(Received for publication, September 13, 1996, and in revised form, November 22, 1996)
From the Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Inactivation of Saccharomyces
cerevisiae poly(A) polymerase in a strain bearing the
temperature-sensitive lethal pap1-1 mutation results in
the synthesis of poly(A) mRNAs that initiate
translation with surprising efficiency. Translation of
poly(A)
mRNAs after polyadenylation shut-off might
result from an increase in the ratio of ribosomes and associated
translation factors to mRNA, caused by the inability of
poly(A)
mRNAs to accumulate to normal levels. To test
this hypothesis, we used ribosomal subunit protein gene mutations to
decrease either 40 or 60 S ribosomal subunit concentrations in strains
carrying the pap1-1 mutation. Polyadenylation shut-off in
such cells results in a nearly normal ratio of ribosomes to mRNA as
revealed by polyribosome sedimentation analysis. Ribonuclease
protection and Northern blot analyses showed that a significant
percentage of poly(A)-deficient and poly(A)
mRNA
associate with smaller polyribosomes compared with cells with normal
ribosome levels. Analysis of the ratio of poly(A)-deficient and
poly(A)
forms of a specific mRNA showed relatively
more poly(A)
mRNA sedimenting with 20-60 S complexes
than do poly(A)+ forms, suggesting a block in an early step
of the translation initiation of the poly(A)
transcripts.
These findings support models featuring the poly(A) tail as an enhancer
of translation and suggest that the full effect of a poly(A) tail on
the initiation strength of a mRNA may require competition for a
limited number of free ribosomes or translation factors.
Virtually all known eukaryotic precursor mRNAs undergo
processing to mature forms by a series of intranuclear
post-transcriptional modifications including intron removal (splicing),
5-end capping, 3
-end cleavage and polyadenylation, and in some cases,
coding sequence editing. Although normal splicing and editing ensure appropriate coding information, both end modifications have a particular impact on regulating expression levels of mature mRNA. Numerous studies have shown that cap structures serve as recognition motifs for translation initiation factors required for protein synthesis (reviewed in Ref. 1). The consistent finding of a poly(A)
tract generated after site-specific cleavage in the mRNA 3
-untranslated region has led to numerous investigations whose results
suggest functions for poly(A) tails in mRNA stability and
translation (2). Recently, evidence for a mRNA turnover pathway in
Saccharomyces cerevisiae and mammalian cells specifically implicates poly(A) tail trimming (deadenylation) as the first and
sometimes rate-determining step in the degradation of some mRNAs
(3-7).
The debate about the importance of poly(A) tails for mRNA
translation reflects an attempt to combine experimental observations in
systems in vitro including wheat germ extracts, rabbit
reticulocyte lysates, and S. cerevisiae extracts (8-11) and
in living cells such as Xenopus laevis oocytes, HeLa cells,
and S. cerevisiae (12-17). In general, the in
vitro studies demonstrated discrimination against the translation
of poly(A)-deficient or poly(A) input mRNAs; however,
the extent of reduced translatability was modest, possibly reflecting
an extract-dependent capacity for multiple rounds of
translation initiation (i.e. reinitiation) as well as the
degree of saturation of the translational machinery by endogenous
mRNAs. In contrast, some in vivo studies showed dramatic
enhancement of the translation of an mRNA after poly(A) tail
addition. In these cases it is unclear whether the injection of
engineered mRNAs into the cell cytoplasm might lead to
mislocalization of the mRNA and perhaps bypass critical
associations with factors normally participating in translational
regulation. Indeed, mRNA expressed from a plasmid injected into the
nucleus of a X. laevis oocyte enters the cytoplasm but is
translationally inactive, whereas the same mRNA directly injected
into the cytoplasm is translationally competent (18).
