(Received for publication, November 8, 1995; and in revised form, January 11, 1996)
From the
Recognition of the 5`-end of eukaryotic mRNA by the ribosomal 43
S preinitiation complex involves the eukaryotic translation initiation
factor eIF-4E (eIF-4). Deletion mutants of the eIF-4E gene of Saccharomyces cerevisiae (CDC33) encoded proteins
with reduced affinity for the 5`-cap. One of these mutant proteins
lacked any detectable binding to a cap analogue binding column, yet was
still able to support cell growth. More than 17% of the total eIF-4E
amino acid sequence could be removed without fully inactivating this
factor. At least 30 of the N-terminal amino acids are not essential for
function. The minimal functional eIF-4E protein segment therefore
comprises at most 176 amino acids. The translation and growth defects
of the deletion mutants could be at least partially compensated by
increases in eIF-4E synthesis, possibly due to a mass-action effect on
mRNA binding. Electroporation of yeast spheroplasts with in vitro synthesized mRNA allowed us to characterize the ability of eIF-4E
mutant strains to distinguish between capped and uncapped mRNAs in
vivo. Our data show that the cap specificity of eIF-4E determines
to what extent the translational apparatus differentiates between
capped and uncapped mRNAs and indicate the minimum relative mRNA (cap)
binding activity of eIF-4E required for yeast cell viability.
The eukaryotic initiation factor eIF-4E ()(or
eIF-4
; see (1) ) is an essential component of the
eukaryotic translational apparatus. eIF-4E constitutes part of the
so-called cap-binding complex eIF-4F (eIF-4), which, in higher
eukaryotes, also contains eIF-4A and p220 (eIF-4
) (2, 3, 4) . This complex, together with
eIF-4B, is thought to mediate binding of the 43 S preinitiation complex
to mRNA(5) . In the yeast Saccharomyces cerevisiae,
eIF-4E has been shown to form a complex with two proteins, p150
(thought to be the homologue of mammalian p220) and p20. The functions
of p20 and p150 are as yet
unknown(6, 7, 8, 9) . However, it is
generally assumed that yeast eIF-4F fulfills the same function(s) as
its mammalian counterparts.
The sequencing of eIF-4E genes from several species has revealed strong homology within the group of known mammalian polypeptide sequences and less extensive but clearly evident homology between the yeast eIF-4E sequence and the sequences of the counterpart proteins of mammals and wheat. The mammalian and yeast proteins are immunologically distinct (10) . Nevertheless, mouse eIF-4E can substitute for its yeast homologue in vivo(11) . Thus, there is also at least partial functional homology between eIF-4E from diverse species. At the same time, the functional role(s) of eIF-4E is not yet clear. The two main lines of evidence for this factor's involvement in yeast translation derive from in vitro experiments. First, addition of a cap analogue (12) or a monoclonal antibody against yeast eIF-4E leads to inhibition of translation in a dose-dependent fashion(10) . Second, in a cell-free system derived from a yeast strain carrying a temperature-sensitive form of eIF-4E, the translation defect could be complemented by addition of purified wild-type eIF-4E(13) . However, the step(s) of in vivo translation in which eIF-4E is involved and its mechanism of action remain uncharacterized. Moreover, other reports have indicated that this protein might be directly or indirectly involved in processes other than translation initiation. For example, the yeast eIF-4E gene has been identified as the locus of a cell cycle mutation (cdc33) that arrests the mitotic cycle at the ``start'' stage(14) . On the other hand, a fraction of the cellular population of eIF-4E in COS cells (15) and yeast (16) localizes to the nucleus.
Much attention has been paid to the fact that eIF-4E can be phosphorylated in the cell. Many apparent correlations between the level of eIF-4E phosphorylation and the rate of cellular protein synthesis have been reported(17) . Clearly, any global regulation of protein synthesis mediated via eIF-4E phosphorylation would have to be based on a specific and precisely controlled mechanism, yet this remains an unresolved issue in mammalian cells. In S. cerevisiae, eIF-4E phosphorylation is unlikely to be regulated by the mechanisms proposed for its mammalian counterparts(18) .
