Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7219-7224, July 1996 Medical Sciences

Ul small nuclear RNA chimeric with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus (catalytic RNA/hammerhead/splicing/Xenopus laevis) ALESSANDRO MICHIENZI, SILVIA PRISLEI, AND IRENE BOZZONI* Istituto Pasteur Fondazione Cenci-Bolognetti and Centro Acidi Nucleici, Dipartimento di Genetica e Biologia Molecolare, Universita "La Sapienza," 00185 Rome, Italy Communicated by Robert P. Perry, Fox Chase Cancer Center, Philadelphia, PA, April 4, 1996 (received for review December 18, 1995)

ABSTRACT The in vivo effectiveness of ribozymes some resides in its base pairing with the 5' splice junction of strongly depends on the correct choice of the vector molecule. the substrate pre-mRNA. In Rev pre-mRNA this sequence is High levels of expression, stability, active conformation, and suboptimal (12) and, together with the 3' splice site (13), correct cellular localization are the most important features reduces splicing efficiency. To increase the specificity of the for a vector. We have exploited the utilization of the U1-ribozyme for the Rev pre-mRNA, mutations were intro- Ul small nuclear RNA (snRNA) as a vector for specifically duced in the 5' splice site pairing region of Ul snRNA such as targeting a ribozyme into the nucleus. The Rev pre-mRNA of to match it with the suboptimal 5' junction of the Rev human immunodeficiency virus type 1 was chosen as target for precursor. Altogether this strategy should confer three fea- testing the activity of the Ul-ribozyme. The catalytic core of tures to the ribozyme: (i) stability, (ii) nuclear localization, and the hammerhead motif, plus the recognition sequences, sub- (iii) increased substrate specificity for the Rev precursor. Ul stituted the stem-loop III of the Ul snRNA. The resulting RNA should carry the ribozyme on the Rev pre-mRNA, construct displays efficient cleavage activity in vitro. In addi- increasing the relative concentration of ribozyme and sub- tion, in the in vivo system of Xenopus laevis oocytes, the strate, thus allowing efficient cleavage. In addition, the use of Ul-chimeric ribozyme accumulates in large amounts in the a Rev-specific Ul snRNA should increase the specificity of the nucleus and produces a considerable reduction of Rev pre- Ul-ribozyme for the Rev precursor in comparison with all the mRNA levels. The Rev-specific ribozyme was also inserted in other endogenous unmodified Ul RNA. a derivative of the Ul snRNA mutated in the region of pairing We have tested the activity of these ribozymes in vitro and with the 5' splice site, such as to match it with the suboptimal in Xenopus laevis oocytes, which represent an ideal in vivo splice junction of the Rev precursor. This construct shows system for the study of RNA compartmentalization and ac- more efficient reduction of Rev pre-mRNA in vivo than the cumulation. We have found that the chimeric ribozymes are wild-type Ul vector. stable and correctly compartmentalize inside the nucleus where they produce a considerable reduction of Rev pre- The ability to target ribozymes to specific in vitro (1-3) mRNA levels. The use of disabled ribozymes has allowed us to has lead to the idea of exploiting their potential therapeutic show that degradation is mainly due to catalytic cleavage value as antiviral agents in vivo. Indeed, several examples have rather than antisense effect. been reported in which ribozymes have been used as anti- human immunodeficiency virus (HIV) molecules (4-6), and in MATERIALS AND METHODS some cases protection against virus replication has been described in different human T-cell populations (7-10). One Construction of Ribozymes. The sequence of the hammer- major problem related to the effectiveness of ribozymes is that head active and inactive core where derived from the studies while in test tubes, these molecules and their substrates can of Uhlenbeck and Haseloff (2, 16, 17). The cleavage site of the diffuse freely, whereas in cells RNAs are sorted to specific Rev sequence corresponds to the C at position 5597 ofthe HIV cellular locations. Viral RNAs are also specifically compart- genome (18). mentalized in vivo, and this feature may reduce their avail- The Ul chimeric ribozymes (U1-Rz) were obtained by ability for ribozyme interaction. It has been demonstrated that inverse PCR on the XlU1.340 clone containing the entire Ul the delivery of a ribozyme to the same cellular location as its RNA gene (19) or on its T7 derivative which allows in vitro target substrate strongly increases the effectiveness of the under the T7 . Both clones were kindly