Plant Cell Physiol. 49(4): 549–556 (2008)
doi:10.1093/pcp/pcn028, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Nascent Peptide-Mediated Translation Elongation Arrest of Arabidopsis
thaliana CGS1 mRNA Occurs Autonomously
Hitoshi Onouchi 1, Yuhi Haraguchi 1, Mari Nakamoto 1, 3, Daisuke Kawasaki 2,
Yoko Nagami-Yamashita 1, 4, Katsunori Murota 2, Azusa Kezuka-Hosomi 1, 5, Yukako Chiba
Satoshi Naito 1, 2, *
1
2
1, 6
and
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, 060-8589 Japan
Introduction
The Arabidopsis thaliana CGS1 gene encodes cystathionine c-synthase, the first committed enzyme of methionine
biosynthesis in higher plants. Expression of CGS1 is feedbackregulated at the step of mRNA degradation in response to
S-adenosyl-L-methionine (AdoMet). A short stretch of amino
acid sequence, termed the MTO1 region, encoded within the
first exon of CGS1 itself acts in cis in the regulation. In vitro
analyses using wheat germ extract (WGE) revealed that
AdoMet induces temporal translation arrest of CGS1 mRNA
prior to mRNA degradation. This translational pausing occurs
immediately downstream of the MTO1 region and is mediated
by the nascent MTO1 peptide. In order to elucidate further
the nature of this unique regulatory mechanism, we have
examined whether a non-plant system also contains the posttranscriptional regulation activity. Despite the fact that
mammals do not carry cystathionine c-synthase, AdoMet
was able to induce the MTO1 sequence-dependent translation
elongation arrest in rabbit reticulocyte lysate (RRL) in a
similar manner to that observed in WGE. This result suggests
that MTO1 peptide-mediated translation arrest does not
require a plant-specific factor and rather most probably occurs
via a direct interaction between the nascent MTO1 peptide
and the ribosome that has translated it. In contrast, decay
intermediates of CGS1 mRNA normally observed upon
induction of CGS1 mRNA decay in plant systems were not
detected in RRL, raising the possibility that CGS1 mRNA
degradation involves a plant-specific mechanism.
Methionine is an essential amino acid for nonruminant animals, which do not have enzymes for
methionine biosynthesis. The Arabidopsis thaliana CGS1
gene (gene ID At3g01120) encodes cystathionine g-synthase
(CGS; EC 2.5.1.48) that catalyzes the first committed
step of methionine biosynthesis in higher plants (Kim
and Leustek 1996, Kim et al. 1999). CGS converts
O-phosphohomoserine and cysteine into cystathionine,
and this step constitutes the key regulatory step in the
methionine biosynthetic pathway. However, unlike many of
the key step enzymes in biosynthetic pathways, CGS is not
an allosteric enzyme (Thompson et al. 1982). Expression of
the CGS1 gene is feedback-regulated post-transcriptionally
at the step of mRNA degradation in response to
S-adenosyl-L-methionine (AdoMet), a direct metabolite of
methionine (Chiba et al. 1999, Chiba et al. 2003). CGS1
mRNA degradation was accelerated when Arabidopsis calli
were fed with methionine to increase the AdoMet level
in vivo. In addition, 50 -truncated CGS1 RNA species
representing a decay intermediate were observed upon
the induction of CGS1 mRNA decay (Chiba et al. 1999,
Lambein et al. 2003). A short stretch of amino acid
sequence encoded by the first exon of CGS1, termed the
MTO1 region, is involved in this regulation by acting in cis
(Chiba et al. 1999, Suzuki et al. 2001, Ominato et al. 2002).
The mto1 mutants of Arabidopsis, which carry single amino
acid sequence alterations within the MTO1 region, are
defective in this regulation and overaccumulate soluble
methionine (Chiba et al. 1999, Ominato et al. 2002).
The post-transcriptional regulation of CGS1 was
recapitulated in the plant in vitro translation system of
wheat germ extract (WGE) (Chiba et al. 2003). When WGE
was programmed with RNA carrying CGS1 exon 1 fused to
a reporter gene, AdoMet induced down-regulation of
reporter activity and accumulation of 50 -truncated CGS1
Keywords: S-Adenosyl-L-methionine — Arabidopsis thaliana
— In vitro translation — Methionine biosynthesis —
mRNA stability — Translation elongation arrest.
