tRNase Z is the endonuclease that is involved in tRNA 3′-end maturation by removal of the 3′-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3′-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.
- 3′-end processing
- post-transcriptional processing
- tRNA precursor (pre-tRNA)
- tRNase Z
All mature tRNAs carry the sequence CCA at their 3′-terminus, which is essential for tRNA aminoacylation and for protein synthesis (for a review, see ). The 3′-CCA end of pre-tRNAs (tRNA precursors) is generated either endonucleolytically or exonucleolytically depending on whether tRNA genes encode 3′-CCA. In bacteria and archaea, the proportion of tRNA genes that encode 3′-CCA varies widely among species. In Escherichia coli, 3′-CCA is encoded in all tRNA genes , whereas in the cyanobacterium Synechocystis sp. PCC6803, none of the tRNA genes encode 3′-CCA . The mature tRNA 3′-end of CCA-containing pre-tRNAs is generated exonucleolytically. In contrast, the 3′-end of the pre-tRNAs that lack CCA is processed by tRNA 3′-endonuclease tRNase Z (also called RNase Z or 3′-tRNase) followed by the addition of CCA by tRNA nucleotidyltransferase (for reviews of tRNase Z, see [4–7]). An exception occurs in Thermotoga maritima, where the CCA-containing pre-tRNAs are processed by tRNase Z . In contrast with the complex situation in bacteria, eukaryotic nuclear and mitochondrial tRNA genes generally do not encode 3′-CCA, which is added post-transcriptionally after the 3′-trailer sequences of pre-tRNAs are removed by tRNase Z. However, unlike nuclear pre-tRNAs, mitochondrial pre-tRNAs are polycistronic . Moreover, mitochondrial tRNAs deviate from the cloverleaf structure typical of nuclear tRNAs. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, pre-tRNA 3′-end processing appears to be reinforced by exoribonucleases [10,11].
tRNase Zs are classified into tRNase ZS (short form of tRNase Z) with 300–400 aa (amino acids) and tRNase ZL (long form of tRNase Z) with 800–900 aa . Sequence analysis suggests that tRNase ZL arose by duplication of tRNase ZS followed by divergence of the sequences . Based on sequence analysis, tRNase ZL can be divided into the N- and C-terminal halves, both of which are related to tRNase ZS, with the N-terminal half being much more divergent in sequence. Both tRNase ZS and the C-terminal half of tRNase ZL contain conserved motifs I–V (motif II is also called the histidine motif), and the PxKxRN, HEAT and HST loops. Most of these motifs are required for catalysis . In contrast, the N-terminal half of tRNase ZL contains the substrate-binding domain termed the flexible arm or exosite  and the pseudo-histidine motif.
tRNase ZL has so far been found only in eukaryotes. Most eukaryotes including S. cerevisiae, Caenorhabditis elegans and Drosophila melanogaster contain only one tRNase ZL, but lack tRNase ZS. In contrast, human cells contain one tRNase ZL, known as ELAC2, and one tRNase ZS, known as ELAC1. ELAC2 was originally identified as the first candidate prostate cancer susceptibility gene . However, how mutations in ELAC2 might increase prostate cancer risk remains unclear. In Arabidopsis thaliana, two tRNase ZLs and two tRNase ZSs have been identified experimentally . Interestingly, our survey of candidate tRNase Zs in nine sequenced higher plants, including A. thaliana and its close relative Arabidopsis lyrata, has shown that only the two Arabidopsis species contain two tRNase ZLs and two tRNase ZSs, whereas all other higher plants contain one tRNase ZL and two tRNase ZSs (Supplementary Table S1 and Supplementary Figures S1 and S2 at http://www.BiochemJ.org/bj/435/bj4350103add.htm).
The existence of one tRNase ZL in most eukaryotes examined to date suggests that a single enzyme is responsible for 3′-end processing of both nuclear and mitochondrial pre-tRNAs. Indeed, Drosophila tRNase ZL, also called JHI-1 (juvenile hormone-inducible protein 1), participates in both nuclear and mitochondrial pre-tRNA 3′-end processing . Both ScTrz1p (S. cerevisiae tRNase ZL) [17,18] and ELAC2 [19,20] are localized to the nucleus and mitochondria. However, their roles in nuclear and mitochondrial pre-tRNA 3′-end processing in vivo remain to be demonstrated.
There are two possible explanations, not mutually exclusive, for why some eukaryotic organisms have more than one tRNase Z. The first explanation is that different tRNase Zs are targeted to different subcellular compartments. A recent study has shown that extra A. thaliana tRNase Zs are targeted to the mitochondria and chloroplasts . The second explanation is that extra tRNase Zs may have unique functions. For example, ELAC1 localizes to the cytoplasm and may function in the degradation of a subset of miRNAs (microRNAs) in the cytoplasm .
