The N-terminal half-domain of the long form of tRNase Z is required fo
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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
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
ABSTRACT
Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300–400 amino acids, and the other is a long form (tRNase ZL) that contains 800–900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNAArg and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the Km values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.
INTRODUCTION
The genome of almost every organism contains the ELAC1 and/or ELAC2 genes, the products of which have been shown to be transfer RNA (tRNA) 3' processing endoribonucleases (1–6). Although these enzymes have been called 3' tRNase or RNase Z, we renamed them tRNase Z (EC 3.1.26.11 ) to avoid confusion. tRNase Z is an enzyme responsible for the removal of a 3' trailer from pre-tRNA which is transcribed as a larger form. In vitro tRNA 3' processing experiments showed that in most cases the enzymes cleave pre-tRNAs immediately downstream of a discriminator nucleotide, onto which the CCA residues are added to produce mature tRNA in vivo, while in some cases additional cleavages occur 1 nt upstream, or 1 or 2 nt downstream (2–4,6–13). We have recently demonstrated that, exceptionally, the tRNase Z from Thermotoga maritima cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature CCA 3' termini of tRNA molecules (5).
The ELAC1 genes encode short forms of tRNase Z (tRNase ZS) that consist of 300–400 amino acids, while the ELAC2 genes produce long forms of tRNase Z (tRNase ZL) that contain 800–900 amino acids (Figure 1). The multiple protein alignment of tRNase Z orthologs has revealed that the C-terminal half region of tRNase ZL has high similarity to the whole region of tRNase ZS (1). These regions contain a well-conserved histidine motif (1), which has shown to be essential for the tRNase Z activity in the T.maritima enzyme (5).
Figure 1. Structures of tRNase ZS and tRNase ZL. (A) Structural difference between tRNase ZS and tRNase ZL and distribution of their genes among various species. The locations of the pseudo-histidine motif, the Walker A motif and the histidine motif are indicated by a gray box, a hatched box and a black box, respectively. The presence of the ELAC1/2 genes in Homo sapiens (Hsa), A.thaliana (Ath), D.melanogaster (Dme), C.elegans (Cel), S.cerevisiae (Sce), Escherichia coli (Eco), T.maritima (Tma), Thermoplasma acidophilum (Tac) and Pyrobaculum aerophilum (Pae) is indicated by their GenBank/EMBL accession number. The sizes of these tRNase ZSs and tRNase ZLs are 280–363 and 789–942 amino acids, respectively. Asterisks denote that the tRNase Z activities are not yet confirmed experimentally. (B) Multiple protein sequence alignment of histidine motif regions in tRNase ZSs, histidine motif regions in the C-terminal domains of tRNase ZLs, and pseudo-histidine motif regions in the N-terminal domains of tRNase ZLs. The computer program ClustalW was used for the alignment. Identical amino acids are shaded in black and similar amino acids are in gray.
Bacteria and archaea genomes contain a gene for the short form of tRNase Z only, while eukaryote genomes encode either only the long form or both the short and long forms (Figure 1). The genomes of Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae encode only tRNase ZL. On the other hand, the genomes of human and Arabidopsis thaliana contain both tRNase ZS and tRNase ZL genes. This raises an interesting question about whether the short and long forms of tRNase Z play differential roles in the cells. It is possible that one is for nuclear tRNA processing, and that the other for the mitochondrial one. Alternatively, one of these enzymes might have an additional role for some RNA metabolism. The fact that tRNase ZL is 2-fold larger than tRNase ZS, and has the extra N-terminal region that is dispensable for the RNA cleaving catalytic reaction, implies that it is the long form that might have the additional role.
The long form of tRNase Z has the following interesting properties. RNAi knockdown experiments against the C.elegans tRNase ZL gene have shown that tRNase ZL plays a role in germline proliferation and that the down-regulation of the tRNase ZL gene results in sterility (14). On the other hand, over-expression of human tRNase ZL in HeLa cells causes a delay in G2-M progression (15). In addition, the human enzyme has been shown to interact physically with the -tubulin complex (15).
The human tRNase ZL gene has been first identified as a candidate prostate cancer susceptibility gene by positional cloning and mutation screening (1). Two mutations in the human tRNase ZL gene, an insertion/frameshift (1641 ins G) and a missense change (Arg781His), segregate with prostate cancer in two pedigrees (1). The frameshift mutation results in premature termination after the miscoding of 67 residues. In addition, two common missense changes, Ser217Leu and Ala541Thr, seem to be associated with the occurrence of prostate cancer (1,16,17).
Mammalian tRNase ZL can function as a four-base-recognizing RNA cutter (RNase 65) through a relatively stable complex between the enzyme and a 3'-truncated tRNA (18–20). Although little is known about the physiological role and substrate of RNase 65, it has been demonstrated that the 3'-truncated tRNA directs substrate specificity via four base pairings. In addition, the RNase 65 activity has led us to develop a technique to cleave any RNA specifically at any site by mammalian tRNase ZL under the direction of small guide RNA (sgRNA) in vivo as well as in vitro (21–24).
To elucidate possible differential roles and functions between tRNase ZL and tRNase ZS, we started by investigating whether the long and short forms have different preferences for RNA substrates. Here we demonstrate that indeed they have different substrate specificities and that the differences are attributed to the N-terminal half of tRNase ZL. Furthermore, we show that the optimal concentrations of NaCl, MgCl2 and MnCl2 for the in vitro pre-tRNA processing differ between tRNase ZSs and tRNase ZLs.
MATERIALS AND METHODS
Preparation of various tRNase Z proteins
Pig tRNase ZL was intensively fractionated from liver through several column chromatography procedures and further purified by glycerol gradient ultracentrifugation as described previously (25).
The recombinant human tRNase ZL and tRNase ZS, and the N-terminal (residues 1–480) and C-terminal (residues 482–826) half-domains of human tRNase ZL were over-expressed from the pTYB11 expression plasmids (New England BioLabs) in Escherichia coli and purified with chitin beads as described previously (3). The recombinant tRNase ZSs from T.maritima and E.coli were produced from the pQE expression plasmids (Qiagen) in E.coli and purified with nickel–agarose as described in (5).
The recombinant yeast tRNase ZL was expressed from pGEX-4T-3/YKR079C in E.coli as described in (3). Harvested cell pellets were suspended in a 10 ml lysis buffer (50 mM Tris–HCl, pH 7.6, 500 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 23 mM AEBSF, 100 mM EDTA, 2 mM bestatin, 0.3 mM E-64, 0.3 mM pepstatin A), sonicated and centrifuged at 1 00 000 g for 1 h. The cleared lysate was incubated with 50 μl glutathione–Sepharose beads at 4°C for 12 h. After exhaustive washing, the recombinant GST-tRNase ZL fusion protein bound to the beads was incubated with 1 U of thrombin in a buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 and 5 mM MgCl2 at 4°C for 12 h. To stop the thrombin reaction, EGTA was added to the solution, and the excised tRNase ZL in the supernatant was collected.