Abbreviations: AdoMet, S-adenosyl-L-methionine; CGS,
cystathionine g-synthase; CTAB, cetyltrimethylammonium bromide; GST, glutathione S-transferase; LUC, firefly luciferase;
ORF, open reading frame; RLUC, sea pansy luciferase; RRL,
rabbit reticulocyte lysate; uORF, upstream ORF; WGE, wheat
germ extract.
3
Present address: Life Science Group, Olympus Corp., Hachioji, 192-8512 Japan.
Present address: Plant Genetic Resources Division, Hokkaido Central Agricultural Experiment Station, Takikawa, 073-0013 Japan.
Present address: Tobacco Science Research Center, Japan Tobacco Inc., Yokohama, 227-8512 Japan.
6
Present address: Research Institute for Bioresources, Okayama University, Kurashiki, 710-0046 Japan.
*Corresponding author: E-mail, [email protected]; Fax, þ81-11-706-4932.
4
5
549
Autonomous translation arrest in Arabidopsis CGS1
RNA species that had the same 50 ends as those detected
in vivo. In addition, we found that translational elongation
is temporally arrested in response to AdoMet prior to
production of the 50 -truncated RNA species (Onouchi et al.
2005). This translational pausing occurs at the Ser94 codon
located immediately downstream of the MTO1 region.
It was also found that the amino acid sequence, rather than
the nucleotide sequence, of the MTO1 region is responsible
for the translation elongation arrest. Together with the cisacting nature of the MTO1 region, these observations led us
to propose a model in which the amino acid sequence
encoded by the MTO1 region in nascent CGS1 polypeptide
acts within the ribosome that has just translated it to cause
translational pausing in response to AdoMet, which then
triggers mRNA degradation (Onouchi et al. 2005).
It remained to be determined whether factors other
than the MTO1 region amino acid sequence are involved in
the post-transcriptional regulation of CGS1. In the present
study, we investigated whether the regulation can be
reproduced in the non-plant in vitro translation system of
rabbit reticulocyte lysate (RRL). If plant-specific factors are
required, the regulation would not be recapitulated in RRL.
Since an amino acid sequence similar to the MTO1 peptide
cannot be found in the public database other than in plant
CGSs (Ominato et al. 2002), it is unlikely that mammalian
cells have a factor that can substitute for the function of
possible plant-specific factors. We show here that when
RRL was programmed with an RNA harboring CGS1 exon
1, translation elongation arrest was induced by AdoMet in
a similar manner to that observed in WGE, although the
RNA decay intermediates were not observed. These results
suggest that no plant-specific factor other than the MTO1
peptide is required to mediate the AdoMet-induced
translation elongation arrest, whereas additional plantspecific factors may be required for the coupled mRNA
degradation.
Results
MTO1 peptide-mediated downregulation of gene expression is
induced in RRL
In previous studies using WGE, MTO1 peptidemediated regulation was investigated using Ex1:Luc
RNAs that contain CGS1 exon 1 fused in-frame to a Luc
gene encoding firefly luciferase (LUC). AdoMet was shown
to induce down-regulation of an Ex1:Luc RNA containing
wild-type CGS1 exon 1 [Ex1(WT):Luc], whereas no such
regulation was observed with a mto1-1 mutant version
[Ex1(mto1-1):Luc] that acted as a negative control
(Onouchi et al. 2005). The mto1-1 mutation is a Gly84 to
serine substitution within the MTO1 region and abolishes
the AdoMet-induced regulation in plants as well as in WGE
(Chiba et al. 1999, Chiba et al. 2003). To determine whether
A
Ex1(WT ):Luc
Ex1
Ex1(mto1–1):Luc
B
LUC
A30
mto1–1 (Gly-84 to Ser)
1.5
Relative reporter activity
(+AdoMet / −AdoMet)
550
1.0
0.5
0
WT
mto1–1
Fig. 1 MTO1 peptide-mediated down-regulation of LUC reporter
activity in RRL. (A) Schematic representation of Ex1(WT):Luc and
Ex1(mto1-1):Luc RNA. (B) In vitro transcripts shown in (A) were
translated in RRL for 60 min in the presence or absence of 1 mM
AdoMet. LUC activity was normalized with control RLUC activity
from co-translated Rluc RNA, and reporter activity relative to
samples without AdoMet (þAdoMet/AdoMet) was calculated.
