©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Catalytic Cleavage of an RNA Target by 25A Antisense and RNase L (*)

Ratan K. Maitra (1), Guiying Li (2), Wei Xiao (2), Beihua Dong (1), Paul F. Torrence (2), Robert H. Silverman (1)(§)

From the (1)Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the (2)Section on Biomedical Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5`-monophosphorylated, 2`,5`-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3`,5`-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (k of approximately 7 s). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3` terminus of the chimera binding site (5` on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.


INTRODUCTION

Selective inhibition of gene expression is possible by the exogenous administration of antisense oligonucleotides(1) . Two of the often cited potential mechanisms of action of such antisense agents are 1) passive or steric blocking of translation and 2) the RNase H-catalyzed degradation of RNA:DNA hybrids formed from the target mRNA and the DNA (or DNA analogue) antisense oligonucleotide. A novel approach to antisense regulation of gene expression, involving 2-5A antisense (2-5A-AS),()was recently reported by us(2, 3, 4) . The 2-5A-AS strategy relies on the recruitment to an RNA target and activation of the protein RNase L (formerly 2-5A-dependent RNase) (5, 6, 7, 8) followed by the selective cleavage of the targeted RNA(2, 3, 4) .

In nature, 2-5A ((pp)p5`(A2`p5`)A) (9) is generated from ATP by any of several isozymes of 2-5A synthetase, which are induced by treatment of cells with interferons and which are activated specifically by double-stranded RNA(10) . In the absence of 2-5A, the ubiquitous RNase L is catalytically inactive(11) ; however, nanomolar concentrations of 2-5A activate RNase L, resulting in the cleavage of single-stranded RNA with only moderate specificity at UpNp sequences (12, 13). The 2-5A system mediates the anti-mengo virus, anti-encephalomyocarditis virus, and possibly the anti-proliferative effects of interferon(8, 14, 15) . Although cellular concentrations of RNase L can be increased in some instances by interferon treatment (16), cell growth arrest(17) , and cellular differentiation(18) , basal concentrations of the enzyme are present in a wide variety of mammalian cell types(19) .

The direct introduction of 2-5A into cells by the use of such techniques as calcium phosphate coprecipitation or hypertonic salt treatment causes a nonspecific, global degradation of mRNA and rRNA (20, 21). In contrast, covalent conjugation of 2-5A to antisense oligodeoxyribonucleotides producing 2-5A-AS imparts a specificity to RNase L, which is lacking in nature(2, 3, 4) . In essence, RNase L becomes adapted for the selective and specific cleavage of a targeted RNA.

The first synthesis (2, 3) of 2-5A-AS chimeras described composite nucleic acids in which a 5`-monophosphorylated 2-5A tetramer was coupled through two phosphodiester bond-linked butanediol residues to the 5`-phosphate of a 3`,5`-oligodeoxyribonucleotide antisense sequence. In a cell-free extract of human Daudi cells, p5`(A2`p)A linked in the preceding manner to (dT)-induced RNase L to cleave among a stretch of adenine deoxyribonucleotides in a modified HIV-1 vif mRNA(2) . More recently, we reported using purified, recombinant RNase L and 2-5A-AS to obtain the selective cleavage of the protein kinase PKR mRNA in the presence of a non-targeted RNA(4) . Moreover, PKR mRNA was ablated from intact HeLa cells after culturing in media containing 2 µM 2-5A-AS(4) . Control experiments with chimeric 2-5A-AS molecules lacking only the 5`-monophosphate moiety, necessary for activation of RNase L, provided clear evidence that the RNase L was responsible for the ablation of PKR mRNA(4) . The utility of 2-5A-AS chimeras was demonstrated by showing that ablation of PKR mRNA and activity effectively blocked induction by double-stranded RNA signaling of transcription factor NF-B(4) . The further development and application of 2-5A-AS technology has been restricted by a lack of specific information concerning the RNA cleavage reaction itself. Here, we present such data that include kinetic parameters, effects of the DNA moiety on non-targeted cleavage reactions, and the precise sites of cleavage in a naturally occurring mRNA sequence.