RNA synthesis
The human pre-tRNAArg R-6TUUU (25), the human micro-pre-tRNAArg R-ATM5 (26), a 40 nt target RNA, the 3'-truncated tRNAArg (20) and a hook RNA were synthesized with T7 RNA polymerase (Takara Shuzo) from the corresponding synthetic DNA templates. The sequences of the target RNA and the hook RNA are 5'-GAAUACGCAUGCUAGCAGGUGCCCGGUGAAAGCUUGAUGU-3' and 5'-GGGCCAGCCAGGUUCGACUCCUGGCU-3', respectively. 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 RNA transcripts for the human pre-tRNAArg, the human micro-pre-tRNAArg, and the 40 nt target RNA were subsequently labeled with fluorescein according to the manufacturer's protocol (Amersham Pharmacia Biotech). Briefly, after the removal of the 5' phosphates of the transcribed RNAs 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.
In vitro RNA cleavage assays
The tRNA 3' processing assays for the fluorescein-labeled human pre-tRNAArg or micro-pre-tRNAArg (0.1 pmol) were performed with tRNase Zs of various origins 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 15 min. The in vitro cleavage assays for the fluorescein-labeled 40 nt target RNA (0.1 pmol) were carried out in the presence of the unlabeled 3'-truncated tRNAArg or hook RNA under the same conditions as above. In assays to determine optimal enzyme conditions, one of the parameters of the conditions was varied and the enzyme amounts used ranged from 10 to 80 ng. 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).
Kinetic analysis
Various amounts (0.5–10 μM) of the T.maritima pre-tRNAArg(CCA) and the human pre-tRNAArg R-6TUUU were assayed at 37°C for 5 min using 0.1 μM of tRNase Z in a mixture (6 μl) containing 10 mM Tris–HCl, pH 7.5, 1.5 mM dithiothreitol and 3.2 mM MgCl2. The reaction was in the linear phase during the 5 min. After resolution of the reaction products on a 10% polyacrylamide–8 M urea gel, cleavage percentages were quantitated with the Typhoon 9210. Km values were obtained from the best-fit line on a Lineweaver–Burk plot.
RESULTS
The long and short forms of tRNase Z from various organisms
To obtain the long and short forms of tRNase Z from human, yeast, E.coli and T.maritima, we over-expressed recombinant tRNase Zs in E.coli and purified them by affinity chromatography. Pig tRNase ZL was purified from pig liver through various column chromatography procedures.
These enzymes were tested for 3' processing of the human pre-tRNAArg that was 5'-end-labeled with fluorescein (Figure 2A). After a 15 min reaction at 50°C, the cleavage products were resolved on a denaturing sequencing gel. All tRNase Zs cleaved the human pre-tRNAArg with the exception of the N-terminal half of human tRNase ZL (Figure 2B). Although T.maritima tRNase ZS cleaved the substrate primarily after U at 75 nt, the cleavages by the other enzymes occurred principally after the discriminator nucleotide (Figure 2B).
Figure 2. Tests of the long and short forms of tRNase Z from various organisms for in vitro tRNA 3' processing activity. (A) A secondary structure of the human pre-tRNAArg R-6TUUU. Discontinuous and continuous arrows denote the primary cleavage sites by T.maritima tRNase ZS and by the other active enzymes, respectively. (B) The in vitro tRNA 3' processing assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled pre-tRNAArg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The pre-tRNAArg and the primary 5' cleavage products are indicated by a bar and arrows, respectively, with their nucleotide size. I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
Optimal catalytic conditions differ between tRNase ZL and tRNase ZS
To find out differences in enzymatic properties between tRNase ZL and tRNase ZS, we investigated the optimal conditions for the 3' processing reaction of the human pre-tRNAArg by each enzyme. We measured percentage cleavage by varying one of the following reaction parameters: temperature, pH and NaCl concentration. All the enzymes showed the highest activities at 50°C except the T.maritima enzyme, which was the most active at 60°C (Figure 3A and D). As for the optimal pH, each tRNase ZL worked best around pH 8, while each tRNase ZS and the human C-terminal-half tRNase ZL cleaved the substrate most efficiently around pH 6 with the exception of the T.maritima enzyme, which was the most active around pH 8 (Figure 3B and E). All three tRNase ZLs worked best at 100 mM NaCl, while T.maritima tRNase ZS and the human C-terminal-half tRNase ZL were the most active at 200 mM NaCl, and human and E.coli tRNase ZSs were the most efficient at 40 and 20 mM NaCl, respectively (Figure 3C and F).
Figure 3. Optimal conditions for in vitro pre-tRNAArg R-6TUUU processing by tRNase Zs from various organisms. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various temperatures. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various pHs. (C) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various NaCl concentrations. (D) Relative cleavage activities of human (diamond), T.maritima (square), and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various temperatures. (E) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various pHs. (F) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various NaCl concentrations.
We also examined the optimal concentrations of Mg2+ or Mn2+ for the activity. Each tRNase ZL was the most efficient 20 mM MgCl2, while human tRNase ZS and human C-terminal-half tRNase ZL was the most active 50 mM MgCl2, and T.maritima and E.coli tRNase ZSs showed the highest activities at 1 and 5 mM, respectively (Figure 4A and C). The optimal MnCl2 concentrations for the tRNase ZLs and the tRNase ZSs were 20 and 1 mM, respectively (Figure 4B and D). The activity of the C-terminal half of human tRNase ZL was not detected in the range of the MnCl2 concentrations we tested.
Figure 4. Enzymatic activities of various tRNase Zs against the human pre-tRNAArg R-6TUUU at various concentrations of MgCl2 or MnCl2. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various MgCl2 concentrations. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human and yeast tRNase ZLs at various MnCl2 concentrations. (C) Relative cleavage activities of human (diamond), T.maritima (square) and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various MgCl2 concentrations. (D) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs at various MnCl2 concentrations.
It should be noted that the optimal conditions for the cleavage of T.maritima pre-tRNAArg(CCA) by T.maritima tRNase ZS were different from those for the cleavage of human pre-tRNAArg. The T.maritima enzyme showed its maximum activity against T.maritima pre-tRNAArg(CCA) at 60°C at pH 9 in 100 mM NaCl in the presence of 10 mM MgCl2 or 0.5 mM MnCl2 (5).
The long form of tRNase Z can process the micro-pre-tRNA substrate
We have already demonstrated that pig tRNase ZL can also cleave micro-pre-tRNA substrates, which consist of the T-stem–loop, the acceptor stem, and a 3' trailer (26). To examine whether such a micro-pre-tRNA is also a substrate for other tRNase ZLs, we tested the recombinant human and yeast tRNase ZLs for cleavage of the human micro-pre-tRNAArg (Figure 5A). These enzymes cleaved the small substrate principally after the discriminator as the pig enzyme did (Figure 5B).
