. 3C, the rate of degradation of Rz2 was lower than those of Rz1 and Rz3. The half-life of Rz2 (100 ± 10 min) was more than twice those of Rz1 (35 ± 2 min) and Rz3 (40 ± 15 min). These results were in good agreement with the results of the transient expression assay and supported our hypothesis that the difference in the steady-state levels of transcripts was due to the stability in cultured cells of each transcript rather than to any differences in the efficiency of transcription.

Intracellular activities of tRNAVal ribozymes. In order to evaluate the intracellular activities of the tRNAVal ribozymes, we performed two types of assay. We first used each tRNAVal ribozyme expression plasmid (pUCdt-Rz) and a target gene-expressing plasmid (pGV-V1) to cotransfect HeLa cells. Ribozyme and target expression vectors were used at a molar ratio of 2:1 for cotransfection of HeLa cells. After transient expression of both genes in each cell lysate, we estimated the intracellular activity of each tRNAVal ribozyme by measuring the luciferase activity. The luciferase activity recorded when we used the control plasmid (pUCdt), with only minimal tRNAVal promoter and terminator sequences instead of the ribozyme expression plasmid, was taken as 100%. As shown in Fig. 4A, Rz2, which had the highest stability in vivo, was the most effective (>60% inhibition), followed by Rz3 (>40% inhibition). Rz1 was not very effective (~10% inhibition), as expected from its low cleavage activity in vitro (Fig. 2B) and low stability in cultured cells (Fig. 3B and C).


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  		FIG. 4.   Inhibition of production of the U5 LTR-luciferase fusion gene in HeLa cells. (A) Transient expression in HeLa cells. Both the target-expressing plasmid and pUCdt-Rz, encoding a ribozyme, were used to cotransfect HeLa cells. (B) Transient expression in stable transformants that express ribozymes. Two independent clones were selected for each construct with similar levels of transcription of the inserted gene (tRNAVal or tRNAVal ribozyme), based on the results of Northern blotting analysis. Only the target-expressing plasmid was used to transfect stable transformants that expressed ribozymes. Bars show standard errors of results from five assays.

Throughout these experiments, the molar ratio of the template DNA coding for the target HIV-1 RNA to that for the ribozyme was kept at 1:2. It is to be noted that greater inhibition could be achieved by choosing a higher molar excess of ribozyme template. Therefore, the rank order of activity should be emphasized in Fig. 3A.

In the second assay, only the target gene-expressing plasmid (pGV-V1) was used to transfect stable transformants that produced almost identical levels of tRNAVal ribozyme (the levels of tRNAVal ribozyme were estimated by Northern blotting analysis [data not shown]). In this experiment (Fig. 4B), with two independent stable transformants for each ribozyme, we observed a trend similar to that described in the preceding paragraph. However, in this case, the effects of all of the ribozymes were stronger than those shown in Fig. 4A, wherein ribozyme and target expression vectors were used at a molar ratio of 2:1, most probably because all stable transformants produced tRNAVal ribozymes prior to the production of the target RNA. Rz2 inhibited expression of the target gene to a significant level, in some cases by as much as 97%.

Although Rz3 had the highest cleavage activity in vitro, it failed to act more effectively than Rz2 in the cellular environment. These results suggest that if a transcribed ribozyme is sufficiently stable within the cell, even if it does not have extremely high cleavage activity, it can have a remarkable effect in cultured cells. As a control in these experiments, an inactive ribozyme that had a single G5right-arrowA5 substitution in the catalytic domain (Fig. 1B) was also tested. Since the inactive tRNAVal ribozyme did not show meaningful inhibitory effects, it is clear that the intracellular activities of the tRNAVal ribozymes originated from their cleavage activities in cultured cells and not from the antisense effects, in agreement with our previous studies (24, 26, 27, 29, 30).