Recent studies support the idea that tRNase ZL itself has additional functions. For example, ELAC2 was found to function in the biogenesis of small noncoding RNA (other than tRNA) [22–25] and viral miRNA [26,27]. In S. cerevisiae, ScTrz1p is probably involved in rRNA processing and mitochondrial maintenance [13,28]. In our previous studies, we have demonstrated that the nuclear-targeted SpTrz1p (S. pombe tRNase ZL) is required for cell viability in the absence of the S. pombe La protein [13,28]. The La protein (which is nonessential in S. cerevisiae and S. pombe) is required for normal 3′-endonucleolytic processing of nuclear pre-tRNAs by blocking exonuclease access to the 3′-ends (for a review, see ). Since in the absence of the La protein, nuclear pre-tRNAs gain access to the 3′-processing exonucleases for 3′-exonucleolytic processing, cells lacking both the La protein and SpTrz1p would be expected to be viable. However, S. pombe cells are unviable in the absence of these two proteins. Together with our finding that both ScTRZ1 and ELAC2 cannot complement the sptrz1 null mutant, our results suggest that SpTrz1p may play an essential function beyond pre-tRNA 3′-end processing, e.g. mRNA or rRNA processing.
tRNase Z belongs to the MBL (metallo-β-lactamase) superfamily [12,30–33]. Members of this superfamily have diverse biological functions, including roles in microbial resistance to β-lactam antibiotics and in RNA metabolism. Besides tRNase Z, a few members of the MBL superfamily with either demonstrated or predicted nuclease activity have been found to participate in nucleic-acid metabolism. Most of these proteins belong to the β-CASP (MBL associated CISF Artemis SNM1/PSO2) family. The best studied member of this family is CPSF-73 (cleavage and polyadenylation specificity factor, 73 kDa subunit), which is an endonuclease required for mRNA 3′-end formation . Other well known nucleases of this family are RNase J involved in 5′-end processing of 16S RNA and mRNA stability in bacteria  and eukaryotic Pso2/Snm1/Artemis proteins that function in DNA repair . The Int11 (integrator complex subunit 11), which participates in maturation of the 3′-end of snRNAs (small nuclear RNAs), is also a predicted nuclease of the MBL superfamily . Despite exhibiting distinct substrate specificities determined by specific domains of tRNase Zs, these enzymes carry highly conserved active-site motifs, including the MBL-superfamily-specific histidine motif (HxHxDH, where x is any hydrophobic residue), and perhaps share a similar catalytic mechanism.
Structural studies of tRNase ZS from E. coli , Bacillus subtilis [38,39] and T. maritima [38,40] have revealed that the protein forms a homodimer, in which each subunit consists of a typical core MBL domain that adopts the four-layer αβ/βα-sandwich fold with two seven-stranded β-sheets flanked on either side by three α-helices. In this core domain, the conserved motifs located in the C-terminal half of the protein constitute the catalytic centre of tRNase ZS. In the catalytic centre, three histidine residues and one aspartate residue from the histidine motif act together with two other histidines and one aspartate from the conserved motifs III–V to co-ordinate two divalent zinc ions, a cofactor required for full activity of tRNase Z . In addition to the core domain, the substrate-binding domain located between motifs III and IV of the protein adopts a flexible arm structure that protrudes from the core domain of tRNase ZS. These studies have also shown that the tRNase ZS dimers contain two binding and catalytic sites for pre-tRNAs and thus can process two pre-tRNA substrates at one time. Interestingly, pre-tRNAs display sigmoidal binding curves, indicating co-operativity in their interaction with tRNase ZS dimers .
Crystal structures for tRNase ZL have not yet been reported. However, structural modelling predicts that both the N-terminal and C-terminal halves of ELAC2 fold into two distinct MBL domains . The MBL domain formed by the C-terminal half contains a catalytic centre but lacks a flexible arm, whereas the other domain formed by the N-terminal half contains a flexible arm but lacks a catalytic site.
We have previously reported the identification of two S. pombe tRNase ZLs, SpTrz1p and SpTrz2p, which are encoded by two different genes and targeted to the nucleus and mitochondria respectively . Furthermore, we have shown that the nuclear-targeted tRNase ZL can promote 3′-end maturation of a suppressor tRNA 3′-end in vivo. In the current study, we biochemically characterized these two proteins. We further delineated targeting signals involved in their subcellular localization. Finally, we evaluated the effects of overexpression of sptrz1+ or sptrz2+ on cell growth and morphology.