Figure 5. In vitro assays for micro-pre-tRNAArg cleavage by tRNase ZL and tRNase ZS from various organisms. (A) A secondary structure of the human micro-pre-tRNAArg R-ATM5. An arrow denotes the primary cleavage site by tRNase Z. (B) The in vitro cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled human micro-pre-tRNAArg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. The micro-pre-tRNAArg and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled substrate; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
The cleavage efficiencies relative to those for the full-length pre-tRNA differed depending on the origins of the enzymes. Pig tRNase ZL cleaved the micro-pre-tRNA with a relative activity of 67.1%, while the human and yeast enzymes processed the substrate with relative activities of 26.6 and 13.6%, respectively (Table 1).
Table 1. Relative activities of tRNase ZLs against various substrates
Next, we assayed the tRNase ZSs from human, E.coli, and T.maritima for the small substrate cleavage. None of these enzymes cleaved the substrate (Figure 5B), even though the full-length pre-tRNAArg was processed under the same conditions (Figure 2). These enzymes were not able to cleave the micro-pre-tRNAArg even in the presence of the N-terminal half of human tRNase ZL with the exception of E.coli tRNase ZS, which became very weakly active (Figure 5B). The small substrate was barely cleaved by the C-terminal half of human tRNase ZL but not by the N-terminal half (Figure 5B).
The N-terminal half of the long form of tRNase Z is essential for the RNase 65 activity
We examined whether the recombinant tRNase ZLs from human and yeast show the RNase 65 activity, which has been detected with respect to mouse and pig tRNase ZLs (18–20). Both recombinant tRNase ZLs cleaved the 40 nt target RNA in the presence of the 3'-truncated tRNA (Figure 6). Each cleavage occurred primarily after the nucleotide in the target corresponding to the discriminator nucleotide when bound to the 3'-truncated tRNA. The activities relative to those for the pre-tRNA cleavage were 84.7, 67.6 and 33.4% for pig, human and yeast tRNase ZLs, respectively (Table 1).
Figure 6. The RNase 65 assays for various tRNase ZLs and tRNase ZSs. (A) A secondary structure of the complex of the target RNA and the 3'-truncated tRNA. The 5'-terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3'-terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase ZL. (B) The RNase 65 assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled 3'-truncated tRNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
The recombinant tRNase ZSs were also tested for the RNase 65 activity, but none of the enzymes cleaved the target (Figure 6B). Neither the N- nor C-terminal half region of human tRNase ZL showed the RNase 65 activity (Figure 6B). Furthermore, the RNase 65 activity was not observed even in coexistence of the tRNase ZSs or the C-terminal half of human tRNase ZL with the N-terminal half (Figure 6B).
The long form of tRNase Z can cleave a separate RNA in the presence of a hook RNA
To further characterize the difference in substrate preference between the long and short forms of tRNase Z, we developed a new RNA cleavage assay system using a new type of sgRNA, hook RNA. From the previous results (22–24,26) and the above data, we deduced that a 26 nt hook RNA would guide cleavage of a separate target RNA by tRNase Z through the micro-pre-tRNA-like complex between the target and the hook RNA (Figure 7A).
Figure 7. Hook RNA-guided specific RNA cleavage by the long form of tRNase Z from various organisms. (A) A secondary structure of the complex of the target RNA and the hook RNA. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase Z. (B) The specific RNA cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled hook RNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
As expected all tRNase ZLs cleaved the target RNA under the direction of the hook RNA, while all short forms did not, regardless of the presence of the N-terminal half of human tRNase ZL (Figure 7B). Each cleavage site was located primarily after the nucleotide corresponding to the discriminator nucleotide. The relative cleavage efficiencies were similar to those for the RNase 65 activity; the relative activities for pig, human, and yeast tRNase ZLs were 100.7, 57.3 and 26.4%, respectively (Table 1).
The Km values for pre-tRNA cleavages by tRNase ZS are much higher than those by tRNase ZL
To assess the affinities of tRNase Zs to substrates, we determined the Km values for pre-tRNA cleavages. Various concentrations of the human pre-tRNAArg R-6TUUU were assayed using human and yeast tRNase ZLs, human and T.maritima tRNase ZSs, and various amounts of the T.maritima pre-tRNAArg(CCA) were tested for cleavages by T.maritima tRNase ZS. The Km values were obtained from the best-fit line on a Lineweaver–Burk plot. The Km values for cleavage of R-6TUUU by human and yeast tRNase ZL were 0.39 and 0.74 μM, respectively, which were much lower than those by human (1.28 μM) and T.maritima (2.40 μM) tRNase ZSs (Table 2). The Km value (2.40 μM) for cleavage of R-6TUUU by the T.maritima enzyme was 1.5-fold higher than that (1.58 μM) for pre-tRNAArg(CCA) cleavage (Table 2). Because T.maritima tRNase ZS appears to have a CCA binding domain (5), the higher value for the R-6TUUU cleavage could be due to the lack of the 74CCA76 sequence in the substrate.
Table 2. Affinities of various tRNase Zs to substrates indicated by Km values
DISCUSSION
A functional role of the N-terminal half of tRNase ZL
We found out that the optimal concentrations of NaCl, MgCl2 and MnCl2 for the in vitro pre-tRNA processing differ between tRNase ZSs and tRNase ZLs (Figures 3 and 4). With respect to the major structural difference between tRNase ZL and tRNase ZS, tRNase ZL has the extra N-terminal domain composed of 500 amino acids, which contains the pseudo-histidine motif (1) and has a similarity in some degree to the C-terminal domain and tRNase ZS (Figure 1). The present observation that only the long forms of tRNase Z can cleave the RNA complexes (Figures 6 and 7) implies that the N-terminal half has an additional important role in enzyme–substrate interactions.
All of the tRNase ZSs did not cleave the micro-pre-tRNA at all, and the C-terminal domain of human tRNase ZL cleaved it inefficiently as compared with the full-length human tRNase ZL (Figure 5). This would be probably because the substrate–enzyme interaction is weakened due to the lack of the D-stem–loop and the anticodon–stem–loop in the substrate. The N-terminal half of tRNase ZL would be involved in the interaction with the substrate at least through the T-stem–loop and the acceptor stem, and would strengthen this interaction. This would allow tRNase ZL to recognize and to cleave the micro-pre-tRNA easily.
This idea is supported by previous (26,27) and present kinetic experiments. The Km values for cleavages of full-length pre-tRNAsArg by pig tRNase ZL were quantitated to be 0.6 μM regardless of the presence of a 5' leader, and the Km values for cleavage of the full-length pre-tRNAArg R-6TUUU by human and yeast tRNase ZLs were 0.39 and 0.74 μM (Table 2). On the other hand, the Km values for cleavages of full-length pre-tRNAsArg by human and T.maritima tRNase ZSs were 1.3–2.4 μM (Table 2). About 3-fold lower Km values for tRNase ZL indicate that its N-terminal half is indeed important for stronger recognition of the substrates. The Km value for cleavage of the micro-pre-tRNAArg R-ATM5 by pig tRNase ZL was 0.57 μM, which was the same as the Km value for cleavage of the full-length pre-tRNAsArg R-UUU (Table 2). This is consistent with the observation that pig tRNase ZL has a strong activity against the micro-pre-tRNAArg (Table 1).
