Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells.

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 step in 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.

Since the discovery of the first two ribozymes (1,2), several new types of ribozyme with self-cleavage activity have been found in nature (3)(4)(5)(6)(7)(8). Small ribozymes that can be designed to cleave RNA strands intermolecularly include hammerhead, hairpin, and HDV 1 ribozymes. These trans-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 (22,23), 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 tRNA Val . Many ribozymes, such as hammerheads and hairpins, have been effectively expressed under the control of promoters of gene for tRNAs (9, 11-15, 20, 21, 24).
A major advantage of our tRNA Val -directed expression system is that, with appropriate modification of the tRNA Val portion, it is possible to colocalize the expressed ribozyme in the cytoplasm with its target mRNA (14,15,22,23,25). Ribozymes expressed under the control of the tRNA Val promoter are exported to the cytoplasm as effectively as natural tRNAs via the action of Xpo(t), 2 a tRNA-binding protein (26,27) 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 in trans 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) (17, 28 -30). 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 5Ј 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 and in 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 the BCR-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 (15,21). 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. 31 and 32) provides a suitable substrate for comparisons of ribozymes. We used six kinds of functional RNA, including hammerhead, hairpin, and HDV ribozymes; our maxizyme and in 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 tRNA Val -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 (22), 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 tRNA Val described above, and the other was a U6 promoter (33,34). 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.

Construction of Vectors for Expression of Ribozymes and EGSs-
The construction of vectors for expression of ribozymes from the tRNA Val promoter using pUC-dt (a plasmid that contains the chemically synthesized promoter for a human gene for tRNA Val between the EcoRI and SalI sites of pUC 19) was described previously (22,23). pUC-dt was double-digested with Csp45I and SalI, and a fragment having a linker sequence with 5Ј Csp45I site and the restriction sites for KpnI and EcoRV and the terminator sequence TTTTT at the 3Ј end with 3Ј SalI site was cloned into the double-digested plasmid to yield pUC-tRNA/ KE. The KpnI 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 (17). The EcoRI and XhoI sites were used for insertion of each ribozyme sequence.
Analysis of the Cleavage Activity of Individual Ribozymes in Vitro-Each ribozyme and two substrates, namely BCR-ABL and ABL RNAs, were prepared in vitro using T7 RNA polymerase. Assays of ribozyme activity in vitro were performed, in 25 mM MgCl 2 and 50 mM Tris-HCl (pH 8.0) at 37°C, under enzyme-saturating (single-turnover) conditions, as described elsewhere (14). Each ribozyme (50 M) was incubated with 2 nM 5Ј-32 P-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.
In Situ Hybridization-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 MgCl 2 , 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 Cy3labeled oligodeoxynucleotide probe with a sequence complementary to the ribozyme and 20 g of tRNA from Escherichia coli MRE 600 (Roche Molecular Biochemicals, Mannheim, Germany), dissolved in 10 l of deionized formamide, were denatured by heating for 10 min at 70°C. The mixture was then chilled immediately on ice, and 10 l of hybridization buffer, containing 20% dextran sulfate and 2% BSA in 4ϫ 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 2ϫ SSC, 50% formamide and in 2ϫ 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).
Northern Blotting Analysis-Cells were grown to ϳ80% confluence (1 ϫ 10 7 cells) and were transfected with a tRNA Val -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 mM HEPES/KOH, pH 7.5, 50 mM potassium acetate, 8 mM MgCl 2 , 2 mM EGTA, and 50 g/ml digitonin) on ice for 10 min. The lysate was centrifuged at 1,000 ϫ 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 with 32 P by T4 polynucleotide kinase (Takara Shuzo Co., Kyoto, Japan).
Measurement of Luciferase Activity-Luciferase activity was measured with a PicaGene ® kit (Toyo-inki, Tokyo, Japan) as described elsewhere (15). In order to normalize the efficiency of transfection by reference to ␤-galactosidase activity, cells were cotransfected with the pSV-␤-galactosidase control vector (Promega, Madison, WI), and then the chemiluminescent signal due to ␤-galactosidase was quantitated with a luminescent ␤-galactosidase genetic reporter system (CLON-TECH, Palo Alto, CA) as described previously (15).