Perhaps the most striking evidence for a translational function for
poly(A) tails comes from developmental studies in frog, mouse, and clam
oocytes (19-24). Translational activation of preexisting maternal
mRNAs carrying, at most, short poly(A) tails constitutes a common
feature during stages of oocyte maturation and postfertilization. Competing protein interactions or possibly the absence of various protein factors required to facilitate translation may mask these mRNAs from the translational apparatus (reviewed in Ref. 25). Regulated extension of their poly(A) tails causes these mRNAs to
become translationally active. In some cases, the mere presence of a
long 3 poly(A) tract signals recruitment, but more recent evidence
suggests that the catalytic act of polyadenylation may trigger cap
methylation, which, in turn, activates translation of some maternal
mRNAs (26). Additional postfertilization studies in slime molds and
sea urchins also revealed an increased translational capacity of many
mRNAs subsequent to the poly(A) addition (27, 28). These
polyadenylated mRNAs displayed a competitive advantage over their
poly(A)-deficient counterparts for assembly into polyribosomes, suggesting that poly(A) tails might increase the probability of translation initiation and, more specifically, ribosomal subunit reinitiation.
Current mechanistic models of poly(A) tail function feature enhancement of an early step in translation initiation. For example, Sachs and Davis (29) observed that a yeast strain depleted of essential poly(A) binding protein (Pab1p) exhibits a larger pool of free 80 S ribosomes as well as an increase in the free 60 S to 40 S subunit ratio, suggesting a block in the 60 S subjoining step during translation initiation (29). Suppressor mutations (spb) that restored viability of these cells provided further evidence for a role for Pab1p in translation (29). The spb mutations decrease the ratio of 60 S to 40 S subunits, and alter 60 S assembly as in the case of spb2, a mutation in the single copy, large subunit ribosomal protein gene, L46 (RPL46). These observations provided a genetic link between poly(A) metabolism and the ribosome and supported a role for Pab1p as a mediator of a poly(A) tail function.
Experiments in rabbit reticulocyte lysates revealed a 2-3-fold greater
translational activity of poly(A)+ to poly(A)
mRNA, a difference that apparently resulted from decreased ability of poly(A)
mRNAs to bind 60 S ribosomes during
initiation (30). These findings provided part of the basis for the
"closed loop model" for poly(A) function in translation, which
features enhancement of 60 S ribosome subunit joining by the
poly(A)-Pab1p complex (2). Tarun and Sachs (11) recently presented
evidence that Pab1p enhances the interaction of poly(A)+,
but not poly(A)
mRNAs, with the 40 S ribosomal
subunit. Although these experiments could not assess the role of 60 S
subunit joining, they do provide a rationalization for the observed
increase in 40 S to 60 S ratios in all spb mutants, since an
increase in 40 S levels might bypass the need for poly(A)-Pab1p at this
step.
Our laboratory recently showed that yeast cells with a conditional
mutation (pap1-1) in the poly(A) polymerase gene
accumulated poly(A)-deficient and poly(A) mRNA
following inactivation of the enzyme by cell growth at a nonpermissive
temperature (35 °C) (31). A 1-h incubation at 35 °C reduced the
total amount of mRNA approximately 2-fold, consistent with an
important role for poly(A) tails in mRNA stability. Surprisingly, we found that some poly(A)
mRNAs associated with at
least the same number of ribosomes as in wild-type cells, suggesting
that poly(A)
mRNAs initiate translation efficiently
under these conditions. The polyribosome profiles from these cells
revealed that the reduction in mRNA levels generated a relatively
large excess of free ribosomes, which could possibly compensate for the
low efficiency of translation of poly(A)
mRNAs. Here
we report experiments designed to test the hypothesis that
discrimination against poly(A)
mRNAs during
translation initiation requires competition for a limiting number of
ribosomes. We effectively reduced the number of ribosomes in
pap1-1 cells by deleting selected nonessential ribosomal
protein genes. Inactivation of polyadenylation in these ribosome-deficient cells produced poly(A)
mRNAs that
translate less efficiently than in cells with normal ribosome levels.
