ABSTRACT
The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A
oncoprotein and it is thought to participate in prevention of the G
0
/G
1
transition during the cell cycle, in activation of certain enhancers and in the
stimulation of differentiation pathways. In order to determine the exact
function of p300, as a first step we constructed a simple assay system for the
selection of a potential target site of a hammerhead ribozyme
in vivo
. For the detection of ribozyme-mediated cleavage, we used a fusion gene (
p300-luc
) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene.
We were also interested in the correlation of the GUX rule, for the triplet
adjacent to the cleavage site, with ribozyme activity
in vivo.
Therefore, we selected five target sites that all included GUX. The rank order of activities
in vitro
indeed followed the GUX rule; with respect to the
k
cat, a C residue as the third base (X) was the best, next came an A residue and
a U residue was the worst (GUC > GUA > GUU). However,
in vivo
the tRNA
Val
promoter-driven ribozyme, targeted to a GUA located upstream of the initiation
codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar
ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities
in vivo
did not conform to the GUX rule, it is unlikely that the rate limiting step for
cleavage of the
p300-luc
mRNA was the chemical step. This kind of ribozyme expression system should be
extremely useful for elucidation of the function of p300
in vivo
.
Elucidation of the functions and the site of interaction with their targets of
transcription factors is of considerable current interest (
1
-
3
). A 300 kDa cellular protein, known as p300, is a nuclear phosphoprotein that
is the binding target of the adenovirus E1A oncoprotein (
4
). The region of E1A required for binding of p300 includes residues at the N-terminus and in CR1 (conserved region 1) of the E1A oncoprotein (
5
). Binding of p300 to E1A is believed to stimulate the G
0
/G
1
transition, to block differentiation and to inhibit the action of certain
transcriptional enhancer elements (
5
). Other data support the view that p300 plays a role as a co-activator (
6
-
10
) and the p300 protein has been shown to activate transcription when fused to a
DNA binding domain (
7
,
8
). However, the functional significance of p300 protein
in vivo
is still unknown. Thus, we decided to construct a system in which expression of
p300 was regulated by a hammerhead ribozyme.
Recently, regulation of gene expression by ribozyme and antisense RNA/DNA has
been performed (
13
-
17
). The principle of catalytic self-cleavage of RNA molecules and of cleavage in
trans
has been well established over the last decade (
18
). Expression of various genes has been suppressed by ribozymes (
13
-
21
) and among the RNA molecules with ribozyme activity, the hammerhead ribozyme is
the best characterized (
22
-
28
). It is well established that ribozymes require divalent metal ions for
cleavage activity
in vitro
and
in vivo
(
29
-
38
). Theoretically, it is possible for one molecule of ribozyme to cleave multiple
target RNAs. On the other hand, for high level activity, an antisense RNA needs
a cellular factor, such as an RNase III-type nuclease, to destroy the target RNA. As a consequence, a ribozyme tends to show a
higher inhibitory effect than does an antisense RNA (
39
).
The activity of ribozymes depends very strongly on the target site and,
furthermore, it is not easy to predict the best cleavable target site. Both
primary (
40
-
43
) and secondary structural rules (
44
) must be considered in selection of the target site. In general, the GUC
triplet should be suitable as a cleavage site, since it conforms to the NUX
rule, where N is A, U, G or C and X is A, U or C (
40
-
47
), and since it is the most popular triplet used in nature (
24
,
43
). In addition, the tertiary structure of the target RNA
in vivo
(including interaction with cellular proteins) should also be considered in
selecting the target site, since it has a strong influence on the activity of
ribozymes and antisense molecules. Unfortunately, it is difficult to predict
the
in vivo
tertiary structure of the cleavage site, even though the selected site is in
accord with the primary and secondary structural rules. As a result, selection
of the best target site remains a matter of trial and error.
