(Received for publication, March 1, 1995; and in revised form, April 24, 1995)
From the
Studies based on experimental strategies that utilized either
inhibitors or structural alterations point to the existence of an
inverse relationship between translation and stability of a given mRNA.
In this study we have investigated the potential link between
translation and stability of the yeast GCN4 mRNA whose
translational rates change with respect to amino acid availability. We
observed that under conditions favoring its translation, the steady
state levels of the GCN4 mRNA were decreased, but this was not
due to a measurable alternation in its decay rate. We have demonstrated
that an extensive destabilization of this message is intimately coupled
with its increased access to heavy polysomes, which occurs transiently
in the process of translational derepression. This transient change in
the stability is what readjusts the steady state levels of the GCN4 mRNA. This study demonstrates in vivo the existence of a
mechanism of mRNA degradation that is coupled with the process of
translation.
The process of genetic information flow involves, among other
things, the turnover of mRNA, the template for translation. In the last
few years, several studies have revealed an underlying relationship
between the decay rates of a large variety of different mRNAs and their
respective translational efficiencies (for reviews, see (1, 2, 3) ).
Using one experimental
approach, it was shown that inhibition of protein synthesis by
antibiotics results in stabilization of many mRNAs as is the case of
histone(4) , tubulin(5) , transferrin
receptor(6) , c-myc(7) ,
c-fos(8) , and granulocyte macrophage
colony-stimulating factor (9) mRNAs. In yeast, it has been
shown that almost all mRNAs are stabilized in the presence of the
translation inhibitor, cycloheximide(10, 11) . The
above studies point to the existence of a mechanism that serves to
couple the intrinsic rate of mRNA decay with its rate of translation.
Two molecular mechanisms, not mutually exclusive, have been proposed to
account for this coupling. First, a ribosome-associated nuclease or
another degradation factor might be responsible, which becomes active
when the ribosome interacts with destabilizing elements within the
mRNA. Alternatively, the degradation mechanism may require labile
cytoplasmic factor(s) that are rapidly lost upon protein synthesis
inhibition.
A different experimental strategy employed alterations
on the mRNA structure, that either introduce premature stop codons or
block translation initiation. Premature translation termination has
been shown to increase the rate of mRNA turnover in a number of cases,
a phenomenon that has been described as nonsense-mediated mRNA
decay(2, 12, 13, 14) . Such studies
have lead to the identification of the UPF1 gene in yeast,
which may be part of a cellular safeguarding system that prevents the
accumulation of potentially deleterious fragmented
polypeptides(12, 13, 14) . Introduction of a
strong secondary structure into the 5`-untranslated region (UTR)
All
of the experimental strategies described so far have the common
disadvantage of utilizing nonnatural conditions, either drugs or
mutated mRNAs. Therefore, it is not surprising to encounter cases of
apparently contradictory results as in the case of c-fos mRNA(15, 8) . In order to overcome this major
disadvantage, we decided to investigate the decay rates of an mRNA that
is translationally regulated in vivo. The yeast GCN4 mRNA is under translational regulation imposed by its primary
structure. This message is poorly translated when cells are grown in
media containing all amino acids (rich media) due to the negative
effects on translation of the four small open reading frames (ORFs)
located in its 5`-UTR(16, 17, 18) . However,
when the cells are grown under amino acid limitation conditions, GCN4 mRNA is translationally derepressed. Therefore, this mRNA
offered us the opportunity to investigate in vivo the
relationship of mRNA stability with translational rates. We have
demonstrated that the GCN4 mRNA is transiently destabilized
when its translation rate is increased.
