Plant Cell Physiol. 49(3): 314–323 (2008)
doi:10.1093/pcp/pcn005, 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]
Ribosome Stacking Defines CGS1 mRNA Degradation Sites During Nascent
Peptide-Mediated Translation Arrest
Yuhi Haraguchi 1, Yoshitomo Kadokura 2, Mari Nakamoto
1
2
1, 3
, Hitoshi Onouchi
1
and Satoshi Naito
1, 2,
*
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589 Japan
Division of Life Science, Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, 060-8589 Japan
(Chiba et al. 1999, Chiba et al. 2003). During this feedback
regulation, a related short RNA species that is truncated at
its 50 end appears, which is probably an intermediate of
CGS1 mRNA degradation (Chiba et al. 1999). The mto1
mutants of Arabidopsis contain mutations within the exon
1 coding region of CGS that renders them insensitive to this
regulation (Chiba et al. 1999). As a result, they overaccumulate CGS1 mRNA, CGS protein and soluble
methionine. Transient and transgenic expression experiments using CGS1 exon 1–reporter fusions demonstrated
that the exon 1 coding sequence of CGS1 is necessary and
sufficient for its post-transcriptional regulation (Chiba et al.
1999, Suzuki et al. 2001). Mutagenesis of CGS1 exon 1
revealed that a short stretch of amino acid sequence, termed
the MTO1 region, encoded within CGS1 exon 1 and covering
the mto1 mutation sites is involved in the regulation. The
MTO1 region amino acid sequence is highly conserved
among the CGS enzymes of multicellular plants (Ominato
et al. 2002).
CGS1 exon 1-mediated post-transcriptional regulation
occurs during translation and is able to be recapitulated in
an in vitro translation system of wheat germ extract (WGE)
(Chiba et al. 2003). When RNA carrying CGS1 exon 1 fused
to a reporter gene was translated in WGE, AdoMet induced
accumulation of 50 -truncated RNA species that were the
same as those detected in vivo (Chiba et al. 2003). These
in vitro experiments also revealed that translation of CGS1
mRNA is temporally arrested prior to mRNA degradation
in response to AdoMet at the Ser94 codon located
immediately downstream of the MTO1 region (Onouchi
et al. 2005). During this translational pausing, the ribosome
is arrested at the translocation step and peptidyl-tRNASer
occupies the A-site of the stalled ribosome. Experiments
using synonymous codon substitutions and frameshift
mutations of the MTO1 region revealed that the amino
acid sequence of the MTO1 region, and not the nucleotide
sequence, is responsible for the translation elongation arrest
(Chiba et al. 1999, Onouchi et al. 2005). Amino acid
substitution experiments showed a close linkage between
the translation arrest and RNA degradation. These
observations led us to propose a model whereby the nascent
Expression of the Arabidopsis CGS1 gene that codes for
cystathionine c-synthase is feedback-regulated at the step of
mRNA degradation in response to S-adenosyl-L-methionine
(AdoMet). This regulation occurs during translation and
involves AdoMet-induced temporal translation arrest prior to
the mRNA degradation. Here, we have identified multiple
intermediates of CGS1 mRNA degradation with different
50 ends that are separated by approximately 30 nucleotides.
Longer intermediates were found to be produced as the
number of ribosomes loaded on mRNA was increased.
Sucrose density gradient centrifugation experiments showed
that the shortest mRNA degradation intermediate was
associated with monosomes, whereas longer degradation
intermediates were associated with multiple ribosomes.
Immunoblot analyses revealed a ladder of premature polypeptides whose molecular weights corresponded to products of
ribosomes in a stalled stack. An increase in smaller premature
polypeptides was observed as the number of ribosomes loaded
on mRNA increased. These results show that AdoMet induces
the stacking of ribosomes on CGS1 mRNA and that multiple
mRNA degradation sites probably correspond to each stacked
ribosome.
Keywords: S-Adenosyl-L-methionine — Arabidopsis
thaliana — Feedback regulation — Methionine biosynthesis
— mRNA stability — MTO1 region.
Abbreviations: AdoMet, S-adenosyl-L-methionine; CGS,
cystathionine g-synthase; GST, glutathione S-transferase; LUC,
firefly luciferase; NGD, no-go decay; WGE, wheat germ extract.
Introduction
The Arabidopsis thaliana CGS1 gene (gene ID
At3g01120) encodes cystathionine g-synthase (CGS; EC
2.5.1.48) (Kim and Leustek 1996) that catalyzes the
first committed step of methionine biosynthesis in higher
plants (Matthews 1999). In Arabidopsis, expression of
CGS1 is negatively feedback-regulated at the step of
CGS1 mRNA degradation in response to S-adenosylL-methionine (AdoMet), a direct metabolite of methionine
3
Present address: Olympus Corp., Life Science Group, Hachioji, 192-8512 Japan.
