From the Department of Chemistry and Biotechnology,
Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo,
Tokyo 113-8656 and the § Gene Discovery Research Center,
National Institute of Advanced Industrial Science and Technology,
1-1-4 Higashi, Tsukuba Science City 305-8562, Japan
Received for publication, November 22, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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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 (3-8). Small ribozymes that can be designed to cleave
RNA strands intermolecularly include hammerhead, hairpin, and
HDV1 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 tRNAVal. 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 tRNAVal-directed expression system
is that, with appropriate modification of the tRNAVal
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 tRNAVal 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 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 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 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 (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
tRNAVal 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 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 (22, 23). 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. 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
MgCl2 and 50 mM Tris-HCl (pH 8.0) at 37 °C,
under enzyme-saturating (single-turnover) conditions, as described
elsewhere (14). 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.
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 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).
Northern Blotting Analysis--
Cells were grown to ~80%
confluence (1 × 107 cells) and were transfected with
a tRNAVal-Rz expression vector with the LipofectinTM
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 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% NuSieveTM
(3:1) agarose gel (FMC Inc., Rockland, ME). After electrophoresis,
bands of RNA were transferred to a Hybond-NTM 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 32P 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 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 tRNAVal 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 (<100 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 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 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 trans-acting
ribozymes (6, 7, 19, 35-37). The stem II region of this 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, 45-47). 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
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-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
tRNAVal-driven maxizyme as a positive control in studies in
cultured cells (15, 21, 25, 45-47). 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
Mg2+ 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 (51-53),
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 5' 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
tRNAVal 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 determined
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
tRNAVal promoter is an internal promoter, in other words
the DNA sequence that corresponds to tRNAVal 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
tRNAVal 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
tRNAVal-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 tRNAVal- 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 tRNAVal-driven
ribozymes with high level of activities are efficiently exported to the
cytoplasm, while similarly expressed tRNA-ribozymes 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.
HeLa cells were transfected with plasmids with a tRNAVal or
U6 promoter. After 36 h, we examined the localization of each
expressed ribozyme by in situ hybridization and Northern
blotting analysis of fractionated cells. For in situ
hybridization, Cy3-labeled probes were incubated with fixed and
permeabilized cells and then the fluorescence of Cy3 was detected by
confocal microscopy. In this way, we confirmed the expected locations
of all 19 different transcripts. Typical examples of our results are
shown in 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, tRNAVal-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
32P-labeled probe after electrophoresis (Fig.
4). Without exception, tRNAVal-driven ribozymes and EGSs expressed under control
of the tRNAVal 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 (tRNAVal or U6 promoter). Since the
steady-state level of the transcript (reflecting its stability in
cells) is a major determinant 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 tRNAVal 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 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 tRNAVal portion by itself had no
inhibitory effect. In all the cases when tRNAVal-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 tRNAVal-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, tRNAVal-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 Mg2+ 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
single-turnover conditions. The rate constants,
kobs, 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 Mg2+ 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 (tRNAVal- 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 Absence of Any Correlation between the Activities of Ribozymes 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 tRNAVal-driven 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 tRNAVal promoter and the U6 promoter were found, as
anticipated, in the cytoplasm and in the nucleus, respectively. The
tRNAVal-driven ribozymes that had been exported to the
cytoplasm had higher activities than the corresponding
tRNAVal-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 tRNAVal-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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 (CLONTECH, Palo Alto, CA)
as described previously (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Design of the functional RNAs directed
against BCR-ABL (B2A2) mRNA and
ABL mRNA. A, the two types
of expression system. The tRNAVal portion was attached to
each ribozyme because the promoters of the gene are internal (A and B
boxes). No extra sequence was attached to the U6-driven transcripts.
However, 17 nt were attached to ribozymes transcribed by T7 polymerase
in vitro for kinetic analysis use because of the design of
the primer used. B, secondary structures of ribozymes and
EGSs. The Wt Rz, minizyme, and maxizyme had the identical target site.
Minizymes A and B were of the same length but had different sequences.
The cleavage sites attached by hairpin ribozymes, HDV ribozymes, and
EGSs A and B were different, as indicated in C. C, secondary structures of the ABL and
B2A2 mRNA substrates. The regions near the splicing
junction are indicated by different colors for each exon.
The substrate-recognition site of each ribozyme is indicated by
underlining in different colors. The
arrows show sites of cleavage.
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Fig. 2.
The cleavage activities of ribozymes in
vitro. The cleavage of the B2A2 substrate by
tRNAVal-driven ribozymes (A) and U6-driven
ribozymes (B) was examined. The 5'-32P-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, kobs, by the
tRNAVal-driven ribozymes and U6-driven ribozymes were
measured and the results are summarized in C and
D, respectively. Mini, minizyme; Hair,
hairpin.
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Fig. 3.
Confocal microscopic images showing the
detection by in situ hybridization of ribozymes and
EGS expressed in mammalian cells. Cy3-labeled probes were used for
detection of the tRNAVal-driven and U6-driven hairpin
ribozymes. Similar images were obtained for all 19 different
constructs, i.e. tRNA-driven ribozymes and EGSs were
transported to the cytoplasm, and U6-driven ribozymes and EGSs were
localized in the nucleus.
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Fig. 4.
Nuclear localization of functional RNAs.
The steady-state levels of tRNAVal-driven ribozymes
(A) and U6-driven ribozymes (B) and their
localization are shown. Approximately the same levels of expression of
functional RNAs from both promoters were observed. N,
nuclear fraction; C, cytoplasm fraction. Without exception,
tRNAVal-driven ribozymes and EGSs were localized in the
cytoplasm and U6-driven ribozymes and EGSs were localized in the
nucleus.
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Fig. 5.
Inhibitory effects in cultured cells of
tRNAVal-driven ribozymes (A) and U6-driven
ribozymes (B) on the expression of chimeric
BCR-ABL-luciferase and
ABL-luciferase genes. A plasmid that encoded a
ribozyme or EGS and a plasmid that encoded the target gene were used to
cotransfect HeLa cells. Decreases in luciferase activity (61) indicate
the cleavage of transcripts by ribozymes in cells. The effects on the
two different substrates are indicated by different colors.
Mini, minizyme; Hair, hairpin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 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
1.
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
1
(24).
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ACKNOWLEDGEMENTS |
---|
We thank Professor Sidney Altman and Dr. Cecilia Guerrier-Takada (Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT) for helpful comments and for the gifts of EGS expression vectors.
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FOOTNOTES |
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* This work was supported in part by grants from the Ministry of Economy, Trade and Industry of Japan and also by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Techology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work.
Recipient of a research fellowship for young scientists from
the Japan Society for the Promotion of Science.
** To whom correspondence should be addressed: Dept. of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel.: 81-3-5841-8828 or 81-298-61-3015; Fax: 81-298-61-3019; E-mail: taira@chembio.t.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010570200
2 T. Kuwabara and K. Taira, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: HDV, hepatitis delta virus; pol, polymerase; EGS, external guide sequence; CML, chronic myelogenous leukemia; nt, nucleotide(s); Wt, wild-type; Rz, ribozyme.
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