Averages SD of triplicate experiments are shown.
CGS1 exon 1 is also able to mediate down-regulation of
gene expression in a non-plant system, these RNAs were
used in the current study to monitor the effect of AdoMet
on reporter gene expression in RRL. All RNA templates
used for in vitro translation assays were prepared from the
DNA constructs by in vitro transcription, were capped and
had an A30 tail (see Materials and Methods).
Ex1(WT):Luc RNA and Ex1(mto1-1):Luc RNA
(Fig. 1A) were translated in RRL in both the presence
and absence of AdoMet. Rluc RNA encoding sea pansy
luciferase (RLUC) was co-translated as an internal control.
Following a 60 min incubation period of the translation
reaction, LUC and RLUC reporter activities were measured
and LUC activity was normalized against RLUC activity.
As shown in Fig. 1B, LUC activity was repressed when
Ex1(WT):Luc RNA was translated in the presence of
AdoMet. In contrast, AdoMet treatment did not influence
reporter activity when Ex1(mto1-1):Luc RNA was used.
This result indicates that AdoMet can induce MTO1
peptide-mediated down-regulation of reporter gene expression in the non-plant RRL system.
MTO1 peptide-mediated translation elongation arrest is
induced in RRL
In order to clarify which steps of the CGS1 posttranscriptional regulation are recapitulated in RRL, we first
Autonomous translation arrest in Arabidopsis CGS1
551
A
GST:Ex1(WT):Luc
GST
GST:Ex1(mto1–1):Luc
Ex1
A30
LUC
mto1–1 (Gly-84 to Ser)
B
C
GST:Ex1(WT ):Luc
0
(kDa)
116
−
30
+
−
60
+
−
+
CTAB
GST:Ex1(mto1–1):Luc
(min)
AdoMet
0
(kDa)
116
−
30
+
−
60
+
−
(min)
+ AdoMet
ppt
(kDa)
sup
+
−
−
−
+ RNase A
1
2
3
4
5
100
66
66
45
45
75
50
37
Fig. 2 Translation elongation arrest in RRL. (A) Schematic representation of GST:Ex1(WT):Luc and GST:Ex1(mto1-1):Luc RNA.
(B) Immunoblot analysis of translation products. In vitro transcripts shown in (A) were translated in RRL for 0, 30 or 60 min in the presence
(þ) or absence () of 1 mM AdoMet, as indicated. Translation products were analyzed by immunoblot analysis using an anti-GST
antibody. The filled arrowhead indicates the position of AdoMet-dependent peptidyl-tRNA (55 kDa), and the open arrowhead indicates the
position of the full-length translation product (110 kDa). The positions of protein size markers are indicated. Representative results of
triplicate experiments are shown. (C) Immunoblot analysis of translation products after CTAB precipitation and/or RNase A treatment.
In vitro transcripts shown in (A) were translated in RRL for 30 min in the presence of AdoMet (lane 4). In lanes 1–3, translation products
were precipitated with CTAB, and supernatants (sup; lane 3) and precipitates (ppt; lanes 1 and 2) were analyzed. In lane 1, precipitates
were treated with RNase A before analysis. In lane 5, the in vitro translation reaction mixture was directly treated with RNase A. Open,
filled and gray arrowheads indicate the positions of full-length translation product (110 kDa), AdoMet-dependent peptidyl-tRNA (55 kDa)
and the AdoMet-dependent band shifted by RNase A treatment (35 kDa), respectively. The positions of protein size markers are indicated.
A representative result of triplicate experiments is shown.
examined whether the translation arrest is induced in RRL.