Moreover, discrimination against poly(A)
mRNAs
occurred regardless of whether we limited 40 or 60 S subunits, consistent with a poly(A)-dependent component to the
binding of each ribosomal subunit.
UR3148-1B (MATa, ade1/ade2, lys2, gal1?,
ura3-52, pap1-1) cells were transformed with a
BglII-EcoRI fragment from the plasmid pAS195
containing the RPL46 gene sequence partially replaced by the
URA3 gene to generate an RPL46 deletion strain,
pap1-1,rpl46 (YAP201). UR3148-6A
(MATa, leu2-3, gal1?, ura3- 52, pap1-1) cells
were used to construct strains bearing deletions in ribosomal protein
genes RPL16B or RPS51A. A
HindIII-SalI fragment from plasmid pGOBLEU2
(kindly provided by M. Rosbash, Brandeis University; Ref. 32)
containing a partial deletion of RPS51A was incorporated into the UR3148-6A genome by homologous recombination to generate the
strain pap1-1,rps51A
(YAP301). Likewise, a
HindIII-BamHI fragment from plasmid pL16B
LEU2
(kindly provided by J. Woolford, Carnegie-Mellon University; Ref. 33)
was used to replace the chromosomal RPL16B gene with a
deleted allele following electrotransformation of UR3148-6A cells to
generate the strain pap1-1,rpl16B
(YAP401). Conversion
to leucine prototrophy by accurate homologous gene replacements in
pap1-1,rps51A
and pap1-1,rpl16B
strains
was confirmed by Southern blotting (data not shown). All strains were propagated in YEPD (2% dextrose) media.
Yeast polyribosome lysates were prepared by glass bead lysis and 25 A254 units layered onto 12-ml low salt sucrose (15-50%) gradients as described previously (34). Following 4 °C centrifugation at 40,000 rpm (Beckman SW40Ti rotor) for 2.5 h, gradient fractions (20 × 0.6 ml) were collected while recording an A254 polyribosome profile by continuous flow measurement using an ISCO model UA-5 absorbance/fluorescence monitor. Isolation of total RNA from polyribosome gradient fractions and subsequent ribonuclease protection and Northern analyses were carried out as described previously (31). Note that for each gradient RNA fractions 18, 19, and 20 were pooled and are proportionally represented in fraction 18 in the experiments illustrated here. All quantitation of the sedimentation pattern of specific mRNAs was done by storage PhosphorImager (Molecular Dynamics) analysis of the ribonuclease protection gels and Northern blots.
To test the hypothesis that the ratio of ribosomes
to mRNA might play an important role in discrimination against
poly(A) mRNAs during translation initiation, we
sought to reduce ribosome levels in pap1-1 cells to
parallel the loss of mRNA resulting from inactivation of poly(A)
polymerase (Pap1p). Normal ribosome subunit assembly requires certain
ribosomal proteins whose loss reduces the levels of the mature subunits
(Refs. 33-37; reviewed in Ref. 38). This effect is usually specific
for the ribosomal subunit, thus allowing alteration of the levels of
one subunit without significantly affecting the production of the other
(39). Accordingly, deletion of certain nonessential ribosomal protein genes or one copy of a duplicated ribosomal protein gene results in a
decrease in the amount of one of the two ribosomal subunits and, in
effect, the number of 80 S ribosomes available for translation. The
ability to specifically decrease 60 or 40 S levels allowed us to
monitor the impact of limiting subunit concentrations on translation of
poly(A)
mRNA.