For this reason efforts are now devoted provided by I. Mattaj (European Molecular Labora- ribozyme (11). many tory, Heidelberg). The utilized are as follows: to setting up new strategies for directing ribozymes to specific A, 5'-GGACTCATCA(G/C)AGACTCATCACGCAGGGG- cellular compartments. TCAGCACAACCGGA-3'; and B, 5'-GTGAGGACGAAA- In this paper we have developed a strategy for the efficient CTGCCTTGACGACTGCATAATTTCTGGTAGT-3'. The expression and compartmentalization of an hammerhead ri- degeneration of the A allows the simultaneous bozyme inside the nucleus and for its specific targeting to a construction of both the active (U1-Rz) and the inactive pre-mRNA. The target chosen for testing ribozyme activity is (Ul-Rzm) ribozymes. The following oligonucleotides were the Rev pre-mRNA of HIV, which undergoes a very peculiar used for constructing the human chimeric ribozymes: A', posttranscriptional regulation (12-15). Ul small nuclear RNA 5'-GGACTCATCA(G/C)AGACTCATCGCAGGGGT- (snRNA) was chosen as vector because it has nuclear local- and 5'-GTGAGGACGAAA- ization and it is known to participate in the splicing reaction. CAGCACATCCGGA-3'; B', The specificity of Ul snRNA interaction inside the spliceo- Abbreviations: snRNA, small nuclear RNA; U1-Rz, active Ul chi- meric ribozymes; U1-Rzm, inactive Ul chimeric ribozymes; AdML, The publication costs of this article were defrayed in part by page charge major late adenovirus. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 7219 Downloaded by guest on September 29, 2021 7220 Medical Sciences: Michienzi et al. Proc. Natl. Acad. Sci. USA 93 (1996) CTGCCTTCGACTGCATAATTTGTGGTAGT-3'. Ul- RESULTS Rz-5' were obtained by inverse PCR on the U1-Rz and Ul-Rzm constructs by using oligonucleotides C (5'- Construction of Ul snRNA Ribozymes. A 42-nt-long frag- TGGGCAGGGGAGATACCATG-3') and D (5'-GTAAGT- ment, containing the catalytic core of the hammerhead ri- ATGAGTTGAACAAG-3'). The ribozyme substitutes nucle- bozyme (17) plus flanking sequences complementary to the otides 95-116 of the Ul snRNA sequence. The tRNA ri- target site, was replaced to a 22-nt-long region ofthe stem-loop bozyme was obtained by inverse PCR on a tRNApro gene (20) III of X laevis Ul snRNA (position 95-116; see ref. 26). The cloned into the EcoRI site of the Bluescript vector. The choice of this region was dictated by the following reasons: (i) ribozyme substitutes 30-33 of the tRNA. The it does not participate in the binding of Sm proteins and of oligonucleotides used are (i) 5'-GGACTCATCAGAGACT- Ul-specific proteins that contribute to the correct compart- CATGAGAATCATACCACTAGACCAT-3', and (ii) 5'- mentalization of the mature Ul snRNP particle (26, 27), and GTGAGGACGAAACTGCCTTTGGGTGCGAGA- (ii) it is not essential for the stability of the Ul snRNA (28). GGTCCCGGGTT-3'. The target sequence is represented by 20 nts of the first coding Construction of Rev Substrates. A 2510-nt-long fragment, exon of the Rev pre-mRNA and the cleavage site corresponds covering the first coding exon (76 nt), the entire (2330 to the C at position 5597 of the BH10 sequence (Fig. 1 and ref. nt), and 104 nt of the second coding exon of Rev was generated 18). From this construct (U1-Rz) a second one was derived by PCR amplification of HIV clone BH10 (18). The oligonu- (U1-Rz-5') in which the 5' terminus of Ul snRNA (5'- cleotides used are as follows: Rev-A, 5'-CCGAATTCATG- GAUACUUACCUGGC-3') was changed into 5'-GAUACU- GCAGGAAGAAGCGGA-3'; and Rev-B, 5'-AAGGATC- UACUGGGC-3'. These substitutions (boldface type), in the CGTTCACTAATCGAATGGA-3'. The primers included the region of complementarity to the 5' splice junction (under- EcoRI and BamHI sites that allow the cloning in the corre- lined), produce a Ul snRNA with perfect complementarity to sponding sites of the pGEM-4Z (Promega) vector to generate the suboptimal 5' splice site of the Rev pre-mRNA (see Fig. 1). clone pGEM-Rev. The BamHI digestion of this clone allows Similar constructs were also made in the human Ul snRNA the in vitro transcription of the entire cloned sequences, while gene and in containing the T7 promoter. The same the AvaII digestion produces a template for the transcription sequence containing the hammerhead catalytic core plus of a 166-nt-long RNA (Rev-AvaII) that was used for the in flanking sequences was also cloned in a tRNA vector (20), vitro analysis of ribozyme activity. upstream of the anticodon loop (tRNA-Rz). The same 2510-nt-long Rev region was inserted in the To control for antisense effect of ribozymes, derivatives of BamHI site of the - 166pTCAT vector (21), between the X U1-Rz and U1-Rz-5', which have the conserved