In the above context, we would also be able to explain why the RNase 65 activity was observed only with respect to tRNase ZL (Figure 6B). The discontinuous acceptor stem in the complex between the target RNA and the 3'-truncated tRNA, and the 5' extra nucleotides in the target before the 4 nt recognition sequence (Figure 6A) would disturb the enzyme–substrate interaction. These anomalies would make it difficult for the tRNase ZS to recognize the RNA complex even though the complex has the D-stem–loop and the anticodon–stem–loop. In contrast, tRNase ZL can recognize the substrate probably with the help of the N-terminal half-domain.
This interpretation would also hold for the interaction of tRNase Z with the complex between the target RNA and the hook RNA. This complex has an additional anomaly in that it does not contain the D-stem–loop and the anticodon–stem–loop (Figure 7A). tRNase ZS would have more difficulty in substrate recognition, while, thanks to the N-terminal half, tRNase ZL would be able to endure even such a difficulty.
The N-terminal half-domain, when added to the reactions, did not cause stronger interactions of human or T.maritima tRNase ZS or the C-terminal half with the micro-pre-tRNA or the RNA complexes, while E.coli tRNase ZS became very weakly active against the micro-pre-tRNA in the presence of the N-terminal half. These observations suggest that the covalent connection between the N- and C-terminal half-domains is important but not necessarily prerequisite for the functional role of the N-terminal half.
The Walker A motif (28) found in the N-terminal domain of human tRNase ZL (Figure 1A) might be involved in this N-terminal function possibly through binding to the 5' nucleotide triphosphate of pre-tRNA. The much lower activities of yeast tRNase ZL against the micro-pre-tRNA and the RNA complexes (Table 1) may be due to the lack of this motif in the yeast enzyme. Consistently, the Km value for the R-6TUUU cleavage by yeast tRNase ZL is 1.9- and 1.3-fold higher than those by the human and pig enzymes, respectively (Table 2).
Other roles of tRNase ZL than the tRNA 3' processing
RNAi knockdown of the tRNase ZL gene in C.elegans causes slow growth and sterility, which results from a drastic reduction in germline proliferation (14). The nematode tRNase ZL is also needed for the germline over-proliferation phenotypes observed in three different alleles (14). In addition, a reduction of tRNase ZL suppresses the multivulva phenotype resulting from two different activating mutations in ras (14). These phenomena would be simply explained through the role of tRNase ZL in the tRNA 3' processing. Because a large number of tRNA molecules are required for early embryogenesis, if a sufficient amount of tRNase ZL that is essential for tRNA biosynthesis is not generated in the germline cells, their proliferations would be severely injured. Indeed, an enormous number of tRNase ZL molecules exist in each Xenopus laevis oocyte (7). This number has been estimated to be 5 x 1011 molecules, which is comparable to 5 x 1012 for the number of tRNA molecules.
It has been shown that in HeLa cells, tRNase ZL physically interacts with the -tubulin complex, suggesting that tRNase ZL might play a role in mitosis as a modulator of centrosome assembly (15). Consistently, cells over-expressing tRNase ZL undergo a delay in cell cycle progression during the G2 phase (15). In contrast to the C.elegans case, the pre-tRNA cleaving activity of tRNase ZL would not be directly related to this phenomenon. Nothing is known about how tRNase ZL interacts with the -tubulin complex and how this enzyme is involved in mitosis. In vitro binding assays for the interactions between -tubulin and various recombinant tRNase ZL fragments would elucidate whether the modulator function is attributed to the N-terminal half region, which is not essential for the enzymatic activity.
We have demonstrated that tRNase ZL shows the RNase 65 activity in vitro in the presence of 3'-truncated tRNAs, and that these RNAs exist ubiquitously in mammalian cell extracts so far tested (18–20). This suggests that tRNase ZL works as 4 nt RNA cutters in other cellular RNA metabolisms. The fact that tRNase ZL can also act as a different type of 4 nt RNA cutter in vitro under the direction of hook RNA implies that this cutter could work in the cells if there exists hook RNA. Recently, a huge number of miRNAs that are believed to work as translational regulators have been discovered (29). The length of miRNAs is 21–23 nt, which is similar to the length of hook RNA, suggesting that some of the miRNAs could work as hook RNAs that direct cellular RNA cleavage by endogenous tRNase ZL. Other unidentified small RNAs might also work as sgRNAs in the cells.
tRNase ZL together with sgRNA can be used as a useful molecular biology tool
We have shown that mammalian tRNase ZL can cleave any RNA at any desired site in vitro under the direction of sgRNA, which includes 5' half tRNA and heptamer RNA (21–24). We have also examined the efficacy of this specific RNA targeting method in the living cells by introducing sgRNAs either as their expression plasmids or as 2'-O-methyl RNAs (24). The expressions of the exogenous reporter genes for E.coli chloramphenicol acetyltransferase and firefly luciferase were down-regulated by appropriately designed sgRNAs in human and dog culture cells, and the down-regulation continued for at least 48 h. A 2'-O-methyl heptamer for targeting the endogenous Bcl-2 mRNA has been also shown to work in Sarcoma 180 cells.
Although these results have indicated that indeed appropriate sgRNAs can repress the targeted gene expressions, no direct evidence exists, yet, demonstrating that the down-regulation is due to specific mRNA cleavage by endogenous tRNase ZL and not due to simple antisense effects or other different mechanisms. We would be able to examine directly whether the in vivo down-regulation is really due to tRNase ZL cleavage by knocking-down either tRNase ZL or tRNase ZS gene expression by RNAi. From the present results which show that tRNase ZL and not tRNase ZS can cleave RNA complexes, we can predict that only when the tRNase ZL gene expression is repressed is the down-regulation by sgRNAs abolished.
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
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
ABSTRACT
Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300–400 amino acids, and the other is a long form (tRNase ZL) that contains 800–900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNAArg and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the Km values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.
INTRODUCTION
The genome of almost every organism contains the ELAC1 and/or ELAC2 genes, the products of which have been shown to be transfer RNA (tRNA) 3' processing endoribonucleases (1–6). Although these enzymes have been called 3' tRNase or RNase Z, we renamed them tRNase Z (EC 3.1.26.11 ) to avoid confusion. tRNase Z is an enzyme responsible for the removal of a 3' trailer from pre-tRNA which is transcribed as a larger form. In vitro tRNA 3' processing experiments showed that in most cases the enzymes cleave pre-tRNAs immediately downstream of a discriminator nucleotide, onto which the CCA residues are added to produce mature tRNA in vivo, while in some cases additional cleavages occur 1 nt upstream, or 1 or 2 nt downstream (2–4,6–13). We have recently demonstrated that, exceptionally, the tRNase Z from Thermotoga maritima cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature CCA 3' termini of tRNA molecules (5).