First, we induced GCN4 mRNA translation by overexpressing, in wild-type
cells, the GCN2 protein kinase that, by phosphorylating the
Figure 1:
Steady state GCN4 mRNA levels
in relation to its translational status. A, the relative
amounts of the GCN4 mRNA in wild-type cells either transformed
with a plasmid overexpressing the GCN2 gene (HGCN2, lane2) or with the vector alone (lane1) are shown. The amount of the total RNA blotted is also
shown (rightpanel). B, wild-type (lanes1 and 2) and gcn2 (lanes3 and 4) cells were either transformed with a
plasmid bearing the
Third, we introduced into a wild-type and a gcn2 strain, a plasmid bearing a GCN4 gene derivative deleted
for the four upstream ORFs and thus relieved from translational
repression (
Figure 2:
Transcription through the GCN4 promoter in relation to the amount of Gcn4. Graphic representation
of the
Figure 3:
Decay rate of GCN4 mRNA under low
and moderate translation rates. Leftpanel, relative
levels of the GCN4 mRNA were measured at different time points
after a shift to 37 °C in a temperature-sensitive RNA polymerase II
mutant (rpb1-1). A, the GCN4 mRNA
decay rate from wild-type cells; B, the endogenous GCN4 mRNA decay rate from cells transformed with the
We addressed the
above hypothesis by monitoring the amount of the GCN4 mRNA
under conditions inducing rapid translational derepression. A shift
from rich to minimal medium, which was previously shown to result in an
acute increase in the translation of the GCN4 mRNA(27) , was imposed to wild-type cells. When
the cells were transferred to minimal medium, the amount of GCN4 mRNA detected within the first 3-5 min after the shift was
decreased (Fig. 4A). In the reverse experiment, when
the cells were returned to rich medium, the relative GCN4 mRNA
amount was increased within the first 5 min.
Figure 4:
GCN4 mRNA levels and decay rates during a
nutritional shift. A, wild-type cells grown in rich
medium (lane1) were shifted to amino acid starvation
medium, and RNA was isolated after 5 min (lane2). In
the reciprocal experiment, cells grown in starvation medium (lane3) were shifted to rich medium, and RNA was isolated
after 5 min (lane4). The respective amount of the
total RNA is depicted in the rightpanel. B, rpb1-1 mutant cells grown in rich medium at 24 °C (0` common for a and b series), underwent
either a single temperature shift to 37 °C (3` and 5` for a series) or an additional nutritional shift
to starvation medium (3` and 5` for b series). Leftpanel, the GCN4 mRNA
levels after 0, 3, and 5 min at 37 °C are shown. Centralpanel, the PGK1 mRNA levels were analyzed as in
the leftpanel. In the rightpanel,
the amount of the total RNA used for the left and centralpanels, is shown. Typical experiments are
presented.
We have revealed the existence of a link between the
stability of the GCN4 mRNA and a transient but drastic change
in its translational status. This finding is in agreement with a
plethora of published reports indicating that decreased rates of mRNA
decay are coupled with a translational blockage imposed either through
inhibitors or structural alterations. Our results present an example of
a natural mRNA whose translational efficiency determines its decay
rates and provide a new natural model through which the mechanism of
translationally coupled mRNA decay rates can be studied.
A first
indication for a link between translational and decay rates came from
measuring the steady state levels of the GCN4 mRNA. Moderate
translational derepression was imposed by increasing in vivo the amount of the Gcn2 protein kinase. The alternative of imposing
amino acid starvation conditions by utilizing inhibitors of amino acid
biosynthesis such as 3-AT (17) was avoided since such treatment
results in stabilization of mRNAs carrying nonsense
codons(28) . In all cases examined, an inverse influence of
translation on the GCN4 mRNA levels was observed, which was
not the result of altered transcriptional rates. Such a drastic
decrease in steady state levels was not easily justified as a
consequence of a change in the translational status, since these
conditions result in only a slight shift of the polysomal distribution
of the GCN4 mRNA toward heavier polysomes(26) .
Indeed, the measured decay rates of the GCN4 mRNA were
unaffected under conditions of low or moderate translation. The
apparent paradox concerning the determinants of the GCN4 mRNA
steady state levels could be resolved only by considering a transient
change in the decay rate that readjusts the final steady state levels.
In order to solve this problem, we followed the GCN4 mRNA
decay rates in a temporal manner. It was known that a transient and
dramatic increase in the rate of the GCN4 mRNA translation is
effected rapidly after the removal of amino acids from the growth
medium(27) . Indeed, by using such a temporal analysis during a
nutritional shift, we revealed that the decay rate of the GCN4 mRNA dramatically increases within a very short period of time
following the initiation of translational derepression. Therefore,
based on the sensitivity of the method employed, we may conclude that a
transient destabilization of the GCN4 mRNA is tightly coupled
with its shift to heavy polysomes. The possibility that the observed
transient phenomenon could be due to unrelated physiological changes is
unlikely, since the decay rates of the reference mRNA (PGK1)
remained constant during the experiment.
