A novel 4-base-recognizing RNA cutter that can remove the single 3' te
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《核酸研究医学期刊》
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niitsu, Niigata 956-8603, Japan
* To whom correspondence should be addressed. Tel: +81 250 25 5119; Fax: +81 250 25 5021; Email: mnashimoto@niigatayakudai.jp
ABSTRACT
Mammalian tRNase ZL shows versatility in substrate recognition. This enzyme can not only process pre-tRNAs by cleaving off their 3' trailer sequences, but also recognize and cleave pre-tRNA-like complexes and micro-pre-tRNAs. Here we demonstrate that 24–27 nt hairpin RNAs (hook RNAs) can guide cleavages of separate target RNAs by tRNase ZL through the micro-pre-tRNA-like complexes between the targets and the hook RNAs and that tRNase ZL together with hook RNA works as 4–7-base-recognizing RNA cutters. The cleavage sites were located only after the nucleotide corresponding to the discriminator nucleotide. Cleavage assays for various substrate/hooker complexes showed that the cleavage efficiency changes depending on the maximum number of substrate/hooker recognition base pairings and the stem length of hook RNA and that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also show that a 4-base RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. These results indicate that this new type of RNA cutter can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.
INTRODUCTION
Almost every genome contains the ELAC1 and/or ELAC2 genes, both of which encode tRNA 3' processing endoribonuclease (tRNase Z; EC 3.1.26.11 ). The ELAC1 genes produce short forms of tRNase Z (tRNase ZS) that contain 300–400 amino acids, while the ELAC2 genes produce long forms of tRNase Z (tRNase ZL) that is composed of 800–900 amino acids (1–5). Both forms of tRNase Z can process pre-tRNAs by cleaving off their 3' trailer sequences. In vitro processing experiments have shown that in most cases these endoribonucleolytic cleavages occur primarily after the discriminator nucleotide (Figure 1A), onto which tRNA nucleotidyltransferase adds the CCA residues to generate mature tRNA in vivo (2–4,6–8).
Figure 1. Various substrates of mammalian tRNase ZL. (A) A secondary structure of human pre-tRNAArg. (B) A secondary structure of the complex between a 3'-truncated human pre-tRNAArg and a target RNA. (C) A secondary structure of the complex between a 5'-half tRNAArg and a target RNA. (D) A secondary structure of human micro-pre-tRNAArg. (E) A secondary structure of the complex between an RNA heptamer and a target RNA. (F) A secondary structure of the complex between a hook RNA and a target RNA. Arrows denote the cleavage sites by tRNase ZL.
Mammalian tRNase ZL shows versatility in substrate recognition. By forming a relatively stable complex between the enzyme and a 3'-truncated tRNA (Figure 1B), tRNase ZL can function as a 4-base-recognizing RNA cutter (RNase 65) (9–12). Although little is known about the physiological substrates and roles of RNase 65, it has been shown that the 3'-truncated tRNA directs substrate specificity via four base pairings. The studies on the RNase 65 activity have provided us an insight to develop a technique to specifically cleave any RNA at any site by tRNase ZL under the direction of small guide RNA (sgRNA) (13); tRNase ZL can cleave a separate target RNA in the presence of a 5'-half tRNA that can form a pre-tRNA-like complex with the target (Figure 1C).
Mammalian tRNase ZL can also cleave various micro-pre-tRNA substrates, which consist of the T-stem–loop, the acceptor stem, and a 3' trailer (Figure 1D) (14). Based on this property of the enzyme, we have successfully demonstrated that a separate target RNA can be cleaved by tRNase ZL under the direction of an RNA heptamer, which binds to a 7 nt sequence immediately upstream of the cleavage site and can form a micro-pre-tRNA-like complex with the target (Figure 1E) (15–17).
From the above observations, we deduced that a short hairpin RNA (hook RNA) would guide cleavage of a separate target RNA by tRNase ZL through the micro-pre-tRNA-like complex between the target and the hook RNA (Figure 1F). Here, we show that expectedly pig tRNase ZL cleaves target RNAs under the direction of the hook RNAs and works as 4–7-base-recognizing RNA cutters. Furthermore, we demonstrate that these RNA cutters can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.
MATERIALS AND METHODS
Preparation of pig tRNase ZL
Pig tRNase ZL was intensively fractionated from liver through several column chromatography procedures and further purified by glycerol gradient ultracentrifugation as described before (18).