Our initial attempt at
reducing the effective concentration of ribosomes was to deplete
pap1-1 cells of the nonessential ribosomal protein L46
(Rpl46p) in order to lower the number of free 60 S ribosomal subunits
(40). Analysis of total RNA from a pap1-1,rpl46 strain
showed a 30-40% reduction in the 25 S to 18 S rRNA ratio (data not
shown) consistent with a requirement for Rpl46p in normal processing of
28 S rRNA. The deletion of the single copy RPL46 gene leads
to the production of lower than normal amounts of ribosomes that lack
ribosomal protein L46. Moreover, RPL46 mutations
(spb2) bypass poly(A)-binding protein (PAB1)
mutations, suggesting a role for Rpl46p in the translational function
of poly(A) tails (29). Therefore, we constructed a second
pap1-1 derivative by deleting RPL16B, one of the
duplicated genes encoding Rpl16p (38). Deletion of only one of two
copies of RPL16 allowed us to lower the 60 S subunit levels
while producing ribosomes with a normal complement of ribosomal
proteins (33, 36, 39).
We assessed the impact of a RPL46 or RPL16B
deletion on overall mRNA translation in pap1-1 cells by
polyribosome profile analyses. In poly(A)+ cells
(pap1-1, 25 °C) we observed (i) a decrease in the height of the largest polyribosome peaks and an increase in the smallest polyribosome peaks suggestive of a decrease in the number of ribosomes per mRNA, (ii) the presence of half-mer polyribosomes, and (iii) a
decrease in the ratio of free 60 S to 40 S ribosomes (Fig.
1). These phenotypes most likely result from a decrease
in the ratio of 60 S subunits to mRNA, which results in a decrease
in the density of polyribosomes and the presence of 48 S initiation
complexes at the translation initiation sites of polyribosomal
mRNAs (half-mer polyribosomes; Ref. 33). Inactivation of Pap1p by
shifting pap1-1 cells to 35 °C for 1 h decreases
mRNA levels about 2-fold (31), resulting in a distribution of
polyribosomes similar in density, although reduced in quantity compared
with cells with normal ribosome levels (Fig. 1, compare A,
B, D, and F). This result suggests that we have achieved the desired adjustment of the ratio of 80 S
ribosomes to mRNA such that it approaches the ratio found under normal conditions.
We compared the translational efficiency of mRNA in
pap1-1,rpl46 or pap1-1,rpl16B
cells with
that in pap1-1 cells after inactivation of polyadenylation
using RNase protection or Northern blot analysis to analyze the
polyribosome size of specific mRNAs. We previously used RNase
protection to show that inactivation of polyadenylation causes
TCM1 mRNA to accumulate as poly(A)-deficient (An < 20) and poly(A)
transcripts (31). We used this assay to monitor the polyribosome size
of these TCM1 mRNAs in pap1-1,rpl46
(35 °C), pap1-1,rpl16B
(35 °C), and
pap1-1 (35 °C) cells. The results show that
TCM1 mRNA produced after polyadenylation shut-off
sediments with fewer ribosomes in the ribosome protein deletion strains
than in cells with normal ribosome levels (Fig. 2). In
particular, the rpl46 and rpl16B deletions cause
TCM1 mRNA to accumulate as 40-60 S complexes at the
expense of the largest polyribosomes, suggesting that
poly(A)
and poly(A)-deficient forms of TCM1
(Fig. 2, A
and A*, respectively) translate
poorly when competing for limiting numbers of ribosomes. Alternatively,
the translation defect apparent in these ribosome-deficient strains
could reflect discrimination acting strictly at the translation initiation site of TCM1. If this were so, then we would
expect poly(A)
and poly(A)-deficient TCM1
mRNAs to suffer the same fate under these conditions. We tested
this by measuring the relative amounts of each form of TCM1
mRNA in each gradient fraction and plotting the results as the
A
/A* ratio (Fig. 3, A and B).
The results indicate that limiting ribosome concentrations causes a
preferential accumulation of poly(A)
TCM1 in
the 20-60 S region of the gradient, a region that includes 43 S, 48 S,
and mRNP complexes. These findings suggest that the absence of a
poly(A) tail plays an important role in limiting the translation of
TCM1 when ribosomes are limiting. Curiously, we do not
observe significant differences in the ratio of poly(A)
to poly(A)+ TCM1 mRNA in any individual
fraction within the polyribosome complex portion of the deletion
gradients (Fig. 3, A and B). This may simply
reflect a marginal functional difference between a short (An < 20) poly(A) tail and a
poly(A)
form of an mRNA, or it may indicate that
poly(A)
mRNAs suffer a disadvantage when loading the
first, but not subsequent ribosomes (see "Discussion"). From these
results, we suggest that TCM1 mRNAs with short poly(A)
tails display a modest translational advantage over
poly(A)
forms and likely compete more effectively for a
rate-limiting step early in translation initiation.