We have previously shown that when RNA harboring
a glutathione S-transferase (GST)-tagged Ex1(WT):Luc
construct [GST:Ex1(WT):Luc] was translated in WGE
supplemented with AdoMet, accumulation of an approximately 55 kDa peptidyl-tRNA was observed due to the
translation elongation arrest that occurred immediately
downstream of the MTO1 region (Onouchi et al. 2005). We
therefore tested for the presence of AdoMet-induced
translation arrest products in RRL using the same GSTtagged constructs.
RNAs
transcribed
from
the
wild-type
[GST:Ex1(WT):Luc] and mutant [GST:Ex1(mto1-1):Luc]
(Fig. 2A) were translated in RRL in the presence or absence
of AdoMet. Translation products were then analyzed by
immunoblot analysis using anti-GST antibody after 30 and
60 min incubation periods. A 55 kDa band was observed
when GST:Ex1(WT):Luc RNA was translated in RRL in
the presence of AdoMet at both time points, whereas no
such specific band was observed with GST:Ex1(mto1-1):
Luc RNA irrespective of AdoMet treatment (Fig. 2B).
This 55 kDa band was similar to the AdoMet-induced
translation arrest product that we observed in WGE
(Onouchi et al. 2005).
In addition to its 55 kDa size, the AdoMet-induced
translation arrest product in WGE is defined by three
additional specific characteristics: (i) the product contains
a tRNA; (ii) the translation elongation arrest is temporal;
and (iii) translation arrest occurs at Ser94 (Onouchi
et al. 2005). If the 55 kDa band observed in the RRL
assay is identical to that in WGE, and the same mechanism is indeed responsible for the translation arrest in
both RRL and WGE, the 55 kDa product induced by
AdoMet in RRL would also have to meet these three
criteria.
As a test for the presence of tRNA, the 55 kDa band
induced in RRL was treated with RNase A (Fig. 2C). This
treatment resulted in a shift of the band to approximately
35 kDa as was the case in WGE (Onouchi et al. 2005).
In addition, the 55 kDa band was precipitable with
cetyltrimethylammonium bromide (CTAB), which precipitates nucleic acids (Fig. 2C). These results corroborate
our notion that this partial translation product is a
peptidyl-tRNA.
552
Autonomous translation arrest in Arabidopsis CGS1
A
B
GST:Ex1(WT)
(kDa)
GST
Ex1
A30
+ AdoMet
(kDa)
75
75
50
50
37
− AdoMet
37
10 15 20 30 40 60 90 120180 min
10 15 20 30 40 60 90 120180 min
Fig. 3 Pulse–chase analysis of translation arrest products. (A) Schematic representation of GST:Ex1(WT) RNA. The positions of
methionine codons are marked with vertical bars. (B) GST:Ex1(WT) RNA shown in (A) was translated in RRL in the presence of
[35S]methionine with (left panel) or without (right panel) 1 mM AdoMet. Edeine was added at 5 min after the start of translation. Aliquots
of the reaction mixtures were withdrawn at the indicated time points, and separated on a 4–12% gradient SDS–polyacrylamide gel. The
filled arrowhead indicates the position of the AdoMet-dependent peptidyl-tRNA (55 kDa), and the open arrowhead marks the position of
the full-length translation product (45 kDa). The positions of protein size markers are indicated. Representative results of duplicate
experiments are shown.
The temporal status of the translation elongation arrest
in RRL was next examined in a pulse–chase experiment
using [35S]methionine and GST:Ex1 RNA without the LUC
reporter (Fig. 3A). The GST coding region carries nine
methionine codons, while the CGS1 exon 1 coding region
has only the first methionine codon (Onouchi et al. 2005).
At 5 min after the start of the translation reaction, edeine,
an inhibitor of translation initiation (Dinos et al. 2004),
was added to inhibit new rounds of translation and the
translation products were then monitored between 10 and
120 min. As shown in Fig. 3B, the presence of AdoMet
resulted in an increase in the 55 kDa peptidyl-tRNA band
up to 15 min, after which it declined to below detection
levels by 120 min. This decrease in the 55 kDa band matched
with an increase in intensity of the full-length 45 kDa
peptide band. Although the intensity of the 55 kDa band
reached a maximum at an earlier time point than that
previously observed in WGE (30 min), the rate of decrease
and thus the temporal status of translation elongation arrest
was similar to that in WGE.
