(Received for publication, April 9, 1997, and in revised form, May 30, 1997)
From the Department of Biochemistry and McGill Cancer Center, Faculty of Medicine, McGill University, Montreal, Quebec, Canada H3G 1Y6
The cap structure, m7GpppN, is
present at the 5-end of all eukaryotic cellular (except organellar)
mRNAs. Initiation of translation is mediated by the multisubunit
initiation factor eIF4F, which binds the cap structure via its eIF4E
subunit and facilitates the binding of mRNA to ribosomes. Here, we
used recombinant proteins to reconstitute the cap recognition activity
of eIF4F in vitro. We demonstrate that the interaction of
eIF4E with the mRNA 5
-cap structure is dramatically enhanced by
eIF4G, as determined by a UV-induced cross-linking assay. Furthermore,
assembly of the eIF4F complex at the cap structure, as well as ATP
hydrolysis, is shown to be a requisite for the cross-linking of another
initiation factor, eIF4B, to the cap structure. In addition, the
stimulatory effect of eIF4G on the cap recognition of eIF4E is
inhibited by the translational repressor, 4E-BP1. These results suggest
that eIF4E initially interacts with the mRNA cap structure as part of the eIF4F complex.
Cap-dependent binding of ribosomes to mRNA is
mediated by several initiation factors, eIF4F, eIF4A, and eIF4B, and
requires energy derived from ATP hydrolysis (1). eIF4F is a
three-subunit complex composed of (i) eIF4E, (ii) eIF4A, and (iii)
eIF4G. eIF4E is a 24-kDa polypeptide that specifically interacts with
the 5-cap structure (m7GpppN; where N is any nucleotide)
(2). eIF4A is a 50-kDa protein that exhibits RNA-dependent
ATPase activity and, in conjunction with eIF4B, RNA helicase activity
(3, 4). eIF4G is a 154-kDa polypeptide that binds to both eIF4E and
eIF4A (5, 6). eIF4G also exhibits sequence-nonspecific RNA binding
activity that is most probably responsible for the RNA binding activity
of eIF4F (7)1.
eIF4E activity is regulated by two proteins, termed 4E-BP1 and 4E-BP2 (8, 9). Interaction of 4E-BP1 with eIF4E inhibits specifically cap-dependent translation (9). 4E-BPs are rapidly hyperphosphorylated in cells following treatment with insulin and growth factors (10, 11). The phosphorylation of 4E-BPs decreases the association of 4E-BP1 with eIF4E (9). Consequently, phosphorylation of 4E-BPs leads to stimulation of translation. 4E-BP1 competes with eIF4G for binding to eIF4E through similar sequence motifs (12). Furthermore, the association of 4E-BP1 with eIF4E prevents the in vitro phosphorylation of eIF4E by protein kinase C, raising the possibility of a temporal relationship between eIF4E binding to 4E-BPs and eIF4E phosphorylation (13).
Two models were proposed for the pathway of eIF4F assembly and
subsequent ribosome binding. One model posits that the first step of
ribosome binding is the interaction between eIF4F and the mRNA cap
structure (1). According to this model, eIF4F in combination with eIF4B
and eIF4A, unwinds secondary structure in the 5-untranslated region of
the mRNA, to create a single-stranded region of RNA, which serves
as a binding site for the 43 S preinitiation complex. eIF4B and eIF4A
were shown to cross-link to the cap structure only in the presence of
eIF4F in a process that requires ATP hydrolysis (14-16). Joining of
the 43 S ribosomal complex is thought to be mediated through an
interaction of the eIF4G subunit and eIF3, the latter being part of the
43 S preinitiation complex. An alternative model for cap recognition
postulates that eIF4E alone binds first the cap structure, which is
then complexed with eIF4G that is already associated with the ribosome
(17). This model is based on the finding that in vitro
translated eIF4G is bound to the 43 S preinitiation complex (17).
Support for the first model stems from the observation that eIF4F
cross-linked much more efficiently to the cap structure than did eIF4E
alone (18). Furthermore, eIF4E in extracts prepared from
poliovirus-infected cells, where the eIF4G subunit is cleaved and as a
result eIF4E is associated with the NH2-terminal fragment
of eIF4G, cross-links extremely inefficiently to the cap structure (19,
20). These results suggest an important function played by eIF4G in the
cap recognition process.
