Nucleic Acids Research

RNA synthesis

RNAs were synthesized with T7 RNA polymerase (Takara Shuzo and Promega) from synthetic double-stranded DNAs containing a T7 promoter (16). The transcription reactions were carried out in the presence or absence of [[alpha]-³²P]UTP (Amersham and Dupont NEN) under the conditions specified by the manufacturers (Takara Shuzo and Promega).

RNA cleavage assay

The specific cleavage reactions for the ³²P-labeled target RNAs (0.1 pmol) were performed with the 3[prime] tRNase fraction (0.4 U) from mouse FM3A cells after the second Blue Sepharose chromatography (16) in the presence of an excess molar amount of a cold 5[prime] half tRNA^Arg or its derivatives in a mixture (10 µl) containing 10 mM Tris-HCl (pH 7.5), 1.5 mM dithiothreitol (DTT), 3.2 mM spermidine for 30 min at 37°C. After resolution of the reaction products on a 10% polyacrylamide-8 M urea gel, the gel was autoradiographed.

Kinetic analysis

Heptamer-directed cleavage of substrate RNAs by 3[prime] tRNase was examined at various concentrations of substrate. More purified 3[prime] tRNase from pig liver was used than from mouse FM3A cells (15). As far as we tested, both enzymes have the same properties (16; data not shown). A reaction mixture (6 µl) contained 10 mM Tris-HCl (pH 7.5), 1.5 mM DTT, 3.2 mM spermidine, 10 µM RNA heptamer and 0.17-1.0 µM target RNA. After pre-incubation at 37°C for 10 min, the reactions were started by adding pig 3[prime] tRNase fraction (1-5 ng) after Mono Q column chromatography (15), and continued at 37°C for 1 min. The reaction products were resolved on a 10% polyacrylamide-8 M urea gel and then quantitated with a Molecular Imager (Bio-Rad). Values of K_m·K_d and V_max were obtained from double-reciprocal plots (16). K_m and K_d represent the Michaelis constant of RNA cleavage by pig 3[prime] tRNase and the dissociation constant of a complex between a substrate RNA and an RNA heptamer, respectively.

RNA 5[prime]-end labeling

The 5[prime]-triphosphates of target RNAs were removed with calf intestine alkaline phosphatase (Promega). After the reaction, the RNAs were extracted with phenol, precipitated with ethanol, and redissolved in water. The target RNAs were 5[prime]-end-labeled with [[gamma]-³²P]ATP (DuPont NEN) using T4 polynucleotide kinase (Gibco BRL) and purified on a denaturing gel.

Structure probing

Partial digestion of the 5[prime]-end-labeled target RNAs was performed in the presence of the unlabeled heptamer T7M7 using RNases A (Sigma), T1 (Boehringer Mannheim) and V1 (Pharmacia)(19). The target RNAs were preincubated with the heptamer at 37°C for 10 min prior to adding RNases. The reaction mixture (6 µl) contained 10 mM Tris-HCl (pH 7.5), 1.5 mM DTT, 3.2 mM spermidine, 1 µg yeast tRNA, 10 µM RNA heptamer, 1.0 µM target RNA, and RNase A, T1 or V1. Reactions were incubated at room temperature (or at 37°C in the case of RNase V1) for 10 min. The samples were analyzed on a 10% polyacrylamide-8 M urea sequencing gel.

An RNA heptamer can direct RNA cleavage by 3[prime] tRNase

To investigate whether 3[prime] tRNase can be used to target specific RNAs under the direction of a smaller 5[prime] half tRNA, five 5[prime] half tRNA^Arg variants (T7M2-T7M6) were analyzed with the wild-type 3[prime] half tRNA^Arg (SPH2) containing a 3[prime] trailer and a 5[prime] artificial extension (Fig. 1A). These variants had 4-16 nt deletions in the D stem-loop domain. The remaining sequences were identical to the wild-type 5[prime] half tRNA^Arg T7M1, which differs from T7H in that T7M1 has no anticodon sequence (16; Fig. 1A). The RNAs were synthesized in vitro using T7 RNA polymerase. These 5[prime] half variants were tested for their ability to direct specific cleavage of ³²P-labeled 3[prime] half tRNA^Arg SPH2 by mouse 3[prime] tRNase. The target SPH2 was specifically cleaved after the discriminator as efficiently as when the wild-type 5[prime] half tRNA^Arg T7H or T7M1 was used (Fig. 1B). T7M3, however, directed cleavage at a lower rate. The efficient cleavage occurred even with T7M5, which had only three nucleotides in the D stem-loop domain. These results indicate that 3[prime] tRNase can readily recognize a pre-tRNA complex even if it lacks the D stem and loop.