C, present in laevis L14 r-protein gene promoter and the simian virus 40 the catalytic core of the hammerhead (CUGAUGA) substi- polyadenylylation site; the insert was obtained by PCR using tuted with a G known to completely abolish cleavage activity the Rev-B and Rev-C (5'-CCGGATCCATGGCAGGAA- (17), were also produced. These constructs, named Ul-Rzm GAAGCGGA-3') oligonucleotides, both including a BamHI and Ul-Rzm-5', should display the same antisense effect as site. This construct (L14-Rev) allows efficient expression of their active counterpart but lack catalytic activity. the fused sequences in a (poly)A+ RNA in X laevis oocytes In Vitro Activity of the Ul Chimeric Ribozymes. T7 tran- (21). scripts of U1-Rz and Ul-Rzm (185 nt) were coincubated with The template used for T7 transcription of the U6 snRNA 32P-labeled Rev-AvaII target. Fig. 2 shows a time course of was obtained by PCR amplification of the U6 X laevis gene incubation of 150 nM of ribozyme and 50 nM of substrate at kindly provided by E. Lund (University of Wisconsin, Madi- 37°C in 10mM MgCl2 and 50mM Tris HCl (pH 7.5). It appears son). that the U1-Rz converts the substrate (band S, 166 nt long) In Vitro Ribozyme Activity. Ribozymes and 32P-labeled very efficiently into the expected cleavage products (bands 5' substrate RNAs were in vitro transcribed from the correspond- and 3'), whereas in the same conditions no cleavage is pro- ing T7 constructs. Ribozyme and substrate (150 nM and 50 duced by the mutant ribozyme (lanes U1-Rzm). Interestingly, nM, respectively, or 400 nM and 100 nM) in 50 mM Tris HCl the tRNA-Rz works less efficiently than the U1-Rz, as dem- (pH 7.5) were heated at 95°C for 2 min and cooled 5 min in ice. onstrated by the reduced conversion of the substrate into MgCl2 (10 mM) was added, and the reactions were allowed to truncated products (lane tRNA-Rz). These data indicate that proceed at 37°C or when specified at different temperature, for inside the Ul vector the ribozyme sequences are available for the indicated times. The RNA was analyzed on 6% polyacryl- interaction with the substrate and that Ut sequences do not amide-7 M urea gels. interfere with catalytic activity. Oocyte Microinjections. , after purification Fig. 2b shows the temperature-dependent activity of the on CsCl gradients, were dissolved in 80 mM NaCl and 10 mM U1-Rz. In this case, 400 nM of ribozyme and 100 nM of Tris (pH 7.0) at a concentration of 400 ng/,ul and 10 nl were substrate was used. It appears that at 250C the activity of the injected into the germinal vesicles of stage VI X laevis oocytes ribozyme is strongly reduced with respect to 37°C. No differ- according to Caffarelli et al. (22). 32P-labeled U6 snRNA ence was found between 25°C and 20°C (not shown). U1- (300,000 cpm/pl) was coinjected as internal control. After ribozyme has shown substrate-specific cleavage also when incubation, RNA was extracted from total oocytes or from incubated in HeLa nuclear extract, indicating that also in the manually dissected nuclei (23). Northern blot analysis was presence of proteins U1-Rz is able to interact with the target performed with probes specific for Rev RNA and Ul snRNA sequence (not shown). The enzyme and substrate concentra- (18, 19). tions used in these two experiments are in the range of those AdML pre-mRNA is 253 nt long and covers part of the used in other similar cases (29). major late transcript of adenovirus; it is obtained by in vitro Expression and Localization of the Ul-Rz Transcripts. The transcription of plasmid pAdML-Ail digested with ScaI (24). genes encoding for the Ul chimeric ribozymes were purified 32P-labeled AdML and U6 RNAs (respectively, 1.5 x 106 and on ethidium bromide-CsCl gradients and injected into oocyte 0.4 x 106 cpm/,ul) were coinjected in oocytes that were nuclei (4 ng/oocyte). After overnight incubation, the nuclei injected the night before with U1-Rz or Ul plasmid DNAs (4 were manually separated from the cytoplasms and RNA ng/oocyte). Incubation was allowed to proceed for an addi- extracted from the two different compartments. Fig. 3 shows tional hour; RNA was extracted and analyzed on a 6% a Northern blot analysis performed with control oocytes (lanes polyacrylamide-urea gel. control) and with oocytes injected with the U1-Rz (lanes Immunoprecipitations with Sm antibodies (25 ,ul) were U1-Rz) and U1-Rz-5' (lanes U1-Rz-5')- genes. The picture performed as described by Hamm et al. (25). shows that both Ul chimeric ribozymes are efficiently ex- Downloaded by guest on September 29, 2021 Medical Sciences: Michienzi et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7221