Design of Ribozymes and
EGSs-In order to express ribozymes in vivo, we used two kinds of pol III promoter (Fig.  1A). Transcripts with the promoter of the gene for tRNA Val can be efficiently transported to the cytoplasm when the appropriate choice of and combination of linker and ribozyme sequence is made (14,15,22,23,25). 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. 1B) directed against sites within a limited region (Ͻ100 nt) of B2A2 and ABL mRNAs ( Fig. 1C; 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 tRNA Val 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 mRNA in vitro and in vivo with extremely high specificity without any damage to normal ABL mRNA (15,21). Since it seemed possible that a hairpin ribozyme might also distinguish abnormal B2A2 mRNA from normal ABL mRNA (even though conventional hammerhead ribozymes fail to do so), we decided to use these two substrates in this study. Using two different substrates, we hoped to gain more insight into the differences among the activities of the various ribozymes in vivo in more general terms.
The hammerhead ribozyme is one of the smallest transacting ribozymes (6, 7, 19, 35-37). The stem II region of this Comparison of Ribozymes in Vitro and in Vivo ribozyme can be varied, and many derivatives with various modifications have been studied (38 -44). The maxizyme is one such derivative and acts as a dimer (Fig. 1B), and, in general, maxizymes have high level activity in vivo (14,15,21,23,25). The term "maxizymes (minimized, active, X-shaped (functions as a dimer), and intelligent (allosterically controllable) ribozymes)" was the name given to the minimized, allosterically controllable dimeric ribozymes with high level activity in vivo (15,21,25,(45)(46)(47). The minizymes shown in Fig. 1B are minimized hammerhead ribozymes with stem II deletions and relatively high activity, and each of them was identified recently by in vitro selection (43,44). These minizymes function in vitro even at low concentrations of Mg 2ϩ ions. Therefore, they may have advantage at low concentrations of Mg 2ϩ ions in vivo; thus, we included them in our study. The maxizyme is an effector-inducible trans-activated ribozyme, and the maxizyme shown in Fig. 1B recognizes the junction region of B2A2; it cleaves B2A2 mRNA but not normal ABL mRNA and, therefore, we used this well characterized tRNA Val -driven maxizyme as a positive control in studies in cultured cells (15,21,25,(45)(46)(47). Hairpin ribozymes, consisting of four helical regions interrupted by two internal bulges, have been used successfully as gene-inactivating agents (9,11,48). The two bulges interact with each other and hairpin ribozymes do not require Mg 2ϩ ions for catalysis, an observation that suggests that a base(s) in this region might be essential for catalysis (49 -52). In this study, we designed two such hairpin ribozymes targeted to two different sites. Although studies of the mechanism of action of HDV ribozymes with a pseudoknot structure indicate that a cytosine base distal to the cleavage site acts as a general acid catalyst (51)(52)(53), very little information is available about the activity of HDV ribozymes in vivo. Both genomic and antigenomic versions of the HDV ribozyme can be generated, and the latter type was made to act in trans (18,54,55). We prepared two such trans-acting HDV ribozymes targeted to two different sites.
Ribonuclease P (RNase P) cleaves tRNA precursors (pre-tRNAs) to generate the 5Ј termini of mature tRNAs (28 -30, 56 -58). Studies of RNase P resulted in the design of EGS RNAs. An EGS is designed to bind to a target RNA to generate a structure that mimics that of a pre-tRNA structure and is recognized as a substrate for RNase P. Upon formation of this structure, the target RNA can be cleaved by RNase P (17,28,30). RNase P is synthesized constitutively in cells and, thus, for use of an EGS as a gene-inactivating agent, it is not necessary to engineer the expression of additional RNase P. An EGS itself does not have cleavage ability. However, in cooperation with endogenous RNase P, it can bring about the cleavage of its target mRNA.
We constructed a total of 19 plasmids for expression of each ribozyme with the exception of the maxizyme under control of the tRNA Val or the U6 promoter. The sequences of ribozymes and the EGS were inserted as shown in Fig. 1A. All sequences in plasmids were confirmed by sequencing. The integrity of each construct was also confirmed by examination of the cleavage activity in vitro of each respective transcript, as described below.