Figure 1. Specific cleavage of a wild-type 3[prime] half tRNAArg, SPH2, by mouse 3[prime] tRNase directed by 5[prime] half tRNAArg variants. (A) Plausible secondary structures of complexes of SPH2 with various 5[prime] half tRNAArgs (T7M1-T7M7). Only the D stem-loop domains are shown in the cases of T7M2-T7M6; the other domains are identical to T7M1. Arrows indicate 3[prime] tRNase cleavage sites. Asterisks denote nucleotide deletions. The sequences 5[prime]-AGCAGGGUCGUUU-3[prime] and 5[prime]-GCACUAAA-3[prime] are omitted from the 3[prime] and 5[prime] regions, respectively, of SPH2. (B) Specific cleavage assays of SPH2 are shown: intact SPH2 (lane 1), RNA products after cleavage reactions with mouse 3[prime] tRNase in the absence (lane 2) and in the presence of 0.1 µM (1.0 µM in the case of T7M7; odd lanes) and 1.0 µM (10 µM in the case of T7M7; even lanes) of the indicated cold 5[prime] half tRNAArg variants. The specific cleavage reactions for the uniformly 32P-labeled SPH2 (0.1 pmol) were performed. After resolution of the reaction products on a 10% polyacrylamide-8 M urea gel, the gel was autoradiographed. Bars and arrowheads with a nucleotide length denote substrates and cleavage products, respectively.

To determine whether a much smaller 5[prime] tRNA could also direct cleavage, T7M7 lacking the anticodon stem as well as the D stem and loop was tested for its ability to direct specific cleavage (Fig. 1A). In the presence of 7 nt T7M7 RNA, 3[prime] tRNase cleaved the 3[prime] half tRNA^Arg SPH2 as efficiently as in the presence of the wild-type 5[prime] half tRNA^Arg (Fig. 1B, lanes 6 and 17). This indicates that the acceptor stem and the T stem-loop plus a 3[prime] trailer are sufficient for efficient substrate recognition and cleavage by 3[prime] tRNase.

The T stem structure is important for cleavage

We further examined whether the RNA heptamer T7M7 can direct specific cleavage of target RNAs that form less stable T stem-loop structures. We tested four target RNAs for cleavage: a wild-type 3[prime] half tRNA (T3H) and three of its variants (T7HM1, T7HM2 and T7HM3) (Fig. 2A). These 3[prime] half tRNAs have potentially 5, 4, 3 and 0 bp T stems, respectively. The calculated values for the free energy [Delta]G of the hairpin structures of T3H, T7HM1 and T7HM2 were -5.2, -2.3 and -0.5 kcal/mol, respectively (20).

Figure 2. Heptamer-directed cleavage of a wild-type 3[prime] half tRNAArg, T3H, and its variants, T7HM1, T7HM2 and T7HM3, by mouse 3[prime] tRNase. (A) Plausible secondary structures of complexes of T7M7 with the substrate RNAs. Arrows indicate 3[prime] tRNase cleavage sites. Asterisks denote nucleotide deletions. The sequence 5[prime]-AGCAGGGUCGUUUU-3[prime] is omitted from the 3[prime] region of the target RNAs. The bases substituted for the original bases in tRNAArg are underlined. The calculated [Delta]G values are for the T stem-loop domains. (B) Cleavage assays for 0.1 pmol of the 32P-labeled target RNAs T3H (lanes 1-3), T7HM1 (lanes 4-6), T7HM2 (lanes 7-9) and T7HM3 (lanes 10-12) were performed with mouse 3[prime] tRNase in the absence (lanes 2, 5, 8 and 11) and in the presence (lanes 3, 6, 9 and 12) of 0.5 µM of T7M7. Bars and arrowheads with a nucleotide length denote substrates and cleavage products, respectively.