U1 -RNA Canonical 5' splice site ml3?pp - -AG GUAAGU- --CGGUC CAUUCA UApppG3m-5' Ul -RNA Rev 5' splice site --CA GUAAGU- --CGG GU CAUUCA UApppG3m-S' Utl -5 '-RNA

Rev pre-mRNA U1-Rz RNA FGGPpp 5'-UCAAGGCAGUCAGACUCAUCA-----CA GUAAGU- AGUUCCGUCA UCUGAGUAGU GU CAUUCA-5' 3 AFC "' A U G G CGA Ul -Rz-5'

A-UG-C G-C A G G U

FIG. 1. Construction and sequences of the Ul chimeric ribozymes. (Left) Schematic representation of the structure of wild-type and U1-Rz snRNAs. The hammerhead sequence (bold) substitutes part of stem-loop III (nt 95-116; see ref. 26). (Right) Sequences of the canonical and Rev 5' splice sites are shown together with the Ul snRNA sequences. Exons are represented as open boxes; underlined nucleotides indicate the substitutions introduced in the Ul snRNA. The pairing of Rev pre-mRNA and U1-Rz-5' is shown below; the arrows indicate the cleavage site and the C to G substitution of the disabled ribozymes.

pressed and stably accumulate inside the nucleus. High levels tional activity of the Ut promoter, should ensure an excess of of expression and correct nuclear compartmentalization are Ul chimeric ribozymes over the Rev transcripts. 32P-labeled observed for the human (lanes Ulh-Rz) and mutant constructs U6 snRNA, known to be specifically retained inside the (not shown). nucleus (31), was also coinjected. This RNA represents an Ul snRNA is known to be associated with a specific set of internal control for successful nuclear injections of plasmid proteins; in particular, the proteins constituting the core DNAs and for normalization of different injections. particle are in common with other snRNAs and are recognized Single injected oocytes (or pools of them) of three indepen- by Sm antibodies (30). Immunoprecipitation with these anti- dent experiments were analyzed by Northern blot for the bodies was used to test whether the Ul chimeric ribozymes are accumulation of Rev, Ul-ribozymes, and U6 RNAs. Densito- able to form Ul snRNP-like complexes. Fig. 3 shows that both metric analysis was performed on a total of 30 oocytes and the ribozymes react to some extent with Sm antibodies: U1-Rz and results are diagrammed in Fig. 4b. An example of the Northern U1-Rz-5' are immunoprecipitated one-half and one-fifth as analysis is shown in Fig. 4a). efficiently as wild-type Ul RNA. These results indicate that In comparison with control injections (lanes -), the Rev Ul-chimeric ribozymes are able to assemble into Sm- pre-mRNA signal is 5 timeS lower in oocytes where plasmid containing particles, even though less efficiently than wild-type L14-Rev was coinjected with U1-Rz (lanes U1-Rz) and 10 Ul snRNA. times lower in injections of U1-Rz-5' (lanes U1-Rz-5'). The In Vivo Ribozyme-Catalyzed Cleavage of Rev pre-mRNA. inactive versions of Ul-ribozymes