Download The fission yeast Schizosaccharomyces pombe has two distinct

Biochem. J. (2011) 435, 103–111 (Printed in Great Britain)
103
doi:10.1042/BJ20101619
The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZL s
encoded by two different genes and differentially targeted to the nucleus
and mitochondria
Xuhua GAN, Jing YANG, Jun LI, Haiyan YU, Hongmei DAI, Jinyu LIU and Ying HUANG1
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, School of Life
Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
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 ZL s 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.
INTRODUCTION
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 [12]. Sequence analysis suggests
that tRNase ZL arose by duplication of tRNase ZS followed by
divergence of the sequences [12]. 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 [13]. In
contrast, the N-terminal half of tRNase ZL contains the substratebinding domain termed the flexible arm or exosite [14] 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 [12]. However, how mutations in
ELAC2 might increase prostate cancer risk remains unclear.
In Arabidopsis thaliana, two tRNase ZL s and two tRNase
ZS s have been identified experimentally [15]. Interestingly, our
survey of candidate tRNase Zs in nine sequenced higher plants,
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 [1]). 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 [2], whereas in
the cyanobacterium Synechocystis sp. PCC6803, none of the
tRNA genes encode 3 -CCA [3]. 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 [8].
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 [9]. Moreover, mitochondrial tRNAs deviate
from the cloverleaf structure typical of nuclear tRNAs. In
the yeasts Saccharomyces cerevisiae and Schizosaccharomyces
Key words: endonuclease, 3 -end processing, post-transcriptional
processing, tRNA precursor (pre-tRNA), tRNase Z.
Abbreviations used: 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.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
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X. Gan and others
including A. thaliana and its close relative Arabidopsis lyrata,
has shown that only the two Arabidopsis species contain two
tRNase ZL s and two tRNase ZS s, whereas all other higher plants
contain one tRNase ZL and two tRNase ZS s (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 [16]. 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 [15]. 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 [21].
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 [29]). 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 [34]. Other well known nucleases of this family are
RNase J involved in 5 -end processing of 16S RNA and mRNA
stability in bacteria [35] and eukaryotic Pso2/Snm1/Artemis
proteins that function in DNA repair [33]. 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 [36]. Despite exhibiting distinct substrate specificities determined by specific domains of
tRNase Zs, these enzymes carry highly conserved active-site
c The Authors Journal compilation c 2011 Biochemical Society
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 [37], 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/βαsandwich 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 [41]. 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 [6].
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 [6]. 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 ZL s, SpTrz1p and SpTrz2p, which are encoded by two
different genes and targeted to the nucleus and mitochondria
respectively [13]. Furthermore, we have shown that the nucleartargeted 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) [42]. Yeast cells
carrying a plasmid were grown in EMM (Edinburgh minimal
medium) plus appropriate supplements [42]. Standard protocols
for genetic manipulation of fission yeast were used as described
in [42].