Two major conclusions can
be drawn from the combined interpretation of the above experimental
results. One is that the apparent increased decay rate of GCN4 mRNA is coupled with its increased access to heavy polysomes. The
second is that the apparent decrease in the steady state levels of this
message under conditions of moderate translation should be the
consequence of a transient destabilization inherited in the physiology
of translational derepression of the GCN4 mRNA. This
interpretation emphasizes the fact that the understanding of steady
state phenomena should consider the dynamic process of their
establishment.
It is worthwhile to discuss our observations
concerning the decay rates of the
The
coupling of translational derepression with changes in mRNA decay rates
poses several questions regarding the nature of this phenomenon. Is it
just the consequence of a physical association between the GCN4 mRNA and the ribosomes on which the decay machinery might reside,
or is GCN4 mRNA degradation an additional step in GCN4 regulation? Given the body of information that has been
accumulated, we believe that the former is true. The apparent
contradiction in the biology of the response between the translational
derepression and the increased decay rates for the overall increase in
the amount of Gcn4, could be resolved by considering the recently
demonstrated increased stability of this protein under amino acid
starvation conditions(30) .
We thank Allan Jacobson for making certain strains and
plasmids available to us as well as for critical reading of the
manuscript and helpful suggestions. We also thank our colleagues Paulos
Alifragis, Alekos Athanassiadis, Elsa Maniataki, and Christos Ouzounis
for helpful suggestions; Georgia Houlaki for the artwork; and Katerina
Michelidaki and Costas Chiotelis for essential services.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)immediately adjacent to the cap site of the mRNAs,
blocks ribosome binding, and therefore inhibits translation in cis. Using this approach, it was shown that the selective
degradation of the granulocyte macrophage colony-stimulating factor
mRNA is coupled to ongoing translation of this mRNA(9) .
Strains and Media
All yeast strains used in this
study were derivatives of S288C cured for the GAL2 deficiency. In detail, these were as follows: MATa
ura3-52 leu2-2, MATa gcn2-11 ura3-52
leu2-2, MATa rpb1-1 ura3-52. The rpb1-1 strain was kindly provided by A. Jacobson. GCN4 derepresing conditions were accomplished by growing
leucine auxotroph cells in minimal medium for 6 h. Minimal medium
contained yeast nitrogen base (Difco) and 2% glucose. All 20 amino
acids were supplemented when necessary.
Plasmids
The construction of GCN4-LacZ, ORFGCN4 and
ORFGCN4-LacZ has been described
elsewhere(16) . The DED1-LacZ plasmid was constructed
by inserting the ScaI/SalI DNA fragment from
ORFGCN4-LacZ, which contains the GCN4-LacZ gene,
from the initiation of transcription of GCN4 to the end of LacZ, into the HindIII site of Ycp88, which is
located just downstream of the promoter of DED1(19) .
The GCREHIS3-LacZ fusion contains the GCR element within
unique BamHI and EcoRI sites in the promoter of HIS3-LacZ, which was modified to include only the regulated
TATA element 18 base pairs downstream. This reporter gene is on a
centromeric yeast plasmid carrying URA3 for selection and was
obtained from K. Struhl. Construction of Yep24-GCN2 has been
described previously(20) .
RNA Procedures
Total yeast RNA was extracted from
the indicated strains grown under the appropriate conditions as
described previously(21) . Northern blotting, hybridization,
and signal quantitation have been described
previously(21, 28) . Total RNA loading and transfer
were monitored by visualization of the ethidium bromide-stained RNA on
the membrane after transfer. The DNA probes used where as follows: the
1.1-kilobase BamHI-PvuII DNA fragment of the GCN4 gene and the 2.1-kilobase BamHI-HindIII DNA
fragment from pRIP1 (22) kindly provided by A. Jacobson,
containing the PGK1 gene. mRNA decay rates were measured in a
strain harboring the rpb1-1 allele as described
previously(2, 10) .
Other Methods
Transformations were carried out
with the lithium acetate method(23) . -Galactosidase
assays were performed as described previously(17) .