MATERIALS AND METHODS
S. pombe strains, media and genetic procedures
The fission yeast strain used in the present study was yAS56 (h−S leu1-32 ura4-D18). Untransformed yeast cells were grown in YES (yeast extract medium plus supplements) . Yeast cells carrying a plasmid were grown in EMM (Edinburgh minimal medium) plus appropriate supplements . Standard protocols for genetic manipulation of fission yeast were used as described in .
Construction of plasmids for overexpression of recombinant proteins
The genes encoding wild-type and mutant S. pombe tRNase ZLs along with the sequence of the nmt1+ (no message in thiamine) terminator were amplified by PCR from the respective plasmids  using primers SpTRZ1-b1 (5′-GAAGATCTATGTCGAAAACTGTTAATTTTAGGGC-3′) and Nmt1term3′ (5′-GCGAGCTCGAGCTCGCATTACTAATAGAAAGGA-3′) for the wild-type and mutant sptrz1+ genes, primers Sptrz2-a4 (5′-GAAGATCTATGAAAGCTTCTCTTCTGGTTCCA-3′) and Nmt1term3′ (5′-GCGAGCTCGAGCTCGCATTACTAATAGAAAGGA-3′) for sptrz2+, and primers Sptrz2-a3 (5′-GAAGATCTAAATCCAAAAGAAACACGAATAGAATTATG-3′) and Nmt1term3′ (5′-GCGAGCTCGAGCTCGCATTACTAATAGAAAGGA-3′) for sptrz2ΔN38. The PCR products were digested with BglII and SacI and subcloned into the BamHI and SacI sites of the S. pombe expression vector pESP2 (Stratagene).
Construction of C-terminal EGFP (enhanced green fluorescent protein) fusion proteins
A plasmid for the expression of EGFP-tagged proteins expressed under the control of the intermediate-strength nmt1+ promoter  was constructed as follows: the PCR fragment containing the EGFP expression cassette was amplified from pFA6a-GFPKanMX6 (S65T) (primer sequences are available upon request) and subcloned into the ApaI and KpnI sites of pJK148 . Then, the PCR fragment containing the intermediate-strength nmt1 promoter (pREP42X) was inserted into the SacI and PstI sites of the plasmid, yielding pYJ18. To construct full-length and truncated forms of SpTrz1p–EGFP fusion proteins, full-length SpTrz1p and various DNA fragments of SpTrz1p were subcloned into the PstI and SalI sites of pYJ18, resulting in C-terminal EGFP fusions. The SpTrz1p208AAAA211–EGFP mutant was constructed by using overlapping PCR to convert amino acid residues K208KRK211 of SpTrz1p to AAAA.
To generate full-length SpTrz2p–EGFP and a truncated version lacking the N-terminal 38 aa predicted to harbour a MTS (mitochondrial-targeting signal), SpTrz2pΔN38–EGFP, DNA sequences coding for the full-length and N-terminal-truncated SpTrz2p proteins were amplified by PCR and subcloned into the PstI and SalI sites of pYJ18. The resulting plasmids were linearized with NruI and transformed into yAS56 cells. Individual Leu+ colonies were streaked on the same medium and the correct integration of the fusion genes was confirmed by PCR.
Protein expression and purification
Recombinant tRNase ZLs were produced by using Stratagene's ESP® yeast expression and purification system, following the manufacturer's instructions. Briefly, the S. pombe expression vectors encoding the wild-type or mutant tRNase ZLs were introduced individually into S. pombe yAS56 cells by the lithium acetate method . A single colony from a fresh transformation was grown overnight in 5 ml of YES and used to inoculate 10 ml of YES to a final D600 of 0.2–0.4. After growth at 30 °C for 5 h to a D600 of 0.7–1.0, the cells were harvested, washed and resuspended in 10 ml of EMM containing 225 mg/l uracil prior to inoculation into EMM containing 225 mg/l uracil. The cells were harvested approx. 18 h after induction in EMM without thiamine. Cell extracts were prepared using a French press. tRNase ZLs were expressed as GST (glutathione transferase)-fusion proteins and affinity-purified to near homogeneity using glutathione–Sepharose 4B resin (BioWorld). Purified tRNase ZLs were subjected to SDS/8% PAGE analysis.