Construction of plasmids for overexpression of recombinant
proteins
The genes encoding wild-type and mutant S. pombe tRNase
ZL s along with the sequence of the nmt1+ (no message
in thiamine) terminator were amplified by PCR from the
respective plasmids [13] using primers SpTRZ1-b1 (5 -GAand
AGATCTATGTCGAAAACTGTTAATTTTAGGGC-3 )
Nmt1term3 (5 -GCGAGCTCGAGCTCGCATTACTAATAGAAAGGA-3 ) for the wild-type and mutant sptrz1+ genes, primers
Sptrz2-a4
(5 -GAAGATCTATGAAAGCTTCTCTTCTGGT
TCCA-3 ) and Nmt1term3 (5 -GCGAGCTCGAGCTCGCATTACTAATAGAAAGGA-3 ) for sptrz2+ , and primers Sptrz2-a3
tRNA 3 -end processing
(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
[43] was constructed as follows: the PCR fragment containing
the EGFP expression cassette was amplified from pFA6aGFPKanMX6 (S65T) (primer sequences are available upon
request) and subcloned into the ApaI and KpnI sites of pJK148
[44]. Then, the PCR fragment containing the intermediatestrength nmt1 promoter (pREP42X) was inserted into the SacI
and PstI sites of the plasmid, yielding pYJ18. To construct fulllength 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 K208 KRK211 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), SpTrz2pN38–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 ZL s 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 ZL s were
introduced individually into S. pombe yAS56 cells by the lithium
acetate method [42]. 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 ZL s were expressed as GST (glutathione transferase)fusion proteins and affinity-purified to near homogeneity using
glutathione–Sepharose 4B resin (BioWorld). Purified tRNase ZL s
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 -AAAACGACC-
105
CTGCTTACACCGAG-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-32 P]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 110nt-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 H2 O. The in vitro pretRNA 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 -32 P-labelled by T4 polynucleotide
kinase using [γ -32 P]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 [43] containing the ura4+ -selectable marker was
described previously [13]. The sptrz2+ gene was subcloned
into the XhoI and SmaI sites of the S. pombe expression
vectors pREP82X, pREP42X and pREP4X, generating pEP82Xsptrz2, 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.
Fluorescence microscopy
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 midlog phase in EMM at 30 ◦ C. After centrifugation, cells were
first washed and then suspended in a DAPI (4 ,6-diamidino-2phenylindole) 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).
c The Authors Journal compilation c 2011 Biochemical Society
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X. Gan and others
RESULTS
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 ZL s: SpTrz1p and SpTrz2p [13].
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 ZS s 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 ZS s (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 ZL s. 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
(SpTrz2pN38) 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-32 P-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 pretRNAArg (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
(SpTrz2pN38) could process pre-tRNAArg , although the tRNase
c The Authors Journal compilation c 2011 Biochemical Society
Figure 1 Recombinant S. pombe tRNase ZL s possess the pre-tRNA 3 -end
processing activity in vitro
Recombinant proteins were expressed as GST-fusion proteins in S. pombe cells and purified by
GST-affinity chromatography. In vitro processing reactions were carried out under conditions
described in the Materials and methods section. Products were resolved by SDS/6 % PAGE and
detected by autoradiography. (A) A Coomassie Blue-stained gel shows purified recombinant
GST-tagged wild-type and mutant SpTrz1p and SpTrz2p proteins resolved by SDS/8 %
PAGE. The positions of protein molecular-mass markers in kDa are indicated on the left.
(B) Processing of internally 32 P-labelled human pre-tRNAArg by GST-tagged wild-type and
H574A mutant SpTrz1p proteins. RNA molecular-size markers (nt) are shown on the left
and products on the right; the rectangles represent mature tRNA and the horizontal lines depict
the 3 -trailer. (C) In vitro processing assay of GST-tagged wild-type and H574A mutant SpTrz1p
proteins with 5 -end-labelled human pre-tRNAArg . (D) Processing of internally 32 P-labelled
human pre-tRNAArg by GST-tagged N-terminal-truncated SpTrz2p lacking the N-terminal MTS
(SpTrz2pN38) and GST-tagged full-length SpTrz2p (SpTrz2p). The dividing line indicates
splicing caused by the grouping of images from different parts of the same gel.
Z activity exhibited by full-length GST-tagged SpTrz2p was
weak when compared with GST-tagged SpTrz2pN38, 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
tRNA 3 -end processing
107
Figure 2 Schematic representation of full-length and deletion constructs
of SpTrz1p generated to determine the NLS
In SpTrz1p208AAAA211 , all basic residues of the basic motif were mutated to alanine residues. All
constructs were fused to the N-terminus of EGFP, expressed under the control of the attenuated,
medium-strength nmt1 promoter (pREP42X) and integrated into the chromosome.
global analysis of protein localization in S. pombe [47].