The exact position of translation arrest in RRL was
next determined by introducing stop codon substitutions
around the MTO1 region within GST:Ex1:Luc constructs.
A stop codon placed at or upstream of the arrest site should
abolish accumulation of the partial translation product,
whereas a stop codon placed downstream of the arrest site
should not affect the translation arrest. For clarity, the
positions of the amino acid residues are numbered from
the first methionine of CGS. As shown in Fig. 4, when the
amber stop codon (UAG) was introduced at residues Ile88
(I88UAG), Lys92 (K92UAG), Trp93 (W93UAG) or Ser94
(S94UAG), the AdoMet-dependent 55 kDa band was
not detected. In contrast, UAG substitutions at Asn95
(N95UAG), Asn96 (N96UAG) and Pro97 (P97UAG) did not
affect the induction of the 55 kDa band detected in the wildtype construct. These results indicate that translation arrest
occurs at Ser94 in RRL, as is the case in WGE.
The above results confirm that the 55 kDa AdoMetinduced translation arrest product detected in RRL is
indeed the same as that observed in WGE, indicating that
MTO1 peptide-mediated translation elongation arrest is
induced in RRL in a similar manner to that in WGE.
mRNA degradation is not coupled with the MTO1
peptide-mediated translation arrest in RRL
In WGE, translation elongation arrest mediated by
MTO1 peptide is followed by production of short RNA
species that lack the 50 region and are thought to be decay
intermediates (Chiba et al. 2003, Onouchi et al. 2005).
We next examined whether production of such 50 -truncated
RNA species is also recapitulated in RRL.
To detect 50 -truncated RNA species in RRL, primer
extension analysis was carried out. In WGE, primer
extension analysis detected a ladder of 50 ends of the
50 -truncated RNA species upstream of the Ser94 codon, the
translation arrest site. These 50 end points are thought to be
produced by RNA degradation events that occur near the 50
edge of the stalled ribosome as well as those that are stacked
behind the initial stalled ribosome (Haraguchi et al. 2008).
GST:Ex1(WT):Luc RNA was translated in the presence
and absence of AdoMet in both WGE and RRL, after
which poly(A) RNA was extracted and analyzed by primer
extension analysis. The GST tag sequence is not necessary
for detection of the RNA degradation intermediates; however, the 50 -truncated RNA ladder is more
clearly detected with GST:Ex1(WT):Luc RNA in WGE
Autonomous translation arrest in Arabidopsis CGS1
WT
P97UAG
N96UAG
N95UAG
S94UAG
K92UAG
I88UAG
WT
25
P97UAG
25
N96UAG
37
N95UAG
37
S94UAG
50
K92UAG
50
W93UAG
(kDa)
W93UAG
− AdoMet
+ AdoMet
(kDa)
I88UAG
553
Fig. 4 Determination of the position of translation elongation arrest. GST:Ex1(WT):Luc RNA or its derivatives that harbor UAG stop codon
substitutions were translated in RRL for 30 min in the presence (left panel) or absence (right panel) of 1 mM AdoMet. Translation products
were analyzed by immunoblot analysis using an anti-GST antibody. The solid arrowhead indicates the position of the AdoMet-dependent
peptidyl-tRNA (55 kDa), and the open arrowhead marks the position of a partial translation product that terminated at the premature stop
codon in each of the stop codon substitution constructs. The positions of protein size markers are indicated. Representative results of
duplicate experiments are shown.
(Haraguchi et al. 2008). As shown in Fig. 5, the previously
reported 50 ends of the 50 -truncated RNA were evidently
observed in WGE (Haraguchi et al. 2008), whereas no
specific bands representing 50 ends of the 50 -truncated RNA
species were detected at the corresponding sites in RRL,
suggesting that AdoMet does not induce production of the
50 -truncated RNA species in RRL. Similar experiments
using total RNA also failed to detect the 50 -truncated RNA
species in RRL (data not shown). The result corroborates
the notion that the specific 50 -truncated CGS1 RNA is not
produced in response to AdoMet in RRL.
