Received 25 January 1999/Returned for modification 11 March
1999/Accepted 23 April 1999
 |
INTRODUCTION |
The differential turnover of mRNAs
is an important aspect of the modulation of gene expression in the
eukaryotic cell (for reviews, see references 9, 15,
29, and 45). It is now clear that, at
least in Saccharomyces cerevisiae, mRNAs with different decay rates are degraded primarily by a single general pathway of mRNA
turnover. In this pathway, mRNAs are first deadenylated, which allows
the transcript to become a substrate for a decapping reaction (18,
28, 40-42), and once decapped, they are susceptible to 5'-to-3'
exonucleolytic degradation by the Xrn1p exoribonuclease (28, 41,
42). Several observations suggest that a similar pathway of
degradation is likely to exist in mammalian cells. For example,
deadenylation can be the first step in mammalian mRNA turnover (see,
e.g., references 48 and 53).
Moreover, deadenylated decapped intermediates of the decay process can
be detected (17), and mammalian homologs of the yeast Xrn1p
exoribonuclease (7) and the yeast-decapping enzyme (encoded
by the DCP1 gene [10]) have been identified
(51).
The basis for differential decay rates of individual yeast mRNAs is
that the transcripts differ in their rates of deadenylation and
decapping. For example, at 24°C the unstable MFA2
transcript (t1/2 = 4 min) deadenylates more
rapidly (~13 A's/min) than does the stable PGK1
transcript (t1/2 = 45 min; ~4 A's/min)
(18). In addition, the MFA2 transcript is
decapped rapidly after deadenylation whereas the PGK1
transcript is decapped slowly (41, 42). Given these
differences, in order to understand differential mRNA degradation it
will be critical to determine the sequences and properties of mRNAs
that modulate the rates of deadenylation and decapping.
In addition to being the site of decapping, the cap structure is
crucial for the ability of a mRNA to initiate translation efficiently
(5, 44). This dual role of the cap structure has led to the
hypothesis that the rate of decapping is specified by the nature of the
cap binding complex or by proteins that interact with this complex of
proteins. Given this view, a likely set of proteins to affect decapping
are those that make up the cytoplasmic cap binding complex, also
referred to as the eIF-4F complex (22, 49). In addition, a
second set of proteins known as eIF-3 may play a role in decapping,
since this complex recruits the 40S ribosomal subunit to the 5' end of
the mRNA by binding to both the eIF-4F complex and the mRNA itself
(25, 43). An important issue is how these proteins influence
the ability of a mRNA to be decapped.
Prior examination on the effects of mutations in translation initiation
factors on mRNA stability have been mixed. For example, mutations in
the Prt1 protein, which is a component of the eIF-3 complex, have been
reported to accelerate the decay of the SSA1 and Ip mRNAs
under specific conditions (6, 16). Conversely, specific
mutations in the translation initiation factor eIF-5A have been
reported to block 5'-to-3' exonucleolytic digestion (55).
Finally, it has been reported that mutations in the eIF-4E protein that
lead to a partial decrease in translation in vivo have no effect on the
degradation rate of the PGK1 mRNA in yeast (37),
which is known to be degraded primarily by deadenylation-dependent decapping (42). This observation has raised the possibility that the eIF-4E protein and possibly other initiation factors do not
play a major role in mRNA turnover (37). However, it is
possible that the eIF-4E alleles examined, which were
partial-loss-of-function alleles, were simply not strong enough to show
an effect.
To examine more completely the role of the translation initiation
factors in controlling mRNA turnover, we have examined the stability of
the MFA2 and PGK1 mRNAs in strains carrying
alleles of several of the components of eIF-4F and eIF-3 that were
known to cause a defect in translation initiation. These two mRNAs were ideal for comparison in this analysis for several reasons. First, they
represent the range of mRNA stability in yeast, since the MFA2 mRNA is unstable (t1/2 = 4
min) and the PGK1 transcript is stable
(t1/2 = 45 min) (27). Most
importantly, the decay of these mRNAs has been well characterized, and
both are degraded by the deadenylation-dependent decapping pathway of
mRNA turnover (41, 42). Moreover, the different rates of
mRNA degradation of these two transcripts can be attributed to specific
differences in deadenylation and decapping (18, 41, 42). We
observed that mutations in either the eIF-4F complex or the eIF-3
complex can influence the rate of mRNA turnover by increasing both the rate of deadenylation and the rate of decapping. These observations have implications for understanding the control of mRNA turnover and
suggest that the status of the translation initiation complex on the
mRNA dictates the rates of both deadenylation and decapping.
| |
MATERIALS AND METHODS |
Yeast strains.
The genotypes of all the strains used are
listed in Table 1, and the strains were
grown in standard media. All strains except yRP1323 and yRP1324 have
GAL1 upstream activating sequence-regulated PGK1pG and MFA2pG genes as well as the
LEU2 gene, collectively termed LEU2pm, integrated
at the CUP1 locus (26). The CAF20 and eIF-4G2
disruption plasmids were obtained from Michael Altmann and used to
disrupt the CAF20 and eIF-4G2 loci with a URA3 gene in the
yRP841 background (23, 34). Both integrations were verified
by Southern blot analysis (data not shown). The eIF-4G1
strain and
the cdc33-42 strains were obtained from Nahum Sonenberg and repeatedly
crossed into the same genetic background by using strain yRP841
(2, 23). The prt1-63 strain was obtained from Alan
Hinnebusch and repeatedly crossed to yRP841 (20, 21). The
mutant eIF-4A (SS8-3A) strain was obtained from Patrick Linder (47) and transformed with pRP469 and pRP485 for mRNA
analysis. The yeast strains were transformed by standard techniques,
and plasmids were maintained when necessary by growth in selective media.
mRNA analysis.
Steady-state transcriptional shutoff
experiments were performed as described previously with slight
modifications (13). Briefly, the cells were grown to mid-log
phase in galactose medium, shifted to 38°C for 1 h, harvested,
and shifted to medium containing dextrose to inhibit transcription,
with an aliquot removed at each time point (specified on figures).