In this report we reconstituted the eIF4F cap recognition activity in vitro using recombinant components. In a photochemical cross-linking assay, we demonstrate directly that eIF4G increases the affinity of eIF4E for the cap structure. Binding of the eIF4F complex to the cap structure, as well as ATP hydrolysis, is shown to be a requisite for the cross-linking of eIF4B to the cap structure. In addition, 4E-BP1 is shown to inhibit cap binding activity of the eIF4E-eIF4G complex.
Murine eIF4E protein was expressed in
Escherichia coli K38 and purified as described previously
(21). Recombinant flag-eIF4G was expressed in Sf9 insect cells and
purified as described previously (22). Glutathione
S-transferase
(GST)2 fusion proteins of
HMK-4E-BP1 and HMK-4E-BP1 were expressed in E. coli BL21
and purified as described previously (6). Recombinant eIF4B was
expressed in Sf9 insect cells as follows: for the construction of the
baculovirus transfer vector, eIF4B cDNA was excised from the
plasmid pGEM3-eIF4B (23) with BamHI and subcloned blunt into
the NheI site of the p10 transfer vector (24). Recombinant baculovirus was generated by cationic liposome cotransfection of
p10eIF4B construct with the linearized genomic AcMNPV DNA according to
the manufacturer's instructions (Invitrogen). Recombinant virus was
isolated (25), and eIF4B was purified as described previously (26).
Uncapped RNA encoding
chloramphenicol acetyltransferase was capped and methylated with 6 units of vaccinia virus guanylyltransferase (Life Technologies, Inc.)
in the presence of 0.4 mM
S-adenosyl-L-methionine and
[-32P]GTP (100 µCi). UV-induced
cross-linking was performed in the presence of 1 mM ATP as
described previously (20). Briefly, 2 × 104 cpm of
[32P]mRNA was incubated with initiation factors in a
total volume of 20 µl in 20 mM HEPES (pH 7.5), 0.5 mM magnesium acetate, 2 mM dithiothreitol, 3%
glycerol, 100 mM potassium acetate at 30 °C for 10 min.
Reaction mixtures were irradiated at 4 °C at a distance of 4 cm with
a G15T8 germicidal lamp for 45 min. The RNA was next digested for 30 min at 37 °C with 20 µg of RNase A. Samples were analyzed on
acrylamide gels followed by autoradiography. Quantitations were
performed using a Fuji BAS2000 phosphorimager.
To study the requirements for the interaction of eIF4E
with the mRNA cap structure, purified recombinant initiation
factors (Fig. 1) were used in a
photochemical cross-linking assay (20). mRNA labeled with
32P in the cap structure was incubated with protein
factors, irradiated with UV light, and RNase-digested. Labeled proteins
were then analyzed by SDS-polyacrylamide gel electrophoresis followed
by autoradiography. In the photochemical cross-linking experiments, RNA
is the limiting component in the reaction mixtures. No detectable signal was observed in the absence of protein (Fig.
2A, lane 1). Similarly, no
cross-linking of eIF4E to the cap-labeled mRNA was observed with 10 and 50 ng of purified eIF4E (lanes 2 and 3,
respectively). Cross-linking was observed with 100 ng of eIF4E, albeit
very inefficient (lanes 4), consistent with previous data
(18, 20, 27). We next examined the effect of eIF4G on the cross-linking
of eIF4E to the cap structure. No cross-linking of eIF4G to the cap
structure was observed (lane 5). Cross-linking of eIF4E to
the cap structure was dramatically enhanced in the presence of eIF4G
(lanes 6-8). As little as 10 ng of eIF4E was efficiently
cross-linked to the cap structure in the presence of flag-eIF4G.
Comparison of lanes 4 and 8 reveals a ~7-fold
increase in the cross-linking of eIF4E to the cap structure in the
presence of eIF4G. The cross-linking of eIF4E was cap-specific as the
interaction was inhibited with 0.6 mM m7GDP
(lane 9). To determine the stoichiometry between eIF4E and eIF4G required for efficient cap binding, 10 ng of eIF4E was
preincubated with increasing amounts of flag-eIF4G before the addition
of the other components (Fig. 2B). While eIF4E alone did not
cross-link to the cap structure (lane 1), addition of
Increasing amounts of flag-eIF4G enhanced eIF4E cross-linking to the
cap structure in a dose-dependent fashion (lanes
2-6). Under these conditions, ~10 ng of flag-eIF4G enhanced
significantly the cross-linking of eIF4E to the cap (lane
2), with optimum binding occurring at a stoichiometry of 1:1
(lane 4). Cross-linking was inhibited by m7GDP
(lane 7), but not by GDP (lane 8).