The ELAC1 genes encode short forms of tRNase Z (tRNase ZS) that consist of 300–400 amino acids, while the ELAC2 genes produce long forms of tRNase Z (tRNase ZL) that contain 800–900 amino acids (Figure 1). The multiple protein alignment of tRNase Z orthologs has revealed that the C-terminal half region of tRNase ZL has high similarity to the whole region of tRNase ZS (1). These regions contain a well-conserved histidine motif (1), which has shown to be essential for the tRNase Z activity in the T.maritima enzyme (5).
Figure 1. Structures of tRNase ZS and tRNase ZL. (A) Structural difference between tRNase ZS and tRNase ZL and distribution of their genes among various species. The locations of the pseudo-histidine motif, the Walker A motif and the histidine motif are indicated by a gray box, a hatched box and a black box, respectively. The presence of the ELAC1/2 genes in Homo sapiens (Hsa), A.thaliana (Ath), D.melanogaster (Dme), C.elegans (Cel), S.cerevisiae (Sce), Escherichia coli (Eco), T.maritima (Tma), Thermoplasma acidophilum (Tac) and Pyrobaculum aerophilum (Pae) is indicated by their GenBank/EMBL accession number. The sizes of these tRNase ZSs and tRNase ZLs are 280–363 and 789–942 amino acids, respectively. Asterisks denote that the tRNase Z activities are not yet confirmed experimentally. (B) Multiple protein sequence alignment of histidine motif regions in tRNase ZSs, histidine motif regions in the C-terminal domains of tRNase ZLs, and pseudo-histidine motif regions in the N-terminal domains of tRNase ZLs. The computer program ClustalW was used for the alignment. Identical amino acids are shaded in black and similar amino acids are in gray.
Bacteria and archaea genomes contain a gene for the short form of tRNase Z only, while eukaryote genomes encode either only the long form or both the short and long forms (Figure 1). The genomes of Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae encode only tRNase ZL. On the other hand, the genomes of human and Arabidopsis thaliana contain both tRNase ZS and tRNase ZL genes. This raises an interesting question about whether the short and long forms of tRNase Z play differential roles in the cells. It is possible that one is for nuclear tRNA processing, and that the other for the mitochondrial one. Alternatively, one of these enzymes might have an additional role for some RNA metabolism. The fact that tRNase ZL is 2-fold larger than tRNase ZS, and has the extra N-terminal region that is dispensable for the RNA cleaving catalytic reaction, implies that it is the long form that might have the additional role.
The long form of tRNase Z has the following interesting properties. RNAi knockdown experiments against the C.elegans tRNase ZL gene have shown that tRNase ZL plays a role in germline proliferation and that the down-regulation of the tRNase ZL gene results in sterility (14). On the other hand, over-expression of human tRNase ZL in HeLa cells causes a delay in G2-M progression (15). In addition, the human enzyme has been shown to interact physically with the -tubulin complex (15).
The human tRNase ZL gene has been first identified as a candidate prostate cancer susceptibility gene by positional cloning and mutation screening (1). Two mutations in the human tRNase ZL gene, an insertion/frameshift (1641 ins G) and a missense change (Arg781His), segregate with prostate cancer in two pedigrees (1). The frameshift mutation results in premature termination after the miscoding of 67 residues. In addition, two common missense changes, Ser217Leu and Ala541Thr, seem to be associated with the occurrence of prostate cancer (1,16,17).
Mammalian tRNase ZL can function as a four-base-recognizing RNA cutter (RNase 65) through a relatively stable complex between the enzyme and a 3'-truncated tRNA (18–20). Although little is known about the physiological role and substrate of RNase 65, it has been demonstrated that the 3'-truncated tRNA directs substrate specificity via four base pairings. In addition, the RNase 65 activity has led us to develop a technique to cleave any RNA specifically at any site by mammalian tRNase ZL under the direction of small guide RNA (sgRNA) in vivo as well as in vitro (21–24).
To elucidate possible differential roles and functions between tRNase ZL and tRNase ZS, we started by investigating whether the long and short forms have different preferences for RNA substrates. Here we demonstrate that indeed they have different substrate specificities and that the differences are attributed to the N-terminal half of tRNase ZL. Furthermore, we show that the optimal concentrations of NaCl, MgCl2 and MnCl2 for the in vitro pre-tRNA processing differ between tRNase ZSs and tRNase ZLs.
MATERIALS AND METHODS
Preparation of various tRNase Z proteins
Pig tRNase ZL was intensively fractionated from liver through several column chromatography procedures and further purified by glycerol gradient ultracentrifugation as described previously (25).
The recombinant human tRNase ZL and tRNase ZS, and the N-terminal (residues 1–480) and C-terminal (residues 482–826) half-domains of human tRNase ZL were over-expressed from the pTYB11 expression plasmids (New England BioLabs) in Escherichia coli and purified with chitin beads as described previously (3). The recombinant tRNase ZSs from T.maritima and E.coli were produced from the pQE expression plasmids (Qiagen) in E.coli and purified with nickel–agarose as described in (5).
The recombinant yeast tRNase ZL was expressed from pGEX-4T-3/YKR079C in E.coli as described in (3). Harvested cell pellets were suspended in a 10 ml lysis buffer (50 mM Tris–HCl, pH 7.6, 500 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 23 mM AEBSF, 100 mM EDTA, 2 mM bestatin, 0.3 mM E-64, 0.3 mM pepstatin A), sonicated and centrifuged at 1 00 000 g for 1 h. The cleared lysate was incubated with 50 μl glutathione–Sepharose beads at 4°C for 12 h. After exhaustive washing, the recombinant GST-tRNase ZL fusion protein bound to the beads was incubated with 1 U of thrombin in a buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 and 5 mM MgCl2 at 4°C for 12 h. To stop the thrombin reaction, EGTA was added to the solution, and the excised tRNase ZL in the supernatant was collected.
RNA synthesis
The human pre-tRNAArg R-6TUUU (25), the human micro-pre-tRNAArg R-ATM5 (26), a 40 nt target RNA, the 3'-truncated tRNAArg (20) and a hook RNA were synthesized with T7 RNA polymerase (Takara Shuzo) from the corresponding synthetic DNA templates. The sequences of the target RNA and the hook RNA are 5'-GAAUACGCAUGCUAGCAGGUGCCCGGUGAAAGCUUGAUGU-3' and 5'-GGGCCAGCCAGGUUCGACUCCUGGCU-3', respectively. 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 RNA transcripts for the human pre-tRNAArg, the human micro-pre-tRNAArg, and the 40 nt target RNA were subsequently labeled with fluorescein according to the manufacturer's protocol (Amersham Pharmacia Biotech). Briefly, after the removal of the 5' phosphates of the transcribed RNAs 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.