Another challenge for
in vivo
application of ribozymes is construction of a gene for a ribozyme that allows
continuous expression of the ribozyme in a particular cell. Despite an increasing number of successful studies, based on general rules for antisense sequences
and on sophisticated constructs for the expression of ribozymes, the design of
ribozymes that cleave RNA
in vivo
is also a matter of trial and error.
Our previous kinetic studies
in vitro
demonstrated that in reactions catalyzed by a
trans
-acting hammerhead ribozyme, mutant substrates that contained a GUA or GUU
triplet at the cleavage site were cleaved less efficiently than a wild-type substrate with the GUC triplet (
42
). For our analysis of the function of p300 using a ribozyme that would
significantly suppress expression of p300
in vivo
, we needed an appropriate combination of a good target site and a ribozyme
expression system. We chose luciferase activity as a reporter and linked the
sequence that encoded the N-terminal region (285 nt in length) of p300 to the gene for luciferase. In
general, the N-terminal region is the first choice for the target site of ribozymes and
antisense molecules (
39
). We were also interested in the correlation of the GUX (X = A, U or C) rule,
derived on the basis of studies
in vitro
(
42
), with ribozyme activity
in vivo
. Therefore, we chose five different target sites which contained the GUC, GUA
or GUU triplet within this N-terminal region and the efficiency of cleavage at each site was examined
in vitro
and
in vivo
in a transient co-transfection assays in HeLa S3 cells.
We report here that all ribozymes whose synthesis was driven by the tRNA
Val
promoter significantly suppressed expression of the
p300-luc
fusion gene, with the exception of the inactive ribozyme control. Since the
inactive ribozyme control did not have any inhibitory effect, the observed
activities appeared to originate from the cleavage activities of the ribozymes. Moreover, since the rank order of activities
in vivo
did not conform to the recently established GUX rule (
41
,
42
), it is unlikely that the rate limiting step for cleavage of the
p300-luc
mRNA
in vivo
was the chemical step. Most importantly, in this study a significant inhibitory
effect was observed when the molar ratio of the DNA template for the target
p300
mRNA to that for the ribozyme was only 1:1. Therefore, this kind of ribozyme
system should be a useful tool to elucidate the function of p300
in vivo
.
Oligonucleotides were synthesized with a DNA/RNA synthesizer (model 392; Applied Biosystems, Foster City, CA) and purified on OPC columns.
Ribozymes and their corresponding substrates used for kinetic measurements
in vitro
were synthesized with a DNA/RNA synthesizer (model 392; Applied Biosystems) and
purified by polyacrylamide gel electrophoresis as described previously (
36
,
38
,
42
,
48
,
49
). Reagents for manipulation of RNA were purchased from either ABI or American
Bionetics Inc. (ABN; Foster City, CA). Sequences of the synthetic ribozymes and
their corresponding substrates were as follows: Ribozyme 1 (R1), 5'-GAG GAA CUG AUG AGG ACC GAA AGG UCG AAA CCA GA-3'; Ribozyme 2 (R2), 5'-CGG AGA CUG AUG AGG ACC GAA AGG UCG AAA
CAA GC-3'; Ribozyme 3 (R3), 5'-CUG GCG CUG AUG AGG ACC GAA AGG UCG AAA CGC CG-3'; Ribozyme 4 (R4), 5'-UGC CAA CUG AUG AGG ACC GAA
AGG UCG AAA CUU GU-3'; Ribozyme 5 (R5), 5'-AUC UUG CUG AUG AGG ACC GAA AGG UCG AAA CCA UG-3'; Substrate 1 (S1), 5'-UCU G
GU U
UU CCU C-3'; Substrate 2 (S2), 5'-GCU U
GU A
UC UC CG-3'; Substrate 3 (S3), 5'-CGG C
GU C
CG CCA G-3'; Substrate 4 (S4), 5'-ACA A
GU C
UU GGC A-3'; Substrate 5 (S5), 5'-CAU G
GU A
CA AGA U-3'.