RNA synthesis
Four different 40 nt RNA substrates and four different RNA hookers were synthesized with T7 RNA polymerase (Takara Shuzo) from the corresponding synthetic DNA templates. The sequences of the RNA substrates, Substrate-1, Substrate-2, Substrate-3, and Substrate-4 are 5'-GAAUACGCAUGCUAGCAGGUGCCCGGUGAAAGCUUGAUGU-3', 5'-GAAUACGCAUGCUAGCAGGGGCCCGGUGAAAGCUUGAUGU-3', 5'-GAAUACGCAUGCUAGCAGUGGCCCGGUGAAAGCUUGAUGU-3' and 5'-GAAUACGCAUGCUAGCACUGGCCCGGUGAAAGCUUGAUGU-3', respectively (the nucleotides substituted for those of Substrate-1 are underlined). The sequences of the hook RNA, Hooker-1, Hooker-2, Hooker-3, and Hooker-4 are 5'-GGGCCAGCCAGGUUCGACUCCUGGCUG-3', 5'-GGGCCAGCCAGGUUCGACUCCUGGCU-3', 5'-GGGCCAGCCAGGUUCGACUCCUGGC-3' and 5'-GGGCCAGCCAGGUUCGACUCCUGG-3', respectively (the nucleotides added to the 3' terminus of Hooker-4 are underlined). The transcription reactions were carried out under the conditions recommended by the manufacturer (Takara Shuzo), and the transcribed RNAs were purified by denaturing gel electrophoresis.
The 40 nt RNA substrates were subsequently labeled with fluorescein according to the manufacturer's protocol (Amersham Pharmacia Biotech) (3,5). Briefly, after the removal of the 5'-phosphates of the RNA substrates with bacterial alkaline phosphatase (Takara Shuzo), the RNAs were phosphorylated with T4 polynucleotide kinase (Takara Shuzo) and ATPS. Then a single fluorescein moiety was appended onto the 5'-phosphorothioate site. The resulting fluorescein-labeled RNAs were gel-purified before assays.
The RNA substrate Substrate-5 that was 5' end labeled with fluorescein was chemically synthesized with an RNA synthesizer and purified through high-performance liquid chromatography by Nippon Bioservice. The sequence of Substrate-5 is 5'-AAUGGCCCGG-3'.
In vitro RNA cleavage assays
The in vitro cleavage assays for the fluorescein-labeled 40 nt RNA substrates (0.1 pmol) were carried out in the presence of the unlabeled hook RNAs with pig tRNase ZL in a mixture (6 μl) containing 10 mM Tris–HCl (pH 7.5), 1.5 mM dithiothreitol and 3.2 mM MgCl2 at 50°C for indicated time periods (13,15,16). After resolution of the reaction products on a 10% polyacrylamide–8 M urea gel, the gel was analyzed with a Typhoon 9210 (Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
Generation of novel 4-base-recognizing RNA cutters
Hooker-1 is an extensively shortened form of human tRNAArg is and composed of the T-stem–loop and the acceptor stem lacking the 3' terminal 4 nt. Substrate-1 is a 40 nt RNA containing the sequence 5'-GCCC-3', which is complementary to the 5' terminal sequence of Hooker-1. Thus, Hooker-1 has a potential to form a micro-pre-tRNA-like complex with Substrate-1 via four base pairings (Figure 2A). We examined whether the Hooker-1/Substrate-1 complex can be recognized and cleaved by tRNase ZL (Figure 2A). As expected from the previous observations (12,14,15), the RNA complex was processed by pig tRNase ZL, and 50% of Substrate-1 molecules were cleaved (Figure 2B and C). The cleavage was dependent on the presence of Hooker-1, and was not detected without additional RNAs or in the presence of yeast total tRNA. From the high-resolution analysis of the 5' cleavage product on a sequencing gel, the cleavage site was located only after the nucleotide G corresponding to the discriminator nucleotide (Figure 3). This indicates that this RNA cutter can be utilized to generate 3' homogeneous RNA molecules from 3' heterogeneous RNA transcripts. The recombinant human tRNase ZL also worked in the same fashion (data not shown).
Figure 2. Generation of 4-base-recognizing RNA cutters from tRNase ZL. (A) Secondary structures of the Substrate-1 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-1. An arrow denotes the cleavage site by tRNase ZL. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-1 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-1 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-1 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 3. Determination of the Substrate-1cleavage sites by tRNase ZL. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-1 (0.1 pmol) in the presence of 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. I, input RNA; L, the alkaline ladder of the fluorescein-labeled Substrate-1. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. Substrate-1 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively.