Transcriptional pulse-chase experiments used to track a synchronous
pool of mRNAs were done as previously described with slight modifications (18, 41). Briefly, cells were grown to mid-log phase in medium containing raffinose, shifted to 38°C for 1 h, harvested, and resuspended in medium containing galactose to induce the
transcripts; finally dextrose was added to shut off transcription.
mRNA was isolated as described previously (13). Agarose
Northern assays were done with 10 µg of RNA, and RNase H and
polyacrylamide Northern (RNA) assays were done with 40 µg of RNA as
previously described (40). Northern assay results were
quantitated on a Molecular Dynamics PhosphorImager and standardized
to 7S RNA, a polymerase III transcript (13).
In vivo translation.
In vivo [35S]methionine
incorporation was performed as previously described (2) with
some modifications. Briefly, yeast strains were grown to mid-log phase
(optical density at 600 nm 
0.4) at 24°C in medium containing
dextrose, at which point half of each culture was shifted to 38°C for
1 h. The cells were then harvested, resuspended in prewarmed
methionine-minus medium containing dextrose, and supplemented with 25 µCi of [35S]methionine. At various time points,
aliquots were removed and the cells were harvested and resuspended in 1 ml of ice-cold 10% trichloroacetic acid. The samples were heated at
95°C for 5 min and cooled on ice. The precipitated protein was
collected on glass fiber filters, washed with 10 ml of 5%
trichloroacetic acid, and washed again with 10 ml of 70% ethanol and
finally with 1 ml of acetone. The filters were air dried for 30 min and
then counted in a liquid scintillation counter.
| |
RESULTS |
Defects in the cytoplasmic cap binding complex (eIF-4F) lead to
higher mRNA decay rates.
To test the hypothesis that translation
initiation factors binding to the 5' end of a mRNA play a role in
controlling decapping, we first examined the effects of mutations in
the components of the cap binding complex (eIF-4F) on mRNA turnover.
This complex in yeast is generally described as consisting of eIF-4E
(cdc33), the cap binding protein (1), and an eIF-4G subunit,
which is encoded by two genes in yeast, TIF4631, which
encodes eIF-4G1, and TIF4632, which encodes eIF-4G2
(23). In addition, we examined the effects of mutations in
eIF-4A, a DEAD box helicase required for translation (47)
and known to physically associate with eIF-4F in mammals (19,
24), and p20, encoded by the CAF20 gene, that is
thought to bind the cap binding protein in a negative regulatory role
(3). If deletion of a given gene was lethal, temperature-sensitive mutants were used in this analysis. All temperature-sensitive alleles used were alleles that had previously been shown to have effects on translation rate in vivo (2, 20, 21,
47) (see below). If a strain with deletion of a particular gene
was viable, strains with disruptions of the gene were used.
The translation initiation mutations could be placed into two
categories based on their effects on mRNA half-life. For example, disruption of the CAF20 gene had little or no consequence on
the decay rate of either the PGK1 mRNA or the
MFA2 mRNA (Fig. 1A and Table
2). Since this protein does not
significantly affect growth rate or translation rate (34),
it was not surprising that a caf20 mutation failed to alter mRNA
decay rates.
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 1.
Disruption of translation initiation factor function
leads to a decrease in the half-lives of mRNAs. (A) mRNA half-life
measurements from strains containing mutations in translation
initiation factors from the eIF-4F complex. (B) mRNA half-life
measurements from strains containing a mutation in PRT1, a
translation initiation factor from the eIF-3 complex. (C) mRNA
half-life measurements from a strain containing mutations in both
eIF-4E and PRT1. (D) mRNA half-life measurements from
strains containing mutations in both PRT1 and
UPF1. Steady-state half-life measurements of the
PGK1pG mRNA were determined from agarose Northern gels. The
numbers above the lanes indicate minutes after transcriptional
repression. A representative gel for each strain is shown, and the
average value (minutes) of the half-life for the full-length transcript
from at least three experiments in each strain is given on the right.
The experimental variation for the half-life measurements of each
strain was less than ±15%. Northern gels were probed with
oligonucleotide oRP141 (5'-AATTGATCTATCGAGGAATTCC-3'), which
is complementary to the poly(G) and flanking 3' sequence in the
PGK1pG mRNA. ts, temperature sensitive.
|
|
In contrast, following a shift to the restrictive temperature for
1 h, temperature-sensitive mutations in eIF-4E and eIF-4A and a
deletion of eIF-4G1 all led to higher rates of mRNA degradation. For
example, in wild-type cells the PGK1 mRNA decayed with an average half-life of 17 min at 38°C (Fig. 1A). It should be noted that this is a shorter half-life than has been previously reported at
lower temperatures and presumably reflects the faster decay of yeast
mRNAs that is seen at high temperatures (27). Notably, the
half-life of the PGK1 mRNA from a strain containing the
temperature-sensitive mutation in eIF-4E/cdc33-42, was decreased to
about 6 min, almost a threefold reduction compared to that of the
wild-type strain at 38°C (Fig. 1A). Similarly, the PGK1
mRNA decayed faster in the temperature-sensitive eIF-4A
(t1/2 = 8.5 min) and eIF-4G1 (t1/2 = 8.2 min) mutants (Fig. 1A and Table
2), although the magnitude of the change was not as great. Control
experiments done at the permissive temperature showed that
PGK1 and MFA2 mRNA from the conditional
eIF-4E/cdc33-42 allele decayed with wild-type kinetics. In contrast,
the eIF-4G1 showed an increased decay rate at high and low temperatures.
Faster mRNA turnover was also observed for the unstable MFA2
mRNA in translation initiation mutant strains. In a wild-type strain at
38°C, the MFA2 half-life was 4 min (Table 2), whereas in
strains containing the temperature-sensitive eIF-4E/cdc33-42, temperature-sensitive eIF-4A, or eIF-4G1 mutations, the
MFA2 half-life was decreased to 3, 3.5, and 2 min
respectively. In addition, the MFA2 mRNA showed accelerated
decay in an eIF-4G2 strain, even though the decay of the
PGK1 mRNA in this strain was only slightly affected (Table
2). These smaller changes in the MFA2 mRNA decay rate were
extremely reproducible, but because of their smaller magnitude, they
should be interpreted with caution. Moreover, it might be difficult to
accelerate the decay rate of the MFA2 transcript greatly
since this mRNA is already highly unstable.