The percent cleavage decreased as the number of T stem base pairs decreased (Fig. 2B). A target with no T stem (T7HM3) was not cleaved under the direction of the heptamer T7M7. The kinetic parameters K_m·K_d and V_max for each cleavage reaction using purified pig 3[prime] tRNase were determined. The relative catalytic efficiency V_max/K_m·K_d of the cleavage decreased as the stability of the T stem-loop structures decreased (Table 1). Using a least-squares method, this relation can be represented by the empirical formula: V_max/K_m = -0.068 × [Delta]G [where a calculated K_d value (0.31 µM) for the complex (20) was used, and [Delta]G denotes the free energy of the hairpin structure]. These results indicate that the T stem structure is important for cleavage by 3[prime] tRNase of pre-tRNA derivatives lacking the D stem-loop and the anticodon stem.

Table 1. Kinetic parameters of RNA cleavage by pig 3[prime] tRNase directed by RNA heptamers

Substrate	Km·Kda (µM2)	Vmaxa (pmol/min)	Relative Vmax/Km·Kd
T7M7-T3H	8.6	1.2	1.0
T7M7-T7HM1	13.1	1.1	0.60
T7M7-T7HM2	18.0	0.20	0.11
T7M7-T7HM3b	ND	ND	ND
GENV7-Env	11.8	0.73	0.44
GGAG7-Gagb	ND	ND	ND

^aOnly the product of K_m and K_d was obtained from this study. The maximum velocity per nanogram of pig 3[prime] tRNase fraction after Mono Q column chromatography is shown. Each measurement was from averages of three trials with a standard deviation of 3-10%.
^bNo cleavage was detected. The lower limit of detection was ~0.01 pmol/min.

To confirm that the predicted secondary structures shown in Figure 2A exist, structure probing assays were performed. 5[prime]-end-labeled target RNAs were partially digested in the presence of unlabeled RNA heptamers with RNases A, T1 and V1, and analyzed on a denaturing sequencing gel (Fig. 3). The digestion pattern of the four targets was nearly identical except in the T stem-loop domain (Fig. 3). The acceptor stem domain was resistant to attack by RNases A and T1, and efficiently cleaved by RNase V1 in each RNA target. The 3[prime] trailer region was cleaved by RNase V1 efficiently at 37G and 47G, probably due to stacking with the G-C base pair of the acceptor stem and unknown self-folding, respectively. The region upstream of 11U was only digested by RNase T1. The T stem domain of the wild-type was not cut by single-strand-specific RNases, but cleaved by RNase V1, while RNase T1 cleavage occurred in the T loop after 21G, supporting the proposed secondary structure (Fig. 3). The digestion patterns of T7HM1 and T7HM2 were similar to that of T3H, but 16G in the T stem was attacked by RNase T1, and 14C and 12U of T7HM1 and 12U of T7HM2 were digested by RNase A, suggesting less stable stem structures (Fig. 3). RNase V1 cleavage after 13A may be due to base stacking. As for T7HM3, 12G and 17G were cleaved by both RNases T1 and V1, suggesting unstable alternative secondary structures in the T stem domain (Fig. 3). RNase V1 digestion of each target in the T loop at 20C might be due to the interaction with the 3[prime] trailer. Although we were not able to ascertain the proposed single-stranded structure in the T stem-loop domain of T7HM3, these results indicated that the wild-type can indeed form the stable T stem structure, and that the T stems of T7HM1 and T7HM2 are less stable.

Figure 3. Structure probing for the target RNAs T3H, T7HM1, T7HM2 and T7HM3. Assays were performed with RNase A at 0.2 and 0.4 U/ml, RNase T1 at 0.1 and 0.2 U/ml, or RNase V1 at 3 and 6 U/ml. Cleavage sites by RNases A, T1 and V1 are indicated by hollow arrows, filled arrows and arrowheads, respectively. Length of the symbols reflects intensity of cleavage. The sequence 5[prime]-AGCAGGGUCGUUUU-3[prime] is omitted from the 3[prime] region of the target RNAs. The bases substituted for the original bases in tRNAArg are underlined. Asterisks denote nucleotide deletions.