The discrimination against poly(A) TCM1
mRNA during translation initiation implies that it competes with
other poly(A)+ mRNAs. Indeed, the normal shapes of the
polyribosome profiles in pap1-1;
pap1-1,rpl46
; and pap1-1,rpl16B
cells
shifted to 35 °C suggests that some mRNAs translate efficiently
despite the ribosome deficiencies. We reasoned that polyadenylation
shut-off should have a more modest impact on mRNAs with long
half-lives than on those with short half-lives, since a smaller
fraction of the former will have been synthesized as
poly(A)
transcripts in the hour between inactivation of
poly(A) polymerase and harvest of the cells for polyribosome analysis.
Accordingly, we monitored the polyribosome density of PGK1
(t1/2
45 min) and PAB1
(t1/2
11 min), which have lengths similar to
TCM1 (t1/2
11 min; Ref. 41). We
showed previously that, at 35 °C in a pap1-1 background,
PAB1 mRNA exists without detectable poly(A) tails, while
PGK1 mRNA exists as a mixture of mostly
poly(A)-deficient and poly(A)+ species as determined by
oligo(dT) cellulose selection (31). Fig. 4, A
and B, shows the percentage distribution of PAB1
mRNA (upper panels) and PGK1 mRNA
(lower panels) in pap1-1,rpl46
(35 °C) and
pap1-1,rpl16B
(35 °C) strains, respectively. The
results indicate that both mRNAs exhibit a reproducible shift
toward smaller polyribosomes in ribosome-deficient cells (Fig. 4,
A and B, lower panels). The impact of
ribosome reduction on PAB1 mRNA translation is similar
to TCM1 mRNA, which also has a short half-life (10-15 min) and competes less well for ribosomes (compare Figs. 2,
A and B, and Fig. 4, A and
B). Taken together, these results suggest that the
translational apparatus discriminates against poly(A)
mRNA when the ratio of mRNA to ribosomes is normalized in
pap1-1 cells. In addition, the presence of large
polyribosome complexes in the deletion strains most probably reflects
the population of poly(A)+ mRNA produced before or soon
after inactivation of poly(A) polymerase.
Deletion of the Small Subunit Ribosomal Protein Gene, RPS51A, Reduces 80 S Ribosome Levels and Causes Translational Discrimination against poly(A)
The decrease in
translation efficiency of poly(A) mRNAs resulting
from a reduction in the number of 60 S ribosomal subunits supports the
idea that discrimination between poly(A)+ and
poly(A)
mRNAs occurs at the 60 S joining step during
translation initiation (2, 30) On the other hand, Tarun and Sachs (11)
presented the results of experiments that suggested a role for poly(A)
tails in enhancing mRNA interaction with 40 S ribosomes. If
poly(A)+ mRNAs compete more effectively than
poly(A)
mRNAs for 40 S ribosome binding, then
limitation of 40 S levels should decrease the rate of initiation of
poly(A)
mRNAs. We tested this hypothesis by deleting
one of the duplicated copies of the gene (RPS51A) encoding
the small ribosomal subunit protein, Rps51p (32, 34). Polyribosome
profile analyses of a RPS51A deletion strains reveals a
similar decrease in polyribosome density observed for the large
ribosomal subunit deletions, but with a large decrease in the number of
free 40 S subunits and a parallel increase in the number of 60 S
subunits (Fig. 1G). Inactivation of Pap1p causes an increase
in average polyribosome size consistent with an increase in the ratio
of ribosomes to mRNA. We monitored the translational efficiency of
TCM1, PAB1, and PGK1 mRNAs as for
the 60 S subunit deletion strains. Limitation of 40 S ribosomes causes
a fraction of TCM1 mRNA to associate with the smallest
of polyribosome complexes (Fig. 2C, upper panel) and increases the ratio of poly(A)
to poly(A)-deficient
TCM1 in fractions containing 48 S, 43 S, and mRNP complexes
(Fig. 3C). Comparison of Northern blots of polyribosome
gradient fractions from poly(A)-deficient cells with normal ribosome
levels (pap1-1) and those with decreased 40 S levels
(pap1-1,rps51A
) reveals a decrease in the amount of
PAB1 mRNA in large polyribosomes, while PGK1
mRNA remains associated with heavy polyribosomes (Fig.