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
(K208 KRK211 ), 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 (P126 KKKRKV132 ) [48]. To investigate
the nuclear localization property of K208 KRK211 , 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 SpTrz1pN138–EGFP, SpTrz1pN199–EGFP, and
SpTrz1pN218–EGFP, which lack the N-terminal 138, 199
and 218 amino acid residues respectively (Figure 2). Two
constructs, SpTrz1pN138–EGFP and SpTrz1pN199–EGFP
(Figures 3F and 3G), which contain the basic motif, were localized
to the nucleus, whereas SpTrz1pN218–EGFP, which lacks the
Figure 3 The N-terminus of SpTrz1p contains an SV40-like nuclear targeting
signal (NLS)
Cells expressing the constructs depicted in Figure 2 were grown to mid-exponential phase in
EMM supplemented with uracil. EGFP signals were detected by fluorescence microscopy and
photographed. Nuclei were visualized in live cells by staining with DAPI. EGFP alone shows a
diffuse distribution within the cytoplasm (A). Both the full-length and N-terminal half of SpTrz1p
proteins localize to the nucleus (B,C), whereas the C-terminal half of SpTrz1p localizes to the
cytosol (D). Mutation of all four basic residues within the basic motif causes the protein to
localize predominantly to the cytosol (E). The truncated proteins deleted for N-terminal residues
138 or 199 are predominantly localized in the nucleus (F,G), whereas truncated SpTrz1p lacking
N-terminal residue 218 is cytosolic (H).
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;
[47]). 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 (SpTrz2pN38–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.
c The Authors Journal compilation c 2011 Biochemical Society
108
X. Gan and others
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
Figure 4
(MTS)
SpTrz2p contains an N-terminal mitochondrial targeting signal
S. pombe cells expressing C-terminal EGFP-tagged full-length SpTrz2p (SpTrz2p–EGFP) or its
N-terminal truncated protein lacking the 38 N-terminal residues (SpTrz2pN38–EGFP) were
grown to mid-exponential phase in EMM supplemented with uracil. After staining with the
mitochondria-specific marker MitoTracker Red, aliquots from each culture were photographed
under the fluorescence microscope to detect EGFP signals.
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 SpTrz2pN38–
EGFP did not localize to the mitochondria, but instead showed
diffuse localization in the cytosol, indicating that the signal for
Figure 5
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 [13], 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
sptrz2+ , but not sptrz1+ , overexpression is toxic to wild-type cells
Wild-type S. pombe cells transformed with a control empty plasmid (pREP4X), plasmids expressing sptrz1+ at low (pREP82X-sptrz1) and high (pREP4X-sptrz1) levels, or plasmids expressing
sptrz2+ at low (pREP82X-sptrz2), medium (pREP42X-sptrz2) and high (pREP4X-sptrz2) levels were streaked on EMM containing leucine with (to repress overexpression of sptrz1+ and sptrz2+ )
or without thiamine (to induce overexpression of sptrz1+ and sptrz2+ ). SpTrz1p was tagged with an N-terminal FLAG epitope to facilitate the detection of the protein. The overexpressed SpTrz2p
proteins did not contain a FLAG epitope tag since the tag interferes with their function.
c The Authors Journal compilation c 2011 Biochemical Society
tRNA 3 -end processing
Figure 6
Morphological changes caused by sptrz2+ overexpression
Wild-type S. pombe cells transformed with a control empty vector (pREP4X) or sptrz2+
overexpression vector (pREP4X-sptrz2) were examined by DIC microscopy. Scale bar, 10 μm.
anti-FLAG antibody [13]. 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.
DISCUSSION
In the present study, we show that both the nuclear- and
mitochondrial-targeted tRNase ZL s 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 tRNAmediated nonsense suppression through promoting suppressor
pre-tRNA 3 -end processing [13].