MATERIALS AND METHODS

Chemical Syntheses of 2-5A and 2-5A Chimeric Oligonucleotides

Chimeric antisense and control oligonucleotides were synthesized using modifications of previously published procedures(2, 3, 4) . All of the chimeric oligonucleotides have the general formula, p5`(A2`p)5`A2`p[O(CH)Op]p(5`N3`p)p5`N. Specifically, the chimeras used in this study were as follows: p5`(A2`p)5`A-anti-PKR, p5`(A2`p)5`A2`p[O-(CH)Op]p5`-GTACTACTCCCTGCTTCTG-3`; (A2`p)5`A-anti-PKR, p5`(A2`p)5`A2`p[O(CH)Op]p5`-GTACTACTCCCTGCTTCTG-3`; p5`(A2`p)5`A-sense PKR, 5`(A2`p)5`A2`p[O(CH)Op]p5`-CAGAAGCAGGGAGTAGTAC-3`; p5`(A2`p)5`A-anti-HIV, 5`(A2`p)5`A2`p[O(CH)Op]p5`-ACACCCAATTCTGAAATGAA-3`.

Ribonuclease Assays

Recombinant, human RNase L was produced in insect cells from a baculovirus vector and was then purified to homogeneity by chromatography through three successive fast protein liquid chromatography columns (Pharmacia Biotech Inc.) as described(11) . The PKR mRNA was synthesized, 5`-labeled, and purified exactly as previously described(4) . Briefly, human PKR cDNA (Ref. 22, a gift of Dr. Bryan Williams, Cleveland) was transcribed in vitro with T7 RNA polymerase. PKR mRNA was labeled at its 5` terminus with [-P]ATP to specific activities of 10,000-20,000 cpm/µg of RNA and was then purified by electrophoresis in 6% polyacrylamide, 8 M urea gels followed by elution as described(4) . Ribonuclease assays were performed as described with modifications(4) . Oligonucleotides were mixed with PKR mRNA in buffer containing 25 mM Tris-HCl pH 7.4, 10 mM magnesium acetate, 8 mM -mercaptoethanol, and 100 mM KCl on ice. After 10-15 min, 5-30 ng of RNase L was added to a final volume of 20 µl. Incubations were at 37 °C except where indicated. Reactions were terminated with the addition of gel sample buffer (U. S. Biochemical Corp.). Degradation of P-labeled PKR mRNA was monitored according to published procedures (4) by electrophoresis in 6% polyacrylamide, 8 M urea gels, autoradiography, and analysis in a PhosphorImager (Molecular Dynamics).

Primer Extension Assays

The precise sites of PKR mRNA cleavage were determined by primer extension assay by a modification of the method of Driscoll et al.(23) as described(2) . The primer extension reactions were performed after incubations containing unlabeled PKR mRNA and different oligonucleotides in the presence of 30 ng of RNase L in a final volume of 20 µl at 30 °C for 30 min. The cleavage sites within PKR mRNA were determined by comparing the migration in 6% polyacrylamide, 8 M urea gels of the primer extension products to DNA sequencing products (performed with the same primer, 5`-GATCTACCTTCACCTTCTGG-3`) on a template of PKR cDNA. DNA sequencing of PKR cDNA was performed using sequenase version 2.0 (U. S. Biochemical Corp.), and deoxyadenosine 5`-[-S]thiotriphosphate and primer extension was with murine leukemia virus reverse transcriptase (Boehringer Mannheim).