Reaction rates
in vitro
were measured at 37oC in 10 mM MgCl
2
and 50 mM Tris-HCl, pH 6.0, under ribozyme saturating (single turnover; the final
concentration of each ribozyme was 100 nM) conditions with 5'-
32
P-labeled substrate (the final concentration of each substrate was 10 nM).
Reactions were stopped by removal of aliquots from the reaction mixture after
10 min incubation and mixing with an equivalent volume of a solution of 100 mM
EDTA, 9 M urea, 0.1% xylene cyanol and 0.1% bromophenol blue. Substrates and 5'-cleaved products were separated by electrophoresis on a 20%
polyacrylamide-7 M urea denaturing gel and were detected by autoradiography (Fig.
2
). The extent of cleavage was determined by quantitation of radioactivity in the
bands of the substrate and product with a Bio-Image Analyzer (BA2000; Fuji Film, Tokyo).
For the detection of ribozyme-mediated cleavage, we used a fusion gene (
p300-luc
) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene.
The plasmid for expression of the
p300-luc
fusion gene was constructed from the Pica Genetm Luciferase Control Vector (Control Vector; Toyoinki, Tokyo). In brief,
the DNA fragment encoding the N-terminal region of p300 (nt 1146-1430) was amplified from pCMVb p300 (
6
) by PCR with 5'-AAT TCG ATA AGC TTG AGA TTT CCT GAG GAT TCT GGT TTT-3' as the 5' primer and 5'-TAG GCC GCT CTA GAG GAT AGA ATG
GCG CCG GGC CTT TCT TTA TGT TTT TAG AAG CTG CAT CTT GTA CCA TG-3' as the 3' primer. After digestion with
Hin
dIII and
Xba
I, this fragment was inserted into the
Hin
dIII and
Xba
I sites of the Control Vector. The nucleotide sequence of the
p300-luc
fusion gene was confirmed by sequencing.
Chemically synthesized oligonucleotides encoding ribozyme 2, inactive ribozyme 2
or antisense 2 (Fig.
1
C) and the pol III termination sequence (
50
) were convered to double-stranded sequences by PCR. After digestion with
Csp
45I and
Sal
I, the fragments were cloned downstream of the tRNA promoter of pUC-tRVP (which contains the chemically synthesized promoter for human tRNA
Val
between the
Eco
RI and
Sal
I sites of pUC19). The sequences of all constructs were confirmed by direct
sequencing.
HeLa S3 cells were cultured in Dulbecco's modified Eagle's medium containing 10%
(v/v) fetal bovine serum, 100 [mu]g/ml penicillin and 100 [mu]g/ml streptomycin. Transfections with plasmid DNA were performed using Lipofectin (Gibco BRL, Gaithersburg, MD) in accordance with the manufacturer's protocol. In brief, 3 * 10
5
cells were plated in 6-well plates 1 day before transfection. After cells had been washed twice
with phosphate-buffered saline (PBS), 0.8 ml OPTI-MEM I medium (Gibco BRL) was added to each well. A solution of plasmid DNA (6 [mu]g plasmid DNA in 100 [mu]l OPTI-MEM I medium) and a solution of Lipofectin (4 [mu]l Lipofectin reagent in 100 [mu]l OPTI-MEM I medium) were mixed gently and the
mixture was kept at room temperature for 20 min to allow formation of
Lipofectin-DNA complexes. The solution of Lipofectin-DNA complexes was added to the cells. After 12 h, the medium was removed and 2 ml of the growth medium were added.
The cultures were incubated for an additional 24 h.