Cleavage Activities of Ribozymes in Vitro-We first deter-mined activities of the various ribozymes in vitro. In order to compare chemical cleavage activities rather than association and/or dissociation kinetics, we measured the activities of the functional RNAs in vitro in the presence of a saturating excess of each ribozyme (single-turnover conditions). Ribozymes and substrate RNAs were transcribed in vitro by T7 RNA polymerase. As substrates, we used RNAs of 92 and 121 nt, which corresponded to regions that spanned the junctions of ABL RNA and B2A2 RNA, respectively (Fig. 1C). Because the tRNA-Val promoter is an internal promoter, in other words the DNA sequence that corresponds to tRNA Val contains the promoter region, transcripts from this promoter are always linked to a portion of the tRNA, which might or might not interfere with the ribozyme's activity. Therefore, ribozymes tested in vitro included a modified tRNA promoter region (about 90 nt) or about 20 nt of the U6 promoter (Fig. 1A). As shown in Fig. 2, wild-type hammerhead ribozymes (Wt Rz) had the highest activity in the case of both the tRNA Val and U6 promoter-driven transcripts and against both the B2A2 and ABL substrates. Since the majority of the target sites are located in the exon 2 region of ABL mRNA and since the computer-predicted secondary structure of this region is almost the same for both substrates (Fig. 1C, dark blue), with the exception of the region upstream of the junction, no significant differences between the rates of cleavage by the ribozymes of B2A2 and ABL substrates were expected or observed. In general, rates of cleavage by tRNA Val -driven ribozymes were slightly higher than those by U6-driven ribozymes, demonstrating that the tRNA portion did not hinder the activity of the ribozyme to any great extent. In terms of the rate of chemical cleavage, no other ribozyme approached the efficiency of the hammerhead ribozyme; the activity of the majority of ribozymes against the relatively long substrates (92 and 121 nt) was 2 or more orders of magnitude lower than that of Wt Rz in vitro.
Localization of tRNA Val -and U6-driven Transcripts-Colocalization of a ribozyme with its substrate is an important determinant of the activity of the ribozyme (10,15,22,23). A transcribed ribozyme might be expected to cleave pre-mRNAs in the nucleus or to be exported to the cytoplasm to cleave mature mRNAs (19). Our earlier data indicate that tRNA Valdriven ribozymes with high level of activities are efficiently exported to the cytoplasm, while similarly expressed tRNAribozymes with low level of activities are accumulated in the nucleus (22). However, there has been no systematic attempt to identify the cellular compartment in which a ribozyme acts most effectively. Given that it should be necessary for a ribozyme to be transported to the cytoplasm in mammalian cells for colocalization with its target mRNA, we developed our expression system for cytoplasmic expression of ribozymes, because mature mRNAs are exported to the cytoplasm for translation. By contrast, an EGS is likely to be operative in the nucleus because cleavage depends on RNase P, which is active only in the nucleus. In order to confirm this hypothesis (see next section), ribozymes were expressed from both types of promoter and their localization was determined both by in situ hybridization and by Northern blotting analysis of fractionated cell lysates.  Fig. 3, in which the red signals indicate the presence of a hairpin ribozyme (top) or an EGS (bottom). In all cases examined, without exception, tRNA Val -driven transcripts were transported to the cytoplasm and U6-driven transcripts localized in the nucleus.
For Northern blotting analysis, HeLa cells were fractionated to yield nuclear and cytoplasmic fractions and RNA was extracted from each fraction. This RNA was allowed to hybridize with an appropriate 32 P-labeled probe after electrophoresis (Fig. 4). Without exception, tRNA Val -driven ribozymes and EGSs expressed under control of the tRNA Val promoter were found in the cytoplasmic fraction and U6-driven transcripts were found in the nuclear fraction. The levels of all transcripts were very similar (they differed by less than 20%), irrespective of the type of ribozyme expressed and the expression system (tRNA Val or U6 promoter). Since the steady-state level of the transcript (reflecting its stability in cells) is a major determi-nant of ribozyme activity in vivo, if levels of transcripts had not been similar, our comparison of activities in vivo would have been more difficult (see the next section).
The Activities of Various Functional RNAs in Cultured Cells-We cotransfected HeLa cells with an expression plasmid that encoded an appropriate ribozyme unit(s) under the control of the tRNA Val or U6 promoter, and a plasmid that encoded the target BCR-ABL or ABL sequence fused with a gene for luciferase (luc), to evaluate the intracellular activity of ribozymes. The plasmid pB2A2-luc contained a sequence of B2A2 mRNA, while pABL-luc contained a sequence of 300 nt that encompassed the same target cleavage site and the junction between exon 1 and exon 2 of normal ABL mRNA. After transient expression of the ribozyme, substrate, and luciferase in individual cell lysates, we estimated the intracellular activity of each ribozyme by measuring luciferase activity.