RESULTS

A Comparison of Targeted and Non-targeted Cleavage of RNA by RNase L

To determine the effect of the DNA moieties of 2-5A chimeras on the selectivity of RNA decay, assays were done with purified, recombinant human RNase L(11) . The substrate was mRNA encoding the protein kinase PKR, produced by in vitro transcription and radiolabeled with 5`-[P]phosphate. To measure the initial cleavage reactions, we monitored loss of intact PKR mRNA by gel electrophoresis followed by analysis in a PhosphorImager. The unlinked RNase L activator, p(A2`p5`)A, caused a concentration-dependent loss of intact PKR mRNA and a 60% decrease at 10 nM (Fig. 1). The chimeric oligonucleotide, p5`(A2`p)5`A-anti-PKR, containing p(A2`p5`)A attached to a 19-nucleotide antisense DNA sequence against PKR mRNA (nucleotide numbers +55 to +73 relative to the start codon) at 30 nM caused nearly complete cleavage of PKR mRNA (Fig. 1). In contrast, p5`(A2`p)5`A-sense PKR, with a DNA sequence that was not complementary to any sequence in PKR mRNA, possessed a greatly decreased RNA cleavage ability, a 50% decrease in intact PKR mRNA that required nearly 400 nM oligonucleotide (Fig. 1). Therefore, the ability of 2-5A linked to oligodeoxyribonucleotide to cause efficient cleavage of RNA was dependent on a complementary DNA sequence in the chimera. In addition, chimeras containing a non-complementary DNA sequence were much less effective at causing RNA cleavage than either p5`(A2`p)5`A-anti-PKR or 2-5A itself ( Fig. 1and Fig. 2).


Figure 1: Cleavage of PKR mRNA (50 nM) by RNase L (12 nM) as function of p(A2`p5`)A (), p(A2`p5`)-anti-PKR (), or p(A2`p5`)A-sensePKR () concentration (as indicated). Incubations were at 37 °C for 15 min. Loss of intact PKR mRNA was measured by gel electrophoresis and phosphorImage analysis (See ``Materials and Methods'').




Figure 2: Catalytic cleavage of PKR mRNA in response to p(A2`p5`)A-anti-PKR. Either p(A2`p5`)A-anti-HIV (50 nM), lanes1-5, or p(A2`p5`)A-anti-PKR (50 nM), lanes 6-10, were incubated with 750 nM of PKR mRNA and 18 nM of RNase L for different periods of time (as indicated). Autoradiograms of the dried gels are shown.



2-5A-AS Induces Catalytic Cleavage of the RNA Target by RNase L

To determine if 2-5A-AS functions repetitively, RNA cleavage assays were performed with a 15-fold molar excess of substrate to oligonucleotide (Fig. 2). After a 30-min incubation at 37 °C, there was no detectable cleavage (<1%) of PKR mRNA in response to p(A2`p5`)A-anti-HIV (50 nM). In contrast, p(A2`p5`)A-anti-PKR (50 nM) caused a 98% loss of intact PKR mRNA in less than 5 min. Therefore, each molecule of p(A2`p5`)A-anti-PKR was responsible for the cleavage of multiple molecules of substrate.

The kinetics of the PKR cleavage reactions were determined by varying the concentration of substrate, holding invariant the concentration of p(A2`p5`)A-anti-PKR (50 nM) and RNase L (3 nM) ( Fig. 3and 4). A rapid loss of intact PKR mRNA as a function of time was observed in autoradiograms of the dried gels (Fig. 3). Quantitation of these results with a PhosphorImager showed that the rate of PKR mRNA cleavage was accelerated by increasing the substrate concentration (Fig. 4). Analysis of these data in an Eadie-Hofstee plot (24, 25) showed that substrate concentrations used were less than the K for the reaction (Fig. 4B). The value of the catalytic constant (k) indicated that the cleavage reaction occurred a maximum of about 10 times per active site per second. Because of limitations in measuring very low levels of RNA, the data obtained with the lowest amount of RNA (0.25 µM) was not used in Fig. 4B. Averages of the kinetic parameters from four separate experiments resulted in estimates of k = 7 ± 2.1 s and K = 1.8 ± 1.1 µM.


Figure 3: Kinetics of RNA cleavage at different ratios of PKR mRNA to p(A2`p5`)A-anti-PKR. Reactions contained 3 nM of RNase L and 50 nM of p(A2`p5`)A-anti-PKR and 1.5, 1, 0.75, 0.5, and 0.25 µM of PKR mRNA. The [RNA] to [p(A2`p5`)A-anti-PKR] ratios were 30:1, 20:1, 15:1, 10:1, and 5:1. Incubations were at 37 °C for different periods of time (as indicated). Autoradiograms of the dried gels are shown.