produce a 2-fold reduction The in vivo activity of the chimeric ribozymes was tested by of the Rev pre-mRNA (lanes U1-Rzm and Ul-Rz-5'm); this coinjection of plasmid DNAs coding for the Rev pre-mRNA effect has not been analyzed in more detail, but it is very likely (clone L14-Rev) and for the different Ul-ribozymes into the due to antisense activity (5). The comparison of the effect of nuclei of X laevis oocytes. After overnight incubation, the the disabled ribozymes with that of the wild types indicates that levels of accumulation of the different transcripts were ana- catalytic activity is higher than antisense effect and strongly lyzed by Northern blotting. The transcription of U1-ribozymes contributes to the degradation of Rev pre-mRNA. It is im- is driven by the homologous Ul snRNA gene promoter, while portant to remember that oocytes grow at 20°C, which is not Rev transcription is driven by the promoter of the X laevis L14 the optimal temperature for hammerhead activity. In addition, ribosomal protein gene of plasmid - 166pTCAT (see Materials low temperatures favor antisense effect, especially when the and Methods). This vector, inX laevis oocytes, ensures efficient pairing region is long such as in our constructs (20 nt). expression of the fused sequences in (poly)A+ RNA (21). The fact that U1-Rz-5' shows the strongest effect indicates Following this protocol, U1-ribozyme RNAs (185 nt) and Rev that the mutation introduced in the Ul vector increases the pre-mRNA [2510 nt plus - 200 nt of (poly)A tail] are expressed recognition of the substrate, thus enhancing catalytic activity. simultaneously. The molar excess of Ul plasmid with respect Differently from the in vitro situation, the 5' and 3' cut-off to the Rev plasmid (5:1) and the expected higher transcrip- molecules originating from ribozyme cleavage are not ex- Downloaded by guest on September 29, 2021 7222 Medical Sciences: Michienzi et al. Proc. Natl. Acad. Sci. USA 93 (1996) a) b) Ul -Rz U1 RzmtRNA-Rz 370C 300C 250C M 115 60o 18 115 60 181115 60o 10

S S (166)

3' 3' (111)

. . i~U

5' 5' (55)

m30ppp 3' 3-

S 5' 3

FIG. 2. In vitro analysis of ribozyme activity. (a) Unlabeled ribozymes (0.15 ,M; U1-Rz, UlRzm, and tRNA-Rz) were incubated for the indicated time (in minutes) with 0.05 ,uM of 32P-labeled Rev substrate (166 nt long). The products of the reaction (5', 55 nt long; and 3', 111 nt long, schematically represented below) were analyzed on a 6% acrylamide-urea gel. Lane M, molecular weight markers (pBR322 plasmid DNA, MspI digested). The structures of the Ul and tRNA ribozymes are represented below. (b) Unlabeled U1-Rz (0.4 ,uM) and of 32P-labeled Rev (0.1 gM) substrate were coincubated at three different temperatures (25°C, 30°C, and 37°C) for the indicated time (in minutes).