In vitro RNA cleavage assays
The tRNA 3' processing assays for the fluorescein-labeled human pre-tRNAArg or micro-pre-tRNAArg (0.1 pmol) were performed with tRNase Zs of various origins 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 15 min. The in vitro cleavage assays for the fluorescein-labeled 40 nt target RNA (0.1 pmol) were carried out in the presence of the unlabeled 3'-truncated tRNAArg or hook RNA under the same conditions as above. In assays to determine optimal enzyme conditions, one of the parameters of the conditions was varied and the enzyme amounts used ranged from 10 to 80 ng. 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).
Kinetic analysis
Various amounts (0.5–10 μM) of the T.maritima pre-tRNAArg(CCA) and the human pre-tRNAArg R-6TUUU were assayed at 37°C for 5 min using 0.1 μM of tRNase Z in a mixture (6 μl) containing 10 mM Tris–HCl, pH 7.5, 1.5 mM dithiothreitol and 3.2 mM MgCl2. The reaction was in the linear phase during the 5 min. After resolution of the reaction products on a 10% polyacrylamide–8 M urea gel, cleavage percentages were quantitated with the Typhoon 9210. Km values were obtained from the best-fit line on a Lineweaver–Burk plot.
RESULTS
The long and short forms of tRNase Z from various organisms
To obtain the long and short forms of tRNase Z from human, yeast, E.coli and T.maritima, we over-expressed recombinant tRNase Zs in E.coli and purified them by affinity chromatography. Pig tRNase ZL was purified from pig liver through various column chromatography procedures.
These enzymes were tested for 3' processing of the human pre-tRNAArg that was 5'-end-labeled with fluorescein (Figure 2A). After a 15 min reaction at 50°C, the cleavage products were resolved on a denaturing sequencing gel. All tRNase Zs cleaved the human pre-tRNAArg with the exception of the N-terminal half of human tRNase ZL (Figure 2B). Although T.maritima tRNase ZS cleaved the substrate primarily after U at 75 nt, the cleavages by the other enzymes occurred principally after the discriminator nucleotide (Figure 2B).
Figure 2. Tests of the long and short forms of tRNase Z from various organisms for in vitro tRNA 3' processing activity. (A) A secondary structure of the human pre-tRNAArg R-6TUUU. Discontinuous and continuous arrows denote the primary cleavage sites by T.maritima tRNase ZS and by the other active enzymes, respectively. (B) The in vitro tRNA 3' processing assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled pre-tRNAArg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The pre-tRNAArg and the primary 5' cleavage products are indicated by a bar and arrows, respectively, with their nucleotide size. I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
Optimal catalytic conditions differ between tRNase ZL and tRNase ZS
To find out differences in enzymatic properties between tRNase ZL and tRNase ZS, we investigated the optimal conditions for the 3' processing reaction of the human pre-tRNAArg by each enzyme. We measured percentage cleavage by varying one of the following reaction parameters: temperature, pH and NaCl concentration. All the enzymes showed the highest activities at 50°C except the T.maritima enzyme, which was the most active at 60°C (Figure 3A and D). As for the optimal pH, each tRNase ZL worked best around pH 8, while each tRNase ZS and the human C-terminal-half tRNase ZL cleaved the substrate most efficiently around pH 6 with the exception of the T.maritima enzyme, which was the most active around pH 8 (Figure 3B and E). All three tRNase ZLs worked best at 100 mM NaCl, while T.maritima tRNase ZS and the human C-terminal-half tRNase ZL were the most active at 200 mM NaCl, and human and E.coli tRNase ZSs were the most efficient at 40 and 20 mM NaCl, respectively (Figure 3C and F).
Figure 3. Optimal conditions for in vitro pre-tRNAArg R-6TUUU processing by tRNase Zs from various organisms. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various temperatures. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various pHs. (C) Relative cleavage activities of pig, human, and yeast tRNase ZLs at various NaCl concentrations. (D) Relative cleavage activities of human (diamond), T.maritima (square), and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various temperatures. (E) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various pHs. (F) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs and the C-terminal half region of human tRNase ZL at various NaCl concentrations.
We also examined the optimal concentrations of Mg2+ or Mn2+ for the activity. Each tRNase ZL was the most efficient 20 mM MgCl2, while human tRNase ZS and human C-terminal-half tRNase ZL was the most active 50 mM MgCl2, and T.maritima and E.coli tRNase ZSs showed the highest activities at 1 and 5 mM, respectively (Figure 4A and C). The optimal MnCl2 concentrations for the tRNase ZLs and the tRNase ZSs were 20 and 1 mM, respectively (Figure 4B and D). The activity of the C-terminal half of human tRNase ZL was not detected in the range of the MnCl2 concentrations we tested.
Figure 4. Enzymatic activities of various tRNase Zs against the human pre-tRNAArg R-6TUUU at various concentrations of MgCl2 or MnCl2. (A) Relative cleavage activities of pig (circle), human (square) and yeast (triangle) tRNase ZLs at various MgCl2 concentrations. In each case, activities are normalized against the maximum activity as 100%. (B) Relative cleavage activities of pig, human and yeast tRNase ZLs at various MnCl2 concentrations. (C) Relative cleavage activities of human (diamond), T.maritima (square) and E.coli (triangle) tRNase ZSs and the C-terminal half region (circle) of human tRNase ZL at various MgCl2 concentrations. (D) Relative cleavage activities of human, T.maritima, and E.coli tRNase ZSs at various MnCl2 concentrations.
It should be noted that the optimal conditions for the cleavage of T.maritima pre-tRNAArg(CCA) by T.maritima tRNase ZS were different from those for the cleavage of human pre-tRNAArg. The T.maritima enzyme showed its maximum activity against T.maritima pre-tRNAArg(CCA) at 60°C at pH 9 in 100 mM NaCl in the presence of 10 mM MgCl2 or 0.5 mM MnCl2 (5).
The long form of tRNase Z can process the micro-pre-tRNA substrate
We have already demonstrated that pig tRNase ZL can also cleave micro-pre-tRNA substrates, which consist of the T-stem–loop, the acceptor stem, and a 3' trailer (26). To examine whether such a micro-pre-tRNA is also a substrate for other tRNase ZLs, we tested the recombinant human and yeast tRNase ZLs for cleavage of the human micro-pre-tRNAArg (Figure 5A). These enzymes cleaved the small substrate principally after the discriminator as the pig enzyme did (Figure 5B).