Transfected cells were washed twice with PBS and lysed in lysis buffer (Tris-PO
4,
pH 7.8, 8 mM MgCl
2
, 1 mM DTT, 1 mM EDTA, 1% Triton X-100, 1% BSA and 15% glycerol). After removal of cell debris by
centrifugation in a microcentrifuge, the luciferase activity in the supernatant
was measured by the method of Alam and Cook (
51
). One hundred microliters of luciferase assay reagent [20 mM Tricine, 1.07 mM
(MgCO
3
)
4
Mg (OH)
2
[middot]5H
2
O, 2.67 mM MgSO
4
, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 [mu]M coenzyme A, 470 [mu]M luciferin and 530 [mu]M ATP] were added to 20 [mu]l of the supernatant in a test tube. The integrated light output was measured with a
luminometer (Lumat LB9501; Berthold, Bad Wildbad, Germany).
Total cellular RNA was isolated from transfected cells as described by
Chomczynski and Sacchi (
52
). RNA (10 [mu]g), denatured with glyoxal (
53
), was fractionated on a 2.2% agarose gel, transferred to a nylon membrane
(Hybond-N; Amersham, UK) and allowed to hybridize with a 5'-
32
P-end-labeled oligonucleotide probe (5'-CTC GCT TGT TTC GGA CCT TT-3', specific for ribozyme 2 and inactive
ribozyme 2, or 5'-CTC GCT TGT ATC TCC GAA A-3', specific for antisense 2; Fig.
1
C).
The exact function of the cellular protein p300 remains obscure. In order to
suppress the expression of p300, we tried to identify the best target site within the p300 transcript for ribozyme-mediated cleavage. Since the phenotype of suppressed p300 clones cannot be used
for quantitation of the efficiency of ribozyme-mediated cleavage and, moreover, since the cleavage products, in general,
cannot be detected
in vivo
, we constructed a rapid assay system for selection of the best target site by
fusing the N-terminal region of the gene for p300 (corresponding to nt 1146-1430, according to the numbering of Eckner
et al
.;
6
) in-frame with the gene for luciferase (Fig.
1
A). The fused gene product was produced under the control of the SV40 promoter
and its enhancer.
In a previous study, we examined the generality of the NUX rule (where N is A,
U, G or C and X is A, U or C) and we demonstrated from kinetic studies
in vitro
that in reactions catalyzed by
trans
-acting hammerhead ribozymes, mutant substrates that contained the GUA or
GUU triplet were cleaved less efficiently than the wild-type substrate with the GUC triplet (GUG could not be cleaved;
42
). In choosing potential target sites for examination of the efficiency of their
ribozyme-mediated cleavage
in vivo
, we made sure that each of the selected conserved sites contained one of all
possible GUX triplets.
The five selected target sites are indicated in Figure
1
A and the corresponding ribozymes are numbered 1-5 in the upstream to downstream direction. Each of the ribozymes was
transcribed under the control of the human tRNA
Val
promoter (Fig.
1
B). Each ribozyme had the same 24 nt catalytic domain as that refined by
Haseloff and Gerlach (
43
) and each was equipped with nine bases on both substrate binding arms that were
targeted to the relatively well-conserved sequences of p300 mRNA.
Since ribozyme 2 targeted to the GUA site was found to be the most active (see
below), constructs with an inactive ribozyme and an antisense control targeted to the same site were generated (Fig.
1
C). The inactive ribozyme differed from the active ribozyme by a single G
5
-> A mutation in the catalytic core (the numbering system follows the rule
for hammerhead ribozymes;
54
). This single base change should diminish the cleavage activity while the
antisense effect, if any, should be unaffected (
39
,
40
). As a second control for comparison of the activity of ribozyme 2 with that of
an antisense RNA, we synthesized an antisense construct in which the entire
catalytic domain of ribozyme 2 was replaced by a single uracil moiety (Fig.
1
C).
In a previous study, in order to examine in detail the generality of the NUX
rule for the GUC triplet adjacent to the cleavage site in hammerhead ribozymes,
kinetic parameters were determined for substrates with mutations only within
this triplet (
42
). In the present study, sequences of the selected five target sites differed
not only in the GUX triplet, but also in the binding sites. In order to examine
whether activities
in vitro
of the five ribozymes selected in this study might follow the GUX rule, we
chemically synthesized five ribozymes (R1-R5) and their corresponding substrates (S1-S5) and relative activities were measured under single turnover
conditions. In this case, each synthetic ribozyme was equipped with six bases
on both substrate binding arms (complete sequences are listed in Materials and
Methods).