We also tested three shorter hook RNAs for the ability to guide the cleavage of Substrate-1 by tRNase ZL. These hook RNAs Hooker-2, Hooker-3 and Hooker-4 lack 1, 2 and 3 nt, respectively, at the 3' ends, compared with Hooker-1 (Figure 2A). The efficiency of Substrate-1 cleavage directed by Hooker-2 was the same as by Hooker-1, while cleavage percentages decreased to 25% under the direction of Hooker-3 and Hooker-4 (Figure 2B and C). The cleavage sites of Substrate-1 were not altered by the shorter hook RNAs (Figure 3).
Because Substrate-1 can bind to each hook RNA through four base pairings at the maximum, only the complex with Hooker-1 can form a 12 bp stem (Figure 2A). The Substrate-1 complexes with the other hook RNAs can form only 9–11 bp discontinuous stems. The Substrate-1/Hooker-2 complex with the 11 bp stem was recognized and cleaved by the enzyme as well as the Substrate-1/Hooker-1 complex, while the cleavage efficiency for the Substrate-1 complex with Hooker-3 or Hooker-4 containing the 10 or 9 bp stem was drastically reduced (Figure 2). These results suggest that when a 4 nt sequence is used as a recognition sequence, a hook RNA should have a 7 to 8 bp stem to achieve the optimal cleavage efficiency.
Cleavage efficiency differs depending on the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings
To investigate how the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings affect the substrate cleavage efficiency, we synthesized three different RNA substrates, Substrate-2, Substrate-3 and Substrate-4, which contain 1, 2 and 3 base substitutions, respectively, upstream of the hook RNA binding site (Figures 4A, 5A and 6A).
Figure 4. Cleavages of the Substrate-2 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-2 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-2. An arrow denotes the cleavage site by tRNase ZL. A Substrate-2 base that can potentially base pair with Hooker-1 in an alternative conformer is shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-2 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-2 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-2 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 5. The cleavages of the Substrate-3 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-3 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-3. An arrow denotes the cleavage site by tRNase ZL. Substrate-3 bases that can potentially base pair with Hooker-1 or Hooker-2 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-3 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-3 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-3 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 6. The cleavages of Substrate-4 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-4 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-4. An arrow denotes the cleavage site by tRNase ZL. Substrate-4 bases that can potentially base pair with Hooker-1, Hooker-2 or Hooker-3 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-4 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-4 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-4 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Substrate-2 can interact with the hook RNAs via five base pairings at the maximum, and the Substrate-2 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4 can form 12, 12, 11 and 10 bp stems, respectively (Figure 4A). The Hooker-1/Substrate-2 complex can form two alternative conformers in theory: one contains four base pairings between the hooker and the substrate and the other contains five base pairings. Substrate-2 was tested for cleavage by tRNase ZL under the direction of the hook RNAs. Percentages of Substrate-2 cleavage directed by Hooker-1 and Hooker-4 were 50%, and those by Hooker-2 and Hooker-3 were 60% (Figure 4B and C). These results imply that a hook RNA containing a 6 or 7 bp stem works better when a 5 nt sequence is utilized as a recognition sequence.
Substrate-3 can bind to the hook RNAs through six base pairings at the maximum (Figure 5A). As in the case of the Hooker-1/Substrate-2 complex, the Substrate-3 complex with Hooker-1 or Hooker-2 can form alternative conformers. Substrate-3 was cleaved less efficiently than Substrate-2 under the direction of every hook RNA, and each cleavage percentage was only 30% (Figure 5B and C), suggesting that any hook RNA can be used for targeting RNA substrates with a 6 nt recognition sequence.
Substrate-4 interactions with hook RNAs are through seven base pairings at the maximum, and the Substrate-4 complex with Hooker-1, Hooker-2 or Hooker-3 can form alternative conformers (Figure 6A). Percentages of Substrate-4 cleavage directed by Hooker-1 and Hooker-2 were 30%, while those by Hooker-3 and Hooker-4 were 50% (Figure 6B and C), implying that a hook RNA should contain a 5 or 6 bp stem to achieve the best efficiency when a 7 nt sequence is used as a recognition sequence.
Taken together, the above observations indicate that substrate cleavage efficiency changes depending on the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings. The differences in the substrate/hooker complex structure would cause differences in the substrate/hooker association rate and the kinetic parameters Km and kcat for tRNase ZL cleavage and affect the cleavage efficiency. The present data suggest that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also confirmed that substrate/hooker complexes with different recognition sequences can be recognized and cleaved by tRNase ZL (data not shown).