GCN4 Translational Derepression Results in Decreased
GCN4 Steady State mRNA Levels
To delineate the possible
relationship between the GCN4 mRNA translational status and
the corresponding steady state mRNA levels, we took advantage of the
available genetic means to affect GCN4 mRNA translation. In
total, three different approaches were undertaken.
subunit of the eIF2, relieves the translational repression of the GCN4 mRNA(20) . This is shown in Fig. 1C (wt, wt/HGCN2, +Leu), where the
translational status of the GCN4 mRNA was monitored by
assaying for
-galactosidase activity produced by a reporter gene
driven by the GCN4 promoter and bearing the 5`-UTR of GCN4. As shown in Fig. 1A, the GCN4 mRNA levels were decreased approximately 3-fold (±0.2) in
this genetic background as compared with a wild-type strain (GCN4 mRNA, lane1versuslane2).
ORFGCN4 gene derivative (lanes2 and 4) or with the vector alone (lanes1 and 3). The longer endogenous (a) GCN4 mRNA migrates slower than the
ORFGCN4 message (b). As in A, the total RNA amount is
depicted in the rightpanel. C, graphic
representation of the
-galactosidase activity from a GCN4 based reporter fusion, in the genetic backgrounds depicted under
rich (+Leu) or poor (-Leu), nutritional
conditions. Typical experiments are presented. Values are Miller units
and represent the average of three independent
experiments.
Second, we investigated whether a blockage in the
translation derepression of the GCN4 mRNA had the opposite
effect on its levels. The above described analysis was performed using
a gcn2 mutant strain, where the Gcn2 protein kinase has been
inactivated. In such strains, translation of GCN4 mRNA is low
and cannot be derepressed (20, 24, Fig. 1C, gcn2, +Leu, -Leu). In this background, the
steady state amount of GCN4 mRNA was increased 2.5-fold
(±0.3) above the levels of a wild-type strain (Fig. 1B, lanes1 and 3).
ORFGCN4). As it can be seen in Fig. 2(GCN4-LacZversus
ORFGCN4-LacZ, +Leu), a GCN4-LacZ reporter gene devoid of its 5`-UTR produces under repressing
conditions a 50-fold excess of
-galactosidase compared with the
one containing the 5`-UTR. It was expected that the resulting increase
in the amount of Gcn4 will activate transcription of the GCN2 gene and thus will induce translational derepression of the GCN4 mRNA (20) . Indeed, in this
ORFGCN4-transformed wild-type strain, the
endogenous GCN4 mRNA was translationally derepressed (Fig. 1C, wt, wt/
ORFGCN4, +Leu). By contrast and as anticipated, the
ORFGCN4-induced translational derepression of the
endogenous GCN4 mRNA did not occur in a gcn2 strain (Fig. 1C, gcn2, gcn2/
ORFGCN4, +Leu). We again observed that the endogenous GCN4 mRNA was decreased approximately 2-fold (±0.1) in a
ORFGCN4-transformed wild-type, as compared with
a gcn2 strain (Fig. 1B, lanes2 and 4). An interesting observation from this series of
experiments was that the steady state levels of the highly translatable
ORFGCN4 mRNA were 1.5-fold (±0.2) lower than those
of the endogenous GCN4 mRNA (Fig. 1B, lanes2 and 4). The reduction in the GCN4 mRNA levels could not be attributed to the Gcn2 protein
kinase specifically, since the elevation of the Gcn2-independent GCN4 mRNA translation that results from overexpression of a
variant tRNA
(25) , also effected the same
reduction in the GCN4 mRNA levels (not shown).
-galactosidase activity from the depicted reporter gene
fusions under either repressing (+Leu), or derepressing (-Leu), conditions. The levels of activity a strain
transformed with the
ORFGCN4 derivative exhibits
(+
ORFGCN4), are also depicted. Values are Miller
units and represent the average of three independent
experiments.
The totality
of the evidence presented above, revealed a relationship between the GCN4 mRNA translational status and its steady state levels.
Whenever GCN4 mRNA was translationally derepressed, its mRNA
levels were decreased.