4C). The impact of the RPS51A deletion on these
mRNAs is not as dramatic as seen in pap1-1,rpl46
or pap1-1,rpl16B
strains. This may reflect the degree of
reduction of ribosome levels in the various strains, or it may suggest
that 40 S ribosomes do not play as important a role in enhancing
translation of poly(A)+ mRNAs as 60 S subunits.
We utilized a yeast strain harboring a temperature-sensitive
mutation (pap1-1) in the poly(A) polymerase gene to monitor
the chemical and functional properties of poly(A)-deficient and
poly(A) mRNAs. We demonstrated previously that
inactivation of Pap1p resulted in the inability of cells to accumulate
poly(A)+ mRNAs (34). Many mRNAs also fail to
accumulate as poly(A)
species, consistent with a role for
poly(A) tails in the synthesis and maintenance of stable mRNA (4,
7). The poly(A)-deficient and poly(A)
mRNA that
accumulate in pap1-1 (35 °C) cells associate with
polyribosomes of at least the same size as those observed in wild-type
(PAP1) cells, suggesting that poly(A) tails are not required
for efficient translation. However, our results also indicated that
mRNA loss in poly(A)-deficient cells resulted in a significant
increase in the ratio of ribosomes to mRNA. We suggested that the
excess of translational components such as ribosomes and their
associated factors might overcome any mechanism for translational
discrimination against poly(A)-deficient or poly(A)
mRNA. Hence, we tested whether discrimination against translation of poly(A)
mRNA would occur in poly(A)-deficient
cells with a normal ratio of mRNA to ribosomes. Our results show
that deletions of ribosomal protein genes RPL46,
RPL16B, or RPS51A in pap1-1 cells
results in a nearly normal ratio of ribosomes to mRNA after
polyadenylation shut-off. Under these conditions, poly(A)
TCM1 and PAB1 mRNAs form smaller
polyribosomes, suggesting that they may not compete for ribosomes as
well as existing polyadenylated mRNAs. Candidates for the latter
mRNAs include relatively stable, polyadenylated messages
(half-life > 40 min), the bulk of which remain after 1 h at
35 °C. We monitored the polyribosome sedimentation pattern of
PGK1 mRNA (t1/2
45 min) and found
that a large portion of it exists in polyribosomes comparable in size
with those found in cells with normal ribosome levels. Finally, our
ability to distinguish between poly(A)+ and
poly(A)
forms of TCM1 mRNA allowed us to
show that the poly(A)
forms appear to exhibit a defect in
entering polyribosomes.