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 pretRNA as a substrate in vitro. Similarly, the potato mitochondrial
tRNase Z has been reported to process a nuclear pre-tRNA in
vitro [49]. 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 ZL s towards
their authentic in vivo substrates.
109
In the present study, we also show that unlike S. cerevisiae
tRNase ZL , which lacks a recognizable NLS, and D. melanogaster
and human tRNase ZL s, 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
ZL s, hinting that the nuclear-localization function of tRNase ZL
could have evolved differently in these organisms. Nevertheless,
because tRNase ZL s 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 [13], the
potential NLSs of S. cerevisiae and human tRNase ZL s 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
[50]. Like ELAC2, C. elegans tRNase ZL has also been suggested
to play a role in cell-cycle control [51]. In S. cerevisiae, tRNase
ZL is up-regulated during mitosis [17].
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 [28]. Interestingly, a recent study has
shown that destruction of mitochondria in human cells through
depletion of mitochondrial DNA is associated with a downregulation of ELAC2 and delays cell-cycle progression [19]. 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
[28]. In contrast, overexpression of ELAC2 induces a delay in
cell-cycle progression [50]. 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 [52]. 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 ZL s.
Interestingly, our recent comprehensive survey of tRNase Zs from
fungal genomes reveals that two tRNase ZL s 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
[53]. Furthermore, it is most likely that these two tRNase ZL s from
each Schizosaccharomyces species are targeted to the nucleus and
mitochondria respectively. Our phylogenetic analysis based on the
c The Authors Journal compilation c 2011 Biochemical Society
110
X. Gan and others
amino acid sequences of fungal tRNase Zs further suggests that
these two distinct tRNase ZL s 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 ZL s is unknown. There are several non-mutually exclusive
possibilities to account for the presence of a mitochondriaspecific 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 [13].
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 ZL s
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 ZL s 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.
AUTHOR CONTRIBUTION
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.
ACKNOWLEDGEMENTS
We are grateful to the anonymous reviewers for helpful and constructive comments on
the manuscript. We thank Yingying Yu for initial efforts on this work, Louis Levinger for
reagents and Ciarán Condon (Institut de Biologie Physico-Chimique, Paris, France)
for molecular modelling.
FUNDING
This work was supported by the National Science Foundation of China [grant number
30771178] and Nanjing Normal University [grant number 2007104XGQ0148].
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Received 6 October 2010/17 December 2010; accepted 6 January 2011
Published as BJ Immediate Publication 6 January 2011, doi:10.1042/BJ20101619
c The Authors Journal compilation c 2011 Biochemical Society
Biochem. J. (2011) 435, 103–111 (Printed in Great Britain)
doi:10.1042/BJ20101619
SUPPLEMENTARY ONLINE DATA
The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZL s
encoded by two different genes and differentially targeted to the nucleus
and mitochondria
Xuhua GAN, Jing YANG, Jun LI, Haiyan YU, Hongmei DAI, Jinyu LIU and Ying HUANG1
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, School of Life
Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
See the pages that follow for Supplementary Figures S1–S5 and
Table S1.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
X. Gan and others
Figure S1
Multiple sequence alignment of candidate tRNase ZL s from nine higher plants
Plant tRNase ZL s are from A. thaliana (AthTrz3 and AthTrz4) [1], A. lyrata (AlyTrz3 and AlyTrz4), Carica papaya (CpaTrz3), Glycine max (GmaTrz3), Oryza sativa ssp. japonica (OsaTrz3), Medicago
truncatula (MtrTrz3), Populus trichocarpa (PtrTrz3), Vitis vinifera (VviTrz3) and Zea mays (ZmaTrz3). Non-plant tRNase ZL s are from S. pombe (SpoTrz1 and SpoTrz2) [2], D. melanogaster (DmeTrz1)
[3] and Homo sapiens (HsaTrz2/ELAC2) [4]. Protein accession numbers are given in Supplementary Table S1. Numbers in parentheses are the positions of the amino acid sequences. The alignment
was constructed using ClustalW [5]. Identical residues are on a black background and similar residues are shaded in grey. Also indicated above the sequence alignment are the conserved motifs in
substrate binding and catalysis. The conserved motifs are labelled according to previous papers [6–8]. The detailed analysis of similarities and dissimilarities between plant tRNase ZL s and their
counterparts in other eukaryotes will be described fully elsewhere (H. Dai and Y. Huang, unpublished work).