The Transcription of the GCN4 Gene Is Not Affected by
Induction of Its Translation
One possible explanation for the
above observed variations in the GCN4 mRNA levels is a
decrease in the transcription of the GCN4 gene effected by the
known squelching properties of high levels of Gcn4(26) . In
order to address this possibility, we examined the expression of a LacZ reporter gene driven by the GCN4 promoter. Cells
were grown under nonstarvation conditions for low, starvation
conditions for moderate, and transformed with ORFGCN4 for
high Gcn4 levels. The intracellular levels of Gcn4 were indirectly
monitored by assaying for
-galactosidase produced by a LacZ reporter driven by a promoter bearing the Gcn4 DNA binding site (GCRE-LacZ, Fig. 2). As shown in Fig. 2, the
transcription driven by the GCN4 promoter was only slightly
affected by the intracellular levels of Gcn4. Therefore, the observed
differences in the GCN4 mRNA levels must, for the most part,
be the consequence of alterations in the mRNA half-lives under the
translation conditions imposed.
The Decay Rate of the GCN4 mRNA Is Not Affected by the
GCN4 mRNA Translational Derepression
To determine the GCN4 mRNA half-life when translated with low or moderate rates, we
employed the rpb1-1 mutant strain (kindly provided by
Allan Jacobson). A time course analysis of the levels of a given mRNA
in this strain, following a shift from 24 to 37 °C, provides an
estimate for its decay rates(2) . Two strains were used for
such an analysis: the rpb1-1 strain and the rpb1-1 strain, transformed with the ORFGCN4 gene derivative to induce the translation of the endogenous GCN4 mRNA to moderate rates. As shown in Fig. 3, we did
not observe any significant differences in the decay rates of the GCN4 mRNA in these two strains. In both strains, the half-life
was approximately 15 min (±1), as it has been reported
previously(15) . Interestingly enough, the
ORFGCN4 mRNA, although highly translated, had a decay rate similar to that
of the wild-type message.
ORFGCN4 gene derivative; C, the exogenous
ORFGCN4 mRNA decay rate in the above transformed cells. Rightpanel; the amount of the total RNA loaded in each lane. A
typical experiment is presented.
GCN4 mRNA Translational Derepression Results in Transient
Destabilization of the GCN4 mRNA
Based on our previous results,
reporting that during a nutritional downshift GCN4 mRNA is
transiently loaded with heavy polysomes(27) , we envisioned a
situation where a transient change in the decay rate of the GCN4 mRNA would take place only immediately after the change in its
translational status. When the translation rate would settle to a
certain equilibrium, then the GCN4 mRNA decay rate would be
readjusted to its original value. This transient destabilization would
then account for the decreased steady state levels.
The short period of
time (relative to the normal half-life) required to observe these
changes implied an abrupt change in the decay rate of the message. In
order to confirm this, we measured decay rates under the above
conditions. rpb1-1 mutant cells, growing at 24 °C in
rich medium, were shifted to 37 °C and simultaneously to starvation
medium. In a parallel control experiment, only the temperature shift
was imposed to the cells. The amount of GCN4 mRNA detected at
the various time points after the double and the single shift is shown
in Fig. 4B. When just the temperature shift was
imposed, the typical decay rate (15 ± 1 min) was observed, as
expected (Fig. 4B, rowa). However,
when the cells underwent an additional nutritional shift, the GCN4 mRNA decay rate was increased 2.6-fold (±0.3) within the
first 3-5 min (Fig. 4B, rowb).
This effect was not a general consequence of the nutritional shift
since the decay rates of the PGK1 mRNA were not similarly
affected (Fig. 4B). We conclude that the GCN4 mRNA is destabilized in parallel with a drastic increase in its
translational rate.
ORFGCN4 mRNA. This
message is degraded with the same rate as the endogenous GCN4 mRNA, even though it is always associated with a larger number of
ribosomes(27) . It should be noted however, that this is a
different mRNA, lacking most of the 5`-UTR containing the upstream
ORFs. One could consider that this region contains a destabilizing
element that adds to the observed decay rate of the GCN4 mRNA,
whereas the
ORFGCN4 mRNA is subjected mainly to the
ribosome associated degradation machinery. It is tempting to speculate
that this instability is mediated by the proposed nonsense mRNA decay
mechanism (29) due to the presence of the four small ORFs. We
suggest that the net effect of these two opposing mechanisms
contributes to the overall decay rate of the GCN4 mRNA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.