Our results are shown in Fig. 5. The luciferase activity recorded when we used each target gene-expressing plasmid (pB2A2-luc or pABL-luc) was taken as 100%. The data presented are the results of three to six independent experiments. However, the various sets of experiments were performed on different days and transfection efficiencies varied, depending on the conditions of cells on each specific day. As a consequence, standard errors (error bars) were relatively large. However, when experiments were carried out on the same day, standard FIG. 2. The cleavage activities of ribozymes in vitro. The cleavage of the B2A2 substrate by tRNA Val -driven ribozymes (A) and U6-driven ribozymes (B) was examined. The 5Ј-32 P-labeled substrate (112 nt) and the cleavage products were detected by autoradiography. Reactions were performed under single-turnover conditions. The rates of cleavage of the B2A2 substrate and the ABL substrate, k obs , by the tRNA Val -driven ribozymes and U6-driven ribozymes were measured and the results are summarized in C and D, respectively. Mini, minizyme; Hair, hairpin. errors were in the range of 10 -20%. Nonetheless, the rank order of the activities of ribozymes always remained the same; thus, the data presented in Fig. 5 can be compared at least qualitatively.
Expression of the tRNA Val portion by itself had no inhibitory effect. In all the cases when tRNA Val -ribozymes were directed against B2A2 target (results indicated by purple colors in Fig.  5) and ABL target (indicated by blue colors), the luciferase activity decreased (the U6-driven maxizyme was not constructed in this study). As expected and in accord with previous findings (15, 20, 21, 25, 45-47), the tRNA Val -driven maxizyme showed high level specificity, cleaving only B2A2 mRNA without damaging ABL mRNA (Fig. 5A). No other ribozyme was able to distinguish between these two substrates. The extent of the decrease in luciferase activity was almost the same when in vitro selected minizymes were tested; they were slightly less effective than other ribozymes in cultured cells. As expected, tRNA Val -driven EGSs were ineffective since they were exported to the cytoplasm and their intracellular actions are known to depend on nuclear RNase P (Fig. 5A). We had expected that in vitro selected minizymes might be more active than their parental ribozymes because the minizymes were selected for the ability to act at low concentrations of Mg 2ϩ ions (43). However, our expectations were not confirmed in cultured cells. Importantly, our data in cells demonstrate that those ribozymes, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited significant activity in cells when an appropriate, high level expression system, which allows transport of ribozyme transcripts to the cytoplasm, was used.
When EGSs were expressed under control of the U6 promoter, they did demonstrate intracellular activity (Fig. 5B). The various other U6-driven ribozymes did not have any significant negative effects on expression of the luciferase gene. These results demonstrated clearly that only exported ribozymes (EGSs are not ribozymes per se) have significant cleavage activity because of the requirement for their colocalization with the target mRNA in the cytoplasm. Our data demonstrate that the target mRNAs in the cytoplasm are significantly more accessible to ribozymes than are the corresponding nuclear pre-mRNAs.

Kinetics of Reactions in Vitro Reveal the Superior Cleavage Activity of a Hammerhead Ribozyme against Relatively Long
Substrates-To investigate the actions of various ribozymes, we first determined kinetic parameters in vitro under singleturnover conditions. The rate constants, k obs , which are summarized in Fig. 2 (C and D) and were obtained in the presence of excess ribozyme, reflected the rate of the chemical cleavage step because almost all of each substrate had been captured by each ribozyme when reactions were started by the addition of Mg 2ϩ ions to the pre-heated and cooled ribozyme-substrate mixtures. Thus, the rate of the association step, which is a second-order reaction and was low under these conditions, could be ignored. In our studies, we used two relatively long substrates (92 and 121 nt) because kinetic parameters for short substrates have been well established and our purpose was to compare the activities of various ribozymes in vitro and in vivo against structured RNA substrates. Fig. 2 shows that the activity of the hammerhead ribozyme (Wt Rz) was significantly higher than that of all the other ribozymes. This was true for both substrates and for two different transcripts (tRNA Val -and U6-driven). The activity against the structured long substrate of the hammerhead ribozyme was about 2 orders of magnitude higher than that of the most of the other ribozymes. Nevertheless, the absolute activity, with the rate constants of 0.01-0.02 min Ϫ1 for the cleavage by the hammerhead ribozyme of the long substrate, was 2 orders of magnitude lower than the absolute activity against a short substrate. Under similar conditions, short RNA substrates can be cleaved by hammerhead ribozymes with rate constants of 1-2 min Ϫ1 . The difference reflects the fact that longer RNA substrates tend to form structures that limit access by ribozymes (60). With our long substrates we showed that the hammerhead ribozyme was the best ribozyme for cleavage of such structured RNAs despite the fact that the hairpin ribozyme can cleave short substrates as efficiently as hammerhead ribozymes, with rate constants of 1-2 min Ϫ1 (24).