Figure 5. In vitro assays for micro-pre-tRNAArg cleavage by tRNase ZL and tRNase ZS from various organisms. (A) A secondary structure of the human micro-pre-tRNAArg R-ATM5. An arrow denotes the primary cleavage site by tRNase Z. (B) The in vitro cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled human micro-pre-tRNAArg (0.1 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea gel. The micro-pre-tRNAArg and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled substrate; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
The cleavage efficiencies relative to those for the full-length pre-tRNA differed depending on the origins of the enzymes. Pig tRNase ZL cleaved the micro-pre-tRNA with a relative activity of 67.1%, while the human and yeast enzymes processed the substrate with relative activities of 26.6 and 13.6%, respectively (Table 1).
Table 1. Relative activities of tRNase ZLs against various substrates
Next, we assayed the tRNase ZSs from human, E.coli, and T.maritima for the small substrate cleavage. None of these enzymes cleaved the substrate (Figure 5B), even though the full-length pre-tRNAArg was processed under the same conditions (Figure 2). These enzymes were not able to cleave the micro-pre-tRNAArg even in the presence of the N-terminal half of human tRNase ZL with the exception of E.coli tRNase ZS, which became very weakly active (Figure 5B). The small substrate was barely cleaved by the C-terminal half of human tRNase ZL but not by the N-terminal half (Figure 5B).
The N-terminal half of the long form of tRNase Z is essential for the RNase 65 activity
We examined whether the recombinant tRNase ZLs from human and yeast show the RNase 65 activity, which has been detected with respect to mouse and pig tRNase ZLs (18–20). Both recombinant tRNase ZLs cleaved the 40 nt target RNA in the presence of the 3'-truncated tRNA (Figure 6). Each cleavage occurred primarily after the nucleotide in the target corresponding to the discriminator nucleotide when bound to the 3'-truncated tRNA. The activities relative to those for the pre-tRNA cleavage were 84.7, 67.6 and 33.4% for pig, human and yeast tRNase ZLs, respectively (Table 1).
Figure 6. The RNase 65 assays for various tRNase ZLs and tRNase ZSs. (A) A secondary structure of the complex of the target RNA and the 3'-truncated tRNA. The 5'-terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3'-terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase ZL. (B) The RNase 65 assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled 3'-truncated tRNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
The recombinant tRNase ZSs were also tested for the RNase 65 activity, but none of the enzymes cleaved the target (Figure 6B). Neither the N- nor C-terminal half region of human tRNase ZL showed the RNase 65 activity (Figure 6B). Furthermore, the RNase 65 activity was not observed even in coexistence of the tRNase ZSs or the C-terminal half of human tRNase ZL with the N-terminal half (Figure 6B).
The long form of tRNase Z can cleave a separate RNA in the presence of a hook RNA
To further characterize the difference in substrate preference between the long and short forms of tRNase Z, we developed a new RNA cleavage assay system using a new type of sgRNA, hook RNA. From the previous results (22–24,26) and the above data, we deduced that a 26 nt hook RNA would guide cleavage of a separate target RNA by tRNase Z through the micro-pre-tRNA-like complex between the target and the hook RNA (Figure 7A).
Figure 7. Hook RNA-guided specific RNA cleavage by the long form of tRNase Z from various organisms. (A) A secondary structure of the complex of the target RNA and the hook RNA. The 5' terminal sequence 5'-GAAUACGCAUGCUAGC-3' and the 3' terminal sequence 5'-AAAGCUUGAUGU-3' are omitted in the target RNA. An arrow denotes the primary cleavage site by tRNase Z. (B) The specific RNA cleavage assays. Each recombinant protein (50 ng) or pig liver tRNase ZL (20 ng) was incubated with the fluorescein-labeled target RNA (0.1 pmol) in the presence of the unlabeled hook RNA (5 pmol) in the absence or presence of the N-terminal half (50 ng) of human tRNase ZL at 50°C for 15 min under the standard assay conditions. The cleavage reactions were analyzed on a 10% polyacrylamide–8 M urea sequencing gel. The target RNA and the primary 5' cleavage product are indicated by a bar and an arrow, respectively, with their nucleotide size. L, the alkaline ladder of the fluorescein-labeled target RNA; I, input RNA; P, pig; H, human; Y, yeast; Tm, T.maritima; Ec, E.coli; N, the C-terminal half of human tRNase ZL; C, the N-terminal half of human tRNase ZL.
As expected all tRNase ZLs cleaved the target RNA under the direction of the hook RNA, while all short forms did not, regardless of the presence of the N-terminal half of human tRNase ZL (Figure 7B). Each cleavage site was located primarily after the nucleotide corresponding to the discriminator nucleotide. The relative cleavage efficiencies were similar to those for the RNase 65 activity; the relative activities for pig, human, and yeast tRNase ZLs were 100.7, 57.3 and 26.4%, respectively (Table 1).
The Km values for pre-tRNA cleavages by tRNase ZS are much higher than those by tRNase ZL
To assess the affinities of tRNase Zs to substrates, we determined the Km values for pre-tRNA cleavages. Various concentrations of the human pre-tRNAArg R-6TUUU were assayed using human and yeast tRNase ZLs, human and T.maritima tRNase ZSs, and various amounts of the T.maritima pre-tRNAArg(CCA) were tested for cleavages by T.maritima tRNase ZS. The Km values were obtained from the best-fit line on a Lineweaver–Burk plot. The Km values for cleavage of R-6TUUU by human and yeast tRNase ZL were 0.39 and 0.74 μM, respectively, which were much lower than those by human (1.28 μM) and T.maritima (2.40 μM) tRNase ZSs (Table 2). The Km value (2.40 μM) for cleavage of R-6TUUU by the T.maritima enzyme was 1.5-fold higher than that (1.58 μM) for pre-tRNAArg(CCA) cleavage (Table 2). Because T.maritima tRNase ZS appears to have a CCA binding domain (5), the higher value for the R-6TUUU cleavage could be due to the lack of the 74CCA76 sequence in the substrate.
Table 2. Affinities of various tRNase Zs to substrates indicated by Km values
DISCUSSION
A functional role of the N-terminal half of tRNase ZL
We found out that the optimal concentrations of NaCl, MgCl2 and MnCl2 for the in vitro pre-tRNA processing differ between tRNase ZSs and tRNase ZLs (Figures 3 and 4). With respect to the major structural difference between tRNase ZL and tRNase ZS, tRNase ZL has the extra N-terminal domain composed of 500 amino acids, which contains the pseudo-histidine motif (1) and has a similarity in some degree to the C-terminal domain and tRNase ZS (Figure 1). The present observation that only the long forms of tRNase Z can cleave the RNA complexes (Figures 6 and 7) implies that the N-terminal half has an additional important role in enzyme–substrate interactions.
All of the tRNase ZSs did not cleave the micro-pre-tRNA at all, and the C-terminal domain of human tRNase ZL cleaved it inefficiently as compared with the full-length human tRNase ZL (Figure 5). This would be probably because the substrate–enzyme interaction is weakened due to the lack of the D-stem–loop and the anticodon–stem–loop in the substrate. The N-terminal half of tRNase ZL would be involved in the interaction with the substrate at least through the T-stem–loop and the acceptor stem, and would strengthen this interaction. This would allow tRNase ZL to recognize and to cleave the micro-pre-tRNA easily.