The conclusion that the mRNA decay rate was higher in the various
translation initiation mutants was also supported by the observation
that the total levels of individual mRNAs in strains containing
translation initiation mutations were decreased. For example, after
1 h at the restrictive temperature, the level of the full-length
PGK1 mRNA in the eIF-4E/cdc33-42 strain was approximately 28% of the level in a wild-type strain under similar conditions (data
not shown). In combination, these results indicated that lesions in the
cap binding complex can accelerate mRNA degradation for yeast
transcripts and suggest a role for these proteins in modulating mRNA
turnover rates (see Discussion).
Defects in the eIF-3 complex lead to higher mRNA decay rates.
In addition to the cap binding complex, a second complex of translation
factors, termed eIF-3, plays a crucial role in interacting with the
eIF-4F complex and the 40S-eIF-2 complex to initiate assembly at the
5' end of the mRNA. The eIF-3 complex is made up of about eight
proteins that physically interact with both the mRNA and the eIF-4F
complex (33, 38). These interactions place eIF-3 in the
immediate vicinity of the 5' cap structure. For this reason, components
of the eIF-3 complex were also tested for a role in mRNA turnover. The
Prt1 protein was tested for its effect on mRNA turnover by using a
temperature-sensitive mutation of the protein.
An interesting result was that a mutation in the Prt1 protein also gave
faster degradation of the PGK1 and MFA2
transcripts (Table 2 and Fig. 1B). For example, the PGK1
half-life was decreased from a wild-type value of 17 min to 6 min in
prt1-63 mutant strains. Mutations in the Gcd10 protein, which has been
hypothesized to be a component of the eIF-3 complex in addition to
having other functions (21), gave similar results (data not
shown). These results showed that defects in the eIF-3 complex can lead
to an acceleration of mRNA turnover.
The above experiments indicated that the PGK1 and
MFA2 mRNAs were degraded more rapidly in strains that
carried lesions in several different translation initiation factors.
The decrease in mRNA stability corresponded to lesions known to
decrease translation initiation. To magnify the effect on mRNA decay
rate, we reasoned that a double mutant defective in both eIF-4F and
eIF-3 function might show an even stronger phenotype with respect to
mRNA decay rates. To test this hypothesis, we constructed a strain
carrying both the temperature-sensitive eIF-4E/cdc33-42 and the prt1-63 mutations. This strain showed an increased sensitivity to higher temperatures and failed to grow above 24°C, whereas either single mutant strain grew well at 30°C. The half-lives of both
PGK1 and MFA2 mRNAs in the double mutant were
shorter than those in either single mutant, suggesting that blocking
multiple steps of translation initiation leads to a more severe mRNA
turnover defect (Fig. 1C). These results demonstrate that lesions that
alter the normal assembly and function of the translation initiation
complex on the 5' end of the transcript result in faster mRNA degradation.
The effect of mutations on the mRNA decay rate generally
corresponds to their effect on the translation rate.
Our analysis
of the effect of various translation initiation mutations on mRNA decay
compared several mutations whose effects on translation rate have been
individually determined by a number of different laboratories using
different types of assays. To relate our observations on the effects on
mRNA decay, it was necessary to compare the effects of all these
mutations on translation rate by using the same experimental protocol.
To do this, we measured the rate of incorporation of
[35S]methionine into trichloroacetic acid-precipitable
counts in the various strains both at 24°C and after a shift to
38°C for 1 h (see Materials and Methods). As can be seen in Fig.
2, with the exception of the caf20
mutants, all the mutants had a decreased translation rate to some
extent. The rates for the tif4631 and tif4632 strains were
decreased to approximately 40% of wild-type rates at both 24 and
38°C. All of the temperature-sensitive strains showed a
temperature-dependent inhibition of translation rate, with the residual
translation at 38°C being 19, 12, 7.5, and 3% for the
eIF-4E/cdc33-42, temperature-sensitive eIF-4A, prt1-63, and
eIF-4E/cdc33-42 prt1-63 mutants, respectively. These results indicate a
strong correlation between translation rate and mRNA stability (see
Discussion).
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 2.
Relative rates of translation in various translation
initiation mutants. The rate of [35S]methionine
incorporation was measured either at 24°C or after a 1-h temperature
shift to 38°C. The rates of translation are given relative to the
wild-type strain at 24°C (where rates were originally measured as the
rate of incorporation per optical density unit after 30 min). The rate
of translation for each strain is the average of at least two
independent experiments. The numbers in parentheses are the rates of
incorporation of the strains at 38°C relative to the wild-type strain
at 38°C. ts, temperature sensitive.
|
|
The faster mRNA turnover seen in translation initiation mutants
occurs by the normal 5'
3' pathway of decay.
An important
question was whether the faster decay seen in the various translation
initiation mutants was due to an acceleration of the normal pathway of
deadenylation-dependent decapping or occurred because the transcript
was being degraded rapidly by alternative degradation mechanisms, which
are known to exist in yeast (4). To interpret the effects of
the lesions in the translation initiation factors, we determined if the
accelerated decay required the Dcp1p decapping enzyme and the Xrn1p
5'-to-3' exoribonuclease, which are known to function in the normal
degradation of these mRNAs (10, 41, 42). To this end,
strains were made which combined the temperature-sensitive eIF-4E
lesions with a dcp1 or a xrn1 mutation.
We observed that the deletion of either the DCP1 gene or the
XRN1 gene prevented the faster degradation seen in the
temperature-sensitive eIF-4E/cdc33-42 strain. For example, deletion of
the DCP1 gene in combination with the cdc33-42 lesion led to
a stabilization of the mRNA, since the decapping step was prevented,
giving the PGK1 mRNA an increased half-life of 27 min (Fig.
3). The eIF-4E/cdc33-42 dcp1 double
mutant had a half-life identical to that of the dcp1 mutant, i.e.,
four to five times longer than that seen in the temperature-sensitive
eIF-4E/cdc33-42 mutant alone. Analysis of the mRNA from the
temperature-sensitive eIF-4E/cdc33-42 xrn1 double-mutant strains
showed very similar results to the temperature-sensitive eIF-4E/cdc33-42 dcp1 double mutant (Fig. 3), although the mRNA from
the temperature-sensitive eIF-4E/cdc33-42 xrn1 double mutant was not
quite as stable as the mRNA from the xrn1 mutant, presumably due to
residual alternative 5'-to-3' exonuclease activity subsequent to the
decapping step (42). We interpreted these results to suggest
that the faster decay seen in the various translation initiation
mutants was due to an increase in the rates of specific steps in the
5'-to-3' mRNA decay pathway.