In vitro pre-tRNA processing assay
The DNA template for in vitro synthesis of pre-tRNAArg was generated by PCR using a plasmid encoding human tRNAArg as a template (generously given by Louis Levinger, Department of Biology, City University of New York, New York, U.S.A.) with primers hArg5 (5′-TAATACGACTCACTATAGGGCCAGTGGCGCAATGG-3′) and hArg3 (5′-AAAACGACCCTGCTTACACCGAG-3′). Pre-tRNAArg used as the tRNase Z substrate in the in vitro pre-tRNA processing assay was synthesized with T7 RNA polymerase. The in vitro transcription reaction was carried out at 37 °C for 4–6 hrs in a total volume of 20 μl in a reaction mixture containing 40 mM Tris/HCl, pH 7.9, 6 mM MgCl2, 10 mM DTT (dithiothreitol), 10 mM NaCl, 2 mM spermidine, 5 mM ATP, CTP and GTP, 10 μCi of [α-32P]UTP (3000Ci/mmol; Beijing Furui Biotechnology) (1 Ci=3.7×1010 Bq), 40 units of RNasin, 100 ng of DNA template and 20 units of T7 RNA polymerase. The reaction products were separated by SDS/8% PAGE, and the primary band corresponding to the 110-nt-long pre-tRNAArg transcripts were excised from the gel, crushed and eluted overnight at 37 °C in 500 mM NaCl. The resulting RNA was ethanol precipitated and resuspended in H2O. The in vitro pre-tRNA processing reaction was carried out at 37 °C for 30 min in 10 μl of the reaction mixture containing 25 mM Tris/HCl, pH 7.0, 1 mM MgCl2, 1 mM DTT, 50 mM KCl, 5% (v/v) glycerol, 100 μg/ml BSA, 1 μl of pre-tRNAArg transcripts, 20 units of RNasin and 100 ng of recombinant proteins. The reaction was stopped by adding 5 μl of RNA loading buffer (5% glycerol, 1 mM EDTA, 0.025% Bromophenol Blue and 20 μg/ml Xylene Cyanol FF). Processing products were separated by SDS/6% PAGE. The gels were vacuum dried and exposed to X-ray film. For 5′-end labelling, pre-tRNAArg was first dephosphorylated using alkaline phosphate and then 5′-32P-labelled by T4 polynucleotide kinase using [γ-32P]ATP (5000 Ci/mmol, 10 mCi/ml; Beijing Furui Biotechnology) according to manufacturer's instructions (MBI Fermentas). The labelled RNA was gel purified by SDS/6% PAGE as described above.
Overexpression of sptrz1+ and sptrz2+ using different-strength thiamine-regulatable nmt1 promoters
Cloning of the FLAG-tagged sptrz1+ gene into pREP82X or pREP4X  containing the ura4+-selectable marker was described previously . The sptrz2+ gene was subcloned into the XhoI and SmaI sites of the S. pombe expression vectors pREP82X, pREP42X and pREP4X, generating pEP82X-sptrz2, pREP42X-sptrz2 and pREP4X-sptrz2 respectively. These constructs were transformed into yAS56 cells, and Ura4+ transformants were selected on EMM containing 15 μM thiamine. Transformants were re-streaked on EMM without thiamine.
Yeast strains carrying pREP4X (control) or pREP4X-sptrz2 were grown in EMM supplemented with 225 mg/l leucine for 20 h. Living cells were examined by Nomarski DIC (differential interference contrast) microscopy. For localization of full-length and truncated SpTrz1p proteins, cells were grown to mid-log phase in EMM at 30 °C. After centrifugation, cells were first washed and then suspended in a DAPI (4′,6-diamidino-2-phenylindole) solution (1 μg/ml in methanol). After incubated for 15 min at 30 °C, the cells were harvested and suspended in PBS. For localization of full-length and truncated SpTrz2p proteins, the cells were stained with 50 nM MitoTracker Red CMXRos (Invitrogen) in EMM at 30 °C for 25 min. The green fluorescence of EGFP, the blue fluorescence of DAPI and the red fluorescence of MitoTracker Red were detected using λex of 488 nm, 372 nm and 556 nm respectively. All images were obtained on a Zeiss Axio Imager A1 microscope equipped with a PCO Sensicam CCD (charge-coupled-device) camera, and data were analysed using MetaMorph image processing software (Universal Imaging).