A common property of all known eIF-4E proteins is recognition of the mRNA cap structure(19) . Interestingly, the estimated binding affinity is at least 4 orders of magnitude lower than that of the most tightly binding RNA-binding proteins for their targets(20) , yet the significance of this binding activity for the cytoplasmic (and possibly nuclear) functions of eIF-4E remains unclear. A key question here is how the affinity of eIF-4E for the cap relates to the ability of ribosomes to bind and translate mRNA efficiently. This relationship is central to the potential function of eIF-4E as a regulatory factor, whose activity might be modulated by phosphorylation or via its interaction with other proteins. Furthermore, the availability and/or activity of eIF-4E might not influence the translation of all mRNAs equally, yet it remains uncertain whether specific mRNA populations can be differentially activated via this factor. It has been suggested that eIF-4E is ``rate-limiting'' for translation initiation on at least some eukaryotic mRNAs because of its low abundance in the cell relative to other initiation factors(19, 21, 22, 23) . This might be especially applicable to those mRNAs whose translation is restricted by intramolecular structure(19, 23) . In certain higher cells, the overproduction of eIF-4E results in a transformed phenotype, an effect proposed to be attributable to relaxation of the inhibitory effect imposed by secondary structure on the translation of growth-controlling mRNAs(24, 25) .
In this paper, we address the above issues by varying both the abundance and the specific activity of eIF-4E in yeast, making use of a series of deletion mutations that progressively reduce its cap binding affinity. A newly developed experimental strategy has allowed us to characterize how altered interactions of these mutant proteins with mRNA influence the translational apparatus in vivo.
Figure 1:
Expression constructs used in this
work. The wild-type or mutant eIF-4E genes were expressed using a CEN4-ARS1 plasmid (A). The promoter was either the GPF1 fusion promoter (33) or the TRP1 promoter(18) . The start codon of the eIF-4E gene was part
of an NdeI site engineered at the beginning of the reading
frame (16) . Transcription was terminated by the PGK1 terminator. The cleavage sites of the following restriction
enzymes are indicated: HindIII (H), BamHI (B), and NdeI (N). B/G indicates
the site of a BamHI/BglII fusion. The eIF-4E reading
frame was shortened at the N and C termini (B). New start
codons were engineered by introducing NdeI sites at various
positions downstream of the authentic initiation codon. Premature stops
were in most cases introduced as double termination codons (TAG.TAG)
upstream of the wild-type stop codon. In 196, this was achieved by
cleavage with EcoRI followed by religation, which in this
vector results in extension of the reading frame at position 196 by two
extra codons (+2). Shown in C is an amino acid
sequence comparison of the N- and C-terminal ends of eIF-4E from man
and the yeast S. cerevisiae, showing the regions removed in
the deletion mutants listed in B.
We tested the viability of strains dependent on
these eIF-4E mutants for translation. After transformation of the
diploid strain GEX1, tetrad analysis was used to assess the viability
of haploid cells lacking the wild-type gene but carrying each deletion
mutant (Fig. 2). This analysis revealed that all of the deletion
mutants except 183 allowed growth (see Table 1and Table 2). Tetrad analysis with
196 gave very poor spore
survival rates when expressed using the TRP1 promoter (see
legend to Fig. 2), but since plasmid shuffling was possible (see
below), this is not an indicator that
196 normally cannot support
growth. In a parallel strategy, the mutant eIF-4E expression plasmids
were transformed into strain 4-2, and the resulting transformants were
cured of pMDA101. This produced haploid strains that each contained
only a mutant form of cdc33. Each of the mutant genes was
expressed at low levels using the TRP1 promoter and at high
levels using the GPF promoter.
30/206 allowed growth only
when expressed from the GPF promoter. Complementation and
growth were poor with
196. Western blotting was used to estimate
the relative amounts of mutant eIF-4E proteins synthesized using the
two promoters (Fig. 3A). Quantitative analysis revealed
that expression from the GPF promoter yielded a steady-state
level of eIF-4E
16-fold greater than that supported by the TRP1 promoter (Fig. 3B). Moreover, the
steady-state level of eIF-4E was
2-fold lower using the TRP1 promoter plasmid than was observed in a haploid strain
(YPM150A195-4C) bearing the chromosomal eIF-4E gene expressed from its
own promoter (Fig. 3A). In further experiments, we
compared the abundance of eIF-4E in extracts from the strains YPM150A,
4-2 [pMDA101], and 4-2 [YCpSupexTRP1
CDC33] (i.e. the wild-type plasmid described in Fig. 1) (data not shown). The plasmid-borne cdc33 genes
supported a steady-state level of eIF-4E that was
50% less than
that of the strain with a chromosomal CDC33 gene and also
apparently less than that of strain 4-2. This is consistent with an
earlier comparison between strains T93C and 4-2(13) .