A 4-base-recognizing RNA cutter can remove the single 3' terminal nucleotide
Furthermore, we examined whether a 4-base-recognizing RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. We chemically synthesized a 10 nt RNA substrate 5' end labeled with fluorescein, Substrate-5, and tested it for removal of the single 3' terminal nucleotide through four base pairings with Hooker-1 (Figure 7A). Although the Hooker-1/Substrate-5 complex can form alternative conformers, in which Hooker-1 interacts with Substrate-5 via five or six base pairings (Figure 7A), we call this Hooker-1/tRNase ZL combination a 4-base RNA cutter. Substrate-5 (0.1 pmol) was incubated with tRNase ZL in the presence of Hooker-1, and the 5' cleavage product was analyzed on a denaturing polyacrylamide gel. As expected, this 4-base RNA cutter removed the single 3' terminal nucleotide G from Substrate-5 (Figure 7B). Although the removal was not complete in 60 min in the presence of 5 pmol of Hooker-1, the 3' terminal G was eliminated from every molecule in the presence of 15 pmol of Hooker-1.
Figure 7. The single 3' terminal nucleotides can be removed by a 4-base RNA cutter. (A) A secondary structure of the Hooker-1/Substrate-5 complex. An arrow denotes the cleavage site by tRNase ZL. Substrate-5 bases that can potentially base pair with Hooker-1 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-5 (0.1 pmol) in the absence (–) or presence of 5 (+) or 15 (++) pmol of Hooker-1 at 50°C for the indicated time period. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-5 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively.
A new strategy to generate homogeneous 3' termini of RNA molecules
Bacteriophage RNA polymerases such as T7 and SP6 RNA polymerases are widely used to produce RNA transcripts in vitro (19,20). In a standard method for in vitro run-off transcription, RNA polymerase starts transcription by recognizing a specific promoter sequence and terminates it at the end of a DNA template by running off the template. When the polymerase leaves the DNA template, the enzyme usually adds one or more non-encoded nucleotides to the 3' end of the RNA chain (19,20).
This causes 3' heterogeneity of in vitro RNA transcripts, and it is not very easy to separate a couple of nucleotides longer RNA molecules from the desired RNA molecules by polyacrylamide gel electrophoresis or chromatographic methods. It has been shown that the use of self-cleaving RNA sequences is effective to avoid unwanted by-products and to produce RNA molecules with homogeneous 5' and 3' ends (21–23). However, construction of DNA templates containing such ribozyme sequences would be often time- and cost-consuming.
Here, we demonstrated that the 4–7-base RNA cutters can be utilized to homogenize, at their 3' termini, RNA molecules synthesized in vitro with T7 RNA polymerase or with an RNA synthesizer. To the best of our knowledge, the method to use this new type of RNA cutter is the only way to remove single 3' terminal nucleotides. By covalently attaching a hook RNA to tRNase ZL, 3' homogeneous RNA products without extra 3' terminal non-encoded nucleotides will be easily purified through phenol/chloroform extraction to remove the hooker/enzyme complex. This consideration implies that our new strategy to generate homogeneous 3' termini of RNA molecules by the 4–7-base RNA cutters would be much easier to perform.
Another potential application of the 4-base RNA cutter
Because Hooker-1, which we used here, can direct RNA cleavage after the sequence 5'...GCCCN...3', the cleavages occur at every 256 nt sequences theoretically. It should be noted that ribozymes can hardly cleave target RNAs with such a low sequence specificity. This new type of 4-base RNA cutter could be applied to analysis of whole gene expression patterns. Total mRNA isolated from the cells will be digested with the 4 nt cutters, subsequently reverse-transcribed and PCR-amplified with sets of specific primers. With this strategy, we will be able to detect not only differences in mRNA expression levels but also single-nucleotide polymorphisms, because the 4 nt RNA cutters should be able to distinguish single-nucleotide differences in mRNAs (12).
ACKNOWLEDGEMENTS
We thank M. Takeda for technical assistance. This work was supported in part by the Science Research Promotion Fund and the Academic Frontier Research Project Grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
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* To whom correspondence should be addressed. Tel: +81 250 25 5119; Fax: +81 250 25 5021; Email: mnashimoto@niigatayakudai.jp
ABSTRACT
Mammalian tRNase ZL shows versatility in substrate recognition. This enzyme can not only process pre-tRNAs by cleaving off their 3' trailer sequences, but also recognize and cleave pre-tRNA-like complexes and micro-pre-tRNAs. Here we demonstrate that 24–27 nt hairpin RNAs (hook RNAs) can guide cleavages of separate target RNAs by tRNase ZL through the micro-pre-tRNA-like complexes between the targets and the hook RNAs and that tRNase ZL together with hook RNA works as 4–7-base-recognizing RNA cutters. The cleavage sites were located only after the nucleotide corresponding to the discriminator nucleotide. Cleavage assays for various substrate/hooker complexes showed that the cleavage efficiency changes depending on the maximum number of substrate/hooker recognition base pairings and the stem length of hook RNA and that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also show that a 4-base RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. These results indicate that this new type of RNA cutter can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.