Both GST-tagged SpTrz1p and SpTrz2p fusion proteins exhibit tRNase Z activity
We have shown previously that S. pombe is unique in that it has two candidate tRNase ZLs: SpTrz1p and SpTrz2p . Although homology modelling using HHpred [Homology detection and structure prediction by HMM (Hidden Markov Model)–HMM comparison, http://toolkit.tuebingen.mpg.de/hhpred] could not predict the topology of the N-terminal halves of these two candidates, which harbour the potential substrate-binding domain, because they have very low sequence similarity to tRNase ZSs of known structure, homology modelling revealed that both the C-terminal halves of the two proteins, which contain the putative catalytic centre for pre-tRNA cleavage, adopt a topology similar to those of E. coli, B. subtilis and T. maritima tRNase ZSs (Supplementary Figures S3 and S4 at http://www.BiochemJ.org/bj/435/bj4350103add.htm). To verify whether these two proteins indeed have tRNase Z activity, we performed the in vitro pre-tRNA processing assay using recombinant tRNase ZLs. Our initial attempts to purify soluble and active His6-tagged SpTrz1p and SpTrz2p using the bacterial pET28a expression system failed due to the formation of inclusion bodies in E. coli cells. However, we were able to express N-terminally GST-tagged SpTrz1p and SpTrz2p in S. pombe and to purify them to near homogeneity and with high yield using the ESP yeast expression system (Figure 1A). To evaluate the requirement of the absolutely conserved histidine motif for the pre-tRNA 3′-end processing activity of SpTrz1p, we also purified a GST-tagged H574A mutant (SpTrz1p-H574A), which contains a point mutation, changing the first histidine residue (His574) of the histidine motif to alanine. Since SpTrz2p is targeted to mitochondria, we also purified an N-terminally truncated SpTrz2p (SpTrz2pΔN38) lacking a MTS (aa 1–38), which is required for the mitochondrial localization of the protein (Figure 4).
The human nuclear-encoded pre-tRNAArg, which is 91 nt long and contains a mature 5′-end and an 18 nt trailer ending in UUUU-3′OH, was used as a substrate for tRNase Z in in vitro pre-tRNA processing assays. This took advantage of the fact that tRNAArg is very similar in S. pombe and humans (see Supplementary Figure S5 at http://www.BiochemJ.org/bj/435/bj4350103add.htm) and that tRNA 3′-end-processing enzymes recognize features shared by all tRNA molecules. Indeed, studies in other laboratories have shown that this pre-tRNA and its variants having various lengths of 3′-trailers could be processed by recombinant tRNase Zs from a variety of species, including E. coli, T. maritima, S. cerevisiae and humans [45,46]. As expected, incubation of recombinant GST-tagged SpTrz1p with internally α-32P-labelled pre-tRNAArg under the conditions described in the Materials and methods section resulted in the generation of two products: a 73-nt-long RNA that corresponds to the size of mature tRNAArg and an 18-nt-long RNA that corresponds to the size of the 3′-trailer (Figure 1B). It should be noted that nearly all of the pre-tRNAs were processed after prolonged incubation times under these conditions (results not shown). Incubation with 5′-end-labelled pre-tRNAArg produced only an RNA of 73 nt in length corresponding to 3′-end-processed tRNAArg, confirming that the shorter fragment is derived from the 3′-end of pre-tRNAArg (Figure 1C). The recombinant histidine motif mutant SpTrz1p-H574A could not cleave pre-tRNAArg, indicating that the histidine motif is required for the endonuclease activity of SpTrz1p (Figure 1B).
Similar to recombinant SpTrz1p, both full-length GST-tagged SpTrz2p and an N-terminal truncated mutant lacking the MTS (SpTrz2pΔN38) could process pre-tRNAArg, although the tRNase Z activity exhibited by full-length GST-tagged SpTrz2p was weak when compared with GST-tagged SpTrz2pΔN38, indicating that the MTS might interfere with the endonuclease activity of GST-tagged SpTrz2p in vitro (Figure 1D). Cleavage of pre-tRNAArg at its 3′-end by full-length and truncated SpTrz2p proteins was confirmed by the 5′-end labelling of pre-tRNAArg (results not shown). Taken together, these results confirmed that both SpTrz1p and SpTrz2p indeed possess the pre-tRNA 3′-end endonuclease activity in vitro and implied that they are directly responsible for nuclear and mitochondrial pre-tRNA 3′-end processing respectively in S. pombe.
The N-terminal half of SpTrz1p contains a sequence that is responsible for the nuclear localization of the protein
Initial evidence for the nuclear localization of SpTrz1p and the mitochondrial localization of SpTrz2p arose from global analysis of protein localization in S. pombe . However, a search with the computer program PredictNLS (http://www.predictprotein.org/) failed to predict the NLS (nuclear localization signal) in SpTrz1p. To identify the NLS in SpTrz1p, we first generated constructs expressing C-terminally EGFP-tagged full-length SpTrz1p, and N-terminal and C-terminal halves of SpTrz1p (Figure 2). To avoid disruption of sptrz1+, which is an essential gene, the EGFP fusion constructs were individually integrated into the chromosomal leu1 locus of S. pombe strain yAS56 with a wild-type sptrz1+ chromosomal allele, and EGFP-fusion proteins were expressed under the control of the intermediate-strength thiamine-repressible nmt1 promoter (pREP42X). The growth and morphological properties of these strains were similar to those of the wild-type strain (results not shown). EGFP alone was localized to only the cytosol (Figure 3A). The merged EGFP and DAPI images clearly show that EGFP-tagged full-length SpTrz1p was localized in the nucleus, confirming the nuclear localization of SpTrz1p (Figure 3B). The EGFP-tagged N-terminal half of SpTrz1p (SpTrz1pN–EGFP), but not the EGFP-tagged C-terminal half of SpTrz1p (SpTrz1pC–EGFP), was also localized in the nucleus (Figures 3C and 3D), indicating that the N-terminal half of SpTrz1p is sufficient to drive nuclear localization of the protein.