c The Authors Journal compilation c 2011 Biochemical Society
tRNA 3 -end processing
Figure S1
Continued
c The Authors Journal compilation c 2011 Biochemical Society
X. Gan and others
Figure S2
Multiple sequence alignment of candidate tRNase ZS s from nine higher plants
Plant tRNase ZS s are from A. thaliana (AthTrz1 and AthTrz2) [1], A. lyrata (AlyTrz1 and AlyTrz2), C. papaya (CpaTrz1 and CpaTrz2), G. max (GmaTrz1 and GmaTrz2), O. sativa ssp. japonica (OsaTrz1
and OsaTrz2), M. truncatula (MtrTrz1 and MtrTrz2), P. trichocarpa (PtrTrz1 and PtrTrz2), V. vinifera (VviTrz1 and VviTrz2) and Z. mays (ZmaTrz1 and ZmaTrz2). Non-plant tRNase ZS s are from B.
subtilis (BsuTrz1) [9] and H. sapiens (HsaTrz1/ELAC1) [4]. Protein accession numbers are given in Supplementary Table S1. Numbers in parentheses are the positions of the amino acid sequences.
The alignment was constructed using ClustalW [5]. Identical residues are highlighted by a black background and similar residues are shaded in grey. Motifs I–V are labelled according to previous
papers [6–8]. The detailed analysis of similarities and dissimilarities between plant tRNase ZS s and tRNase ZS s in other organisms will be described fully elsewhere (H. Dai and Y. Huang, unpublished
work).
c The Authors Journal compilation c 2011 Biochemical Society
tRNA 3 -end processing
Figure S3 Predicted secondary structure and topology of the C-terminal
half of SpTrz1p
(A) Predicted secondary structure of the C-terminal half of SpTrz1p using
HHpred (Homology detection and structure prediction by HMM–HMM comparison,
http://toolkit.tuebingen.mpg.de/hhpred). The numbers above the sequence indicate the positions
of amino acid residues. The secondary structure elements labelled sequentially are indicated
below the sequence. Blue rectangles represent α-helices and green arrows represent β-strands.
Additional α-helices inserted in the metallo-β-lactamase fold (two opposing seven-strand
β-sheets flanked by α-helices on each side) are indicated in red. (B) Topology diagram showing
the secondary-structure elements that form the metallo-β-lactamase fold and insertions (in red).
Figure S4 Predicted secondary structure and topology of the C-terminal
half of SpTrz2p
See the legend to Supplementary Figure S3 for a description.
c The Authors Journal compilation c 2011 Biochemical Society
X. Gan and others
Figure S5
Comparison of S. pombe and human tRNAArg sequences and secondary structures
(A) Sequence alignment of human nuclear tRNAArg (hutRNAArg ) [6], S. pombe nuclear tRNAArg (spnutRNAArg ; accession number SPATRNAARG.02) and S. pombe mitochondrial tRNAArg (spmitRNAArg ;
accession number SPMITTRNAARG.02). S. pombe nuclear and mitochondrial tRNAArg sequences were obtained from the S. pombe genome database at the Wellcome Trust Sanger Institute
(http://old.genedb.org/genedb/pombe/). The alignment was constructed using ClustalW [5]. Numbers are positions in tRNA sequences. Identical nuclei acids are shaded in black. (B) Cloverleaf
structures of predicted human nuclear tRNAArg and S. pombe nuclear and mitochondrial tRNAArg . The tRNA secondary structures were predicted using tRNAscan-SE [10]. Nucleotide modifications
are not indicated.