Discussion
Plant-specific trans-acting factor is not required for MTO1
peptide-mediated translation elongation arrest of CGS1
mRNA
Our previous studies using WGE revealed that
translation of CGS1 mRNA is temporally arrested in
response to AdoMet prior to mRNA degradation, and
that the MTO1 region amino acid sequence is responsible
for the translation elongation arrest (Onouchi et al. 2005).
Here, we demonstrated that AdoMet induces MTO1
peptide-mediated translational pausing in the mammalian
RRL system in a similar manner to that in WGE. When
RNA carrying wild-type CGS1 exon 1 was translated in
RRL supplemented with AdoMet, translation paused at
exactly the same position as in WGE, and resumed similarly
to the case in WGE. These results suggest that no plantspecific factor other than the cis-acting MTO1 peptide is
required for the AdoMet-induced translation elongation
arrest. Although we cannot rule out the involvement of
additional factors conserved between plants and animals,
they would probably be general factors that are not
specifically involved in the CGS1 regulation, because
mammals do not have CGS, and an amino acid sequence
similar to the MTO1 region cannot be found in the public
database other than in plant CGSs (Ominato et al. 2002),
and therefore it is unlikely that CGS1-specific regulation is
conserved in mammals. A plausible model is that both
AdoMet and the MTO1 peptide interact directly with the
translation elongation complex to cause the translation
arrest. Translation arrest occurs at the Ser94 codon located
immediately downstream of the MTO1 region, suggesting
that the nascent MTO1 peptide resides in the stalled
ribosome when translation is arrested (Onouchi et al.
2005). This supports the idea that the nascent MTO1
peptide interacts directly with the ribosome that has
translated it to mediate the translation arrest.
One possible mode of direct action of AdoMet on the
translation elongation complex could be methylation of
a translational machinery component using AdoMet as a
methyl donor. This modification could then make the
ribosome sensitive to the inhibitory effect of the MTO1
peptide, resulting in translation elongation arrest. An
observation that argues against this possibility is that
S-adenosyl-L-homocysteine, a competitive inhibitor of the
AdoMet-dependent methylation reaction, did not exhibit
any competitive effect with AdoMet on the MTO1 peptidemediated regulation in WGE (Chiba et al. 2003). Another
possibility is that, without the methyltransfer reaction,
direct binding of AdoMet to the translation elongation
complex including the nascent peptide causes translation
arrest by modulating sensitivity of the ribosome to the
MTO1 peptide or acting cooperatively with the MTO1
peptide within the translating ribosome.
Several examples of nascent peptide-mediated ribosomal regulation have been reported (for reviews, see
Lovett and Rogers 1996, Tenson and Ehrenberg 2002).
554
Autonomous translation arrest in Arabidopsis CGS1
WT
WGE
120 0
60 120 (min)
− + − + − + − + AdoMet
MTO1 region
T G C A
RRL
Fig. 5 Analyses of degradation of CGS1 exon 1-containing RNA
in RRL. GST:Ex1(WT):Luc RNA was translated in RRL for 0, 60 or
120 min in the presence (þ) or absence () of 1 mM AdoMet, as
indicated. As a control, the same RNA preparation was also
translated in WGE for 120 min in the presence or absence of 1 mM
AdoMet. Poly(A) RNA was extracted and analyzed by primer
extension analysis using 50 -32P-labeled TO4L primer (Chiba et al.
2003). Lanes T, G, C and A represent the sequence ladder
synthesized using the same primer. The open arrowhead marks the
position of full-length RNA. The filled arrowheads with a bracket
indicate the positions of the 50 ends of the 50 -truncated RNA species
detected in WGE. The MTO1 region is indicated beside the
sequence ladder. A representative result of quadruple experiments
is shown.
Interestingly, nascent peptides encoded by the Escherichia
coli secM and tnaC genes have been shown to interact
directly with components of the ribosomal exit tunnel to
cause translation arrest (Nakatogawa and Ito 2002,
Cruz-Vera et al. 2005). Furthermore, it has been suggested
that a small inducer molecule, tryptophan, binds directly
to a ribosome to induce TnaC peptide-mediated ribosomal
inhibition at the step of translation termination by
interfering with the activity of the peptidyltransferase
center (Gong and Yanofsky 2002, Cruz-Vera et al. 2006,
Cruz-Vera et al. 2007).