INTRODUCTION
Almost every genome contains the ELAC1 and/or ELAC2 genes, both of which encode tRNA 3' processing endoribonuclease (tRNase Z; EC 3.1.26.11 ). The ELAC1 genes produce short forms of tRNase Z (tRNase ZS) that contain 300–400 amino acids, while the ELAC2 genes produce long forms of tRNase Z (tRNase ZL) that is composed of 800–900 amino acids (1–5). Both forms of tRNase Z can process pre-tRNAs by cleaving off their 3' trailer sequences. In vitro processing experiments have shown that in most cases these endoribonucleolytic cleavages occur primarily after the discriminator nucleotide (Figure 1A), onto which tRNA nucleotidyltransferase adds the CCA residues to generate mature tRNA in vivo (2–4,6–8).
Figure 1. Various substrates of mammalian tRNase ZL. (A) A secondary structure of human pre-tRNAArg. (B) A secondary structure of the complex between a 3'-truncated human pre-tRNAArg and a target RNA. (C) A secondary structure of the complex between a 5'-half tRNAArg and a target RNA. (D) A secondary structure of human micro-pre-tRNAArg. (E) A secondary structure of the complex between an RNA heptamer and a target RNA. (F) A secondary structure of the complex between a hook RNA and a target RNA. Arrows denote the cleavage sites by tRNase ZL.
Mammalian tRNase ZL shows versatility in substrate recognition. By forming a relatively stable complex between the enzyme and a 3'-truncated tRNA (Figure 1B), tRNase ZL can function as a 4-base-recognizing RNA cutter (RNase 65) (9–12). Although little is known about the physiological substrates and roles of RNase 65, it has been shown that the 3'-truncated tRNA directs substrate specificity via four base pairings. The studies on the RNase 65 activity have provided us an insight to develop a technique to specifically cleave any RNA at any site by tRNase ZL under the direction of small guide RNA (sgRNA) (13); tRNase ZL can cleave a separate target RNA in the presence of a 5'-half tRNA that can form a pre-tRNA-like complex with the target (Figure 1C).
Mammalian tRNase ZL can also cleave various micro-pre-tRNA substrates, which consist of the T-stem–loop, the acceptor stem, and a 3' trailer (Figure 1D) (14). Based on this property of the enzyme, we have successfully demonstrated that a separate target RNA can be cleaved by tRNase ZL under the direction of an RNA heptamer, which binds to a 7 nt sequence immediately upstream of the cleavage site and can form a micro-pre-tRNA-like complex with the target (Figure 1E) (15–17).
From the above observations, we deduced that a short hairpin RNA (hook RNA) would guide cleavage of a separate target RNA by tRNase ZL through the micro-pre-tRNA-like complex between the target and the hook RNA (Figure 1F). Here, we show that expectedly pig tRNase ZL cleaves target RNAs under the direction of the hook RNAs and works as 4–7-base-recognizing RNA cutters. Furthermore, we demonstrate that these RNA cutters can be utilized to homogenize at their 3' termini RNA transcripts synthesized in vitro with a bacteriophage RNA polymerase.
MATERIALS AND METHODS
Preparation of pig tRNase ZL
Pig tRNase ZL was intensively fractionated from liver through several column chromatography procedures and further purified by glycerol gradient ultracentrifugation as described before (18).
RNA synthesis
Four different 40 nt RNA substrates and four different RNA hookers were synthesized with T7 RNA polymerase (Takara Shuzo) from the corresponding synthetic DNA templates. The sequences of the RNA substrates, Substrate-1, Substrate-2, Substrate-3, and Substrate-4 are 5'-GAAUACGCAUGCUAGCAGGUGCCCGGUGAAAGCUUGAUGU-3', 5'-GAAUACGCAUGCUAGCAGGGGCCCGGUGAAAGCUUGAUGU-3', 5'-GAAUACGCAUGCUAGCAGUGGCCCGGUGAAAGCUUGAUGU-3' and 5'-GAAUACGCAUGCUAGCACUGGCCCGGUGAAAGCUUGAUGU-3', respectively (the nucleotides substituted for those of Substrate-1 are underlined). The sequences of the hook RNA, Hooker-1, Hooker-2, Hooker-3, and Hooker-4 are 5'-GGGCCAGCCAGGUUCGACUCCUGGCUG-3', 5'-GGGCCAGCCAGGUUCGACUCCUGGCU-3', 5'-GGGCCAGCCAGGUUCGACUCCUGGC-3' and 5'-GGGCCAGCCAGGUUCGACUCCUGG-3', respectively (the nucleotides added to the 3' terminus of Hooker-4 are underlined). The transcription reactions were carried out under the conditions recommended by the manufacturer (Takara Shuzo), and the transcribed RNAs were purified by denaturing gel electrophoresis.
