The fowl adenovirus type 1 (CELO) virus-associated RNA-encoding gene: a new ribozyme-expression vector

Gene. 161 (1995) 1X9-193 10 1995 Elsevier Science B.V. All rights reserved.

GENE

189

0378-I 119, Y5SO9.50

08969

The fowl adenovirus type 1 (CELO) virus-associated RNA-encoding gene: a new ribozyme-expression vector (Catalytic

RNA; secreted alkaline

A.N. Zakharchuk,

phosphatase;

K.K. Doronin,

Received by H. van Ormondt:

24 October

hammerhead

V.A. Karpov,

1994; Accepted:

Rz)

V.A. Krougliak”

I6 February

and B.S. Naroditsky

1995; Received at publishers:

7 April 1995

SUMMARY

A new system virus-associated secreted between

for hammerhead RNA (CELO

ribozyme VA RNA)

(Rz) expression

was examined

was used as a vector

for the incorporation

type 1 (CELO)

of Rz to target

the mRNA

of

alkaline phosphatase (SEAP) both in vitro and in vivo. The Rz gene was integrated into the CELO VA RNA the internal promoter boxes A and B; apparently this did not interfere with its transcription. Rz integrated

into CELO

VA RNA

RNA sequences

and, lacking

did not inhibit

293 cells by co-transfection We conclude

the viral sequences,

the integrated

with plasmids

that CELO

exhibited

the same activity

VA RNA may be used for effective expression

of catalytic

RNAs that cleave target

RNAs in a sequence-specific manner (Cech. 1987) they have been proven to be a potentially powerful instrument in the control of gene expression and in gene therapy (Rossi and Sarver, 1990; Altman, 1993). The so-called Correspondence

hammerhead

to: Dr.

ribozymes

B.S. Naroditsky,

Timiryazevskaya

(Rz), which are

Institute

Street 42, Moscow

Tel. (7-095) 977-0829;

of Agricultural 127550, Russian

Fax (7-095) 977-0947;

e-mail: [email protected] address:

Department

Main Street, West Hamilton.

of Btology, Ontario

McMaster

University,

L8S 4K1, Canada.

1280

Tel. (t-416)

525-9140. Abbreviations:

Ad. adenovirus;

bp, base pair(s);

1; CELO VA RNA, VA RNA from CELO;

ribozyme(s);

Rz, gene encoding

CELO.

fowl Ad type

CELO VARNA, gene encod-

ing CELO VA RNA: MCS, multiple cloning minimum essential medium; m.u., map unit(s); Rz: SEAP,

site; MEM, Eagle’s nt, nucleotide(s); Rz.

human

secreted

placental

alkaline phosphatase; SEAP, gene encoding SEAP: VA RNA, virusassociated RNA; VA1 RNA. VA1 RNA from human Ad2: VAI RNA, gene encoding VA1 RNA.

SSDI 037X-111’)(95100251-0

VA

Rz activity in vitro. In vivo experiments were carried out with human Rz and SEAP. Inhibition of enzyme activity was 50% in 48 h.

(Forster

Federation.

CELO

of hammerhead

Rz.

found in some plant viroids and plant viral satellite

Since the discovery

Biotechnology.

in vitro. Consequently,

containing

INTRODUCTION

*Present

in which fowl adenovirus

and Symons,

1987), have a common

structure and cleave the substrate N is any nucleotide, U is uridine

RNA

secondary

at an NUH site, where and H may be A, C or

U (Ruffner et al.. 1990). Effective gene suppression by Rz in vivo requires a substantial molar excess of the Rz over the target RNA (Cotten

and Birnstiel,

1989; Cameron

which can be achieved by increasing stability of Rz. A promising solution merase

III-driven

expression

and Jennings,

1989)

the amount or the is the use of poly-

advanced

by Cotten

and

Birnstiel (1989). Genes under control of an RNA polymerase III promoter are efficiently transcribed in various tissues (Geiduschek and Tocchini-Valentini, 1988). They usually have a rigid secondary structure that ensures transcript stability. The genes for tRNA (Cotten and Birnstiel, 1989; Yuyama et al., 1992; Ohkawa et al., 1993) and human Ad2 virus-associated RNA (VA1 RNA) (Ventura et al., 1993) have already been used to express hammerhead Rz. In this work, we examined an Rz-producing system based on CELO VA RNA. Like VA1 RNA. the CELO VA

190

RNA is transcribed by RNA polymerase III; however, it differs dramatically from I/AZ RNA in both length and sequence. It can be abundantly expressed in a broad range of tissues from different hosts (Larsson et al., 1986). It has a high-free-energy stem-loop structure (Fig. 1B) which makes it very stable.