*Corresponding author: E-mail, [email protected]; Fax, þ81-11-706-4932.
314
Ribosome stacking and mRNA degradation
GS
T G C A
+
−
x1
(m
T:E
GS
T:E
x1
(W
T)
to1
-1)
MTO1 peptide-dependent translation elongation arrest
triggers mRNA degradation (Onouchi et al. 2005).
Primer extension analysis of CGS1 exon 1-containing
RNA after in vitro translation in the presence of AdoMet
revealed two different 50 ends of the 50 -truncated RNA
species that were approximately 30 nucleotides apart (Chiba
et al. 2003). This led us to hypothesize that another
ribosome is stalled behind the initially stalled ribosome
and that an mRNA degradation event also occurs at the
secondary stalled ribosome. In the present study, we have
tested this hypothesis and demonstrate that AdoMet
induces the stacking of ribosomes on CGS1 mRNA,
which probably determines the mRNA degradation points.
315
+
−
AdoMet
FL
Results
V
IV
III
MTO1 region
Multiple 50 -truncated RNA species are produced in CGS1
exon 1-mediated post-transcriptional regulation
Previous in vitro analyses of CGS1 post-transcriptional
regulation revealed the presence of two CGS1 degradation
intermediates with 50 ends located approximately 30
nucleotides apart (Chiba et al. 2003, Onouchi et al. 2005).
Since these 50 ends are located near the predicted 50 edge of
stalled ribosomes, we hypothesized that ribosome location
and mRNA degradation are closely linked; however, direct
evidence for this was still lacking. These studies were based
on an RNA construct that contained wild-type CGS1 exon
1 fused in-frame to a firefly luciferase (LUC) reporter gene
(Ex1:Luc). In order to enable the analysis of premature
protein products together with RNA intermediates during
translation arrest, we have developed a new RNA construct,
GST:Ex1, which contains a glutathione S-transferase (GST)
tag sequence fused in-frame to the N-terminus of CGS1
exon 1.
GST:Ex1(WT), a wild-type CGS1 exon 1 version of
GST:Ex1, was translated for 90 min in WGE in the presence
of 1 mM AdoMet. This translation period was chosen as
previous Northern hybridization experiments showed that
accumulation of 50 -truncated RNA species increased to
60 min and then remained relatively constant until 120 min
(Onouchi et al. 2005). In the current translation reaction,
primer extension analysis of poly(A)-selected RNA revealed
five different 50 -truncated RNA species with distinct 50
ends (RNA species I–V, Fig. 1). The two shortest RNA
species representing the downstream 50 -truncation sites
(RNA species I and II) had the same 50 ends as those
previously reported (Chiba et al. 2003), whereas RNA
species III–V were not detected in earlier studies. Similar
experiments using RNA without poly(A) selection produced
the same set of 50 -truncated RNA species. No such
truncated bands were detected with equivalent RNA
constructs containing the mto1-1 mutation within the
CGS1 exon 1 [GST:Ex1(mto1-1)] irrespective of AdoMet
II
I
Fig. 1 Detection of multiple 50 ends of the AdoMet-dependent
50 -truncated RNA species by primer extension analysis.
GST:Ex1(WT) RNA or GST:Ex1(mto1-1) RNA (2 fmol ml–1) was
translated for 90 min in the presence (þ) or absence (–) of 1 mM
AdoMet, as indicated. Poly(A) RNA was extracted and analyzed by
primer extension analysis with 50 -32P-labeled TO4L primer. The
numbered solid arrowheads (I–V) indicate the positions of the 50
ends of the 50 -truncated RNA species. Lanes T, G, C and A
represent a sequence ladder synthesized using the same primer
(shown in the sense strand sequence). Full-length primer extension
products (FL) are marked with an open arrowhead. The MTO1
region is indicated beside the sequence ladder. A representative
result of quadruple experiments is shown.
316
Ribosome stacking and mRNA degradation
MTO1 region
nucleotide position
230
260
290
200
CCU CCU AAU UUC GUC CGU CAG CUG AGC AUU AAA GCC CGU AGA AAC UGU AGC AAC AUC GGU GUU GCA CAG AUC GUG GCG GCU AAG UGG UCC AAC AAC CCA UCC UCC GCG
Trp Ser
III
II
I
P A
Fig. 2 The 50 -end points of 50 -truncated RNA species I–III determined by primer extension gel analysis. Reverse-transcribed products of
multiple 50 -truncated RNA species were fractionated in a sequencing gel as described in Materials and Methods. The 50 ends of the cDNA
fragments were determined by comparing the mobility of each fragment with dideoxy sequencing markers. Regions where major 50 -end
points are clustered are marked with filled boxes. An open arrowhead indicates the position of the toeprint signal (Onouchi et al. 2005).