Apart from the CGS1 gene, eukaryotic examples of
nascent peptide-mediated ribosomal regulation involve
peptides encoded by upstream open reading frames
(uORFs) that cause ribosome stalling at the stop codon of
the uORF (Cao and Geballe 1996, Wang and Sachs 1997,
Wang et al. 1999, Law et al. 2001, for a review see Morris
and Geballe 2000). In these cases, the stalled ribosome
blocks translation initiation of a downstream ORF. Among
them, uORF peptide-mediated regulation of Neurospora
crassa arg-2 (and its Saccharomyces cerevisiae ortholog,
CPA1) and mammalian AdoMet decarboxylase is induced
by arginine and polyamine, respectively, which are the
products of metabolic pathways in which these genes are
involved (Wang et al. 1999, Law et al. 2001, Mize and
Morris 2001). In these systems, these low molecular weight
compounds can induce the regulation even when the uORFs
were introduced into heterologous systems, namely
N. crassa arg-2 and S. cerevisiae CPA1 genes in WGE
(Wang et al. 1999), the arg-2 gene in RRL (Fang et al. 2004)
and the mammalian AdoMet decarboxylase gene in
S. cerevisiae (Mize and Morris 2001), in which regulation
of the corresponding gene is not conserved. Thus, similarly
to the CGS1 mRNA translation arrest, specific factors do
not appear to be required in these systems. The unique
feature of the CGS1 regulation is that the nascent
regulatory peptide is encoded by the same ORF that
codes for the CGS enzyme, although the arg-2 uORF
peptide was shown to be capable of inducing translation
elongation arrest when the uORF was fused in-frame with
another ORF (Fang et al. 2004). It remains to be elucidated
whether these uORF systems share a common mechanism
with the CGS1 regulation. The data presented in the present
study not only add to the repertoire of heterologous
conservation of translation arrest mechanisms, but also
provide means by which we may explore the complex nature
of the CGS1 regulation system.
Possible uncoupling of CGS1 mRNA degradation from
translation arrest in RRL
We have previously shown that nascent MTO1
peptide-mediated translation arrest is coupled with production of CGS1 mRNA decay intermediates in WGE
(Onouchi et al. 2005). Strong correlation between these
two events was observed by mutational analysis of the
MTO1 region amino acid sequence, suggesting that the
translation arrest triggers CGS1 mRNA degradation in
plant cells (Onouchi et al. 2005). In contrast, such decay
intermediates were not detected in RRL even under
Autonomous translation arrest in Arabidopsis CGS1
conditions that resulted in translation arrest. Although a
possibility remains that the 50 -truncated RNA species,
whose half-life in Arabidopsis calli is about 30 min
(Lambein et al. 2003), is very unstable in RRL, the results
obtained here suggest that a plant-specific mechanism may
be required for CGS1 mRNA degradation to be triggered
by the translation arrest. Alternatively, mammalian cells
could contain a functionally equivalent mechanism that is
inactive in a cell-free condition or in specialized cells such as
reticulocytes.
Materials and Methods
Chemicals
AdoMet was purchased from Sigma-Aldrich (St Louis, MO,
USA). Edeine sulfate was a gift from National Cancer Institute
(Bethesda, MD, USA). Other chemicals used are described in
Chiba et al. (2003).
Plasmids
Plasmids pMI21(WT) and pMI21(mto1-1), which carry the
Ex1(WT):Luc and Ex1(mto1-1):Luc DNA in the pSP64 Poly(A)
vector (Promega, Madison, WI, USA), respectively, were described
previously (Chiba et al. 2003). Plasmids pYN10(WT),
pYN10(mto1-1), pMN1(WT) and pMI21(mto1-1), which carry
the GST:Ex1(WT):Luc, GST:Ex1(mto1-1):Luc, GST:Ex1(WT)
and GST:Ex1(mto1-1) DNA in the pSP64 Poly(A) vector
(Promega), respectively, were described previously (Onouchi
et al. 2005). Plasmids carrying amber stop codon (TAG) substitutions in the GST:Ex1(WT):Luc construct were also described
previously (Onouchi et al. 2005).