Heptamer-directed cleavage of an HIV-1 RNA

The above results suggested that generally we can cleave a target RNA containing a T stem-loop-like structure after the eighth nucleotide downstream of the structure using mammalian 3[prime] tRNase and an RNA heptamer complementary to a 7 nt sequence immediately 3[prime] to the T stem-loop-like structure. We applied this heptamer method to two HIV-1 RNAs previously shown to be cleaved by their corresponding 5[prime] half tRNAs (16). One was the RNA target Env that potentially forms a stable hairpin structure with a 6 bp stem having a [Delta]G of -4.6 kcal/mol (Fig. 4A). As expected, the RNA heptamer GENV7, which is specific to the target Env, directed its cleavage very efficiently (Fig. 4C). The cleavage directed by GENV7 was as efficient as that by the 36 nt wild-type 5[prime] half tRNA GENV (16). A relative V_max/K_m·K_d value for this cleavage reaction was 0.44 (Table 1). When calculated K_d (0.46 µM) for the GENV7-Env complex (20) is taken into account, a relative V_max/K_m value (0.20) is obtained, and it is 0.11 lower than a value calculated from the [Delta]G value (-4.6 kcal/mol) with the above formula. This may be principally due to the unusual `T-loop' sequence and/or the reduced `T loop'. The non-specific heptamers GGAG7 and T7M7, however, did not direct the cleavage at all (Fig. 4C). The fact that T7M7, which was a successful heptamer for targeting T3H, did not work for targeting Env (Figs 2 and 4) indicates that `acceptor-stem' formation between a target RNA and its complementary heptamer is prerequisite for 3[prime] tRNase recognition.

Figure 4. Assays for specific cleavage of the RNA substrates Env and Gag by mouse 3[prime] tRNase directed by various RNA heptamers. (A) A complex of Env with an RNA heptamer, GENV7. The sequences 5[prime]-GACAAUUA-3[prime] and 5[prime]-GCAGCAGGAA-3[prime] are omitted from the 3[prime] and 5[prime] regions of Env, respectively. (B) A complex of Gag with an RNA heptamer, GGAG7. The sequences 5[prime]-AAACAUCA-3[prime] and 5[prime]-GGGCA-3[prime] are omitted from the 3[prime] and 5[prime] regions of Gag, respectively. Arrows indicate major cleavage sites. Asterisks denote nucleotide deletions. The calculated [Delta]G values are for the `T stem-loop' domains. (C and D) Specific cleavage assays using the 32P-labeled targets Env (C) and Gag (D) are shown: the intact target (lane 1), RNA products after cleavage reactions with 3[prime] tRNase in the absence (lane 2) and in the presence of 0.5 µM (lane 3) and 5.0 µM (lanes 4-6) of the indicated unlabeled RNA heptamers. Arrowheads with a nucleotide length denote cleavage products.

The second HIV-1 target was the Gag RNA, which is unlikely to form any stable hairpin structure as indicated by a positive [Delta]G value (Fig. 4B). The resulting GGAG7-Gag complex may only have a simple 7 bp double-stranded region. In contrast with the target Env, neither GGAG7, which is specific to the substrate Gag, nor the non-specific heptamers GENV7 and T7M7 directed cleavage of the target Gag (Fig. 4D). It is noteworthy that the cleavage of Gag occurred in the presence of the 36 nt wild-type 5[prime] half tRNA GGAG, and that 3[prime] tRNase can recognize a pre-tRNA-like complex without a stable T stem, given a D stem-loop and an anticodon stem (16).