View larger version (74K):
[in this window]
[in a new window]
|
FIG. 3.
mRNAs from strains containing a mutation in eIF-4E
undergo turnover through the general 5'3' pathway. mRNA half-lives
were measured in strains containing mutations in both eIF-4E and
DCP1 and in strains containing mutations in both eIF-4E and
XRN1. Steady-state half-life measurements of the
PGK1pG mRNA were determined from agarose Northern gels. The
numbers above the lanes indicate minutes after transcriptional
repression. The average value (minutes) of the half-life for the
full-length transcript from at least three experiments in each strain
is given on the right. Northern gels were probed with oligonucleotide
oRP141. ts, temperature sensitive.
|
|
Defects in translation initiation increase the rate of both
deadenylation and decapping.
To determine the basis for the faster
mRNA decay seen in the various translation initiation mutants, it was
important to determine what step in mRNA decay was being accelerated.
In principle, the higher observed decay rate could be due either to an
acceleration of deadenylation and/or decapping or to a bypass of the
requirement for deadenylation before decapping (41). In the
following experiments, we examined the rates of deadenylation and
decapping in the various mutant strains.
One method for measuring the rates of deadenylation and decapping is to
monitor the changes in poly(A) tail length and mRNA levels following
repression of transcription from a culture at steady state
(40). Two significant observations were made from this
examination. First, measurement of the rate of deadenylation, as
assessed by the shortening of the longest poly(A) tails in the
population, indicated that the temperature-sensitive eIF-4E/cdc33-42, temperature-sensitive eIF-4A, eIF-4G1 (data not shown), and prt1-63 strains all showed higher rates of deadenylation (Fig.
4; summarized in Table 2). For example,
measurements of the wild-type strain showed that deadenylation of the
mRNA present as full-length mRNA proceeded at a rate of about 3.1 adenylate residues per min (Fig. 4 and Table 2); whereas the
deadenylation of the full-length mRNA from the temperature-sensitive
eIF-4E/cdc33-42 mutant strain proceeded at a rate of 4.1 adenylate
residues per min. The largest change in deadenylation rate was seen in
the eIF-4E/cdc33-42 prt1-63 double mutant, wherein the poly(A) tail of
the PGK1 mRNA now shortened at 5 to 6 adenylate residues per min.
Similar results were obtained by measuring deadenylation rates in
transcriptional pulse-chase experiments, where the deadenylation rate
is measured by monitoring the poly(A) tail length from a synchronous
population of mRNAs (see below) (data not shown). Based on these
observations, we concluded that lesions in the various translation
initiation factors promoted faster deadenylation. This has implications
for understanding the control of the deadenylation rate (see
Discussion).
View larger version (98K):
[in this window]
[in a new window]
|
FIG. 4.
The rate of deadenylation is increased when translation
initiation is impaired. Polyacrylamide Northern gels of transcriptional
repression experiments examining the deadenylation of PGK1
and accumulation of oligoadenylated mRNAs in various translation
initiation mutants are shown. Minutes following transcription
repression are given directly above each sample. The top fragment was
produced by cleavage of the full-length mRNA with RNase H and
oligonucleotide oRP70 (5'-CGGATAAGAAAGCAACACCTGG-3'). This
cleavage shortens the mRNA enough to visualize small differences at the
3' end. The bottom fragment is the decay intermediate stabilized by the
poly(G) insertion in the 3' UTR. The arrow indicates the size of the
oligoadenylated mRNA. The numbers after the A's in the cartoons
represent the range of poly(A) tail sizes found on each mRNA species as
determined by comparison with the oligo(dT) lanes, in which the poly(A)
tails have been completely removed by cleavage with RNase H,
oligonucleotide oRP70, and oligo(dT). The blots were probed with
oRP141. ts, temperature sensitive.
|
|
The second important observation from examining the decay of the mRNA
following transcriptional repression was that the deadenylated form of
the PGK1 mRNA, which is relatively stable in wild-type strains due to the low rate of decapping for this transcript, decays
rapidly in all the mutant strains except the eIF-4G2 and the
caf20 strains (Fig. 4 and data not shown). A clear example of this
phenomenon is seen by comparing the wild-type and temperature-sensitive eIF-4E strains (compare panels in Fig. 4). Since decapping is a
prerequisite for 5'-to-3' exonucleolytic digestion, these observations indicate that defects in the translation initiation complex increase the rate of decapping (see Discussion).
Decapping remains dependent on prior deadenylation in translation
initiation mutants.
In the 5'-to-3' mRNA decay pathway, mRNA
turnover proceeds sequentially by deadenylation followed by
decapping and exonucleolytic decay. To determine if the rapid decay
seen in the translation initiation mutants still required
deadenylation, we examined the decay in these strains by using a
transcriptional pulse-chase method (18). These experiments
used the regulatable GAL1 upstream activating sequence to
create a synchronous pulse of mRNA that can be monitored throughout the
decay pathway, thereby allowing an examination of the relationship
between deadenylation and decay.
Transcriptional pulse-chase experiments with the various mutants
provided two observations that argued that decapping still required
prior deadenylation. First, the mRNA levels did not begin to drop until
after the poly(A) tail had been shortened to an oligo(A) length. This
is characteristic of mRNAs that require deadenylation before decay
(18) and is not seen with mRNAs that are degraded
independently of poly(A) tail shortening, which occurs in response to
premature translation termination (41). The second observation, that the rapid decay seen in the translation initiation mutants still required deadenylation, came from the analysis of an mRNA
decay intermediate stabilized by the insertion of a poly(G) tract in
the 3' untranslated region (UTR), which serves to block the Xrn1p
exoribonuclease (18, 52). Consistent with earlier work on
transcripts that require deadenylation before decay (18), this mRNA fragment was produced in the various mutant strains, including the temperature-sensitive eIF-4E/cdc33-42 prt1-63
double-mutant strain, only after a substantial fraction of the mRNA had
been deadenylated to an oligo(A) length (Fig.
5). Moreover, these mRNA decay
intermediates had oligo(A) tails, even at times when the mRNA
population consisted of transcripts with both long and short poly(A)
tails (Fig. 5), arguing that only oligoadenylated transcripts are
substrates for the next step in decay. These results are in strong
contrast to cases where decapping is independent of deadenylation, wherein this mRNA fragment is produced before poly(A) shortening has
reached a oligo(A) tail and so the mRNA fragment itself has long
poly(A) tails (41).