Ability to inhibit replication of HIV-1. Since the above-described studies demonstrated that Rz2 and Rz3 might have significant cleavage activities against the sequence of HIV-1 in vivo, we next compared the abilities of the tRNAVal ribozymes to inhibit replication of HIV-1. Using an HIV vector (Fig. 5 [43]), we obtained transduced cells of the H9 cell line that expressed Rz2 or Rz3 (since Rz1 was inactive in the studies described above, we made no attempts to isolate transduced cells that produced Rz1). Cells transduced with the HIV vector without a ribozyme expression cassette (Fig. 5A) were used as a mock control. Two independent cell lines were used for subsequent analysis, and we detected no obvious changes in their growth rates over a period of 11 days, compared with that of cells that did not produce either ribozyme (data not shown). Therefore, the ribozymes were not detrimental to host cells and probably only cleaved their target RNA with high specificity (26, 27, 29, 30).


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  		FIG. 5.   Schematic representation of the HIV vector. The expression cassette for each tRNAVal ribozyme was inserted into the SalI site immediately upstream of TK-Neor in the HIV-1-derived vector (A) to yield a retroviral vector, HIVRibo.N, that encoded a tRNAVal ribozyme (B). Psi  indicates a packaging signal.

Before the virus challenge assay, we measured the steady-state level of each tRNAVal ribozyme in the transduced H9 cells by quantitative RT-PCR analysis. The results of the transient expression assay in HeLa cells shown in Fig. 3B, namely, that the difference in steady-state levels of Rz2 and Rz3 was about fivefold, were confirmed by RT-PCR analysis (Fig. 6A and B). Clearly, Rz2 was more stable in cells than Rz3.


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  		FIG. 6.   Quantitation of expression of tRNAVal ribozymes in stably ribozyme-transduced H9 cells (CD4+ T cells) and inhibition of production of p24 in the transduced cells. (A) Quantitation of results (shown in panel B) of Southern blotting analysis of the RT-PCR-amplified ribozyme from two independent clones of ribozyme-transduced H9 cells. Products of PCR after 13, 15, and 17 cycles were analyzed by Southern blotting with a 32P-labeled oligonucleotide probe. Squares and circles indicate results with transduced cells of ribozyme 2 (Rz2) and ribozyme 3 (Rz3), respectively. (B) Results of Southern blotting. (C) Cells were cultured for 11 days after infection with HIV-1NL432. Small aliquots of supernatant were prepared from each culture on days 3, 7, and 11. Levels of p24 antigen were determined by HIV-1 antigen capture ELISA. Triangles indicate the mock control with only HIV vector. Squares and circles indicate results with ribozyme 2 (Rz2) and ribozyme 3 (Rz3), respectively.

When we challenged transduced cells of the H9 cell line that produced a tRNAVal ribozyme constitutively with HIV-1 virions, two independent Rz2-producing cell lines inhibited viral replication almost completely (~99%), as determined on day 11 postinfection (Fig. 6C). By contrast, to our surprise, Rz3 failed to inhibit viral replication at all under these experimental conditions. In the HIV-1 challenge assay, the difference between the effects of Rz2 and Rz3 was conspicuous.

Intracellular localization of tRNA ribozymes. Since the colocalization of a ribozyme with its target is clearly an important determinant of the ribozyme's efficiency (6, 7, 46), it was essential to determine the intracellular localization of tRNAVal ribozymes. Total RNA from stable transformants that expressed Rz2 was separated into nuclear and cytoplasmic fractions. Then, transcribed Rz2 was detected by Northern blotting analysis with a probe specific for the ribozyme. As shown in Fig. 7A, Rz2 was found predominantly in the cytoplasmic fraction and was not detected to any significant extent in the nuclear fraction. The other tRNA ribozymes (Rz1 and Rz3) were also localized predominantly in cytoplasmic fractions (data not shown).