The P- and A-sites of the initial stalled ribosome as inferred from the position of the toeprint signal, and the Trp93 and Ser94 codons are
indicated. Nucleotide positions are numbered relative to the first ATG codon of CGS1 exon 1. The 50 -truncated RNA IV and V were too
faint to determine the 50 end positions at the nucleotide level.
application (Fig. 1), consistent with previous studies (Chiba
et al. 1999, Chiba et al. 2003).
The GST:Ex1(WT) RNA construct allowed multiple
50 -truncated RNA intermediates to be identified that were
not previously detected. It was possible that these additional
intermediates were an artifact produced by the GST
tag sequence. However, this does not seem to be the case,
as similar multiple RNA species were also observed when
Luc:Ex1(WT), GST:Ex1(WT):Luc and Ex1(WT) RNAs
were translated in the presence of AdoMet (data not
shown).
Longer degradation intermediates appear as the number of
ribosomes loaded on mRNA increases
The locations of the 50 ends for each of the 50 -truncated
RNA species were mapped using primer extension gel
analysis. In the case of RNA species IV and V, only
approximate nucleotide positions for the 50 ends could be
calculated due to the faint signal strengths in the primer
extension analysis. As shown in Fig. 2, the 50 ends for each
of the 50 -truncated RNA species I–III are located approximately 30 nucleotides apart. In eukaryotes, one ribosome
protects about 30 nucleotides from RNase digestion (Wolin
and Walter 1988). Therefore, we hypothesized that multiple
ribosomes are stacked behind the initially stalled ribosome
and each 50 -end point corresponds to a stacked ribosome.
If this hypothesis is correct, the number of different
50 -truncated RNA species should be affected by the
number of ribosomes loaded on one mRNA.
To test this hypothesis, edeine, an inhibitor of
translation initiation (Dinos et al. 2004), was used to
control the number of ribosomes loaded on mRNA.
GST:Ex1(WT) RNA was translated in the presence of
AdoMet, and edeine was added to the reaction mixture at
different time points before or during the translation
reaction. The translation reaction was continued for
90 min after the last addition (30 min) of edeine and thus
a 120 min reaction period was used for all samples. As
shown in Fig. 3A, only 50 -truncated RNA species I and II
were observed when edeine was added at 10 min after the
start of translation, whereas all five 50 -truncated RNA
species were detected when edeine was added at 30 min after
the start of translation. No 50 -truncated RNA species were
detected if edeine was added at or before initiation of
translation (0 min and –10 min).
In parallel experiments, GST:Ex1(CD4) RNA was
used as a competitor to adjust the number of ribosomes
loaded on GST:Ex1(WT) RNA. GST:Ex1(CD4) RNA
contains codons 1–136 of CGS1 exon 1 sequence including
the MTO1 region and translation arrest site (Ominato et al.
2002), but lacks the region complementary to the TO4L
primer used for primer extension analyses. Translation
reactions containing a constant amount of GST:Ex1(WT)
RNA and different amounts of GST:Ex1(CD4) RNA were
incubated for 90 min and analyzed by primer extension
analysis. Increased amounts of GST:Ex1(CD4) RNA in the
reaction mixture were correlated with decreasing amounts
of longer 50 -truncated species of GST:Ex1(WT) RNA
(Fig. 3B). These results are consistent with the hypothesis
that the longer RNA degradation intermediates appear as
the number of ribosomes loaded on a single RNA increases.
Longer RNA degradation intermediates sediment with
multiple ribosomes
As shown in Fig. 2, the 50 ends of 50 -truncated RNA
species I–III mapped near the 50 edges of the predicted first,
second and third stalled ribosomes. Taken together with our
previous observation that translation arrest is temporal and
that translation resumes thereafter (Onouchi et al. 2005),
the possibility existed that stalled ribosomes remain
associated with mRNA degradation intermediates. If multiple ribosomes are tightly stacked behind the initially stalled
ribosome, and the multiple 50 -end points of the 50 -truncated
RNA species correspond to each stacked ribosome, longer
RNA degradation intermediates would be bound by a
greater number of ribosomes.