Figure 2:
Viability of haploid yeast strains
dependent on each of the newly constructed mutant forms of eIF-4E for
translation. Shown is the tetrad analysis of the C-terminal mutants. S. cerevisiae GEX1 was transformed using derivatives of the TRP1 promoter version of the vector shown in Fig. 1.
After sporulation, the mutants 206 and
200 allowed
complementation of the CDC33::LEU2 disruption, yielding mainly
4:0 viable spores.
183, on the other hand, did not allow
complementation, yielding only 2:0 viable spores.
196 allowed
complementation at only a very low frequency (in 1 out of 24 tetrads),
generally also yielding only 2:0 viable spores. It is therefore shown
in parentheses here to indicate that its results in tetrad
analysis were almost as poor as those of
183. Moreover,
196
supported considerably more rapid growth after plasmid shuffling when
expressed from the GPF promoter than when expressed from the TRP1 promoter (see Fig. 3and Table 2).
Figure 3:
Relative levels of expression of eIF-4E
from the TRP1 and GPF promoters. Extracts from
transformed haploid yeast strains containing only the indicated form of
the eIF-4E gene on the single-copy expression plasmid (Fig. 1)
were subjected to SDS-15% polyacrylamide gel electrophoresis and
Western blotting. The samples were loaded in pairs, each pair
comprising extracts from strains bearing one of the types of eIF-4E
reading frames: wild-type (wt) or one of the deletion mutants,
under the control of either the GPF1 promoter or the TRP1 promoter (A). Equal amounts of each cell extract within
each pair were loaded, permitting direct comparison of the relative
amount produced by the respective promoters. The GPF promoter
reproducibly supported the maintenance of an amount of eIF-4E greatly
exceeding that found in cells using the TRP1 promoter. Serial
dilution of extracts from cells expressing eIF-4E from the GPF promoter allowed us to estimate the presence of an 16-fold
greater amount of eIF-4E in comparison with the TRP1 promoter.
An example of such a comparison is shown in B. This second
Western blot also shows the relationship between the relative level of
eIF-4E in cells producing eIF-4E from the chromosomal wild-type
promoter (P
-4E; in the strain
YPM150A) and that maintained by the TRP1 promoter.
Figure 4:
Elution of wild-type and mutant eIF-4E
proteins from a 7-methyl-GDP-Sepharose column. eIF-4E could be isolated
either as part of the yeast cap-binding complex (A) or from E. coli inclusion bodies (B). The silver-stained gel (A) shows the two main fractions (F1 and F2)
specifically eluted using 7-methyl-GDP after the column had been washed
using buffer A and GDP (see ``Materials and Methods''). The
amount of protein eluted in this way provides a measure of the relative
cap binding affinity of each deletion mutant. Three examples are shown,
illustrating the large reductions in affinity caused by the described
deletions. 196 (not shown here) was not detectably bound in this
assay. Similar elution behavior was seen using inclusion body
preparations from E. coli strains expressing the respective
mutant eIF-4E genes from pCYTEXP1 (B). The latter two
Coomassie Blue-stained gels show fractions from all stages of affinity
chromatography on 7-methyl-GDP-Sepharose. The combination of the
N-terminal deletion
7 with the C-terminal deletion
206
resulted in a further reduction in cap binding affinity over either of
the deletions alone. wt,
wild-type.
In an additional approach to analysis of
the eIF-4E mutants, we used electroporation to introduce in vitro transcribed mRNA into yeast spheroplasts (Fig. 5). This
method allowed us to compare the translation of capped and uncapped
mRNAs in vivo. The results clearly demonstrate that the
deletion mutant 196, when expressed at a level comparable to that
of eIF-4E in wild-type yeast cells, confers reduced selectivity upon
the translational apparatus with respect to capped mRNA. The uncapped
mRNA is able to compete more effectively as a translational template in
the strain containing the mutated eIF-4E. The luciferase activity
encoded by a capped mRNA in a strain containing wild-type eIF-4E was up
to 5 times greater than that encoded by an uncapped mRNA, whereby the
ratio averaged over the four different amounts of mRNA used (Fig. 5A) in 16 independent experiments was 4.3. This
ratio was never more than 2.0 in a strain containing
196 instead
of wild-type eIF-4E, whereby the average for the equivalent set of
experiments was 1.9. These results show that eIF-4E has a major
influence on the ability of ribosomes in vivo to select mRNA
on the basis of the modification status of its 5`-end. However, the
distinction between capped and uncapped mRNAs is also markedly reduced
when wild-type CDC33 is overexpressed (Fig. 5B).