pected to accumulate in vivo because their unprotected 5' and injections of the tRNA-ribozyme have shown that this con- 3' termini are rapidly degraded by endogenous 5'-3' and 3'-5' struct is less active than the Ul constructs even if the expres- exonucleases. Rapid turnover of cleaved RNA molecules has sion driven by the pol III promoter is higher than that of the been indeed observed inX laevis oocytes (23, 32). On the other pol II promoter of the Ul gene (not shown). hand, mature Rev RNA is not visualized since Rev pre-mRNA Effect of Ul-Ribozymes on Splicing. To analyze whether the is a very inefficient splicing substrate (13) and, in the oocyte, excess of a modified form of Ul snRNA could affect the produces very little amounts of mRNA even at long incubation efficiency of the cellular splicing machinery, we tested the times (not shown), in analogy with other cases of inefficient accumulation of splicing products derived from a 32P-labeled splicing substrates (22, 23). AdML pre-mRNA (clone pAdML-Ail; see refs. 21 and 23) Fig. 4a (Middle) shows that the injected Ul chimeric genes injected in oocytes previously incubated with wild-type Ut are efficiently expressed in all the different oocytes. snRNA (lanes Ut) or U1-Rz (lanes Ul-Rz) genes. Also in this The U6 signals in the lower panel derive from the injected case 32P-labeled U6 snRNA was coinjected as an internal 32P-labeled U6 snRNA and witness that the same amount of control. Fig. 5 shows that only a minimal effect on the plasmid DNA was correctly injected inside the nucleus. Oocyte efficiency of splicing is observed when Ul chimeric ribozymes are expressed in the nucleus ofX laevis oocytes in comparison Control Ul-Rz U1Az5' UlhR- with overexpression of wild-type Ut snRNA. This is shown by the slightly lower conversion of pre-mRNA into spliced prod- ucts in lanes Ul-Rz. In the lower panel of Fig. 5, the Northern Ul-Rz analysis of the same oocytes is shown; it indicates that a very high level of expression of the exogenous genes is obtained. In conclusion, the overexpression of U1-Rz only does not visibly affect the overall efficiency of the splicing machinery.

DISCUSSION C N Sm C NSm C N Sm C N In this paper we have used the oocyte system in order to FIG. 3. Cellular compartmentalization of the Ul chimeric ri- analyze whether the Ut snRNA gene can be utilized as a vector bozymes. Plasmid DNAs coding for U1-Rz, U1-Rz-5', and Ulh-Rz system for expressing high levels of chimeric ribozymes and for were injected into the nuclei of X laevis oocytes and incubated their delivery to the nuclear compartment. Ul snRNA genes overnight. Two nuclei (lanes N) and the corresponding cytoplasms would also provide appropriate cassettes for the expression of C) were manually dissected and the RNA analyzed by Northern (lanes ribozyme sequences in a stable and tissue nonspecific manner. blot with a Ul snRNA specific probe. Two additional nuclei were utilized for immunoprecipitation with Sm antibodies before RNA Constructs were specifically designed with the idea of targeting extraction (lanes Sm). The endogenous Ul snRNA and the U1- the ribozyme to a pre-mRNA before it is spliced and the ribozymes (20 nt longer) are indicated. mRNA transported to the cytoplasm. This strategy is well Downloaded by guest on September 29, 2021 Medical Sciences: Michienzi et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7223

a U1 -Rz U1-Rzm U1 -Rz-5' Ul-RZm-5' Ul U1-Rz M

l_'.:s 'm: ~~~~~~~~~~.~~~~~~~~~~~~~~~~~~&':~~~~~~~~~~~~~~~~~~~~~~~~~~ -RevR

IEJ-I: -Ul-Rz

-Ul

'$$>x+''$|.

b c *~~~~~~~~~~~~~~~~~~~~~~~~~~~~...*...... EEii:..'. 0 90 C) 0 80 U6 0 70 e: : 60 z cc 50 a) 40 cc 0 30 20 1 0

- U1-Rz U1-Rzm Ul-Rz-5' Ul-RZm-5'