To test this hypothesis, we performed sucrose density
gradient centrifugation experiments of translation products
Ribosome stacking and mRNA degradation
A
317
B
AdoMet
GST:Ex1(WT)
+
+ −
+
T G C A
−
−10 0 10 30 (min)
GST:Ex1(WT)
+
−
AdoMet
Edeine
T G C A
0
0
FL
FL
V
V
IV
IV
III
MTO1 region
MTO1 region
III
II
II
I
I
1 2 3 4
5 6
Competitor
RNA
1 2 3 4
5 6 7 8
Fig. 3 Accumulation pattern of AdoMet-dependent 50 -truncated RNA species during control of translational efficiency. (A) Effect of
translation initiation inhibitor on accumulation pattern of the 50 -truncated RNA. GST:Ex1(WT) RNA (2.fmol ml–1) was translated in the
presence (þ) or absence (–) of AdoMet. Edeine was added either 10 min before GST:Ex1(WT) RNA addition (–10) or at the indicated time
points after starting the translation reaction (lanes 1–4). The total reaction incubation time was 120 min. Control samples without edeine
supplementation were normally translated for 120 min (lanes 5, 6). Poly(A) RNA was extracted and analyzed by primer extension analysis
with the TO4L primer. Full-length primer extension products (FL) are shown in the upper panel. A representative result of duplicate
experiments is shown. (B) Competition of the 50 -truncated RNA species bands by GST:Ex1(CD4) RNA. GST:Ex1(WT) RNA (2 fmol ml–1) was
translated for 90 min in the absence of competitor (0; lanes 1 and 5) or in the presence of 8 (lanes 2 and 6), 48 (lanes 3 and 7) and
123 fmol ml–1 (lanes 4 and 8) of GST:Ex1(CD4) RNA. Total RNA was extracted and analyzed by primer extension analysis with the TO4L
primer. Total RNA without poly(A) selection was used in this experiment due to the 460-fold difference in input RNA that alters recovery
efficiency of degradation intermediates using poly(A) RNA selection. Full-length primer extension products (FL) are shown in the upper
panel. A representative result of triplicate experiments is shown.
after a 60 min incubation in the presence and absence of
AdoMet (Fig. 4A, B). This shorter incubation time was
chosen because translation arrest is temporal and a stalled
ribosome resumes translation after a certain period
(Onouchi et al. 2005), which may affect the sedimentation
pattern. Primer extension analyses of poly(A) RNA
extracted from each fraction revealed that 50 -truncated
RNA species I sedimented with monosomes (Fig. 4B, lanes
5 and 6) and 50 -truncated RNA species II, III and IV
sedimented in polysome fractions with two, three and four
ribosomes (Fig. 4B, lanes 5–11). The results suggest that
ribosome stalling induces multiple ribosome stacking and
mRNA degradation at multiple sites.
The difference in the distribution of full-length mRNA
in the sucrose gradient in the absence and presence of
AdoMet suggests that ribosome stacking induced polysome
formation. In the absence of AdoMet (Fig. 4A), the relative
amount of full-length mRNA was similar in fractions 7–11
(corresponding to 42 ribosomes), whereas in the presence
of AdoMet (Fig. 4B), the amount of the full-length mRNA
was greatest in the bottom two fractions (45 ribosomes).
It is possible that multiple ribosomes are loaded on
mRNA but are stacked at the termination codon (Wolin
and Walter 1988, Gaba et al. 2005). To determine the
distribution of the premature translation products that
occurred by AdoMet-induced translation arrest, fractions
of the same sucrose density gradient were subjected to
immunoblot analysis using anti-GST antibody (Fig. 4C, D).
The distribution of the full-length translation products in
the polysome fractions (fractions 7–11) is similar to that of
the full-length RNA in both the absence (Fig. 4A, C) and
presence (Fig. 4B, D) of AdoMet. This suggests that
multiple ribosomes can be present on CGS1 mRNA, but
this is independent of AdoMet-induced translation arrest
and probably due to multiple co-translation events or
temporary stacking at the termination codon. On the other
hand, accumulation of the AdoMet-induced translation
arrest product was greatest in the bottom two fractions
318
Ribosome stacking and mRNA degradation
A
Top
−AdoMet
B
Bottom
Top
+AdoMet
Bottom
80S
A 254
A 254
80S
Polysomes
Polysomes
6
2 3 45
2 3 4 56
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Primer extension
Primer extension
FL
FL
IV
MTO1 region
MTO1 region
III
T GC A
II
I
T GC A
1 2 3 4 5 6 7 8 9 10 11
Fraction number
C
Immuno-blotting
Fraction number
D
(kDa)
50
FL peptide
1 2 3 4 5 6 7 8 9 10 11
Immuno-blotting
arrest-PtR
FL peptide
(kDa)
50
37
37
BC 1 2 3 4 5 6 7 8 9 1011
BC 1 2 3 4 5 6 7 8 9 10 11
Fraction number
Fraction number
Fig. 4 Sedimentation of the 50 -truncated RNA species and peptidyl-tRNA in the sucrose gradient. GST:Ex1(WT) RNA (50 fmol ml–1) was
translated for 60 min in the absence (A) or presence (B) of AdoMet and the samples were fractionated by 10–30% (w/v) sucrose density
gradient centrifugation. UV absorbance profile at 254 nm (upper panels) and primer extension analysis of each fraction with 50 -32P-labeled
TO4L primer (middle and lower panels) are shown. Full-length primer extension products (FL) are shown in the middle panels. In the top
panel, the positions of the 80S monosome and polysomes with 2–6 ribosomes are indicated in the UV absorbance profile. Free RNA
sediments in fractions 1–3. Representative results of duplicate experiments are shown. (C and D) Immunoblot analysis of translation
products prepared from the translation reaction mixture used in (A) and (B), respectively. Peptide products were detected using an anti-GST
antibody. The 45 kDa full-length translation product (FL peptide) and the 55 kDa AdoMet-dependent peptidyl-tRNA (arrest-PtR) are
marked. Lanes BC represent samples before centrifugation. Representative results of duplicate experiments are shown.