Taken together, the results from both large and small ribosomal
protein-deleted pap1-1 strains indicate that, in
vivo, poly(A) tails may enhance the translation of mRNA most
effectively when the ratio of total mRNA to ribosomes approximates
the levels observed in normal cells at steady state. Under these
conditions, we observe discrimination against the translation of
poly(A) TCM1, and PAB1 mRNAs
and a competitive advantage favoring the translation of poly(A)+
TCM1 and PGK1 mRNAs. The fractions in
the nonpolyribosomal portions sedimenting lighter than 60 S, but
heavier than approximately 20 S, display the most significant
difference in the A
/A+ TCM1 mRNA ratio. This region
encompasses free 40 S ribosomal subunits and associated preinitiation
complexes (i.e.. 43 and 48 S), suggesting inefficient
formation of 80 S ribosomes (i.e. 60 S subunit joining) by
poly(A)
TCM1 mRNA. Since we do not observe
significant differences in the A
/A+ TCM1 mRNA ratio in
smaller polyribosome fractions, it might be that the partial block in
60 S subunit joining occurs during the loading of the very first
ribosome, while subsequent ribosomes (and subunits) escape the
inhibition, allowing for the formation of relatively large polyribosome
complexes. Alternatively, TCM1 mRNA bearing short
poly(A) tails (An < 20) may have only
a modest competitive advantage in translation compared with
poly(A)+ mRNA. If we could compare the polyribosome
distribution of poly(A)
TCM1 mRNA with its
full-length poly(A) tail (An = 60-70) counterpart, the A
/A+ ratio might be significantly lower in the polyribosome fractions.
A link between poly(A) tails and the translation machinery was proposed
more than 2 decades ago, but the biochemical nature of the interaction
remains obscure. Evidence that poly(A) tails enhance the translational
efficiency (i.e. functional half-life) of a mRNA came
from several in vitro and in vivo experiments
comparing the rate or amount of polyribosome formation of
poly(A)+ mRNA with its poly(A) form in
programmed rabbit reticulocyte lysates or following injection of
synthetic mRNAs into X. laevis oocytes. For instance,
Galili et al. (14) provided evidence that in stage VI
Xenopus oocytes, injected poly(A)+ and
poly(A)
zein mRNAs did not compete for the binding of
the first ribosome, yet only poly(A)+ mRNA associated
with large polyribosome complexes after further incubation. In this
system, the poly(A)
mRNA was as stable as its
poly(A)+ form, leading to the suggestion that poly(A) tails
enhance, in cis, the reinitiation of ribosome loading at the
5
-end of mRNA. One important feature of this experiment was that
stage VI Xenopus oocytes have a saturated translational
capacity, and it remains unclear whether discrimination against
poly(A)-deficient mRNAs would be found at other stages of
development where the translational machinery is in excess of
substrate.
In experiments employing rabbit reticulocyte lysates, Munroe and
Jacobson (30) found that only 50% of input poly(A)
mRNA associated with polyribosomes compared with its
poly(A)+ counterpart and provided evidence that the
decreased efficiency of poly(A)
mRNA translation
resulted from a reduced ability to couple 60 S ribosomal subunits to 48 S preinitiation complexes (30). In this system, endogenous mRNA
removed by nuclease treatment yielded an excess of ribosomes and
translation factors compared with the levels of input mRNAs.
Although translational discrimination was observed, the competitive
advantage of poly(A)+ mRNA might have been greater if
the translational apparatus was saturated with mRNA. Indeed, Gallie
showed that a poly(A) tail increased translation of a reporter mRNA
40-fold in yeast protoplasts, which presumably translate a full
complement of cellular mRNAs (15).
The extent to which the translational machinery in normal S. cerevisiae is saturated by mRNA is unclear. In Escherichia coli, induction of mRNA synthesis from multicopy plasmids decreases the translation of preexisting mRNAs by competition for limiting translation components (42). A similar effect is observed in oocytes of X. laevis, where the injection of exogenous mRNA decreased translation of cellular mRNAs at all concentrations of injected mRNA (43). Furthermore, when polyribosome complexes were introduced into these same oocytes, no effect on endogenous protein synthesis was observed, suggesting that a limiting component for translation is polyribosome-associated (43). In our system, 35 °C-shifted pap1-1 cells carrying deletions in ribosomal proteins appear to have a normal ratio of ribosomes to mRNA. The existing ribosomes and associated translation factors participate in discrimination against poly(A)-deficient mRNAs unlike in a pap1-1 (35 °C) strain, where ribosome levels are in excess of substrate mRNAs. Since reducing the numbers of 60 or 40 S subunits effectively lowers ribosome concentration and in turn promotes translational discrimination, one might speculate that the levels of intact ribosomes alter the abundance or function of an associated discriminatory factor.