EXPERIMENTAL

A. PCVA

pCVArib3

or pCMrib2

AND DISCUSSION

(a) Cloning of Rz into CELO VA RNA

CELO I/ARNA is located at about 90 mu. of the viral genome (Larsson et al., 1986). A MaeI-CfvI genomic fragment including CELO I/ARNA was cloned into pGEM-3zf(-) to generate pCVA (Fig. 1A). Using the method of Haseloff and Gerlach (1988) we have constructed a Rz specifically targeted against the SEAP reporter gene (Zakharchuk et al., 1994). The Rz was inserted into pGEM-3zf(-) downstream from the SP6 promoter, and also into the unique BspMII site of pCVA downstream from the same promoter to generate pR3 and pCVAR3 (Fig. 1A). The unique BspMII site is located between the A and B promoter boxes of CELO WI RNA. In the present work we inserted the Rz between the internal promoter boxes of CELO T/ARNA. Ventura et al. (1993) cloned the Rz downstream from the promoter boxes of T/AZ.The unique BspMII site between the promoter boxes substantially facilitated the cloning into CELO VA RNA. The putative secondary structure of the CELO VA RNA with integrated Rz is shown in Fig. 1B. In the secondary structure of CELO VA RNA with integrated Rz, predicted by the computer program DNASIS, the antisense arms of Rz are in a loop and thus can bind to the target. The fragment of the target gene was also cloned into pGEM-3zf(-) downstream from the SP6 promoter to give pSB (Fig. 1A.).

cleavage

A-U Et

Fig. 1. Plasmid (A) Open

activity in vitro

To evaluate the influence of flanking sequences on the catalytic activity of the Rz, the Rz,CELO I/ARNA with integrated Rz and a fragment of the target gene were transcribed in vitro. Both catalytic RNA transcripts cleaved the target RNA in vitro in the expected manner (Fig. 2): two specific products of predicted size (170 and 70 nt) were observed. The products accumulated while the amount of substrate decreased with time. The cleavage activity of Rz was assayed at two constant temperatures and with the temperature between 80 and 56°C. Cyclic changes of the reaction temperature

and secondary sequences;

structure

sequences;

Promoter

sequence

cated.

boxes and terminator

All enzymes

Lithuania)

and

operations plasmids

together

plasmid

the pGEM-3zf(-) with BamHI

vector.

synthesized treatment,

pCVArib3,

cannot ment

plasmid

Germany).

et al. (1989). All

of the

and A&-linearized contained

was recloned

The

WI, USA) genome

was

site of the

anti-SEAP

chemistry;

were annealed

Rz after

were kinase

to generate

BspMII

into BspMII-linearized

pCVA,

pGEM-3zf-),

yielding

pR3.

the Rz whose antisense

arms

bind with the SEAP RNA sequence. of SEAP

Madison,

of the CELO

into the MCS BarnHI

ends and inserted pCVArib2

(Vilnius,

(Mannheim,

phosphoramidite strands

are indi-

Fermentas

by Sambrook

(Promega,

Pentacontanucleotides

complementary

sequence;

boxes, Rz sequence.

of CELO VA RNA

(MaeI-CfrI) linkers

by automated

and AuaI-compatible Similarly,

out as described

A 190-bp fragment

black

were from

Boehringer-Mannheim

contain

backbone. cloned

used for cloning

were carried

of CELO VA RNA.

dark gray boxes, SEAP

light gray boxes, pGEM-3zf(-)

yielding

(b) Influence of the flanking sequences on the Rz catalytic

constructs

boxes, CELO

The HindIII-BanaH

from pBcl2/RSV/SEAP

frag-

(Berger

et al.,

1988) into pGEM-3zf(-) at the same sites, to give plasmid pSB. All of the constructs were sequenced. (B) Left: secondary structure of CELO VA RNA (Larsson et al., 1986); the insertion site of Rz is indicated. Right:

possible

integrated

secondary

structure

for CELO

VA RNA

with

the

Rz.

enhanced the Rz efficiency in agreement with the data of Dropulic et al. (1993). The enzymatic in vitro activity of the Rz alone was similar to that of Rz incorporated into CELO VA RNA (Fig. 3). Consequently, the extra sequences around Rz

191

A. 4tt3-

65

4

3

2

1

242-

,Pl

llO-

-P2

67-

nt

7

6

5

4

3

2

56<--->80

56

37

B.