Examination of the amino acid sequence of SpTrz1p revealed that the N-terminal half of the protein contains a basic motif (K208KRK211), which is similar to the NLS of the SV40 (simian virus 40) large T-antigen consisting of a cluster of five contiguous positively charged residues (P126KKKRKV132) . To investigate the nuclear localization property of K208KRK211, we next created the mutant SpTrz1p208AAAA211 in which all residues of this basic motif were changed to alanine. As shown in Figure 3(E), substitution of basic residues within this motif with alanine residues led to an impairment of nuclear localization of the fusion protein, as SpTrz1p208AAAA211–EGFP was no longer enriched in the nucleus, but diffused throughout the cytoplasm. These results indicate that the basic motif plays a critical role in the nuclear localization of SpTrz1p.
To determine whether SpTrz1p contains additional NLSs, we also generated three N-terminal truncation mutants, designated SpTrz1pΔN138–EGFP, SpTrz1pΔN199–EGFP, and SpTrz1pΔN218–EGFP, which lack the N-terminal 138, 199 and 218 amino acid residues respectively (Figure 2). Two constructs, SpTrz1pΔN138–EGFP and SpTrz1pΔN199–EGFP (Figures 3F and 3G), which contain the basic motif, were localized to the nucleus, whereas SpTrz1pΔN218–EGFP, which lacks the NLS, was distributed evenly in the cytosol (Figure 3H). These results indicate that SpTrz1p contains only one NLS.
The MTS of SpTrz2p resides within the N-terminal 38 amino acids
SpTrz2p contains a predicted MTS that resides within the N-terminal 38 amino acids (as determined using MITOPROT software: http://ihg.gsf.de/ihg/mitoprot.html), consistent with the observation that SpTrz2p localizes to mitochondria (Figure 4; ). To investigate whether this sequence could function as an MTS, we constructed a full-length EGFP-fusion protein (SpTrz2p–EGFP) and an N-terminally truncated EGFP-fusion protein lacking the first 38 amino acids (SpTrz2pΔN38–EGFP). The EGFP was fused C-terminally to full-length and truncated SpTrz2p proteins. These EGFP fusions were expressed under the intermediate-strength thiamine-repressible nmt1 promoter. To avoid possible interruption of the function of SpTrz2p, we integrated these constructs individually at the chromosomal leu1 locus of strain yAS56 containing the wild-type chromosomal sptrz2+ allele. MitoTracker Red was used to selectively label the mitochondria. As shown in Figure 4, SpTrz2p–EGFP formed string-like structures and was co-localized with MitoTracker Red dye to the mitochondria, whereas the truncated SpTrz2pΔN38–EGFP did not localize to the mitochondria, but instead showed diffuse localization in the cytosol, indicating that the signal for the mitochondrial-targeting resides was located within the first 38 amino acid residues of the protein.
Overexpression of sptrz2+, but not sptrz1+, is toxic to yeast cell growth and affects cell morphology
To investigate the phenotypic consequences of overexpression of S. pombe tRNase ZL genes, we examined the effects of overexpression of sptrz1+ or sptrz2+ on cell growth and morphology. To this end, plasmids expressing sptrz1+ orsptrz2+ under the control of three different-strength thiamine-repressible nmt1 promoters were used to transform yAS56 cells. Full-length SpTrz1p was expressed with an N-terminal FLAG epitope tag to facilitate the detection of protein expression , whereas full-length SpTrz2p was expressed without a tag, since an N-terminal FLAG tag had a detrimental effect on the in vivo function of the protein (results not shown). Ura+ transformants were selected on EMM plates with thiamine (repressed condition) and then re-streaked on EMM plates without thiamine (derepressed condition). Overexpression of sptrz2+ from the weakest nmt1 promoter (pREP82X) modestly inhibited cell growth, but overexpression of sptrz2+ from the nmt1 promoter of intermediate strength (pREP42X) or full strength (pREP4X) strongly inhibited or completely blocked cell growth respectively (Figure 5). Thus growth retardation induced by sptrz2+ overexpression appeared to increase in severity along with the increased expression of sptrz2+. By contrast, cells overexpressing sptrz1+ under the control of the weakest and full-strength nmt1 promoters grew normally (Figure 5). Overexpression of FLAG-tagged SpTrz1p was confirmed by Western blotting using anti-FLAG antibody . Thus overexpression of sptrz2+, but not of sptrz1+, conferred a dosage-dependent growth defect. These data indicate that S. pombe wild-type cells are sensitive to the level of SpTrz2p, but not to the level of SpTrz1p.