In vitro transcription
DNA templates in pSP64 poly(A) vector (Promega) were
linearized with EcoRI, purified, and transcribed in vitro in the
presence of a cap analog m7G[50 ]ppp[50 ]GTP as described (Chiba
et al. 2003).
In vitro translation
In vitro translation reactions in RRL were carried out using
the Flexi Rabbit Reticulocyte Lysate System (Promega). The
standard reaction mixture contained 33 ml of RRL, 3.5 ml of 1 M
KOAc, 1.0 ml of 1 mM amino acid mixture lacking methionine
(Promega), 1.0 ml of 5 mM L-methionine and 50 U of recombinant
RNasin ribonuclease inhibitor (Promega), for a total volume of
50 ml. MgOAc was added to adjust the final concentration of Mg2þ
to 1.2 mM. AdoMet was added at 1 mM final concentration when
its effect was analyzed. In vitro synthesized RNA was added to
the reaction mixture at a final concentration of 2 fmol ml1 for
primer extension experiments, and 50 fmol ml1 for immunoblot
and pulse–chase experiments. For the LUC assay, 2 fmol ml1 of
Ex1:Luc RNA and 1 fmol ml1 of Rluc RNA were used. All
reaction mixtures were incubated at 308C. LUC and RLUC
activities were assayed as described (Chiba et al. 2003). For RNase
A treatment, RNase A was added at a final concentration of
0.5 mg ml1, and the reaction mixtures were incubated for 15 min at
378C. CTAB precipitation was performed as described
(Nakatogawa and Ito 2001).
In vitro translation reactions using WGE (Promega) were
carried out as described (Chiba et al. 2003).
555
Immunoblot analysis
In vitro translation products were separated on a NuPAGE
4–12% Bis–Tris gel (Invitrogen, Carlsbad, CA, USA), transferred
to a Hybond-ECL nitrocellulose membrane (Amersham
Biosciences, Buckinghamshire, UK) and probed with a polyclonal
anti-GST antibody (Sigma-Aldrich or Zymed Laboratories, San
Francisco, CA, USA). The signals were detected using the ECL
Plus Immunoblotting Detection system (Amersham Biosciences),
and visualized using Hyperfilm ECL (Amersham Biosciences).
RNA analysis
Extraction of RNA after the in vitro translation reaction was
accomplished as described (Chiba et al. 2003). Primer extension
analysis was accomplished as described by using the TO4L primer
(Chiba et al. 2003). The radiolabeled image was visualized using
a BAS1000 Bio Image Analyzer (Fuji Photo Film, Tokyo, Japan).
Pulse–chase experiment
Pulse–chase analysis was performed as described (Onouchi
et al. 2005). [35S]Methionine (37 TBq mmol l1; Amersham
Pharmacia Biotech, Uppsala, Sweden) was added instead of nonlabeled methionine at a final concentration of 3.7 kBq ml1 (0.1 mM
methionine). Edeine was used to inhibit new rounds of translation
(Fang et al. 2004).
Funding
The Ministry of Education, Culture, Sports, Science
and Technology of Japan Grant-in-Aid for Scientific
Research on Priority Areas (17026001 to S.N.); Japan
Society for the Promotion of Science Grants-in-Aid for
Scientific Research (16370016 to S.N. and 18570032 to
H.O.); Japan Society for the Promotion of Science Fellows
Grant (9138 to Y.H.); the Asahi Glass Foundation Grant
Program for Natural Science (to H.O).
Acknowledgments
We are grateful to Dr. Derek Goto for critical reading of the
manuscript, Ms. Saeko Yasokawa for skilful technical assistance,
and Ms. Kumi Fujiwara for general assistance. We used the
Radioisotope Laboratory of the Graduate School of Agriculture,
Hokkaido University. Y.H. and D.K. are supported by the Japan
Society for the Promotion of Science.
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(Received January 21, 2007; Accepted February 14, 2008)