Figure 5:
Expression of electroporated mRNA in
spheroplasts of S. cerevisiae. Luciferase activities were
measured in extracts from spheroplasts 5 h after electroporation with
0.5, 1.0, 2.0, or 4.0 µg of in vitro synthesized LUC mRNA. Typical data are shown, each point representing the average
of two parallel measurements. Capped () or uncapped (
)
polyadenylated mRNA was introduced in various amounts into spheroplasts
of a strain with wild-type CDC33 expressed from the relatively
weak TRP1 promoter (A). The same transcripts were
also introduced into spheroplasts of a strain with the
196 gene
expressed from the TRP1 promoter (capped LUC mRNA
(
) and uncapped LUC mRNA (
)). The second set of
results (B) were obtained with spheroplasts derived from
strains in which either CDC33 (capped (
) and uncapped
(
)) or
196 (capped (
) and uncapped (
)) was
expressed from the relatively strong GPF fusion promoter. The
amounts of mRNA added were all below the level of saturation for the
translational apparatus.
The presence of a defective eIF-4E
protein resulted in changes in the profiles obtained from polysomal
gradient analysis. Thus, as clearly evident in the case of 196,
the relative proportion of ribosomes in the 80 S peak increased
markedly (Fig. 6). Salt treatment resulted in the dissociation
of most of the 80 S ribosomes into 40 S and 60 S subunits, thus
indicating that a large proportion of the 80 S ribosome population
comprised nontranslating 80 S ``couples'' (compare with (36) ). Examination of the influence of the promoter used on
the relative sizes of the 80 S peaks reveals that there is again an
eIF-4E dosage effect. The increased abundance of the
196 eIF-4E
mutant synthesized from the GPF promoter compensated partially
for its reduced specific activity, allowing more ribosomes to be
translationally active. A further unusual aspect of the polysomal
profiles was the enlarged 40 S/43 S peak, which was particularly
exaggerated in extracts from cells containing GPF promoter
constructs. One possible explanation of this phenomenon might be the
release of non-scanning 40 S subunits that are normally involved in
complexes (e.g. post-termination complexes) with the mRNA.
Figure 6:
Polysomal gradient analysis of extracts
from yeast cells whose growth is dependent on truncated forms of
eIF-4E. The absorbance profile at 260 nm after sucrose gradient
centrifugation indicates the sizes and positions of the polysome
components. In cells dependent on 196 eIF-4E expressed from the TRP1 promoter (C), the 80 S peak is greatly
exaggerated compared with the profile seen with wild-type (WT)
eIF-4E (A). Addition of salt leads to dissociation of many of
the 80 S ribosomes (compare B and D), indicating that
the majority are nontranslating 80 S couples. Western blot analysis was
performed on the unusual polysomal gradient peak patterns obtained with
196. Typical results are shown in C and D. They
reveal that eIF-4E is distributed throughout the gradient, whereas
eIF-2
and ribosomal protein L15 show more localized distributions,
acting as markers for 43 S preinitiation complexes and 60 S ribosomal
subunits, respectively. Overexpression of
196 resulted in a
profile more typical of cells with a comparatively small amount of
wild-type eIF-4E (compare E and A). This resulted in
the generation of far fewer 80 S couples (compare F and D). All strains were grown with galactose as carbon
source.
Both wild-type eIF-4E and the mutant derivatives were present in all
fractions of the gradient (Fig. 6). This contrasted with the
distribution of eIF-2, which was found mainly in the 40 S/43 S
fractions, with only low levels in the polysomal fractions. This is
consistent with eIF-2 dissociating upon formation of 80 S monosomes and
being present in much reduced amounts in the polysomes. The results
obtained with the eIF-4E antibody indicate that this factor remains
bound to cytoplasmic mRNA at least during translation initiation and
elongation. Moreover, the greatly reduced cap binding activity of
196 does not prevent this truncated form of eIF-4E from
interacting with mRNA in vivo.
Figure 7:
Effects of eIF-4E deletions on protein
synthesis directed by mRNAs whose translation is restricted by
secondary structure in their leaders. The LUC gene was
expressed from the GPF promoter using the YCpLUCEX plasmid (33) . Three different leaders were used: a control leader and
the same leader with a stem-loop structure inserted either close to the
mRNA 5`-end (B3) or close to the start codon (X3). The respective
vectors were expressed in a double transformant of strain 4-2 together
with an expression plasmid bearing either the wild-type (WT)
eIF-4E gene (CDC33) or one of the two deletion mutants
19/206 and
196. The individual values were normalized to the
luciferase activity observed using the wild-type eIF-4E gene and the LUC control leader. Plotted here are averages of measurements
on at least three independently prepared extracts. The error bars indicate the magnitudes of the respective standard
deviations.