The ability of poly(A) tails to enhance translation might be mediated
by the capacity of poly(A) to recruit poly(A)-binding protein and in
turn engage the translational apparatus in some undetermined manner.
Poly(A)-binding protein reverses the inhibitory effect of excess
poly(A) added to reticulocyte lysates, suggesting that poly(A)-binding
protein is a titratable factor that may have a role in translation
(14). Sachs and Davis (29) provided evidence that yeast cells harboring
a temperature-sensitive lethal allele of PAB1
(pab1-f364l) have defects in poly(A) tail shortening and
translation initiation (29). Interestingly, this mutation also gives
rise to an increased ratio of free 60 S to 40 S ribosomal subunits.
Several cold-sensitive suppressors of the pab1-f364l mutant,
including one (spb2) bearing a mutation in ribosomal protein L46, display an inversion of the free subunit ratio but do not restore
normal poly(A) tail lengths (29). Improved growth in spb2
cells is therefore linked to better translation of existing mRNAs
by alteration of 60 S subunit levels. These observations led to the
suggestion that Pab1p is intimately linked to the ribosome via the 60 S
subunit, a notion consistent with recent work from our laboratory
demonstrating a Pab1p association with polyribosomes translating
poly(A)-deficient or poly(A) mRNAs (40).
Recently, Tarun and Sachs (11) used a S. cerevisiae in vitro
translation system to monitor the translational potential of synthetic
poly(A)+ versus poly(A) mRNA
in the presence or absence of poly(A)-binding protein (Pab1p). The
results of their experiments suggested that Pab1p enhances the binding
of poly(A)+ mRNA to 40 S ribosomal subunits, implying
that the Pab1p-poly(A) tail complex serves a role in the translational
recruitment of mRNA from mRNPs to active polyribosome complexes.
However, these experiments, which employed an inhibitor of 60 S subunit
joining could not assess the relative impact of Pab1p on 60 and 40 S
subunit binding.
The results presented here indicate that poly(A) tails may confer a
small competitive advantage to a mRNA at both the 60 and 40 S
binding steps; limitation of either subunit appears to reduce the
efficiency of translation of poly(A) mRNAs more than
poly(A)+ mRNAs. Similarly, some, but not all, mutations
that reduce ribosomal subunit levels reduce the expression of
poly(A)
yeast viral mRNAs (44). In both cases, 60 S
subunit changes produced a more pronounced effect, possibly reflecting
a more critical role of the 60 S subunit joining step in enhancing
translation of poly(A)+ mRNAs. Interestingly, while
these studies suggest that limitation of 60 S ribosomal subunit levels
inhibits the translation of poly(A)
mRNAs, such
changes bypass the need for Pab1p in translation (29, 45). It remains
unclear whether this paradox reflects fundamental aspects of
Pab1p-poly(A) complex function or whether specific ribosomal
alterations may suppress or antagonize different defects in mRNA
3
-end structure. Finally, the Pab1p-poly(A) complex plays a critical
role in determining mRNA levels, since Pab1p defects cause
premature decapping and mRNA decay (46, 47). In this regard, the
small effects of ribosome limitation on poly(A)
mRNA
translation observed here, as well as the large decreases in mRNA
concentration caused by the pap1-1 defect (31), argue that
the regulation of mRNA stability is the primary role of poly(A) tails in yeast. However, the intimate link between the Pab1p, poly(A)
tails, translation rates, and mRNA decay requires careful consideration of each of these facets of mRNA function before we
reach a full understanding of how mRNA structure affects gene expression.
We are grateful to the members of our laboratory for stimulating discussions and comments on the manuscript. We thank Michael Rosbash and John L. Woolford, Jr., for providing plasmids.