View larger version (81K):
[in this window]
[in a new window]
|
FIG. 5.
The rates of both deadenylation and decapping are
increased when translation initiation is impaired. Transcriptional
pulse-chase analysis of the PGK1pG mRNA in various
translation initiation mutant strains is used to examine deadenylation
and fragment production. Time points used after a 6-min transcriptional
induction and subsequent repression are shown above each lane. The top
fragment was produced by cleavage of the full-length mRNA with RNase H
and oligonucleotide oRP70. This cleavage shortens the mRNA enough to
visualize small differences at the 3' end. The bottom fragment is the
decay intermediate stabilized by the poly(G) insertion in the 3' UTR.
The blots were probed with oRP141. ts, temperature sensitive.
|
|
These observations indicate that the ability of the poly(A) tail to act
as an inhibitor of decapping is not prevented even if there is a strong
inhibition to translation. This is inconsistent with the simple model
that the mechanism by which poly(A) tails inhibit decapping is by
bringing the poly(A) binding protein to the mRNA and thereby, through
its interaction with eIF-4G, stabilizing the cap binding complex.
The increased rate of mRNA turnover in translation initiation
mutants is not UPF1 dependent.
The above results
showed that defects in the Prt1 protein led to faster decay of the
PGK1 and MFA2 mRNAs, by promoting increases in
the rates of both deadenylation and decapping. It has also been
recently shown that prt1 mutations can affect the levels of the SSA1
and SSA2 mRNAs in yeast, suggesting that in prt1 mutants these mRNAs
might also be degraded more rapidly (6). Interestingly, the
faster turnover seen in the prt1 mutant strains was dependent on the
Upf1 protein, which is known to be required for the degradation of
mRNAs containing early nonsense codons (36). Given this
requirement for the Upf1p in the faster decay seen in the prt1 mutants,
we determined if the faster decay we observed in the prt1 mutants for
the PGK1 and MFA2 transcripts was also UPF1 dependent.
Strains that contained mutations in both the eIF-4E/CDC33
gene and the UPF1 gene or in the PRT1 gene and
the UPF1 gene were created. Transcriptional shutoffs were
performed as previously described to determine the mRNA half-lives of
PGK1 and MFA2 in the mutant strains. In both the
temperature-sensitive double-mutant eIF-4E/cdc33-42 upf1 and the
prt1-63 upf1 strains, the half-lives resembled those of the
single-translation-initiation mutant, indicating that the Upf1 protein
was not required for the faster decay of these mRNAs in response to
mutations in the translation initiation factors (Fig. 1D).
These results indicated that the faster decay observed for the PGK1 and
MFA2 mRNAs in response to the mutations in the PRT1 or
eIF-4E/CDC33 gene is somehow different from the proposed
faster decay seen with the SSA1 and SSA2 mRNAs in
the PRT1 strain. Unfortunately, since the actual mechanism
by which the SSA1 and SSA2 transcripts are
degraded is not known, it is difficult to evaluate these differences. It is possible that the SSA1 or SSA2 mRNAs are
subjected to a different mechanism of turnover and therefore give
different results. Alternatively, since SSA1 and
SSA2 encode heat shock proteins, these mRNAs might have
methods for enhancing their translation during heat shock and as such
might have a different dependence on the Upf1p.
| |
DISCUSSION |
Mutations in translation initiation factors increase the rate of
decapping.
Several observations indicated that defects in the
translation initiation factors eIF-4E, eIF-4G, eIF-4A, and Prt1 (a
component of eIF-3) led to an increase in the rate of decapping. First, in strains carrying mutations in these translation initiation factors,
the decay rate of the PGK1 and MFA2 transcripts
was higher. Second, oligoadenylated PGK1 mRNA species
produced following deadenylation were degraded rapidly in the
translation initiation mutants (Fig. 4 and 5). In contrast, such
oligoadenylated PGK1 transcripts persisted in the wild-type
strain due to the normally low rate of decapping for this mRNA. These
results support the conclusion that the ability of the decapping enzyme
to cleave a mRNA substrate is affected by the ability of the
translation initiation complex to form or to persist on the transcript.
This interpretation is also supported by the prior observation that
altering the translation initiation process in cis by the
insertion of secondary structures in the 5' UTR increases the rate of
decapping of the PGK1 mRNA (42).
Comparison of translation rates in the various mutants allowed us to
relate the effects on mRNA turnover to changes in the translation rate.
In general, the stronger the inhibition of observed translation rate,
the higher the observed mRNA decay rate. For example, the tif4631
and tif4632 mutations had the smallest effects on PGK1
mRNA decay, and these strains showed the highest level of residual
translation. It is interesting and notable that the tif4632 mutation
caused a decrease in the translation rate without significantly
affecting the decay rate of the PGK1 mRNA, although the MFA2
mRNA did show accelerated decay in this strain (Table 2 and Fig. 2).
This suggests that there will be some mRNA-specific effects of
mutations in particular translation initiation factors. In the other
extreme, the strongest defect in the translation rate was seen in the
eIF-4E/cdc33-42 prt1-63 double mutant, which showed the highest mRNA
decay rates (Table 2 and Fig. 2).
The increase in decapping rate we observed in a defective allele of the
eIF-4E protein is seemingly different from reports that small N- and
C-terminal deletions of the eIF-4E protein had no effect on the
half-lives of the mRNAs in strains containing those mutant proteins
(37). To compare our results with this prior work, we
examined the turnover of mRNAs from two of these eIF-4E deletion
mutants (cdc337/209 and cdc33202) and also found no change in the
half-lives of PGK1 and MFA2 mRNA (data not
shown). This suggests that these particular mutations in eIF-4E are
competent for proper mRNA turnover, possibly due to their partial
ability to assemble a cap binding complex. The mutation used in the
current work, temperature-sensitive eIF-4E/cdc33-42, leads to a more
severe defect in cap binding than do the nonlethal eIF-4E truncation mutations and also leads to a more significant decrease in translation of mRNAs at the restrictive temperature (2). This more
severe defect in eIF-4E function manifests itself as an increase in the rate of mRNA turnover.