We next analyzed the effect of other initiation factors on the
cross-linking of eIF4E to the cap structure (Fig.
3). eIF4E alone did not cross-link to the
cap structure (Fig. 3, lane 1), as observed above. The
interaction of eIF4E with the cap structure was not affected by the
presence of eIF4A (lane 5), eIF4B (lane 6), or a
combination of eIF4B and eIF4A (lane 7). Cross-linking of
eIF4A alone was not observed either (lane 3). Cross-linking of eIF4A to the cap structure can be detected only when using the
chemical cross-linking assay, where periodate oxidized mRNA is used
(14-16, 20). Similarly, eIF4B failed to cross-link to the cap
structure under these conditions (lanes 2, 6, and
7). As expected, cross-linking of eIF4E to the cap structure
was dramatically enhanced in the presence of flag-eIF4G (lane
8). Flag-eIF4G did not promote, however, the cross-linking of
either eIF4A or eIF4B when present alone (lanes 9 and
10, respectively), or in combination (lane 11),
to the mRNA cap structure. Furthermore, a combination of flag-eIF4G
and eIF4E failed to promote cross-linking of eIF4B (lane 12)
or eIF4A (lane 13) to the cap structure. Cross-linking of
eIF4B was observed only in the presence of all the subunits of the
eIF4F complex (eIF4A, eIF4E, and flag-eIF4G; lane 14), as
shown earlier (18). As expected, the specific interaction of eIF4E and
eIF4B with the cap structure was insensitive to 0.6 mM GDP
(lane 15) and was inhibited by 0.6 mM
m7GDP (lane 16). This confirms earlier findings
that cross-linking of eIF4B to the cap structure is dependent on eIF4F
and ATP hydrolysis (16, 19, 20). Taken together, these results provide
direct evidence for the stimulatory effect of eIF4G on the interaction of eIF4E with the cap structure.
4E-BP1 Prevents the Stimulatory Effect of eIF4G on the Cap Binding Activity of eIF4E
The activity of eIF4E is inhibited by 4E-BP1, which binds to eIF4E and prevents its interaction with eIF4G to form the eIF4F cap-binding protein complex (8, 9, 12). Earlier reports showed that binding of 4E-BP1 to eIF4E did not prevent the interaction of eIF4E with a cap-bound matrix (9, 12). However, the effect of 4E-BP1 on eIF4E binding to the cap structure as part of the mRNA has not been determined. As we have shown above, the interaction of eIF4E with the mRNA cap structure is dramatically enhanced when it is bound to eIF4G. This is consistent with the finding that following poliovirus infection, which leads to the cleavage of eIF4G, the cross-linking of eIF4E to the mRNA cap structure is drastically reduced (18, 19, 20). Under these conditions, eIF4E complexed to the amino-terminal cleavage product of eIF4G binds a cap-bound matrix (18). Similarly, eIF4E complexed to 4E-BP1 can efficiently bind the cap affinity column (9).
Based on the above observations, it is predicted that the cross-linking
of eIF4E in an extract (where it binds tightly as part of eIF4F) should
be diminished in the presence of 4E-BP1 (12). To examine this,
photochemical cross-linking to the mRNA cap structure was performed
in a rabbit reticulocyte lysate. Cross-linking was done in the presence
of Mg2+-ATP to detect also eIF4B binding. UV irradiation
induced cross-linking of polypeptides of 24, 65, and 80 kDa (Fig.
4A, lane 1), as shown previously (20). The 24- and 80-kDa polypeptides correspond to eIF4E
and eIF4B, respectively, while the identity of the 65-kDa polypeptide
is not known (20). The cross-linking of eIF4E and eIF4B was insensitive
to 0.6 mM GDP (lane 2), but was inhibited by the same
concentration of m7GDP (lane 3). In contrast,
the cross-linking of the 65-kDa polypeptide was not affected by either
nucleotide. Strikingly, preincubation of the reticulocyte lysate with
GST-4E-BP1 drastically reduced the cross-linking of eIF4E and eIF4B to
the cap structure (lane 4). Since eIF4B cross-linking is
dependent on eIF4F, it is inhibited in the presence of 4E-BP1, which
prevents eIF4F complex formation. These results indicate that eIF4E as
a complex with 4E-BP1 interacts weakly with the cap structure, as
compared with eIF4E as a subunit of eIF4F.