Absence of Any Correlation between the Activities of Ri-bozymes in Vitro and in Cultured Cells-Each functional RNA exhibited cleavage activity both in vitro and in cultured cells but with varying efficiency. As expected, the hammerhead ribozyme had significant activity in cultured cells (Fig. 5). We did not expect 100% inhibition in our transient expression assays because not all cells would have been transfected by the various respective plasmids. As seen from Fig. 5, there was no correlation between the trends in the ribozyme activity in vitro (Fig. 2) and in those in cultured cells (Fig. 5). The kinetic parameters obtained in vitro indicated that the hammerhead ribozyme was superior to other ribozymes, but the activities of other tRNA Valdriven ribozymes in cultured cells were significantly improved relative to that of the hammerhead ribozyme (Fig. 5A). It has been suggested that the rate-limiting step in vivo of a reaction mediated by a catalytic RNA, such as a ribozyme, is the substrate-binding step (59,60). Our analysis is clearly consistent with this suggestion because the efficacies of ribozymes in vivo depend more strongly on the expression system and the localization within cells than on cleavage activities in vitro. It is now apparent that the rate-limiting step in ribozyme-mediated reactions in vivo is not the cleavage step. Thus, hairpin and other ribozymes with limited activities in vitro can have significant inhibitory effects in vivo, as demonstrated previously (9,11). Even HDV ribozymes, which are very inefficient in vitro, had clear inhibitory effects in cultured cells.
Cytoplasmic mRNAs Were Cleaved Significantly More Effectively than Nuclear Pre-mRNAs-Ribozymes expressed under the control of the tRNA Val promoter and the U6 promoter were found, as anticipated, in the cytoplasm and in the nucleus, respectively. The tRNA Val -driven ribozymes that had been exported to the cytoplasm had higher activities than the corresponding tRNA Val -driven ribozymes that retained in the nucleus (Fig. 5). Nuclear pre-mRNAs might be less accessible to ribozymes than cytoplasmic mature mRNAs because pre-mRNAs form complexes with heterogeneous nuclear proteins and small nuclear ribonuclear proteins and interact with various RNA-binding proteins, for example, proteins involved in splicing and in the export of processed mRNAs. It is also likely that higher ordered structures of mRNAs are disrupted more effectively in the cytoplasm by various RNA helicases (60). Thus, a ribozyme can attack its target site during the breathing of the cytoplasmic target mRNA.
The present analysis confirmed that cleavage by various ribozymes occurs more efficiently in the cytoplasm than in the nucleus. Without exception, the tRNA Val -driven ribozymes that had been exported to the cytoplasm (Figs. 3 and 4) had inhibitory effects (Fig. 5A), whereas U6-driven ribozymes that had remained in the nucleus were completely ineffective (Fig. 5B), despite the fact that both types of ribozyme were targeted to the identical site (Fig. 1C) and both had similar activity in vitro (Fig. 2, C and D). By contrast, the EGS in the nucleus mediated cleavage more effectively than the EGS that had been exported to the cytoplasm because the action of the EGS requires RNase P, which is localized in the nucleus. It should be noted also that RNase P might have an RNA unwinding activity.
We confirmed unambiguously that mature mRNAs in the cytoplasm were more accessible to ribozymes than pre-mRNAs in the nucleus. Thus, if we are to exploit ribozyme activity in cells, ribozymes must be concentrated in the cytoplasm while EGSs must remain in the nucleus. Our findings should be useful for the selection of expression systems and the future design of intracellularly active ribozymes.