A straightforward mechanistic interpretation of these observations is
that decapping requires the destabilization or disassembly of the cap
binding complex, to allow the decapping enzyme to gain access to the
cap structure. In the simplest view, the cap binding complex and the
decapping enzyme could be viewed as being in direct competition for the
5' cap structure. However, it is not unreasonable to expect that there
will be specific transitions or states of the cap binding complex that
promote interactions of the decapping enzyme with this complex and
ultimately lead to decapping of the transcript. It should be noted that
this view of decapping predicts that there should be specific alleles
of translation initiation factors that lead to an inhibition of the
decapping rate. However, one possibility that cannot be formally ruled
out is that translation initiation indirectly protects the mRNA from
decapping. For example, the mere presence of elongating ribosomes bound
to the mRNA could be hypothesized to prevent decapping.
Mutations in translation initiation factors increase the rate of
deadenylation.
We also observed that the same lesions in
translation initiation factors that gave higher rates of decapping also
led to an increase in the rate of deadenylation. The critical
experiment was that direct measurement of deadenylation rate in
wild-type and mutant strains showed that in strains containing
mutations in translation initiation factors (eIF-4E, eIF-4A, Prt1
[Fig. 4 and 5], and eIF-4G1 [data not shown]), deadenylation was
faster than in a wild-type strain. The most extreme example of this
effect was observed in the temperature-sensitive eIF-4E/cdc33-42
prt1-63 double mutant, whose deadenylation rate was increased threefold over that of the wild-type strain (Table 2). These observations led us
to propose that the status of the translation initiation complex plays
a major role in modulating the deadenylation rate for yeast mRNAs.
The conclusion that the status of the translation initiation complex
plays a significant role in determining the deadenylation rate is
supported by several prior observations in the literature. First,
insertion of a stem-loop into the PGK1 5' UTR to inhibit ribosome assembly leads to a higher rate of deadenylation
(42). Second, alterations of the PGK1 5' UTR and
AUG context which decrease the translation initiation rate also
increase the rate of deadenylation (31). Third, the AU-rich
elements known to promote deadenylation in mammalian cells have also
been observed to inhibit translation initiation rate (30).
Similarly, the human ferritin L-chain mRNA also shows changes in
poly(A) tail length based on the translational status of the mRNA
(39). Finally, deadenylation of the c-myc mRNA is
inhibited by the addition of the translation elongation inhibitor
cycloheximide (32).
Why do changes in translation initiation affect the deadenylation rate?
The simplest hypothesis is that the rate of translation initiation
affects the nature of the physical interaction between the 5' and 3'
ends of the mRNA and that changes in the nature of this 5'-3'
interaction in turn affect the rate of deadenylation by altering the
availability of the poly(A) tail to the deadenylating nuclease(s).
There are two ways in which this could be occurring. In one model, the
poly(A) binding protein (PAB) is an inhibitor of deadenylation and must
dissociate from the poly(A) tail for deadenylation to occur. This view
is suggested by the observation that in both mammalian cell extracts
and Xenopus oocytes PAB appears to be an inhibitor of
deadenylation (11, 54). Interestingly, PAB interacts with
the cap binding complex in several systems (50), and such an
interaction can increase the ability of PAB to bind the poly(A) tail
(35). This suggests that decreases in the stability of the
cap binding complex could lead to destabilization of PAB bound to the
poly(A) tail and thereby activate deadenylation. It should be noted
that this model of deadenylation occurring in the absence of Pab1p is
at odds with the observation that yeast strains deficient for PAB have
lower rates of deadenylation (14, 46). This observation in
yeast leads to an alternative model in which PAB bound to the poly(A)
tail is required for proper deadenylation but that poly(A) shortening
is slowed by the process of translation. Transient dissociation between
the PAB protein and translation initiation factors, possibly between
rounds of translation initiation, allows for periods of
deadenylation to occur. This model explains both the pab1
phenotypes and the increase in deadenylation seen in yeast translation
initiation mutants. Further experiments are required to resolve these issues.
What determines the mRNA half-life?
Our results imply that the
relative translational efficiency of a transcript will be a major
determinant of the mRNA half-life. In strains carrying strong defects
in various translation initiation factors, the mRNA half-lives
were significantly decreased, and both mRNAs examined showed
relatively similar decay rates. For example, in a wild-type strain
there is about a fivefold difference in mRNA half-life between
PGK1 and MFA2, but in the translation initiation
mutants there is only about a twofold difference in half-life between
the two mRNAs, implying that stability differences may arise due to
differences in the translational efficiency of the individual mRNAs.
This observation suggests that the PGK1 mRNA is stable in
the wild-type strain because it is efficiently translated and that the
MFA2 mRNA is highly unstable because it is poorly
translated. Consistent with this hypothesis, replacing the context of
the PGK1 AUG with the MFA2 AUG context, which
decreases the translation initiation rate of the PGK1 mRNA,
also leads to faster mRNA degradation (31). Moreover,
inhibiting PGK1 translation with a stem-loop leads to faster
decay, due to the loss of efficient translation (42). In
contrast, insertion of a stem-loop into the MFA2 5' UTR has
little effect on the decay rate, presumably because this mRNA is
already so poorly translated (8).
The view that the translation efficiency of a mRNA is a major
determinant of the decay rate has important implications for understanding the function of sequences that ultimately dictate differential rates of mRNA degradation. This hypothesis leads to the
prediction that at least some sequences that promote the mRNA decay
rate will function by decreasing the rate of translation initiation,
with corresponding downstream changes on the decapping and
deadenylation rates. Interestingly, several cis-acting
elements that can destabilize yeast transcripts have been described as affecting the rates of both deadenylation and decapping (15, 31,
40, 42). This effect of such instability sequences on both steps
in decay can now be easily understood if they primarily affect the rate
of translation initiation.
We thank Michael Altmann, Alan Hinnebusch, Patrick Linder, and
Nahum Sonenberg for providing many of the yeast strains used in this
work. In addition, we thank the members of the Parker laboratory for
their support and contributions in the preparation of the manuscript.
This work was supported by the Howard Hughes Medical Institute and by a
grant to R.P. from the National Institute of Health (GM45443).
| 1.
|
Altmann, M.,
C. Handschin, and H. Trachsel.
1987.
mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae.
Mol. Cell. Biol.
7:998-1003[Abstract/Free Full Text].
|
| 2.
|
Altmann, M.,
N. Sonenberg, and H. Trachsel.
1989.
Translation in Saccharomyces cerevisiae. Initiation factor 4E-dependent cell-free system.