(“C)

Temperature

1 Fig. 3. Comparison

of the Rz enzymatic

temperatures.

boxes. activity

Gray

RNA; open boxes, activity constant

temperatures

equihbrdte

80-

of heating

by cooling two

of cleavage

for

VA

was conducted

at

to the maximum

I min. Allowing

temperatures,

each

the available

target cleaved during the reaction.

-P2

by counting

the radioactivity

took

4 min.

the percentage

of

This was accomplished

of gel slices corresponding

Each value is the mean of five independent

temperature

for the time to

round

was based on determining

-R

cleavage of SEAP RNA substrate hy the Rz. (A) substrate (S) by the Rr (R) integrated into CELO VA RNA

reaction

into CELO

(37’C or 56°C) or cycled between 56°C and 80-C

between

Quantitation

at various

of Rz alone. The cleavage

for 1 h. Each cycle consisted for 1 mm. followed

activities

of Rz integraled

to the bands.

experiments.

Confidence

bars (95%) are shown.

Fig. 2. In vitro Cleavage

of

produces products Pl and P2. The reaction was conducted at 56’C. Incubation times: 15 min (lane 3). 30 min (lane 4). 60 min (lane 5); the Rz in these lanes was unlabeled. substrate

without

Lanes: 1. [r-3’P]

R7: 6, molecular

strate (S) by the Rz (R) without PI and PI?. The reaction

size marker.

Rz; 2. [r-32P]-labeled (B) Cleavage

the viral sequences

was conducted

produces

at 56’C. Incubation

min (lane 2), 20 min (lane 3 I, 40 min (lane 4), 50 min (lane 6): molecular Rz without

reaction.

size marker

of subproducts times: 10

(lane 5), 60 min

(lane 7). Lane 1, labeled substrate

and

Methods: Plasmids pCVArib3 and pR3 were linear-

ized in the MCS with XhuI or EwRI;

pSB was linearired

with EcoRI.

The corresponding transcript lengths were 270, 90 and 240 nt, respectively. All linearized DNAs were transcribed in vitro with the Riboprobe II System cleavage

(Promega)

and [a-32P]UTP

was conducted

10 mM MgCI.

(PEI,

in the presence

The RNAs

were initially

Obninsk,

Russia).

of 10 mM TrisHCl heat-denatured

at 95°C for

1 min. and than quickly cooled on ice. The substrdte’ribozyme was 1:l. The reaction loading

buffer

was terminated

(95”/0

formamide/‘lO

by adding mM

The

pH 7.5’ ratio

an equal volume of gel

Na,EDTA.‘O.O?%

xylenc

cyanolei0.02”C bromophenol blue). Samples were then heat-denatured at X0 C for 2 min prior to running on 8 M urea-lo% polyacrylamidc

However,

Ventura

vitro catalytic

et al. (1993) have shown

activity

of Rz encoded

rated into the 3’ region of I/AZ downstream moter

boxes is inhibited

by flanking

that the in

by sequences

integ-

from the pro-

sequences

of VAI.

The secondary structure of CELO VA RNA may be more convenient for hammerhead Rz than that of VAI. The choice of insertion

site between

the promoter

boxes may

also have played an important role. The relatively low catalytic activity of the Rz at 37°C may be explained by the appreciable length (22 nt) of the flanking antisense arms which bind tightly to the substrate and hinder the release of the Rz from the substrate complex, thereby attenuating its turnover number. However, the Rz with longer arms may be more efficient within cells (Crisell et al.. 1993).

gels. The gels were autoradiographed.

were inert with regard to the enzymatic activity of the Rz in vitro. These data are in agreement with those of Cotten and Birnstiel (1989) and Yuyama et al. (1992).

(c) Transcription of CELO VA RNA with integrated Rz in 293 cell line We attempted to evaluate the activity of the CELO VA RN.4 promoter after insertion of the Rz.To this end, total RNA from a pCVArib3-transfected 293 cell line

192

(Graham et al., 1977) was subjected to primer extension analysis, with a labeled oligodeoxyribonucleotide from one of the Rz DNA strands. Fig. 4A demonstrates that lOO-nt extension product corresponding to the 5’region of the CELO VA RNA is transcribed in the cells. Total RNA from pCVArib3- or pCVA-transfected cells was examined by slot-blot hybridization (Fig. 4B). The transcription of VA RNA with the Rz insert was not less than its native counterpart. The results obtained suggest that the 50-nt insert between the promoter boxes A and B does not affect the transcription of CELO VA RNA. Expression of a Rz active against SEAP RNA should attenuate the amount of the protein product and thus decrease the enzymatic activity. This was tested

TABLE

I

Expression

B.

118lOO-

a.

82-

b. c. d.