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  		FIG. 7.   Intracellular localization of the tRNAVal ribozyme. Northern blotting analysis was performed with RNA from each intracellular fraction. Nucleic and cytoplasmic RNAs were prepared separately from stable transformants that expressed tRNAVal ribozyme (the same transformants had been used in the experiments for which results are given in Fig. 4B). Nucleic and cytoplasmic RNAs were loaded in amounts of 30 and 10 µg, respectively. (A) Results obtained with an appropriate 32P-labeled probe specific for each transcript. (B) Controls. Contamination of the cytoplasmic fractions was examined with a probe specific for the transcript of the natural U6 gene.

Since recent reports have indicated that similarly transcribed tRNAiMet ribozymes remain in the nucleus (7, 18), as a control we constructed the same tRNAiMet ribozyme (designated Rz-BR [Fig. 1E]) and analyzed its location. In agreement with the finding by Bertrand et al. and Good et al. (7, 18), about 90% of the transcripts indeed remained in the nucleus (Fig. 7A, right panel). One type of tRNAVal ribozyme (Rz-N [Fig. 1D]), which we constructed for other purposes, also remained in nucleus (Fig. 7A, center panel) despite the fact that the same tRNAVal expression system as for Rz1 to Rz3 was used in this construct (see below). U6 snRNA, which remains in the nucleus, was included as a second control in these studies (Fig. 7B).


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

A ribozyme is a potentially useful tool for suppression of the expression of specific genes, since it can be engineered to act on other RNA molecules with high specificity (21, 51). Although many trials have been successful (13, 42, 50, 52), it remains difficult to design an effective ribozyme expression system that can be used in vivo. One major challenge related to the use of ribozymes and antisense RNAs as therapeutic or genetic agents is the development of suitable expression vectors (5, 7, 25, 45, 49). Two kinds of expression systems have been used to date, as discussed in the introduction, namely, the Pol II and Pol III systems. In this study, we used the Pol III system and the promoter of a human gene for tRNAVal for transcription of ribozymes (55). This promoter is not only suitable for transcription of small RNAs, but its use also facilitates prediction of secondary structure by computer folding. More importantly, if properly designed, it allows export of transcribed ribozymes from the nucleus to the cytoplasm so that the tRNAVal ribozymes can find their mRNA targets.

Design of expression cassettes. The secondary structure of a target mRNA determines its susceptibility to ribozyme-mediated cleavage, and the ribozyme must also fold into appropriate secondary and tertiary structures for maximal activity. Although there is no guarantee that a computer-predicted secondary structure really represents the corresponding structure after transcription, the structures predicted in this study (Fig. 1A to C) were well correlated with cleavage activities in vitro (Fig. 2). In the expression cassettes, the last seven bases of the mature tRNAVal (Fig. 1, bottom right panel) had been removed, without any effect on transcription, in order to block 3' end processing of the transcript (2). They were replaced by a linker followed by a ribozyme (Fig. 1). The freedom or availability of the substrate recognition arms was adjusted by the linker sequence via formation of stable stem structures in combination with the sequence of tRNAVal, which accounted for about two-thirds of the whole sequence. Thus, it was relatively easy to predict, by computer folding, the secondary structure and the accessibility of each recognition arm. Furthermore, even if the sequence of the substrate recognition arm is changed, as long as the same rules for predicting overall secondary structure are used, it is still possible to predict the accessibility of recognition arms. Indeed, we have succeeded in constructing a similar ribozyme expression system for inhibition of the expression of other genes (26, 27, 29, 30). Our expression system, as shown in Fig. 1A to C, facilitates the design of an effective ribozyme expression cassette.