RNA heptamers can direct efficient RNA cleavage with a higher specificity than expected

Here we demonstrated that mammalian 3[prime] tRNase can recognize and cleave an RNA target under the direction of an RNA heptamer if the target has a stable T stem-loop-like structure (Fig. 5). If the target has no stem-loop structure, no cleavage occurs. This finding sheds new light on antisense techniques; i.e. an RNA heptamer can efficiently direct targeted RNA cleavage with a higher specificity than expected for an RNA oligomer consisting of only seven nucleotides. Although there may be some other restrictions on what 3[prime] tRNase can cut, roughly speaking, a heptamer can direct efficient RNA cleavage with a specificity of a 7 (from the `acceptor stem') plus 5 (from the need for a stable `T stem') = 12 nt sequence and not merely a 7 nt sequence. This means that if G-U pairs are not taken into account, T-stem-loop-like structures should appear once every 4⁵ nucleotides in an mRNA pool. The probability of this occurrence is the same as that in which we would find a specific 5 nt sequence. It should be noted that the shortest sequence that is likely to be unique within a typical eukaryotic RNA pool of 20 million nucleotides is estimated to be 13 nt (9).

Figure 5. Schematic presentation for heptamer-guided RNA targeting. Mammalian 3[prime] tRNase can efficiently cleave only a heptamer-bound site that is adjacent to a stable T stem-loop-like hairpin structure. Roughly speaking, the potential target site appears once every 4¹² nucleotides in an mRNA pool (see text).

Mammalian RNase H is believed to be involved in conventional antisense effects, although its physiological role is not clear (21). RNase H is comparable with 3[prime] tRNase in several aspects. The proteinaceous enzyme RNase H can cleave any RNA under the direction of oligodeoxynucleotides (21). Recently, RNase H has been shown to cleave an RNA target in the presence of a propynyl heptanucleotide with a higher specificity than expected by 7 nt antisense oligonucleotides (22). The specificity appeared to be achieved by targeting a site of RNA that happened to be highly accessible to the heptanucleotide due to the absence of stable secondary and tertiary interactions within the RNA. This specificity of the targeting strategy using RNase H, however, originates from circumstances of a target RNA and not from a property of RNase H itself, so that the increased specificity may not be maintained in a broad sense.

Our results suggest that some of the antisense oliogodeoxynucleotide inhibition reported in vivo may be due to 3[prime] tRNase-catalyzed cleavage of a target RNA that happened to be folded into a pre-tRNA-like structure through the assistance of an antisense oligodeoxynucleotide. This may explain the seemingly random effectiveness of some antisense oligodeoxynucleotides. Both oligodeoxynucleotides and phosphorothioate-modified oligodeoxynucleotides, which are more stable than RNA oligomers, can also direct RNA cleavage by 3[prime] tRNase, although less efficiently than RNA oligomers (data not shown). Our current efforts are to apply this heptamer strategy to cultured cells. If we can generate efficient cleavage of target RNAs within living cells, this technology may be applicable to treat diseases such as AIDS or cancer.

Another role of 3[prime] tRNase?

Small RNA molecules play an important role in essential cellular processes such as RNA splicing (23), modification and processing of rRNAs (24), RNA editing (25) and translation control (26,27). Of particular note, temporally-regulated decreases in the lin-14 and lin-28 gene products are critical for normal development in Caenorhabditis elegans (26,27). A 22 nt RNA, encoded by the gene lin-4, is believed to function as a post-transcriptional regulator by hybridizing to the 3[prime] untranslated regions of the lin-14 and lin-28 mRNAs (26,27). The resulting RNA complexes resemble a pre-tRNA structure (16), suggesting that these may be substrates for 3[prime] tRNase. Indeed, we demonstrated that mammalian 3[prime] tRNase can recognize portions of the lin-14 mRNA in the presence of the lin-4 RNA and cleave them in vitro (data not shown). It remains to be shown, however, if nematode 3[prime] tRNase is involved in developmental regulation and whether the mammalian counterpart of this system exists. The flexibility of substrate recognition by mammalian 3[prime] tRNase, shown here and in previous studies (15,16), suggests that this enzyme may play a role in post-transcriptional regulation of expression of a certain gene by cleaving its mRNA under the direction of a small complementary RNA transcribed from another gene.

ACKNOWLEDGEMENTS

We thank K.Maeda for excellent technical assistance and Dr L. Levinger for helpful discussion. This work was supported in part by the Bireley Foundation. A part of this study was performed in JT Life Science Research Laboratory, Yokohama, Japan.

REFERENCES

1. Murray,J.A.H. and Crockett,N. (1992) In Murray,J.A.H. (ed.),Antisense RNA and DNA. Wiley-Liss Inc., New York, NY, pp 1-49.