c The Authors Journal compilation c 2011 Biochemical Society
tRNA 3 -end processing
Table S1
Candidate tRNase Zs in nine higher plants
All candidate tRNase Z protein sequences were obtained by BLAST searches using known tRNase Z protein sequences as queries against plant genome databases including the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/mapview/static/MVPlantBlast.shtml?10), Phytozome (http://www.phytozome.net/) and UniProt (http://www.uniprot.org/). All candidate
proteins were verified and the mispredicted sequences were corrected as described in [11]. Species abbreviations used in this study are shown in parentheses. The plus sign (+) indicates the number
of amino acids in plant tRNase Zs. Asterisks indicate that the mispredicted sequences obtained from the database have been corrected.
Species
Taxon
Protein name
Form
Accession number
Database
No. aa+
A. thaliana (Ath)
Dicot
AthTrz1
AthTrz2
AthTrz3
AthTrz4
tRNase ZS
tRNase ZS
tRNase ZL
tRNase ZL
NP_177608.2
NP_178532.2
NP_175628.2
NP_188247.2
NCBI
NCBI
NCBI
NCBI
280
354
890
942
A. lyrata (Aly)
Dicot
AlyTrz1
AlyTrz2
AlyTrz3
AlyTrz4
tRNase ZS
tRNase ZS
tRNase ZL
tRNase ZL
476606
480092
474332
341756
Phytozome
Phytozome
Phytozome
Phytozome
280
353
833
946
C. papaya (Cpa)
Dicot
CpaTrz1
CpaTrz2
CpaTrz3
tRNase ZS
tRNase ZS
tRNase ZL
Evm.TU.supercontig_17.217
Evm.TU.supercontig_132.51
Evm.TU.supercontig_34.31
Phytozome
Phytozome
Phytozome
306
396
872*
G. max (Gma)
Dicot
GmaTrz1
GmaTrz2
GmaTrz3
tRNase ZS
tRNase ZS
tRNase ZL
Glyma20g01480
Glyma02g43400
Glyma13g43270
Phytozome
Phytozome
Phytozome
279
354
923*
O. sativa ssp. japonica (Osa)
Monocot
OsaTrz1
OsaTrz2
OsaTrz3
tRNase ZS
tRNase ZS
tRNase ZL
NP_001046280.1
LOC_Os09g30466
B9EUI7
NCBI
Phytozome
UniProt
302
365
964
M. truncatula (Mtr)
Dicot
MtrTrz1
MtrTrz2
MtrTrz3
tRNase ZS
tRNase ZS
tRNase ZL
Medtr5g101340
Medtr5g092020
Medtr2g122460
Phytozome
Phytozome
Phytozome
323*
357
950*
P. trichocarpa (Ptr)
Dicot
PtrTrz1
PtrTrz2
PtrTrz3
tRNase ZS
tRNase ZS
tRNase ZL
POPTR_0015s07570
B9IAM1
POPTR_0001s18750
Phytozome
UniProt
Phytozome
301
366
941*
V. vinifera (Vvi)
Dicot
VviTrz1
VviTrz2
VviTrz3
tRNase Z S
tRNase Z S
tRNase ZL
D7SIF1
D7TEG8
GSVIVT01017040001
UniProt
UniProt
Phytozome
330
354
841
Z. mays (Zma)
Monocot
ZmaTrz1
ZmaTrz2
ZmaTrz3
tRNase ZS
tRNase ZS
tRNase ZL
GRMZM2G147727
GRMZM2G125844
GRMZM2G379286
Phytozome
Phytozome
Phytozome
302
357*
930
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Received 6 October 2010/17 December 2010; accepted 6 January 2011
Published as BJ Immediate Publication 6 January 2011, doi:10.1042/BJ20101619
c The Authors Journal compilation c 2011 Biochemical Society