Translocation of tRNAVal ribozymes from the nucleus to the cytoplasm. The ribozyme expression cassettes shown in Fig. 1A to C allowed all of the transcripts to be exported to the cytoplasm (Fig. 7A) where they could find their mRNA targets, and significant inhibition by ribozymes of expression of the target molecules was observed (Fig. 4 and 6C). This was also true in the case of our novel dimeric ribozymes (maxizymes) with extremely high activity (29, 30). In a previous study, deletion of the last 11 bases of mature tRNAiMet not only blocked 3' processing (2) but also inhibited the export of the transcript to the cytoplasm (7, 18). These results suggested that 3' processing might be linked to export to the cytoplasm and that 3'-altered tRNA transcripts are not exported efficiently (8, 9). However, as demonstrated in Fig. 7, deletion of the last seven bases of mature tRNAVal did not inhibit the export of transcripts from the nucleus (see also Fig. 4B and C in reference 30).

A protein, designated Exportin(tRNA), which transports tRNA from the nucleus to the cytoplasm has recently been identified (4). Exportin(tRNA) binds RanGTP in the absence of tRNA, but it does not bind tRNA in the absence of RanGTP. Therefore, a model for the transport of tRNAs was proposed wherein Exportin(tRNA) associates with RanGTP first in the nucleus, and then the complex binds a mature tRNA molecule. This final complex is translocated through a nuclear pore complex to the cytoplasm. There, the Ran-bound GTP is hydrolyzed, releasing the tRNA into the cytoplasm and allowing Exportin(tRNA) to be recycled to the nucleus (4). We do not yet know the minimal sequence or structure within a tRNA that can be recognized by Exportin(tRNA). However, since the ribozymes shown in Fig. 1A to C were successfully translocated to the cytoplasm, it is possible that they were recognized and transported by Exportin(tRNA) despite the deletions and alterations at the 3' end of the natural tRNA.

It is clear from our study that even 3'-altered tRNA transcripts can be transported efficiently to the cytoplasm if their secondary structures resemble those shown in Fig. 1A to C. When we tried similarly to express other kinds of ribozymes (Rz-N and Rz-BR in Fig. 1) in HeLa cells, the transcripts remained in the nucleus (Fig. 7A). The secondary structure of Rz-N (Fig. 1D) is quite different from that of ribozymes Rz1, Rz2, and Rz3, which were cytoplasmic despite the fact that not only the sequence corresponding to the A and B box promoter elements (Fig. 1) but also all of the remaining sequence within the tRNAVal segment were identical in transcripts Rz1, Rz2, Rz3, and Rz-N. This observation suggests that if Exportin(tRNA) can indeed recognize the ribozyme transcript, it is unlikely that it recognizes a specific nucleotide sequence. Rather, Exportin(tRNA) might recognize some specific higher-order structure of tRNA or some sequence within such a higher-order structure.

Indeed, another ribozyme, constructed for other purposes, whose secondary structure resembled that of Rz-N was found only in the nucleus (data not shown). Moreover, the tRNAiMet ribozyme (Rz-BR), which had originally been constructed by Bertrand et al. and Good et al. (7, 18), remained in the nucleus (Fig. 7A), and its secondary structure (Fig. 1E) was quite different from those of cytoplasmic Rz1 through Rz3. We have constructed more than 10 other ribozymes for suppression of several other genes, keeping in mind that their secondary structures should resemble those of Rz1 through Rz3 (Fig. 1) and adjusting linker sequences so that they might be transported to cytoplasm. All of these ribozymes were found in the cytoplasm after transcription. They not only had high activities (>95% inhibition) but also high specificity (<5% inhibition by the inactive control) in mammalian cells. Thus, cytoplasmic ribozymes based on the design shown in Fig. 1A to C seem very attractive (26, 27, 29, 30). We should also mention that those tRNAiMet ribozymes, including Rz-BR (Fig. 1E), which remained in the nucleus were not very active (7).

It will be of interest to determine whether ribozymes such as Rz1 through Rz3 (but not Rz-N or Rz-BR) form complexes with Exportin(tRNA) in the presence of RanGTP, that is, under conditions in which formation of a complex between an export receptor and its cargo would be expected (4).