Figure 8:
Steady-state abundance of luciferase
``reporter'' mRNAs in vivo. Shown is the Northern
analysis of extracts from cells bearing each of the LUC expression vectors together with a vector expressing the wild-type (wt), 196, or
19/206 form of the eIF-4E gene (cdc33) from the TRP1 promoter. The blot was
hybridized with
P-labeled probes specific for LUC and PGK1.
In this work, we have generated and analyzed a series of deletion mutants in CDC33 that encode proteins with reduced affinity for the 5`-cap of mRNA. These mutants have proved to be useful tools in investigations of the in vivo function of eIF-4E, which we have studied using a combination of in vivo and in vitro methods.
First, the binding characteristics of eIF-4E have been shown to determine the selectivity of the cellular translational apparatus with respect to capped and uncapped mRNAs in vivo. The competitiveness of uncapped mRNA as a template for translation can be greatly increased as a result of mutations in eIF-4E that reduce this factor's specificity for the cap. The electroporation method we have used here allows mRNAs to be introduced into the cytoplasm of yeast independently of the normal pathway of (pre-)mRNA synthesis, processing, and transport. Expression of the in vitro synthesized mRNAs reflects primarily the relative rates of their translation. Apart from clearly demonstrating the role of eIF-4E in mediating the interaction between ribosomes and mRNA in vivo, our data show how regulatory changes in eIF-4E binding activity can be expected to influence the global pattern of translation in the cell. For example, potential modulation of the binding activity of eIF-4E due to phosphorylation or via the influence of regulatory proteins would also be expected to change the pattern of mRNA recognition in an analogous fashion. A further striking aspect of these data is that overexpression of eIF-4E (via the GPF promoter) reduces the difference in translation rates between capped and uncapped mRNAs. Saturation of the recognition process is achieved for uncapped mRNA only at higher intracellular activities of eIF-4E. Under normal conditions in the cell, the activity of eIF-4E is set at a level that is optimal for the translation of capped mRNA, but greatly suboptimal for the translation of uncapped mRNA.
Second, while the reductions in cap binding affinity affected the selectivity of ribosomes for capped mRNA, we observed no differential effects related to structure in the mRNA leader. This therefore extends our previous study of the influence of excessive eIF-4E activity in the cell (16) to the region of activity significantly below the normal level. Overall, these data provide no evidence that the activity of eIF-4E is used by the yeast cell to achieve differential regulation of gene expression on the basis of mRNA structure. Moreover, previous investigations yielded no evidence that increases in eIF-4E activity above the wild-type level (however these may be achieved) will either enhance the general translational activity of the cell or differentially increase translation of mRNAs whose translation is restricted by secondary structure in the 5`-UTR ( (16) and this paper). Therefore, the yeast translational apparatus does not seem to respond to variations in eIF-4E abundance or activity in the same way as proposed for higher eukaryotic cells(24, 25) . However, we cannot rule out at this stage that some natural yeast mRNAs respond to changes in eIF-4E activity as a result of properties of the 5`-UTR unrelated to the type of secondary structure used here. On the other hand, it should be stressed that the physiological mechanism of the transforming effects caused by overexpression of eIF-4E in higher eukaryotes has yet to be determined. More information about the role of eIF-4E in the control of higher eukaryotic translation could be expected from detailed studies of the effects of progressive reductions in eIF-4E activity in appropriate cell lines. De Benedetti et al.(39) have already demonstrated that the synthesis of antisense RNA directed against eIF-4E mRNA in HeLa cells results in greatly reduced levels of both eIF-4E and p220, strong inhibition of protein synthesis, and ultimately in cell death.
Third, the data
provide an indication of the minimal eIF-4E cap affinity and/or
activity necessary for cell viability in yeast. The mutant 196 has
only a minimal binding preference for capped, as opposed to uncapped,
mRNA, and the mutant protein allows only drastically reduced absolute
translation rates. Yet cells still survive even with the low level of
this protein obtained using the TRP1 promoter. Strikingly,
however, the overall rate of translation, but not the ability to
differentiate between capped and uncapped mRNAs, is enhanced upon
overexpression of the
196 form. One explanation of the dosage
compensation of poor binding achieved by increased expression may lie
in the mass-action principle. Increasing the cellular concentration of
a less tightly binding mutant of eIF-4E may force more of it into an
mRNA-bound form.