To further substantiate these conclusions, the UV-induced cross-linking
assay was performed using purified components as in Figs. 2 and 3.
eIF4E alone did not cross-link to the cap structure (Fig. 4B,
lane 1). To examine the effect of 4E-BP1 on the cross-linking of
eIF4E to the mRNA cap structure in this reconstituted system, 4E-BP1 was preincubated with eIF4E before the addition of the other
components. 4E-BP1 prevented the efficient eIF4E cross-linking that
occurs in the presence of flag-eIF4G (lane 2). To verify that the inhibitory effect was a result of a direct interaction between
GST-4E-BP1 and eIF4E, a mutant of 4E-BP1 (GST-4E-BP1) was used.
GST-4E-BP1
contains a deletion of the 4E binding domain and does not
repress translation (6, 12). Preincubation of eIF4E with GST-4E-BP1
had no effect on the stimulatory effect of eIF4G on the cross-linking
of eIF4E to the cap structure (lane 3). The cross-linking of
eIF4E to the cap structure in the presence of flag-eIF4G was sensitive
to inhibition by 0.6 mM m7GDP (lanes
5). Taken together, these results demonstrate that 4E-BP1 prevents
the facilitative effect of eIF4G on the interaction of eIF4E with the
mRNA cap structure.
In summary, we have reconstituted the cap recognition step of
eukaryotic translation initiation in vitro using purified
components. We have demonstrated that eIF4G significantly enhances the
cap recognition activity of eIF4E, suggesting that eIF4G plays an important role in the mechanism of mRNA cap recognition during eukaryotic translation initiation. Furthermore, 4E-BP1 inhibited the
stimulatory effect of eIF4G on the cap binding activity of eIF4E.
However, a 4E-BP1·eIF4E complex can be isolated by a cap affinity
column (9, 12). It is conceivable that eIF4G, because of its RNA
binding activity (28),3
interacts with the RNA in the vicinity of the cap structure and facilitates a stable association between eIF4E and the mRNA 5-cap structure. Indeed, eIF4F binds much more avidly to RNA than either eIF4E or eIF4A (7). Our results also indicate that eIF4B can gain
access to the cap structure only in the presence of an intact eIF4F
complex and ATP hydrolysis. In support of this conclusion, disruption
of eIF4F complex by 4E-BP1 interfered with the efficient cross-linking
of eIF4B to the cap structure. Taken together, our results indicate
that eIF4E initially interacts with the cap structure as a subunit of
eIF4F.
Several other observations support the hypothesis that eIF4F complex
assembly occurs prior to the cap recognition step of translational
initiation (18-20). The equilibrium constant
(keq) of m7GpppG·eIF4E (mammalian)
complex formation has been determined by spectroscopic studies to be
4.8 × 105 M (29, 30). This indicates a weak
interaction between eIF4E and the cap structure that is unlikely to be
favored in vivo. eIF4E as a subunit of eIF4F cross-links
20-fold better to the cap structure than eIF4E alone (31). Furthermore,
Pelletier and Sonenberg (20, 32) showed that insertion of secondary structures 38 nucleotides downstream from the cap structure had no
effect on UV cross-linking of initiation factors to the cap structure,
whereas binding of ribosomes to mRNA was impaired, suggesting that
eIF4F interaction with the cap structure precedes ribosome binding to
mRNA. Based on our results and the studies cited above (19, 20,
32), we favor the model where some localized unwinding of the
5
-untranslated region of mRNA precedes ribosome attachment.
Other important parameters have also been implicated in effecting the interaction between eIF4E and the cap structure. Phosphorylation of both eIF4E and eIF4G is enhanced following treatment of cells with growth factors and insulin (33). eIF4E is more phosphorylated as a subunit of eIF4F (34, 35), and phosphorylated eIF4E forms a more stable complex with eIF4G (36). Furthermore, phosphorylated eIF4E was reported to bind better to the cap structure relative to its unphosphorylated form (37), and only the phosphorylated form of eIF4E is present in the 48 S preinitiation complex (38). In addition, the association of 4E-BP1 with eIF4E in vitro prevents the phosphorylation of eIF4E (13). Taken together these findings lend support to a model where prior assembly of eIF4F complex is a requisite for the cap recognition and subsequent ribosome binding steps of translation initiation in eukaryotes.
We thank Greg Cosentino, Andrew Craig, and Kianoush Khaleghpour for critical reading of the manuscript. We gratefully acknowledge Arnim Pause for providing recombinant eIF4A and appreciate the technical assistance of Nathalie Methot in the purification of eIF4B.