in Fig. 4D, suggesting that AdoMet does indeed induce
ribosome stacking on CGS1 exon 1 mRNA, most probably
at the Ser94 codon (numbered from the first methionine of
CGS1) where AdoMet-induced ribosome stalling takes
place (Onouchi et al. 2005).
Identification of premature translation products caused by
ribosome stacking
To examine further the hypothesis of ribosome
stacking during translation arrest, premature translation
products were characterized in greater detail. If ribosome
Ribosome stacking and mRNA degradation
stacking is induced upon translational pausing, multiple
premature translation products should also accumulate
when RNA containing CGS1 exon 1 is translated in the
presence of AdoMet. Since different tRNA species have
different effects on the migration of peptidyl-tRNA on
SDS–PAGE (Onouchi et al. 2005), RNase A treatment was
carried out before immunoblotting to remove attached
tRNA species and enable accurate separation of premature
translation products.
Since a single ribosome covers approximately 30
nucleotides, we may expect that the second and third
ribosome in a stalled stack would produce polypeptides
with a size difference of approximately 1 kDa (10 amino
acids). Following the RNase A treatment, the initial single
peptidyl-tRNA band signal separated into three distinct
bands in the 33–35 kDa region (premature polypeptides I, II
and III; Fig. 5A). The difference between each premature
polypeptide was approximately 1 kDa.
If these three bands are indeed produced by the first,
second and third ribosome in a stalled ribosome stack,
premature polypeptides III and II should disappear as the
number of ribosomes loaded on a single RNA is reduced. In
order to test this, a titration-style experiment was designed
to reduce the number of loaded ribosomes by adjusting the
availability of RNA target. Different amounts of GST:Ex1
RNA were added to the translation reaction mixture and
incubated for 30 min prior to RNase A treatment. This
incubation time was previously shown to represent the
maximum accumulation of peptidyl-tRNASer (Onouchi
et al. 2005). Increased amounts of GST:Ex1 RNA in the
translation reactions were found to be associated with a
reduction in accumulation of premature polypeptides II and
III (Fig. 5B, RNase A treatment), suggesting that premature
polypeptides II and III do indeed correspond to the second
and third ribosome, respectively, stacked behind the initial
stalled ribosome.
The three 33–35 kDa bands were not observed when
GST:Ex1(WT) RNA was translated in the absence of
AdoMet, or when GST:Ex1(mto1-1) RNA was translated
in the presence or absence of AdoMet (Fig. 5A). However,
different bands of about 38 kDa appeared in these reactions
(marked by asterisks in Fig. 5A). A large stem–loop
structure is predicted to exist in the downstream region
starting at the 103rd codon (Hacham et al. 2006), and these
bands disappear if this stem–loop region is deleted (data not
shown). It is therefore likely that these bands in the 38 kDa
region are produced by ribosomes stalled around this stem–
loop structure.
Discussion
In the present study, we have demonstrated that
multiple forms of 50 -truncated RNA species and premature
A
319
GST:Ex1(WT) GST:Ex1(mto1-1)
−
+
(kDa)
−
+
−
−
+
−
+
+
−
AdoMet
+
RNase A
75
arrest-PtR
50
FL peptide
P-I
P-II
P-III
37
25
P-I
P-II
P-III
B
GST:Ex1(WT)
+
−
+
50 250 500
arrest-PtR
AdoMet
(kDa)
RNase A
50 250 500 input RNA
(fmol/µl)
50
FL peptide
FL peptide
P-I
P-II
P-III
37
25
input RNA
(fmol/µl)
50
250
500
P-I
P-II
P-III
Fig. 5 Immunoblot
analysis of
translation
products.