Fourth, comparison of the amounts of wild-type
eIF-4E present in cells in which the gene is transcribed from the
wild-type promoter and from the TRP1 promoter (Fig. 3)
indicates that in wild-type yeast cells, this factor does not restrict
general gene expression by virtue of its limited availability to the
translational apparatus, at least under the chosen laboratory growth
conditions. A reduction in the cellular abundance of eIF-4E by a factor
of 2 does not limit the growth rate of the host cell (compare Fig. 3and Table 2). It is therefore likely that the
cellular concentration of eIF-4E is normally above that which would
have a measurably restrictive effect. This observation helps explain
why the overproduction of eIF-4E has little effect on cell growth. Only
at very high levels is a (negative) effect observed(16) ,
possibly due to the movement of increased amounts of eIF-4E into the
nucleus(16) , the sequestering of proteins that bind eIF-4E, or
other nonspecific effects. However, the fact that eIF-4E levels are not
normally restrictive for translation does not rule out that translation
can be regulated via modulation of eIF-4E activity. It simply means
that greater changes in eIF-4E activity are required in order to
achieve a given regulatory effect than would be the case if eIF-4E
activity were strongly rate-controlling. Finally, a corollary of the
above is that high cap specificity and cap binding activity per se are not necessary for eIF-4E function.
Fifth, large deletions
in yeast eIF-4E do not inactivate this protein, and the described
mutants help define the minimal functional protein structure. We have
shown that up to 30 of the N-terminal amino acids, or up to 17
C-terminal amino acids, can be eliminated without giving rise to a
lethal phenotype. In those mutants where amino acids were removed from
both ends, deletion of a total of 37 amino acids was tolerated.
Evidently, more amino acids can be removed from the N terminus without
inactivating the protein than from the C terminus. The deleted
sequences belong to those regions of yeast eIF-4E showing no
significant amino acid sequence identity to the equivalent regions of
the higher eukaryotic eIF-4E proteins (Fig. 1). None of the
deletions eliminated any of the eight tryptophan residues that are
generally conserved in eIF-4E amino acid sequences from various sources
and that are suspected to be significant for the recognition of the cap
structure(40, 41) . Mutation of either the first
(Trp-43) or the last (Trp-166) of these to phenylalanine was found
previously to abolish cap binding activity(41) . Given that the
mutation of especially these two respectively N-terminal and C-terminal
proximal tryptophans has the most drastic effect on cap
binding(41) , it may be possible to delete further sections of
the interior region of the eIF-4E sequence without fully eliminating
activity. The limits to this type of mutation will be at least
partially dictated by the necessity of individual residues for the
maintenance of the active conformation and/or stability of the protein.
In another study, we have shown that the N-terminal sequence also
contains two phosphorylation sites of yeast eIF-4E, both of which are
recognized by casein kinase II(18) . An important further issue
to be considered here is that cap binding is evidently not the only
property of eIF-4E that is relevant to cell viability. This is
illustrated by comparison of, for example, 30/206 and
196.
196 binds more weakly to the cap analogue column, yet allows
growth at the low level of eIF-4E supported by the TRP1 promoter. In contrast,
30/206 shows measurable cap binding
activity, yet cannot support growth when expressed from the TRP1 promoter. One possible explanation is that the N-terminal deletion
affects the ability of eIF-4E to interact with the ribosome and/or p20
or p150/p130.
In conclusion, we have obtained in vivo evidence that eIF-4E normally mediates the selectivity of yeast ribosomes for capped mRNAs. However, cells can survive with a greatly reduced preference for the capped state, accompanied by an overall reduction in the ability of eIF-4E to promote functional ribosomal binding, provided the selectivity does not drop to a point where uncapped mRNA becomes significantly competitive. The absolute priority for capped mRNA is clearly not essential for cell viability. Overexpression of CDC33 mutants apparently allows the cell to partially compensate for the defective ribosome-mRNA binding pathway. The simplest rationalization of these observations is that eIF-4E directly mediates 40 S ribosomal interactions with the mRNA, whereby the factor itself provides the specificity of cap-dependent binding. However, this does not rule out that eIF-4E can act indirectly, for example, by promoting the localization of (capped) mRNAs to a compartment where ribosomes can bind, by mediating the action of other proteins, or via another step preceding or subsequent to the actual ribosome-mRNA binding interaction.