The 40 nt RNA substrates were subsequently labeled with fluorescein according to the manufacturer's protocol (Amersham Pharmacia Biotech) (3,5). Briefly, after the removal of the 5'-phosphates of the RNA substrates with bacterial alkaline phosphatase (Takara Shuzo), the RNAs were phosphorylated with T4 polynucleotide kinase (Takara Shuzo) and ATPS. Then a single fluorescein moiety was appended onto the 5'-phosphorothioate site. The resulting fluorescein-labeled RNAs were gel-purified before assays.
The RNA substrate Substrate-5 that was 5' end labeled with fluorescein was chemically synthesized with an RNA synthesizer and purified through high-performance liquid chromatography by Nippon Bioservice. The sequence of Substrate-5 is 5'-AAUGGCCCGG-3'.
In vitro RNA cleavage assays
The in vitro cleavage assays for the fluorescein-labeled 40 nt RNA substrates (0.1 pmol) were carried out in the presence of the unlabeled hook RNAs with pig tRNase ZL in a mixture (6 μl) containing 10 mM Tris–HCl (pH 7.5), 1.5 mM dithiothreitol and 3.2 mM MgCl2 at 50°C for indicated time periods (13,15,16). After resolution of the reaction products on a 10% polyacrylamide–8 M urea gel, the gel was analyzed with a Typhoon 9210 (Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
Generation of novel 4-base-recognizing RNA cutters
Hooker-1 is an extensively shortened form of human tRNAArg is and composed of the T-stem–loop and the acceptor stem lacking the 3' terminal 4 nt. Substrate-1 is a 40 nt RNA containing the sequence 5'-GCCC-3', which is complementary to the 5' terminal sequence of Hooker-1. Thus, Hooker-1 has a potential to form a micro-pre-tRNA-like complex with Substrate-1 via four base pairings (Figure 2A). We examined whether the Hooker-1/Substrate-1 complex can be recognized and cleaved by tRNase ZL (Figure 2A). As expected from the previous observations (12,14,15), the RNA complex was processed by pig tRNase ZL, and 50% of Substrate-1 molecules were cleaved (Figure 2B and C). The cleavage was dependent on the presence of Hooker-1, and was not detected without additional RNAs or in the presence of yeast total tRNA. From the high-resolution analysis of the 5' cleavage product on a sequencing gel, the cleavage site was located only after the nucleotide G corresponding to the discriminator nucleotide (Figure 3). This indicates that this RNA cutter can be utilized to generate 3' homogeneous RNA molecules from 3' heterogeneous RNA transcripts. The recombinant human tRNase ZL also worked in the same fashion (data not shown).
Figure 2. Generation of 4-base-recognizing RNA cutters from tRNase ZL. (A) Secondary structures of the Substrate-1 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-1. An arrow denotes the cleavage site by tRNase ZL. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-1 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-1 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-1 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 3. Determination of the Substrate-1cleavage sites by tRNase ZL. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-1 (0.1 pmol) in the presence of 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. I, input RNA; L, the alkaline ladder of the fluorescein-labeled Substrate-1. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. Substrate-1 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively.
We also tested three shorter hook RNAs for the ability to guide the cleavage of Substrate-1 by tRNase ZL. These hook RNAs Hooker-2, Hooker-3 and Hooker-4 lack 1, 2 and 3 nt, respectively, at the 3' ends, compared with Hooker-1 (Figure 2A). The efficiency of Substrate-1 cleavage directed by Hooker-2 was the same as by Hooker-1, while cleavage percentages decreased to 25% under the direction of Hooker-3 and Hooker-4 (Figure 2B and C). The cleavage sites of Substrate-1 were not altered by the shorter hook RNAs (Figure 3).