(A) GST:Ex1(WT) RNA and GST:Ex1(mto1-1) RNA (50 fmol ml–1)
was translated for 30 min in the presence (þ) and absence (–) of
1 mM AdoMet, as indicated. Translation products were analyzed
by immunoblot analysis using an anti-GST antibody. The 55 kDa
AdoMet-dependent peptidyl-tRNA (arrest-PtR), the 45 kDa fulllength translation product (FL peptide) and approximately 35 kDa
triplet bands shifted by the RNase A treatment (P-I, P-II and P-III) are
marked. The positions of protein size markers are indicated (upper
panel). The region indicated by brackets in the upper panel is
enlarged (lower panel). A representative result of duplicate
experiments is shown. (B) Increasing amounts of GST:Ex1(WT)
RNA were translated as indicated and translation products were
analyzed by immunoblot analysis using an anti-GST antibody
(upper panels). The 55 kDa AdoMet-dependent peptidyl-tRNA
(arrest-PtR), the 45 kDa full-length translation product (FL peptide)
and approximately 35 kDa triplet bands shifted by the RNase A
treatment (P-I, P-II and P-III) are marked. The 33–35 kDa region in
the upper right panel is enlarged (lower panel). A representative
result of duplicate experiments is shown.
320
Ribosome stacking and mRNA degradation
translation products are produced in response to AdoMet
during in vitro translation of RNA carrying CGS1 exon 1.
Distances between each of the 50 -truncated RNA species
I–III were about 30 nucleotides, which matches the length
of mRNA covered by a ribosome (Wolin and Walter 1988).
Our data indicated that these RNA degradation intermediates and translation arrest products are caused by the
stacking of multiple ribosomes, and that the initial stalled
ribosome was responsible for production of 50 -truncated
RNA species I and premature polypeptide I. As the number
of ribosomes loaded on mRNA increases, the amount of
longer 50 -truncated RNA species and smaller premature
polypeptides increased. This observation is consistent with a
model where ribosomes are tightly stacked behind the initial
stalled ribosome and mRNA degradation events occur with
a certain possibility near the 50 edge of the stacked
ribosomes. This is the first report of RNA degradation at
multiple upstream sites due to ribosome stacking.
We have previously shown that the 50 ends of
0
5 -truncated RNA species (corresponding to RNA species
I and II in this article) produced in vivo and in vitro were
essentially the same (Chiba et al. 2003). We also found that
the 50 -truncated CGS1 RNA species III is detectable in
methionine-treated wild-type calli (Y. Haraguchi et al.
unpublished results). These data suggest that the in vitro
ribosome stacking and RNA degradation events observed
here are a true representation of those occurring in vivo.
A simple model for the production of multiple
50 -truncated RNA species by stacked ribosomes would be
that these are produced by 50 !30 exoribonuclease digestion.
This model predicts that as the number of ribosomes loaded
on one mRNA increases, the amount of 50 -truncated RNA
species I, which is produced by the initially stalled ribosome,
would decrease. However, Fig. 3B shows that this is not the
case. The amount of 50 -truncated RNA species I remained
constant over a 60-fold difference in the amount of
input RNA (tester RNA plus competitor RNA) (Fig. 3B,
lanes 1–4). This result infers that even though multiple
ribosomes are loaded on mRNA, the production of
50 -truncated RNA species I remains relatively unchanged.
The polysome profile in Fig. 4B shows that at this input
RNA to ribosome ratio, the majority of the RNA (fulllength band) is in fractions that contain 45 ribosomes
bound on RNA (fraction numbers 10 and 11). It should also
be noted that the amount of the input RNA, and thus the
input RNA to ribosome ratio, is the same between Fig. 3B
lane 3 and Fig. 4B. Finally, the 50 end positions of the
50 -truncated RNA I, II and III are located within the
regions that would be covered by the first, second and third
stalled ribosomes, respectively (Fig. 2). It is therefore likely
that an endonucleolytic digestion, rather than an exonucleolytic digestion, is responsible for the production of the
50 -truncated RNA species.
Sucrose density gradient centrifugation experiments
showed that the stalled ribosomes remain associated with
50 -truncated mRNA species (Fig. 4B). Although the precise
position of these ribosomes remains to be determined, it is
likely that the initially stalled ribosome and stacked
ribosomes are located at the 50 end of the 50 -truncated
mRNAs. The 50 -truncated RNA would thus be protected
from 50 !30 exoribonuclease by these stalled ribosomes
(Zicker et al. 2007), explaining why the 50 -truncated RNA
species are relatively stable as RNA degradation intermediates. We have previously determined that the
50 -truncated RNA has a half-life of about 30 min in callus
cultures (Lambein et al. 2003).