Results of such studies are shown in Figure
2
. The level of cleavage (%) in 10 min in 10 mM MgCl
2
and 50 mM Tris-HCl, pH 6.0, at 37oC was 5, 20, 25, 27 and 12% respectively, for R1/S1 (GUU), R2/S2
(GUA), R3/S3 (GUC), R4/S4 (GUC) and R5/S5 (GUA). The order of efficiency of
cleavage was as follows: R4 (GUC) > R3 (GUC) > R2 (GUA) > R5 (GUA) > R1 (GUU),
where the target triplet is indicated in parentheses. Therefore, the rank order
of activities
in vitro
followed the GUX rule; a C residue as the third base (X) was the best, next
came an A residue and a U residue was the worst (GUC > GUA > GUU).
The effects of the ribozymes, produced in
trans
, on the specific target mRNA, namely the transcript of the
p300-luc
fusion gene, were examined by measuring luciferase activity in HeLa S3 cells
that had been co-transfected with the plasmids that encode the ribozymes and the
p300-luc
iferase (pUC-tRR and p300-luc plasmids respectively). To determine the effectiveness of ribozyme-mediated inhibition of expression of the
p300-luc
fusion gene in the transient expression assay, a control plasmid (pUC-tRVP), namely pUC-tRR from which the ribozyme sequence had been deleted (Fig.
1
B), was used to allow generation of luciferase activity that was designated
100%. As shown in Figure
3
, all the active ribozymes were capable of decreasing luciferase activity
in vivo
. The results shown are averages of results from five sets of experiments and are
given as percentages relative to the control value of 100%. The extent of
inhibition by ribozymes expressed from the pol III promoter varied from 47 to
96% when the molar ratio of template DNAs for the target and the ribozyme was
1:4. To the best of our knowledge, this is the lowest ratio ever reported to
yield a significant inhibitory effect and it proves the utility of the pol III
promoter in mammalian cells (
55
,
56
).
Since ribozyme 2 was found to be the most effective ribozyme (Fig.
3
), we examined its antisense effect using the inactive ribozyme control.
Furthermore, the inhibitory effect of the ribozyme was compared with that of antisense RNA targeted to the same site. Since the
antisense RNA was significantly shorter than the ribozyme, levels of expression of ribozyme 2, inactive ribozyme 2 and antisense
2 RNA were examined by Northern blotting (Fig.
4
). The levels of expression were determined by quantitation of the radioactivity
of bands with an image analyzer. The levels of expression were found to be
nearly identical in each of the three cases, within the limits of experimental
error (Fig.
4
).
The three constructs were examined for their ability to suppress expression of
the
p300-luc
fusion gene, as described in the previous section. As shown in Figure
5
A, the inactive ribozyme did not have any inhibitory effect, in accord with our
previous finding in a bacterial system (
39
). The expected stability of RNA duplexes is as follows: antisense > inactive
ribozyme [approx] active ribozyme. The significant inhibition by the antisense RNA was in
accord with this prediction. Moreover, the RNase III-type activity of the host cells should increase the inhibitory effect of
the antisense RNA. Since the inactive ribozyme (which, with its target site
duplex, would not be expected to be a substrate for RNase III-type ribonucleases because of the discontinuous base pairing) did not have
any inhibitory effect, it seems likely that the inhibitory effects of the
ribozyme originated from its cleavage activity.
The authors thank Dr Rchard Eckner and Dr David M. Livingston for the gift of
pCMV[beta] p300 and information of the nucleotide sequence of mouse p300
(unpublished data).
*To whom correspondence should be addressed at: Institute of Applied
Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305, Japan
REFERENCES
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