Because Substrate-1 can bind to each hook RNA through four base pairings at the maximum, only the complex with Hooker-1 can form a 12 bp stem (Figure 2A). The Substrate-1 complexes with the other hook RNAs can form only 9–11 bp discontinuous stems. The Substrate-1/Hooker-2 complex with the 11 bp stem was recognized and cleaved by the enzyme as well as the Substrate-1/Hooker-1 complex, while the cleavage efficiency for the Substrate-1 complex with Hooker-3 or Hooker-4 containing the 10 or 9 bp stem was drastically reduced (Figure 2). These results suggest that when a 4 nt sequence is used as a recognition sequence, a hook RNA should have a 7 to 8 bp stem to achieve the optimal cleavage efficiency.
Cleavage efficiency differs depending on the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings
To investigate how the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings affect the substrate cleavage efficiency, we synthesized three different RNA substrates, Substrate-2, Substrate-3 and Substrate-4, which contain 1, 2 and 3 base substitutions, respectively, upstream of the hook RNA binding site (Figures 4A, 5A and 6A).
Figure 4. Cleavages of the Substrate-2 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-2 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-2. An arrow denotes the cleavage site by tRNase ZL. A Substrate-2 base that can potentially base pair with Hooker-1 in an alternative conformer is shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-2 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-2 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-2 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 5. The cleavages of the Substrate-3 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-3 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-3. An arrow denotes the cleavage site by tRNase ZL. Substrate-3 bases that can potentially base pair with Hooker-1 or Hooker-2 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-3 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-3 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-3 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Figure 6. The cleavages of Substrate-4 complexes with the hook RNAs by tRNase ZL. (A) Secondary structures of the Substrate-4 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in Substrate-4. An arrow denotes the cleavage site by tRNase ZL. Substrate-4 bases that can potentially base pair with Hooker-1, Hooker-2 or Hooker-3 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-4 (0.1 pmol) in the absence (–) or presence of 5 pmol of yeast total tRNA (c), 5 pmol of Hooker-1 (lane 1), 5 pmol of Hooker-2 (lane 2), 5 pmol of Hooker-3 (lane 3) or 5 pmol of Hooker-4 (lane 4), at 50°C for 15 min. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-4 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively. I, input RNA. (C) Percentages of Substrate-4 cleavages by tRNase ZL in the presence of the hook RNAs are shown. Data are the means ± SD of three independent experiments.
Substrate-2 can interact with the hook RNAs via five base pairings at the maximum, and the Substrate-2 complexes with Hooker-1, Hooker-2, Hooker-3 and Hooker-4 can form 12, 12, 11 and 10 bp stems, respectively (Figure 4A). The Hooker-1/Substrate-2 complex can form two alternative conformers in theory: one contains four base pairings between the hooker and the substrate and the other contains five base pairings. Substrate-2 was tested for cleavage by tRNase ZL under the direction of the hook RNAs. Percentages of Substrate-2 cleavage directed by Hooker-1 and Hooker-4 were 50%, and those by Hooker-2 and Hooker-3 were 60% (Figure 4B and C). These results imply that a hook RNA containing a 6 or 7 bp stem works better when a 5 nt sequence is utilized as a recognition sequence.
Substrate-3 can bind to the hook RNAs through six base pairings at the maximum (Figure 5A). As in the case of the Hooker-1/Substrate-2 complex, the Substrate-3 complex with Hooker-1 or Hooker-2 can form alternative conformers. Substrate-3 was cleaved less efficiently than Substrate-2 under the direction of every hook RNA, and each cleavage percentage was only 30% (Figure 5B and C), suggesting that any hook RNA can be used for targeting RNA substrates with a 6 nt recognition sequence.
Substrate-4 interactions with hook RNAs are through seven base pairings at the maximum, and the Substrate-4 complex with Hooker-1, Hooker-2 or Hooker-3 can form alternative conformers (Figure 6A). Percentages of Substrate-4 cleavage directed by Hooker-1 and Hooker-2 were 30%, while those by Hooker-3 and Hooker-4 were 50% (Figure 6B and C), implying that a hook RNA should contain a 5 or 6 bp stem to achieve the best efficiency when a 7 nt sequence is used as a recognition sequence.
Taken together, the above observations indicate that substrate cleavage efficiency changes depending on the stem length of hook RNA and the maximum number of substrate/hooker recognition base pairings. The differences in the substrate/hooker complex structure would cause differences in the substrate/hooker association rate and the kinetic parameters Km and kcat for tRNase ZL cleavage and affect the cleavage efficiency. The present data suggest that a 5 nt recognition sequence and a hook RNA containing a 6 or 7 bp stem are the best combination for the optimal target cleavage. We also confirmed that substrate/hooker complexes with different recognition sequences can be recognized and cleaved by tRNase ZL (data not shown).