Microscopic examination of S. pombe wild-type cells transformed with a control empty vector or a vector overexpressing sptrz2+ from the full-strength nmt1 promoter revealed morphology defects induced by sptrz2+ overexpression (Figure 6). Wild-type cells were rod-shaped, and had variable lengths that were cell-cycle-dependent. In contrast, cells overexpressing sptrz2+ were swollen and some appeared to be round in shape. Overexpression of sptrz1+ did not cause morphological changes (results not shown). Thus overexpression of sptrz2+, but not sptrz1+, led to aberrant cell morphology.
In the present study, we show that both the nuclear- and mitochondrial-targeted tRNase ZLs from S. pombe possess pre-tRNA 3′-end processing activity in vitro. A point mutation of the first histidine residue in the histidine motif of SpTrz1p to alanine (H574A) totally abolishes the activity of the enzyme, suggesting that the histidine motif of SpTrz1p is critical for catalytic activity. These results are in agreement with the in vivo findings that overexpression of wild-type SpTrz1p, but not the mutant (SpTrz1p-H574A), can increase suppressor tRNA-mediated nonsense suppression through promoting suppressor pre-tRNA 3′-end processing .
Our in vitro processing assays with a human nuclear pre-tRNA show that SpTrz2p has 3′-end processing activity that is comparable with SpTrz1p, suggesting that mitochondrial tRNase ZL from S. pombe is able to recognize nuclear pre-tRNA as a substrate in vitro. Similarly, the potato mitochondrial tRNase Z has been reported to process a nuclear pre-tRNA in vitro . However, it is possible that SpTrz2p may exhibit a different substrate preference from its nuclear counterpart. This hypothesis is supported by the observations that: (i) mitochondrial pre-tRNAs are polycistronic and much larger than monocistronic nuclear pre-tRNAs; (ii) mitochondrial tRNAs appear to deviate structurally from nuclear tRNAs; and (iii) SpTrz2p shows weak sequence homology with SpTrz1p (20% identity and 21% similarity). Further experiments are required to determine the substrate specificity of the two S. pombe tRNase ZLs towards their authentic in vivo substrates.
In the present study, we also show that unlike S. cerevisiae tRNase ZL, which lacks a recognizable NLS, and D. melanogaster and human tRNase ZLs, which harbour a predicted NLS overlapping with a predicted MTS at the N-terminus of the proteins, SpTrz1p possesses an SV40-like NLS located within residues 200–218. It appears that the SpTrz1p NLS is not conserved in S. cerevisiae, D. melanogaster and human tRNase ZLs, hinting that the nuclear-localization function of tRNase ZL could have evolved differently in these organisms. Nevertheless, because tRNase ZLs from S. cerevisiae and humans can rescue the temperature-sensitive allele sptrz1–1 and promote 3′-end maturation of a nuclear tRNA suppressor in S. pombe , the potential NLSs of S. cerevisiae and human tRNase ZLs appear to be functional in S. pombe cells.
sptrz2+ overexpression is lethal and causes aberrant cell morphology. The molecular basis for these phenotypic changes is unclear. One possibility is that SpTrz2p may be involved directly or indirectly in cell-cycle control via its role in mitochondrial tRNA 3′-end processing. Therefore, sptrz2+ overexpression may lead to cell-cycle perturbations. Support for this possibility comes from studies of tRNase ZL in other eukaryotes. In HeLa cells, overexpression of ELAC2 delays the cell cycle, suggesting that ELAC2 may be involved in the control of cell-cycle progression . Like ELAC2, C. elegans tRNase ZL has also been suggested to play a role in cell-cycle control . In S. cerevisiae, tRNase ZL is up-regulated during mitosis .
Another possibility is that the action of SpTrz2p may be related to mitochondrial function. It is likely that sptrz2+ overexpression may cause abnormal mitochondrial function (which is vital for S. pombe), thus leading to the observed phenotypes. In support of this hypothesis, it has been suggested that ScTrz1p may participate in mitochondrial function . Interestingly, a recent study has shown that destruction of mitochondria in human cells through depletion of mitochondrial DNA is associated with a down-regulation of ELAC2 and delays cell-cycle progression . These results suggest that ELAC2 may link mitochondrial function and cell-cycle control.