2. Cantor,G.H., McElwain,T.F., Birkebak,T.A. and Palmer,G.H. (1993)Proc. Natl. Acad. Sci. USA, 90, 10932-10936. MEDLINE Abstract

3. Yu,M., Ojwang,J., Yamado,O., Hampel,A., Rapapport,J., Looney,D. and Wong-Staal,F. (1993) Proc. Natl. Acad. Sci. USA, 90, 6340-6344. MEDLINE Abstract

4. Tsuchihashi,Z., Khosla,M. and Hershlag,D. (1993) Science, 262, 99-102. MEDLINE Abstract

5. Hershlag,D., Khosla,M., Tsuchihashi,Z. and Karpel,R.L. (1994) EMBO J., 13, 2913-2924.

6. Roush,W. (1997) Science, 276, 1192-1193. MEDLINE Abstract

7. Couture,L.A. and Stinchcomb,D.T. (1996) Trends Genet., 12, 510-515. MEDLINE Abstract

8. Loke,S., Stein,C.A., Zhang,X.H., Mori,K., Nakanishi,M., Subasinghe,C., Cohen,J.S. and Neckers,L.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 3474-3478. MEDLINE Abstract

9. Woolf,T.M., Melton,D.A. and Jennings,C.G.B. (1992) Proc. Natl. Acad. Sci. USA, 89, 7305-7309. MEDLINE Abstract

10. Fakler,B., Herlitze,S., Amthor,B., Zenner,H.-P. and Ruppersberg,J.-P. (1994) J. Biol. Chem., 269, 16187-16194. MEDLINE Abstract

11. Nashimoto,M. (1997) Nucleic Acids Res., 25, 1148-1154. MEDLINE Abstract

12. Deutscher,M.P. (1995) In Soll,D. and RajBhandary,U.L. (eds), tRNA: Structure, Biosynthesis and Function. American Society for Microbiology Press, Washington, DC, pp 51-65.

13. Nashimoto,M. (1992) Nucleic Acids Res., 20, 3737-3742. MEDLINE Abstract

14. Nashimoto,M. (1993) Nucleic Acids Res., 21, 4696-4702. MEDLINE Abstract

15. Nashimoto,M. (1995) Nucleic Acids Res., 23, 3642-3647. MEDLINE Abstract

16. Nashimoto,M. (1996) RNA, 2, 523-534. MEDLINE Abstract

17. Matteucci,M.D. and Wagner,R.W. (1996) Nature, 384 (suppl.), 20-22. MEDLINE Abstract

18. Altman,S. (1993) Proc. Natl. Acad. Sci. USA, 90, 10898-10900. MEDLINE Abstract

19. Knapp,G. (1989) Methods Enzymol., 180, 192-212. MEDLINE Abstract

20. Turner,D.H., Sugimoto,N., Laeger,J.A., Longfellow,C.E., Freier,S.M. and Kierzek,R. (1987) Cold Spring Harbor Symp. Quant. Biol., 52, 123-133. MEDLINE Abstract

21. Hostomsky,Z., Hostomska,Z. and Matthews,D.A. (1993) In Linn,S.M., Lloyd,R.S. and Roberts,R.J. (eds), Nucleases. Cold Spring Harbor Laboratory Press, New York, NY, pp 341-376.

22. Wagner,R.W., Matteucci,M.D., Grant,D., Huang,T. and Froehler,B.C. (1996) Nature Biotechnol., 14, 840-844.

23. Guthrie,C. (1991) Science, 253, 157-163. MEDLINE Abstract

24. Tollervey,D. (1996) Science, 273, 1056-1057. MEDLINE Abstract

25. Simpson,L. (1990) Science, 250, 512-513. MEDLINE Abstract

26. Lee,R.C., Feinbaum,R.L. and Ambros,V. (1993) Cell, 75, 843-854. MEDLINE Abstract

27. Wightman,B., Ha,I. and Ruvkun,G. (1993) Cell, 75, 855-862. MEDLINE Abstract

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*To whom correspondence should be addressed. Tel: +1 801 378 8667; Fax: +1 801 378 5474; Email: mnashimoto@chemgate.byu.edu

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