Activities of tRNAVal ribozymes in vivo. Sullenger and Cech (46) and Bertrand et al. (7) clearly demonstrated the importance of intracellular colocalization of ribozymes with their targets. In the case of one specific expression cassette, both the ribozyme and its RNA target were located in the nucleus and specific cleavage by the ribozyme of its target was detected (5). Thus, the critical parameter is not the localization of the ribozyme per se but is rather the ability of the ribozyme to colocalize with its target (7). Since various proteinaceous factors are involved in the intracellular processing and transport of mRNAs, and since such factors may bind promptly with (pre-mRNAs immediately after transcription, such factors could inhibit the binding of the ribozyme with its RNA target in the nucleus. Also, in the cytoplasm, polysomes might inhibit binding of the ribozyme with its RNA target. Moreover, since nuclear tRNAiMet ribozymes failed to inactivate a cytoplasmic mRNA that had originally been produced in the nucleus (7), the transport of mRNA from the nucleus to the cytoplasm seems to be much more rapid than the attack by the nuclear tRNAiMet ribozyme. One of the most critical factors determining ribozyme activity in vivo seems to be the association between the ribozyme and its target. A significant fraction of ribozymes must be degraded during transport and also during approach to the target site. For this reason, colocalization of a ribozyme and its target does not, by itself, guarantee the efficacy of ribozymes in vivo.

The ribozyme Rz2, which was most stable in cultured cells (Fig. 3B, 3C, 6A, and 6B), was more effective in the intracellular environment (Fig. 4) than Rz3, which had higher cleavage activity in vitro (Fig. 2). This difference in activity was magnified in the HIV-1 challenge (Fig. 6C). Although cells producing the more stable Rz2 were almost completely resistant to infection by HIV-1, other cells producing the less stable Rz3 were as sensitive as control cells to infection by HIV-1. Although Rz2 had a half-life that was about twice that of Rz3, it is unclear at present which structural feature(s) made Rz2 more resistant to RNases. There were six more nucleotides within the linker in Rz3 than in Rz2, which must have influenced the higher-order structure.

The half-lives of natural tRNAs range from 50 to 60 h (44), while that of Rz2 was only about 100 min. If the half-life of the tRNA ribozyme could be prolonged, a higher inhibitory effect might be expected. While we still cannot predict the relative stabilities in vivo of transcripts, we can design ribozymes that can be transported into the cytoplasm by incorporating secondary structures such as those shown in Fig. 1. Since we cannot accurately predict the stability of a transcript, we usually test several constructs and, in the case of various genes tested to date, we have always been able to obtain a cassette that can inactivate the gene of interest with >95% efficiency (26, 27, 29, 30) as long as we follow the rule described above.

The tRNAVal vector may be useful for expression of functional RNAs other than ribozymes whose target molecules are localized in the cytoplasm. Although colocalization in the cytoplasm cannot by itself guarantee effectiveness (Rz3; also tRNA-Rib5 in Gebhard et al. [17]), we can clearly increase the probability of success. In our hands, tRNAVal ribozymes have consistently high activities, at least in cultured cells. Therefore, properly designed tRNAVal ribozymes appear to be very useful as tools in molecular biology (26, 27, 29, 30), with potential utility in medicine as well.


    ACKNOWLEDGMENTS

S.K. and T.T. contributed equally to this work.

This research was supported by various grants from the Ministry of International Trade and Industry (MITI) of Japan, especially by the Molecular Design & Mechanism project of the National Institute for Advanced Interdisciplinary Research (NAIR), and also by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan. S.K. is a recipient of the predoctoral JSPS research fellowship.


    FOOTNOTES

* Corresponding author. Mailing address: Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305-8572, Japan. Phone: 81-298-54-3015. Fax: 81-298-54-3019. E-mail: taira{at}nair.go.jp.


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Results
Discussion
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Journal of Virology, March 1999, p. 1868-1877, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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