Figure 4: A, rate of cleavage of PKR mRNA by p(A2`p5`)A-anti-PKR and RNase L. The amount of intact PKR mRNA remaining is plotted as a function of the time of incubation. B, Eadie-Hofstee plot of catalytic cleavage of PKR mRNA substrate by p(A2`p5`)A-anti-PKR and RNase L. Data from the 2-min determinations are shown, excluding the curve obtained with 0.25 µM input PKR mRNA. K and k values are shown.



Mapping the Selected Cleavage Sites in PKR mRNA

The precise sites of cleavage in PKR mRNA induced by p(A2`p5`)A-anti-PKR and RNase L were determined by a primer extension assay. Incubations with control chimeric oligonucleotides (p(A2`p5`)A-anti-HIV, p(A2`p5`)A-sense PKR, and (A2`p5`)A-anti-PKR) did not produce detectable cleavage of PKR mRNA under the conditions of the assay (Fig. 5). The lack of cleavage in response to (A2`p5`)A-anti-PKR is due to a requirement for a 5`-phosphoryl group for efficient activation of human RNase L (Ref. 11 and references therein). In contrast, use of p(A2`p5`)A-anti-PKR resulted in a nearly complete loss of intact PKR mRNA (Fig. 5). Seven cleavage sites were detected. Six were 5` to the oligonucleotide binding site, despite the fact that the 2-5A portion of the chimera is at the opposite end of the oligonucleotide binding site (Fig. 5). Five of the upstream sites were clustered 16-20 nucleotides 5` of the mRNA target sequence, while one minor upstream site was 31 nucleotides 5` of the binding site in the mRNA. One cleavage was detected in the middle of the oligonucleotide binding site. The same cleavage sites were observed in three separate experiments. There was no apparent nucleotide specificity in the cleavage sites. Under the conditions of the assay, we observed nearly complete cleavage (to very small fragments migrating to the bottom of the gel) of the PKR mRNA with p(A2`p`5A).


Figure 5: Primer extension assays to determine sites of cleavage in PKR mRNA by RNase L (18 nM) and p(A2`p5`)A-anti-PKR. Lanes1-5, primer extension reactions without or with the 2-5A chimeras (each at 100 nM), as indicated, at 30 °C for 30 min. Lettersoversequencinglanes (rightfourlanes) indicate the nucleotide complementary to those that were sequenced. The portion of the PKR mRNA that was analyzed extends from the 5` termini of the transcripts to about 85 nucleotides 3` of the chimeric oligonucleotide binding site.