A 4-base-recognizing RNA cutter can remove the single 3' terminal nucleotide
Furthermore, we examined whether a 4-base-recognizing RNA cutter can remove the single 3' terminal nucleotides from RNA molecules. We chemically synthesized a 10 nt RNA substrate 5' end labeled with fluorescein, Substrate-5, and tested it for removal of the single 3' terminal nucleotide through four base pairings with Hooker-1 (Figure 7A). Although the Hooker-1/Substrate-5 complex can form alternative conformers, in which Hooker-1 interacts with Substrate-5 via five or six base pairings (Figure 7A), we call this Hooker-1/tRNase ZL combination a 4-base RNA cutter. Substrate-5 (0.1 pmol) was incubated with tRNase ZL in the presence of Hooker-1, and the 5' cleavage product was analyzed on a denaturing polyacrylamide gel. As expected, this 4-base RNA cutter removed the single 3' terminal nucleotide G from Substrate-5 (Figure 7B). Although the removal was not complete in 60 min in the presence of 5 pmol of Hooker-1, the 3' terminal G was eliminated from every molecule in the presence of 15 pmol of Hooker-1.
Figure 7. The single 3' terminal nucleotides can be removed by a 4-base RNA cutter. (A) A secondary structure of the Hooker-1/Substrate-5 complex. An arrow denotes the cleavage site by tRNase ZL. Substrate-5 bases that can potentially base pair with Hooker-1 in alternative conformers are shown in blue. (B) The in vitro RNA cleavage assays. Pig liver tRNase ZL (20 ng) was incubated with fluorescein-labeled Substrate-5 (0.1 pmol) in the absence (–) or presence of 5 (+) or 15 (++) pmol of Hooker-1 at 50°C for the indicated time period. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. Substrate-5 and its 5' cleavage product are indicated by a bar and an arrowhead, respectively.
A new strategy to generate homogeneous 3' termini of RNA molecules
Bacteriophage RNA polymerases such as T7 and SP6 RNA polymerases are widely used to produce RNA transcripts in vitro (19,20). In a standard method for in vitro run-off transcription, RNA polymerase starts transcription by recognizing a specific promoter sequence and terminates it at the end of a DNA template by running off the template. When the polymerase leaves the DNA template, the enzyme usually adds one or more non-encoded nucleotides to the 3' end of the RNA chain (19,20).
This causes 3' heterogeneity of in vitro RNA transcripts, and it is not very easy to separate a couple of nucleotides longer RNA molecules from the desired RNA molecules by polyacrylamide gel electrophoresis or chromatographic methods. It has been shown that the use of self-cleaving RNA sequences is effective to avoid unwanted by-products and to produce RNA molecules with homogeneous 5' and 3' ends (21–23). However, construction of DNA templates containing such ribozyme sequences would be often time- and cost-consuming.
Here, we demonstrated that the 4–7-base RNA cutters can be utilized to homogenize, at their 3' termini, RNA molecules synthesized in vitro with T7 RNA polymerase or with an RNA synthesizer. To the best of our knowledge, the method to use this new type of RNA cutter is the only way to remove single 3' terminal nucleotides. By covalently attaching a hook RNA to tRNase ZL, 3' homogeneous RNA products without extra 3' terminal non-encoded nucleotides will be easily purified through phenol/chloroform extraction to remove the hooker/enzyme complex. This consideration implies that our new strategy to generate homogeneous 3' termini of RNA molecules by the 4–7-base RNA cutters would be much easier to perform.
Another potential application of the 4-base RNA cutter
Because Hooker-1, which we used here, can direct RNA cleavage after the sequence 5'...GCCCN...3', the cleavages occur at every 256 nt sequences theoretically. It should be noted that ribozymes can hardly cleave target RNAs with such a low sequence specificity. This new type of 4-base RNA cutter could be applied to analysis of whole gene expression patterns. Total mRNA isolated from the cells will be digested with the 4 nt cutters, subsequently reverse-transcribed and PCR-amplified with sets of specific primers. With this strategy, we will be able to detect not only differences in mRNA expression levels but also single-nucleotide polymorphisms, because the 4 nt RNA cutters should be able to distinguish single-nucleotide differences in mRNAs (12).
ACKNOWLEDGEMENTS
We thank M. Takeda for technical assistance. This work was supported in part by the Science Research Promotion Fund and the Academic Frontier Research Project Grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
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