FIG. 4. In vivo cleavage activity. (a) A solution (10 nl) containing FIG. 5. Effect of Ul-ribozymes on splicing. Oocytes were injected 1 ng of L14-Rev plasmid was injected alone (lanes -) or in combi- with wild-type Ul (lanes Ul) or U1-Rz (lanes U1-Rz) plasmids; after nation with 3 ng of each one of the different Ul constructs (U1-Rz, overnight incubation, 10 nl of a solution containing 1.5 x 106 cpm/,ul UlbRzm, U1-Rz-5', and Ul-Rz-5'm) into the nuclei ofX laevis oocytes of 32P-labeled AdML pre-mRNA and 0.4 x 106 cpm/,ul of 32P-labeled and incubated overnight. As an internal control, in vitro transcribed U6 snRNA were injected into the germinal vesicles and incubation 32P-labeled U6 snRNA was also coinjected. RNA from single oocytes allowed to proceed for an additional hour. Nuclei from single oocytes was extracted and analyzed by Northern blotting on: 1% agarose- were manually isolated and RNA extracted. Samples were run on a 6% formaldeyde gel [hybridization with Rev probe (Top)] and 6% poly- polyacrylamide-urea gel and blotted. Direct autoradiography of the acrylamide-urea gel [hybridization with the Ul probe (Middle)]. The while allows the visualiza- filter allows the visualization of 32P-labeled RNAs, hybridization direct autoradiography of the polyacrylamide gel with Ul allows to analyze the expression of the injected tion of the U6 RNA signal (Bottom). (b) Autoradiograms of Rev probe plasmids. The different splicing products are indicated on the side. were by hybridizations from three independent experiments analyzed Lane M, molecular weight markers (pBR322 plasmid DNA, MspI densitometric scanning, and the values were expressed as percentage of the Rev pre-mRNA values obtained in controls. digested). substituting an essential of the catalytic core with an suited for those pre-mRNAs that are not splicing efficient inactive one. Because the inactive ribozymes showed only a substrates such as the Rev pre-mRNA, which is poorly spliced (12, 13). The colocalization of the Rev pre-mRNA and its partial effect relative to the wild types, we could conclude that specific ribozyme should increase the relative concentration of the additional activity of the active ribozymes was indeed due to This activity is expected to be much enzyme and substrate and the effectiveness of the ribozyme catalytic cleavage. activity. To increase the specificity of the Ul chimeric ri- higher in cells growing at 37°C than in oocytes since this bozyme for the Rev precursor, mutations were introduced in temperature is optimal for hammerhead cleavage. the region of Ul complementarity with the 5' splice site such We have shown that the Ul chimeric ribozymes only slightly is very as to have a perfect match with the Rev 5' splice site. reduce the splicing efficiency of the oocytes. This likely Experiments of oocyte microinjection have shown that the due to the large nonphysiological excess of the U1-Rz RNA, Ul chimeric ribozymes are efficiently expressed and stably obtained in the oocyte expression system, that competes with accumulated; in addition, their pathway of expression reflects the endogenous Ul snRNA. This excess is unlikely to occur in that of the wild-type Ul snRNA in that it accumulates in the experiments of gene transfer in human cells. nucleus and it is assembled with Sm antigens. In conclusion, these results show that Ul snRNA is an The Ul chimeric ribozymes display a specific effect on the effective vector for the efficient expression and delivery of accumulation of Rev pre-mRNA: U1-Rz reduces 5 times the ribozymes in the nuclear compartment, pointing out to its level of Rev pre-mRNA, while U1-Rz-5' displays an additional general use in cases where nuclear targets should be hit. The 2-fold greater effectiveness relative to the control. This indi- choice of substituting stem-loop III with ribozyme sequences cates that the increased specificity of the ribozyme vector for has resulted in efficient in vitro and in vivo cleavage demon- the Rev substrate favors catalytic activity. strating that the ribozyme sequences do not markedly affect Ribozymes have all the properties of antisense RNA with the stability and compartmentalization of the chimeric con- the additional feature of catalytic cleavage. To separate anti- struct and that they are exposed and accessible for substrate sense from cleavage effect, we created inactive ribozymes by interaction. The efficient activity of the Ul chimeric ri- Downloaded by guest on September 29, 2021 7224 Medical Sciences: Michienzi et al. Proc. Natl. Acad. Sci. USA 93 (1996) bozymes, raised against the Rev pre-mRNA, justify their 15. Fischer, U., Meyer, S., Teufel, M., Heckel, C., Luhrmann, R. & utilization and test in HIV-infectable T lymphocytes; in addi- Rautmann, G. (1994) EMBO J. 13, 4105-4112. tion, the ribozyme activity in these systems is expected to be 16. Uhlenbeck, 0. C. (1987) Nature (London) 328, 596-600. strongly enhanced by the growth temperature of 37°C, which 17. Ruffner, D. E., Stormo, G. D. & Uhlenbeck, 0. C. (1990) Bio- is optimal for hammerhead cleavage. chemistry 29, 10695-10702. 18. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalshi, J. A., Whitehorn, E. A., We are grateful to Massimo Arceci for skillful technical help and to Fabio Riccobono and M-medical Baumeister, K., Ivanoff, I., Petteway, S. R., Pearson, M. L., for oligonucleotide facilities. S.P. is Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., the recipient of an AIDS fellowship and A.M. is the recipient of a Cenci-Bolognetti fellowship. This work has been supported by grants Gallo, R. C. & Wong-Staal, F. 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