DISCUSSION

Targeted Decay of RNA in Response to 2-5A-AS

2-5A-AS chimeras are designed to activate and direct RNase L to cleave specific RNA targets in vitro and in vivo(2, 3, 4) . Here, we have used purified enzyme, RNA, and 2-5A-AS to study the cleavage reaction itself. Cleavage of PKR mRNA in response to p(A2`p5`)A was about two-fold more efficient than with p(A2`p5`)A-anti-PKR (Fig. 1), perhaps due to the fact that the 2-5A-AS chimera restricts RNase L to cleave within a single region in PKR mRNA (Fig. 1). Accordingly, while p(A2`p5`)A causes general RNA decay, p(A2`p5`)A-anti-PKR induced the targeted cleavage of PKR mRNA (Ref. 4, Fig. 5). We previously showed that PKR mRNA could be degraded selectively by purified RNase L in the presence of a non-targeted mRNA(4) . The cleavage of RNA targets by 2-5A-AS is envisioned to occur in discrete steps. The 2-5A moiety binds to and activates RNase L while the antisense part of the chimera binds to complementary sequence in the RNA, permitting cleavage to occur(2) . Therefore, it is the antisense moiety that confers selectivity in this approach. However, the present study demonstrates an additional property of the linker-DNA domain of the chimeras, namely, a suppression of non-targeted RNA cleavage. For example, cleavage of PKR mRNA by p(A2`p5`)A-anti-PKR was about 20-fold more efficient than with p(A2`p5`)A-sense PKR (Fig. 1). In addition, we observed that the presence of four mismatched nucleotides in the 19-nucleotide DNA sequence of p(A2`p5`)A-anti-PKR was also relatively inactive in the cleavage of PKR mRNA.()These observations suggest that the composite nucleic acids of the general formula p5`(A2`p)5`A2`p[O(CH)Op]p(5`N3`p)p5`N are relatively inefficient activa-tors of the RNase L. However, when the deoxyribonucleotide domain of the chimera is complementary (antisense) to a sequence within an RNA molecule, ability to activate RNase L is greatly enhanced. Apparently, cleavage of the RNA substrate is facilitated when held in close proximity to the active site by hybridization with the antisense domain of the chimera. According to this model, the selectivity for a given RNA, the specificity of the nucleotide cleavages sites, and the enhanced potency of 2-5A-AS, compared with similarly constructed chimeras lacking complementary DNA sequence, may all be due to the creation of a new and specific high affinity RNA binding site as well as to a proximity effect (26, 27, 28) on catalysis. In this model, 2-5A performs two critical functions: it activates the RNase L and it functions to anchor the specific substrate binding domain to the RNase.

Kinetics and Selectivity of an RNA Cleavage nduced by 2-5A-AS

Kinetic analysis demonstrated that both the enzyme and the 2-5A-AS chimera are released from the RNA cleavage products, enabling them to be reused. The simplest model based on the data is that the reaction occurs according to Michaelis-Menten kinetics. There is a relatively rapid turnover number (k = 5-10 s) for the targeted cleavage of PKR mRNA in response to p(A2`p5`)A-anti-PKR under the conditions of these assays ( Fig. 3and Fig. 4). It is possible that dissociation of the enzyme from 2-5A-AS after the RNA cleavage reaction could contribute to the turnover number. In addition, the hybridization site may be partly unwound in the complex (as suggested by the cleavage data). A destabilization of the hybrid would make the 2-5A-AS accessible to another RNA molecule, thereby presenting a new RNA substrate to the RNase L.

The pattern of cleavage sites in PKR mRNA induced by p(A2`p5`)A-anti-PKR suggests that it is a combination of secondary structure of the RNA and the three-dimensional structure of the 2-5A-AS-RNase complex that determines where cleavages occur. Surprisingly, most of the cleavages occurred 5` to the oligonucleotide binding site despite the presence at the 3` side of the binding site of the 2-5A moiety of the chimera (Fig. 5). Therefore, the structure of the RNA must have placed these cleavage sites in proximity to the RNase. As previously mentioned, in both this study and in a previous report(2) , RNA cleavage was also detected within the oligonucleotide binding site itself. The nucleotide specificity of RNase L for UpNp dimers is clearly altered by directing the RNase to particular sequences (Refs. 2, 12, and 13 and Fig. 5). Because knowledge of the structure of RNA within cells and the location of RNA binding proteins are usually unknown, uncertainty in predicting precise sites and efficiencies of RNA cleavage in response to 2-5A-AS in intact cells is likely. However, these findings demonstrate that RNase L will cleave near RNA sequences to which it is directed regardless of the sequence.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant 1 PO1 CA 62220-01A1, awarded by the Department of Health and Human Services, NCI, National Institutes of Health (to R. H. S.), and by an award from the National Institutes of Health Director's AIDS Targeted Antiviral Program (to P. F. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Cancer Biology, NN1-06, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9650; Fax: 216-445-6269.

The abbreviations used are: 2-5A-AS, 2-5A antisense; HIV, human immunodeficiency virus; PKR, double-stranded RNA-dependent protein kinase.

A. Maran, R. K. Maitra, R. H. Silverman, W. Xiao, and P. F. Torrence, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. George Stark, Bryan Williams, Dennis Stuehr, Avudaiappan Maran (all from Cleveland), and Brian W. Pontius (University of Oregon) for discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.