It is worth mentioning that the consequences of overexpression of the tRNase ZL gene have also been studied in S. cerevisiae and human cancer cells. Overexpression of the gene encoding tRNase ZL in S. cerevisiae does not appreciably affect cell growth . In contrast, overexpression of ELAC2 induces a delay in cell-cycle progression . Although the consequence of tRNase ZL overexpression in Drosophila cells has not been reported, the mRNA level of Drosophila tRNase ZL is markedly induced by juvenile hormone . These results demonstrate that different organisms may have different sensitivitities to the tRNase ZL levels and suggest that different organisms may use different mechanisms to regulate or constrain tRNase ZL activity.
Most eukaryotes examined to date have only one tRNase ZL, which is targeted to both the nucleus and mitochondria, indicating that a single tRNase ZL is sufficient for 3′-end processing of both nuclear and mitochondrial pre-tRNAs. However, it is not known how the protein is imported into the nucleus and mitochondria, and how the subcellular distribution of the protein is regulated. S. pombe is unique in that it contains two tRNase ZLs. Interestingly, our recent comprehensive survey of tRNase Zs from fungal genomes reveals that two tRNase ZLs encoded by two different tRNase ZL genes are also present in three other sequenced Schizosaccharomyces species (Schizosaccharomyces octosporus, Schizosaccharomyces japonicus and Schizosaccharomyces cryophobus) in the ascomycete subphylum Taphrinomycotina . Furthermore, it is most likely that these two tRNase ZLs from each Schizosaccharomyces species are targeted to the nucleus and mitochondria respectively. Our phylogenetic analysis based on the amino acid sequences of fungal tRNase Zs further suggests that these two distinct tRNase ZLs may have arisen from gene duplication and that subsequent sequence divergence may allow targeting of the two proteins to different subcellular compartments.
The reason that S. pombe and other fission yeasts require two tRNase ZLs is unknown. There are several non-mutually exclusive possibilities to account for the presence of a mitochondria-specific tRNase ZL in addition to nuclear tRNase ZL in S. pombe. The first possibility is that partitioning of nuclear and mitochondrial tRNase ZL activities between two distinct proteins in S. pombe offers the possibility for the independent control of the expression of these two proteins, which is probably important for their proper functions. In this regard, we note substantial differences in the sensitivity of S. pombe cells to SpTrz1p and SpTrz2p. Overexpression of sptrz2+, but not of sptrz1+, can lead to phenotypic changes in growth rate and cellular morphology. Moreover, the extent of growth inhibition clearly correlates positively with the level of sptrz2+ overexpression. A second possibility is that SpTrz1p or/and SpTrz2p may have specialized functions that are incompatible with the dual subcellular localization in the nucleus and mitochondria. This possibility is supported by our previous study showing that sptrz1+ is essential, even though nuclear pre-tRNA 3′-end processing in S. pombe appears to be backed by exoribonucleases . These results suggest that sptrz1+ may have additional essential functions. Finally, it is possible that S. pombe cells may require a dedicated mitochondrial enzyme with different properties that enables different aspects of polycistronic mitochondrial pre-tRNA 3′-end processing. Thus the existence of two distinct tRNase ZLs may reflect the fundamental differences in the mechanisms of pre-tRNA 3′-end processing in the nucleus and mitochondria in S. pombe. Further functional characterization of the two S. pombe tRNase ZLs should help us to understand why S. pombe and other fission yeasts need to separate the nuclear and mitochondrial tRNase Z activities into two distinct proteins. Our fission yeast model should provide a unique tool for increasing our understanding of tRNase Z function and evolution.
Xuhua Gan designed and performed in vitro experiments, analysed in vitro results and performed modelling. Jing Yang and Jun Li carried out localization studies. Haiyan Yu and Hongmei Dai performed overexpression experiments. Hongmei Dai, Jun Li and Jinyu Liu conducted online database searches and carried out the sequence alignment. Ying Huang conceived and designed the experiments, analysed the results and wrote the paper.
This work was supported by the National Science Foundation of China [grant number 30771178] and Nanjing Normal University [grant number 2007104XGQ0148].
Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast; DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; EMM, Edinburgh minimal medium; GST, glutathione transferase; MBL, metallo-β-lactamase; miRNA, microRNA; MTS, mitochondrial-targeting signal; NLS, nuclear localization signal; nmt1, no message in thiamine; pre-tRNA, tRNA precursor; ScTrz1p, Saccharomyces cerevisiae tRNase ZL; SpTrz1p, S. pombe tRNase ZL; SV40, simian virus 40; tRNase, ZL, long form of tRNase Z; tRNase, ZS, short form of tRNase Z; YES, yeast extract medium plus supplements
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