Mol. Cell. Biol.
9:4467-4472[Abstract/Free Full Text].
|
| 3.
|
Altmann, M.,
N. Schmitz,
C. Berset, and H. Trachsel.
1997.
A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.
EMBO J.
16:1114-1121[Medline].
|
| 4.
|
Anderson, J. S. J., and R. Parker.
1998.
The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SK12 DEVH box protein and 3' to 5' exonucleases of the exosome complex.
EMBO J.
17:1497-1506[Medline].
|
| 5.
|
Banerjee, A. K.
1980.
5'-terminal cap structure in eucaryotic messenger ribonucleic acids.
Bacteriol. Rev.
44:175-205.
|
| 6.
|
Barnes, C. A.
1998.
Upf1 and Upf2 proteins mediate normal yeast mRNA degradation when translation initiation is limited.
Nucleic Acids Res.
26:2433-2441[Abstract/Free Full Text].
|
| 7.
|
Bashkirov, V. I.,
H. Scherthan,
J. A. Solinger,
J. Buerstedde, and W. Heyer.
1997.
A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates.
J. Cell Biol.
136:761-773[Abstract/Free Full Text].
|
| 8.
|
Beelman, C. A., and R. Parker.
1994.
Differential effects of translational inhibition in cis and trans on the decay of the unstable yeast MFA2 mRNA.
J. Biol. Chem.
269:9687-9692[Abstract/Free Full Text].
|
| 9.
|
Beelman, C. A., and R. Parker.
1995.
Degradation of mRNA in eukaryotes.
Cell
81:179-183[Medline].
|
| 10.
|
Beelman, C. A.,
A. Stevens,
G. Caponigro,
T. E. LaGrandeur,
L. Hatfield,
D. M. Fortner, and R. Parker.
1996.
An essential component of the decapping enzyme required for the normal rates of mRNA turnover.
Nature
382:642-646[Medline].
|
| 11.
|
Bernstein, P.,
S. W. Peltz, and J. Ross.
1989.
The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.
Mol. Cell. Biol.
9:659-670[Abstract/Free Full Text].
|
| 12.
|
Binder, R.,
J. A. Horowitz,
J. P. Basilion,
D. M. Koeller,
R. D. Klausner, and J. B. Harford.
1994.
Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3' UTR and does not involve poly(A) tail shortening.
EMBO J.
13:1969-1980[Medline].
|
| 13.
|
Caponigro, G.,
D. Muhlrad, and R. Parker.
1993.
A small segment of the MAT 1 transcript promotes mRNA decay in Saccharomyces cerevisiae. A stimulatory role for rare codons.
Mol. Cell. Biol.
13:5141-5148[Abstract/Free Full Text].
|
| 14.
|
Caponigro, G., and R. Parker.
1995.
Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast.
Genes Dev.
9:2421-2432[Abstract/Free Full Text].
|
| 15.
|
Caponigro, G., and R. Parker.
1996.
Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae.
Microbiol. Rev.
60:233-249[Free Full Text].
|
| 16.
|
Cereghino, G. P.,
D. P. Atencio,
M. Saghbini,
J. Beiner, and I. E. Scheffler.
1995.
Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role.
Mol. Biol. Cell
6:1125-1143[Abstract].
|
| 17.
|
Couttet, P.,
M. Fromont-Racine,
D. Steel,
R. Pictet, and T. Grange.
1997.
Messenger RNA deadenylylation precedes decapping in mammalian cells.
Proc. Natl. Acad. Sci. USA
94:5628-5633[Abstract/Free Full Text].
|
| 18.
|
Decker, C. J., and R. Parker.
1993.
A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation.
Genes Dev.
7:1632-1643[Abstract/Free Full Text].
|
| 19.
|
Edery, I.,
M. Humbelin,
A. Darveau,
K. A. Lee,
S. Milburn,
J. W. Hershey,
H. Trachsel, and N. Sonenberg.
1983.
Involvement of eukaryotic initiation factor 4A in the cap recognition process.
J. Biol. Chem.
258:11398-11403[Abstract/Free Full Text].
|
| 20.
|
Evans, D. R. H.,
C. Rasmussen,
P. J. Hanic-Joyce,
G. C. Johnston,
R. A. Singer, and C. A. Barnes.
1995.
Mutational analysis of the PRT1 protein subunit of yeast translation initiation factor 3.
Mol. Cell. Biol.
15:4525-4535[Abstract].
|
| 21.
|
Garcia-Barrio, M. T.,
T. Naranda,
C. R. Vazquez de Aldana,
R. Cuesta,
A. G. Hinnebusch,
J. W. B. Hershey, and M. Tamame.
1995.
GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.
Genes Dev.
9:1781-1796[Abstract/Free Full Text].
|
| 22.
|
Goyer, C.,
M. Altmann,
H. Trachsel, and N. Sonenberg.
1989.
Identification and characterization of cap-binding proteins from yeast.
J. Biol. Chem.
264:7603-7610[Abstract/Free Full Text].
|
| 23.
|
Goyer, C.,
M. Altmann,
H. S. Lee,
A. Blanc,
M. Deshmukh,
J. L. Woolford, Jr.,
H. Trachsel, and N. Sonenberg.
1993.
TIF4631 and TIF4632. Two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.
Mol. Cell. Biol.
13:4860-4874[Abstract/Free Full Text].
|
| 24.
|
Grifo, J. A.,
S. M. Tahara,
M. A. Morgan,
A. J. Shatkin, and W. C. Merrick.
1983.
New initiation factor activity required for globin mRNA translation.
J. Biol. Chem.
258:5804-5810[Abstract/Free Full Text].
|
| 25.
|
Hannig, E. M.
1995.
Protein synthesis in eukaryotic organisms: new insights into the function of translation initiation factor eIF-3.
Bioessays
17:915-919[Medline].
|
| 26.
|
Hatfield, L.,
C. A. Beelman,
A. Stevens, and R. Parker.
1996.
Mutations in trans-acting factors affecting mRNA decapping in Saccharomyces cerevisiae.
Mol. Cell. Biol.
16:5830-5838[Abstract].
|
| 27.
|
Herrick, D.,
R. Parker, and A. Jacobson.
1990.
Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.
Mol. Cell. Biol.
10:2269-2284[Abstract/Free Full Text].
|
| 28.
|
Hsu, C. L., and A. Stevens.