48Fig. 4. CELL isolated

VA RNA transcription

from cells transfected

from cells transfected 3, molecular-size

SEAP activityb

(mu/ml) pCVArib2

0.52 i_ 0.05

pCVArib3

0.26 & 0.07

a The 293 cells (10’ in a 30-mm dish) were co-transfected pBcl2/RSV/SEAP plasmid

The primer extension

Lane 4, RNA isolated control). product

an arrow. (B) Total RNA (20 pg) of cells transfected pCVArib3

(d) was applied to the Bio-Dot

USA), transferred

to Hybond

1 and

is indicated

by

with pCVA (c) or

SF (Bio-Rad,

N (Amersham,

Lanes

Richmond,

Lincoln

CA,

Place, UK) filter,

and probed with a-3zP-labeled pCVA DNA. The specific activity of the probe was lo9 dpm/ug. (a) In vitro transcribed CELO VA RNA with integrated

Rz, positive

control;

b, RNA of untransfected

cells, negative

control. Methods: Human 293 cells (Graham et al., 1977) were grown in MEM medium with 5% fetal calf serum and transfected with plasmid DNA using the Caphosphate

precipitation

der Eb, 1973). For RNA isolation,

protocol

(Graham

and Van

10 ug of pCVA or pCVArib3

was

used to transfect a 60-mm dish containing lo6 cells. Total RNA of the transfected cells was isolated with the TRI REAGENT (MRC, Cincinnati, OH, USA). Primer-extension analysis of the isolated RNA was performed

plasmid

by the Ca.phosphate

which

sampled

served

precipitation

method

with 1 ug of or pCVArib3

(Graham

pCVArib2

contains

Rz with random

as a negative

control.

The culture

der Eb, 1973). Plasmid arms

and 3 ug of either pCVArib2

and Van sequence

medium

was

at 48 h after transfection.

b The level of SEAP activity

was determined

et al. (1988). It is given in milliunits One mU is defined

as the amount

of p-nitrophenylphosphate experiments.

(mu)

by Berger medium.

of SEAP that hydrolyses

1.0 pmol

per min. Each Standard

as described per ml of culture value

deviations

is the mean

of five

are shown.

by co-transfecting 293 cells with pCVArib3 and pBC12/RSV/SEAP (Berger et al., 1988) carrying the SEAP coding sequence under control of Rous sarcoma virus long terminal repeat. As a negative control, 293 cells were cotransfected with pBC12/RSV/SEAP and pCVArib2 where the same Rz has random sequence arms precluding correct interaction with the substrate. The DNA molar ratio of SEAP-producing and Rz-expressing plasmids was 1:lO. The SEAP activity in the culture medium of cells co-transfected with pCVArib3 decreased by half at 48-h post transfection (Table I). The mechanism of target inactivation includes antisense inhibition and enzymatic cleavage. We did not evaluate the contribution of catalytic and antisense effects separately because it was outside of our scope. These questions will be addressed in future studies.

in cell line 293. (A) Lane 2, RNA

with pCVArib3.

with pCVA (the negative

marker.

of Rz in 293 cells

Plasmid”

independent

A.

of SEAP in the presence

with the AMV Reverse Transcriptase

Primer

(d) Conclusions CELO VA RNA was explored as a new vector for expressing the hammerhead Rz. (I) The scheme of Cotten and Birnstiel (1989) can be used for inserting Rz into CELO VA RNA. The surrounding viral sequences are inert to the in vitro Rz activity. (2) The 50-nt insert between the promoter boxes does not affect the transcription of CELO VA RNA in cell culture. The Rz is efficiently transcribed as a part of the CELO VA RNA in human 293 cells and shows bio-activity. (3) CELO VA RNA is an alternative for transfer and effective expression of hammerhead Rz.

Extension

System (Promega). The primer complementary to the Rz sequence (50 nt) was labeled with [y-a’P]dATP (PEI) and T4 polynucleotide kinase; the specific activity was 5 x lo5 dpm/pmol. RNA (6 pg) was annealed with excess primer for 30 min at 70°C. The primer extension was con-

ACKNOWLEDGEMENTS

ducted at 42°C for 30 min. Labeled on a 8 M urea-lo% polyacrylamide

We thank Dr. B.R. Cullen (Howard Hughes Medical Institute, Durham, NC, USA), who kindly supplied plas-

extension gel.

products

were separated

193 mid pBcl2/RSVjSEAP, and Dr. S.V. Zelivyansky for helpful discussions. This work was supported the Russian Foundation for Basic Research grant 930420767 and ISF grant M HGOOO.

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