A plausible model for mRNA degradation caused
by AdoMet-induced ribosome stalling at the Ser94 arrest
site is shown in Fig. 6. The translation of CGS1 mRNA
is temporarily arrested at the Ser94 codon in response
to AdoMet immediately following translation of the
MTO1 region. As a result of this translation arrest, multiple
ribosomes become tightly stacked behind the initial stalled
ribosome, and mRNA degradation events then occur
near the 50 -edge of each of stacked ribosomes with a
certain possibility. If mRNA degradation events were to
occur with high efficiency at each ribosome in a stack,
only the shortest 50 -truncated RNA would be produced.
The fact that multiple forms of 50 -truncated RNA
species were observed indicates that mRNA degradation
occurs with a low efficiency at any of the ribosomes in
the stack. Multiple forms of the 50 -truncated mRNA
species and premature translation products are thus
produced.
The data presented in this study showed that in the
AdoMet-induced ribosomal stalling of CGS1 mRNA, the
major 50 -end point of the 50 -truncated RNA I is located
13–14 nucleotides upstream of the A-site codon (Fig. 2).
End points of mRNA degradation intermediates have been
mapped near stalled ribosomes in Escherichia coli ybeL
(Hayes and Sauer 2003), secM (Sunohara et al. 2004,
Garza-Sánchez et al. 2006) and daaP (Loomis et al. 2001),
and in Bacillus subtilis ermC (Drider et al. 2002). Among
these, E. coli secM and B. subtilis ermC mRNA have their
degradation intermediate end points at 8–11 and 12
nucleotides, respectively, upstream of the A-site codon.
Also in the case of E. coli daaP, mRNA cleavage occurs at
15 nucleotides upstream of an A-site, although additional
experimental evidence is required to clarify the site of
ribosome stalling in this case. Björnsson and Isaksson
(1996) and Li et al. (2007) have reported that ribosome
stalling occurs at the termination codon and mRNA
degradation occurs at 13 and 15 nucleotides, respectively,
upstream of the A-site. It is possible that CGS1 may share a
similar mRNA degradation mechanism with these bacterial
systems.
Ribosome stacking and mRNA degradation
nascent peptide
AUG
5′ cap
321
AdoMet-induced translation arrest
at Ser-94 codon
UGA
AAA
UGA
AAA
AAAA
80S
5′ cap
UGA
AAA
AAAA
UGA
AAA
AAAA
AAAA
UGA
AAA
AAAA
Fig. 6 Model for ribosome pausing and CGS1 mRNA degradation at multiple sites caused by AdoMet-induced translation arrest. When
the leading ribosome stalls at the Ser94 codon under the cellular condition of high AdoMet concentration, other ribosomes stack up behind
the initially stalled ribosome. Following the sequential stacking of the ribosomes, mRNA degradation events occur with a certain possibility
near the 50 edge of a ribosome in a stalled stack. The sum of these degradation events generates multiple 50 -truncated RNAs carrying
different 50 ends that are separated by approximately 30 nucleotides, corresponding to the length occupied by one ribosome. The RNase
(Pacman), presumably an endoribonuclease, responsible for the initial mRNA degradation reaction is yet to be identified.
On the other hand, there are only a limited number of
reports on the stacking of ribosomes and mRNA degradation in eukaryotes. In the case of c-myc mRNA in
mammals, the ribosome is transiently arrested at a rare
codon and the downstream region that is depleted of
translating ribosome becomes sensitive to an endoribonuclease digestion (Lemm and Ross 2002). However, this is the
opposite to that occurring for CGS1 mRNA as destabilization of mRNA upstream of the ribosomal pause site was
not reported. It is intriguing to surmise that plant and
mammals may have different systems to degrade mRNA
upon translation elongation arrest. In the case of CGS1,
translation arrest can be recapitulated in an in vitro
translation system of rabbit reticulocyte lysate, but
50 -truncated RNAs are not detected (H. Onouchi et al. in
preparation).
In Saccharomyces cerevisiae, ‘no-go decay (NGD)’ has
recently been identified that cleaves mRNAs when translation elongation is arrested due to a strong stem–loop
structure, a pseudo-knot or a rare codon (Doma and Parker
2006). Dom34p, an essential component of NGD, has been
shown to have endoribonuclease activity (Lee et al. 2007)
and is thought to interact with an empty A-site of a stalled
ribosome since it is related to a eukaryotic release factor,
eRF1. In the case of CGS1 mRNA decay, the A-site of the
AdoMet-induced stalled ribosome has been suggested to be
occupied by peptidyl-tRNA (Onouchi et al. 2005). It is
therefore unlikely that NGD is responsible for the production of the 50 -truncated RNA species I as the ribosome–
mRNA complex would be inaccessible to the Arabidopsis
Dom34p counterpart.