1993.
Yeast cells lacking 5'3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure.
Mol. Cell. Biol.
13:4826-4835[Abstract/Free Full Text].
|
| 29.
|
Jacobson, A., and S. W. Peltz.
1996.
Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells.
Annu. Rev. Biochem.
65:693-739[Medline].
|
| 30.
|
Kruys, V.,
O. Marinx,
G. Shaw,
J. Deschamps, and G. Huez.
1989.
Translational blockade imposed by cytokine-derived UA-rich sequences.
Science
245:852-855[Abstract/Free Full Text].
|
| 31.
|
LaGrandeur, T., and R. Parker.
1999.
The context of the translation start codon, in combination with other mRNA features, contributes to mRNA decay rates in Saccharomyces cerevisiae.
RNA
5:420-433[Abstract].
|
| 32.
|
Laird-Offringa, I. A.,
C. L. De Witt,
P. Elfferich, and A. J. van der Eb.
1990.
Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation.
Mol. Cell. Biol.
10:6132-6140[Abstract/Free Full Text].
|
| 33.
|
Lamphear, B. J.,
R. Kirchweger,
T. Skern, and R. E. Rhoads.
1995.
Mapping of functional domains in eIF4G with picornaviral proteases. Implication for cap-dependent and cap-independent translational initiation.
J. Biol. Chem.
270:21975-21983[Abstract/Free Full Text].
|
| 34.
|
Lanker, S.,
P. P. Müller,
M. Altmann,
C. Goyer,
N. Sonenberg, and H. Trachsel.
1992.
Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.
J. Biol. Chem.
267:21167-21171[Abstract/Free Full Text].
|
| 35.
|
Le, H.,
R. L. Tanguay,
M. L. Balasta,
C. Wei,
K. S. Browning,
A. M. Metz,
D. J. Goss, and D. R. Gallie.
1997.
Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity.
J. Bio. Chem.
272:16247-16255[Abstract/Free Full Text].
|
| 36.
|
Leeds, P.,
J. M. Wood,
B.-S. Lee, and M. R. Culbertson.
1992.
Gene products that promote mRNA turnover in Saccharomyces cerevisiae.
Mol. Cell. Biol.
12:2165-2177[Abstract/Free Full Text].
|
| 37.
|
Linz, B.,
N. Koloteva,
S. Vasilescu, and J. E. G. McCarthy.
1997.
Disruption of ribosomal scanning on the 5'-untranslated region, and not restriction of translational initiation per se, modulates the stability of nonaberrant mRNAs in the yeast Saccharomyces cerevisiae.
J. Biol. Chem.
272:9131-9140[Abstract/Free Full Text].
|
| 38.
|
Mader, S.,
H. Lee,
A. Pause, and N. Sonenberg.
1995.
The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 and the translational repressors, 4E-binding proteins.
Mol. Cell. Biol.
15:4990-4997[Abstract].
|
| 39.
|
Muckenthaler, M.,
N. Gunkel,
R. Stripecke, and M. W. Hentze.
1997.
Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.
RNA
3:983-995[Abstract].
|
| 40.
|
Muhlrad, D., and R. Parker.
1992.
Mutations affecting stability and deadenylation of the yeast MFA2 transcript.
Genes Dev.
6:2100-2111[Abstract/Free Full Text].
|
| 41.
|
Muhlrad, D.,
C. J. Decker, and R. Parker.
1994.
Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5' to 3' digestion of the transcript.
Genes Dev.
8:855-866[Abstract/Free Full Text].
|
| 42.
|
Muhlrad, D.,
C. J. Decker, and R. Parker.
1995.
Turnover mechanisms of the stable yeast PGK1 mRNA.
Mol. Cell. Biol.
15:2145-2156[Abstract].
|
| 43.
|
Naranda, T.,
S. E. MacMillan, and J. W. B. Hershey.
1994.
Purified yeast translation factor eIF3 is an RNA-binding protein complex that contains the PRT1 protein.
J. Biol. Chem.
269:32286-32292[Abstract/Free Full Text].
|
| 44.
|
Rhoads, R. E.
1988.
Cap recognition and entry of mRNA into the protein synthesis initiation cycle.
Trends Biochem. Sci.
13:52-56[Medline].
|
| 45.
|
Ross, J.
1995.
mRNA stability in mammalian cells.
Microbiol. Rev.
59:423-450[Abstract/Free Full Text].
|
| 46.
|
Sachs, A. B., and R. W. Davis.
1989.
The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation.
Cell
58:857-867[Medline].
|
| 47.
|
Schmid, S. R., and P. Linder.
1991.
Translation initiation factor 4A from Saccharomyces cerevisiae. Analysis of residues conserved in the D-E-A-D family of RNA helicases.
Mol. Cell. Biol.
11:3463-3471[Abstract/Free Full Text].
|
| 48.
|
Shyu, A. B.,
J. G. Belasco, and M. E. Greenberg.
1991.
Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay.
Genes Dev.
5:221-234[Abstract/Free Full Text].
|
| 49.
|
Sonenberg, N.,
M. A. Morgan,
W. C. Merrick, and A. J. Shatkin.
1978.
A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA.
Proc. Natl. Acad. Sci. USA
75:4843-4847[Abstract/Free Full Text].
|
| 50.
|
Tarun, S., and A. B. Sachs.
1996.
Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G.
EMBO J.
15:7168-7177[Medline].
|
| 51.
|
Tharun, S., and R. Parker.
1999.
Analysis of mutations in the yeast mRNA decapping enzyme.
Genetics
15:1273-1285.
|
| 52.
|
Vreken, P., and H. A. Raue.
1992.
The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region.
Mol. Cell. Biol.
12:2986-2996[Abstract/Free Full Text].
|
| 53.
|
Wilson, T., and R. Triesman.
1988.
Removal of poly(A) and consequent degradation of the c-fos mRNA facilitated by 3' AU-rich sequences.
Nature
336:396-399[Medline].
|
| 54.
|
Wormington, M.,
A. M. Searfoss, and C. A. Hurney.
1996.
Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.
EMBO J.
15:900-909[Medline].
|
| 55.
|
Zuk, D., and A. Jacobson.
1998.
A single amino acid substitution in yeast eIF-5A results in mRNA stabilization.
EMBO J.
17:2914-2925[Medline].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