This idea is supported by previous (26,27) and present kinetic experiments. The Km values for cleavages of full-length pre-tRNAsArg by pig tRNase ZL were quantitated to be 0.6 μM regardless of the presence of a 5' leader, and the Km values for cleavage of the full-length pre-tRNAArg R-6TUUU by human and yeast tRNase ZLs were 0.39 and 0.74 μM (Table 2). On the other hand, the Km values for cleavages of full-length pre-tRNAsArg by human and T.maritima tRNase ZSs were 1.3–2.4 μM (Table 2). About 3-fold lower Km values for tRNase ZL indicate that its N-terminal half is indeed important for stronger recognition of the substrates. The Km value for cleavage of the micro-pre-tRNAArg R-ATM5 by pig tRNase ZL was 0.57 μM, which was the same as the Km value for cleavage of the full-length pre-tRNAsArg R-UUU (Table 2). This is consistent with the observation that pig tRNase ZL has a strong activity against the micro-pre-tRNAArg (Table 1).
In the above context, we would also be able to explain why the RNase 65 activity was observed only with respect to tRNase ZL (Figure 6B). The discontinuous acceptor stem in the complex between the target RNA and the 3'-truncated tRNA, and the 5' extra nucleotides in the target before the 4 nt recognition sequence (Figure 6A) would disturb the enzyme–substrate interaction. These anomalies would make it difficult for the tRNase ZS to recognize the RNA complex even though the complex has the D-stem–loop and the anticodon–stem–loop. In contrast, tRNase ZL can recognize the substrate probably with the help of the N-terminal half-domain.
This interpretation would also hold for the interaction of tRNase Z with the complex between the target RNA and the hook RNA. This complex has an additional anomaly in that it does not contain the D-stem–loop and the anticodon–stem–loop (Figure 7A). tRNase ZS would have more difficulty in substrate recognition, while, thanks to the N-terminal half, tRNase ZL would be able to endure even such a difficulty.
The N-terminal half-domain, when added to the reactions, did not cause stronger interactions of human or T.maritima tRNase ZS or the C-terminal half with the micro-pre-tRNA or the RNA complexes, while E.coli tRNase ZS became very weakly active against the micro-pre-tRNA in the presence of the N-terminal half. These observations suggest that the covalent connection between the N- and C-terminal half-domains is important but not necessarily prerequisite for the functional role of the N-terminal half.
The Walker A motif (28) found in the N-terminal domain of human tRNase ZL (Figure 1A) might be involved in this N-terminal function possibly through binding to the 5' nucleotide triphosphate of pre-tRNA. The much lower activities of yeast tRNase ZL against the micro-pre-tRNA and the RNA complexes (Table 1) may be due to the lack of this motif in the yeast enzyme. Consistently, the Km value for the R-6TUUU cleavage by yeast tRNase ZL is 1.9- and 1.3-fold higher than those by the human and pig enzymes, respectively (Table 2).
Other roles of tRNase ZL than the tRNA 3' processing
RNAi knockdown of the tRNase ZL gene in C.elegans causes slow growth and sterility, which results from a drastic reduction in germline proliferation (14). The nematode tRNase ZL is also needed for the germline over-proliferation phenotypes observed in three different alleles (14). In addition, a reduction of tRNase ZL suppresses the multivulva phenotype resulting from two different activating mutations in ras (14). These phenomena would be simply explained through the role of tRNase ZL in the tRNA 3' processing. Because a large number of tRNA molecules are required for early embryogenesis, if a sufficient amount of tRNase ZL that is essential for tRNA biosynthesis is not generated in the germline cells, their proliferations would be severely injured. Indeed, an enormous number of tRNase ZL molecules exist in each Xenopus laevis oocyte (7). This number has been estimated to be 5 x 1011 molecules, which is comparable to 5 x 1012 for the number of tRNA molecules.
It has been shown that in HeLa cells, tRNase ZL physically interacts with the -tubulin complex, suggesting that tRNase ZL might play a role in mitosis as a modulator of centrosome assembly (15). Consistently, cells over-expressing tRNase ZL undergo a delay in cell cycle progression during the G2 phase (15). In contrast to the C.elegans case, the pre-tRNA cleaving activity of tRNase ZL would not be directly related to this phenomenon. Nothing is known about how tRNase ZL interacts with the -tubulin complex and how this enzyme is involved in mitosis. In vitro binding assays for the interactions between -tubulin and various recombinant tRNase ZL fragments would elucidate whether the modulator function is attributed to the N-terminal half region, which is not essential for the enzymatic activity.
We have demonstrated that tRNase ZL shows the RNase 65 activity in vitro in the presence of 3'-truncated tRNAs, and that these RNAs exist ubiquitously in mammalian cell extracts so far tested (18–20). This suggests that tRNase ZL works as 4 nt RNA cutters in other cellular RNA metabolisms. The fact that tRNase ZL can also act as a different type of 4 nt RNA cutter in vitro under the direction of hook RNA implies that this cutter could work in the cells if there exists hook RNA. Recently, a huge number of miRNAs that are believed to work as translational regulators have been discovered (29). The length of miRNAs is 21–23 nt, which is similar to the length of hook RNA, suggesting that some of the miRNAs could work as hook RNAs that direct cellular RNA cleavage by endogenous tRNase ZL. Other unidentified small RNAs might also work as sgRNAs in the cells.
tRNase ZL together with sgRNA can be used as a useful molecular biology tool
We have shown that mammalian tRNase ZL can cleave any RNA at any desired site in vitro under the direction of sgRNA, which includes 5' half tRNA and heptamer RNA (21–24). We have also examined the efficacy of this specific RNA targeting method in the living cells by introducing sgRNAs either as their expression plasmids or as 2'-O-methyl RNAs (24). The expressions of the exogenous reporter genes for E.coli chloramphenicol acetyltransferase and firefly luciferase were down-regulated by appropriately designed sgRNAs in human and dog culture cells, and the down-regulation continued for at least 48 h. A 2'-O-methyl heptamer for targeting the endogenous Bcl-2 mRNA has been also shown to work in Sarcoma 180 cells.
Although these results have indicated that indeed appropriate sgRNAs can repress the targeted gene expressions, no direct evidence exists, yet, demonstrating that the down-regulation is due to specific mRNA cleavage by endogenous tRNase ZL and not due to simple antisense effects or other different mechanisms. We would be able to examine directly whether the in vivo down-regulation is really due to tRNase ZL cleavage by knocking-down either tRNase ZL or tRNase ZS gene expression by RNAi. From the present results which show that tRNase ZL and not tRNase ZS can cleave RNA complexes, we can predict that only when the tRNase ZL gene expression is repressed is the down-regulation by sgRNAs abolished.
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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