In this study, we have provided evidence for AdoMetinduced ribosome stacking by analyzing the mRNA
degradation intermediates and translation arrest products.
The findings allow us to probe the molecular mechanisms of
CGS1 mRNA degradation, and continue to give new
insights into the nascent peptide-mediated regulation of
gene expression. One exciting question yet to be answered
about CGS1 regulation is the identity of the pathway for
mRNA degradation. Based on research done primarily in
yeast and mammal systems, two general pathways for
mRNA turnover have been identified in eukaryotic cells
(Parker and Song 2004). It remains to be determined
whether CGS1 regulation involves these pathways or
represents a specialized degradation mechanism.
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 listed in Chiba
et al. (2003).
Plasmid construction
pMN1(WT) and pMN1(mto1-1) which carry the
GST:Ex1(WT) and GST:Ex1(mto1-1) DNA in the pSP64
poly(A) vector (Promega, Madison, WI, USA), respectively, were
described previously (Onouchi et al. 2005).
The plasmid pSY152(WT) carries the GST:Ex1(CD4) DNA
encoding the C-terminal deletion variant of CGS1 exon 1
(carries amino acids 1–136 of CGS; Ominato et al. 2002). To
construct pSY152(WT), the 0.55 kbp XbaI–BamHI fragment of
pMN1Xba(WT) was replaced with the 0.4 kbp XbaI–BamHI
322
Ribosome stacking and mRNA degradation
fragment of the CD4 version of pMI4(WT) (Ominato et al. 2002).
In pMN1Xba(WT), the XbaI site (50 -TCTAGA-30 ) immediately
50 of the GST coding region of pMN1(WT) was mutated to
50 -ACTAGA-30 to facilitate subcloning.
In vitro transcription
DNA templates in pSP64 poly(A) vector (Promega) were
linearized with EcoRI and purified as described previously (Chiba
et al. 2003). In vitro transcription in the presence of a cap analog
m7G[50 ]ppp[50 ]GTP (Epicentre Technologies, Madison, WI, USA)
was accomplished and poly(A) RNA was purified as described
(Chiba et al. 2003).
In vitro translation
In vitro translation reactions using WGE (Promega) were
carried out as described (Chiba et al. 2003). AdoMet was added at
1 mM final concentration. Edeine sulfate was used to inhibit new
rounds of translation initiation during prolonged incubation. For
RNase A treatment, RNase A was added at a final concentration
of 0.5 mg ml–1, and the reaction mixtures were incubated for 15 min
at 378C.
Primer extension studies
The primer extension reaction was carried out as described
(Chiba et al. 2003) except that the primer used was TO4L
(50 -TACCGGCATGAACAGTGAGGCTCCCAT-30 ), and the
reverse transcription reaction was carried out at 588C. The 30 end
of the TO4L primer is located at the 524 bp position from the first
ATG of CGS1 exon 1. The radiolabeled image was visualized using
a BAS1000 Bio Image Analyzer (Fuji Photo Film, Tokyo, Japan).
Sucrose density gradient centrifugation
GST:Ex1 RNAs were translated in vitro in a 50 ml reaction
mixture. Sucrose density gradient centrifugation and collection of
fractions was then performed as described (Onouchi et al. 2005).
For the primer extension reaction, total RNA was extracted from
each fraction and poly(A) RNA was purified as described (Suzuki
et al. 2001, Chiba et al. 2003).
Immunoblot analysis
After in vitro translation of GST:Ex1 RNA in a 20 ml reaction
mixture, immunoblot analysis was performed as described
(Onouchi et al. 2005) except that monoclonal anti-GST antibody
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used and
the signals were detected using Immobilon Western Detection
Reagents (Millipore, Billerica, MA, USA).
Funding
Ministry of Education, Culture, Sports, Science and
Technology of Japan Grants-in-Aid for Scientific Research
(16370016 and 17026001 to S.N.; 18570032 to H.O.); the
Asahi Glass Foundation Grant Program for Natural
Science (to H.O.); Hokkaido University Grant Program
for Leading Edge Research (to S.N.); the Japan Society for
the Promotion of Science (9138 to Y.H.).
Acknowledgments
We are grateful to Dr. Derek Goto for critical reading of the
manuscript as well as for valuable discussions, Ms. Saeko
Yasokawa for skillful technical assistance, and Ms. Kumi
Fujiwara for general assistance. We used the Radioisotope
Laboratory of the Graduate School of Agriculture, Hokkaido
University. Y.H. is supported by the Japan Society of Promotion
of Science.
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(Received December 9, 2007; Accepted January 6, 2008)