(Received for publication, October 3, 1995; and in revised form, October 19, 1995)
From the
The eIF4 group initiation factors carry out recognition of the mRNA cap, unwinding of mRNA secondary structure, and binding of mRNA to the 43 S preinitiation complex. Infection by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, an event that severely restricts cap-dependent translation but permits cap-independent initiation to proceed from internal ribosome entry sequences in picornaviral RNAs. The 5`-untranslated region (5`-UTR) of eIF4G mRNA resembles such picornaviral sequences in being unusually long and containing multiple open reading frames and a polypyrimidine tract. When inserted upstream of a luciferase reporter gene, this 5`-UTR served as a translational enhancer in four different cell lines. Mutation of all four upstream ATG codons to AAG did not alter the translational enhancement. The presence of the eIF4G 5`-UTR between an RNA hairpin and the luciferase cistron stimulated expression 119-fold. Similarly, the presence of the 5`-UTR between the two cistrons of a bicistronic mRNA stimulated expression of the downstream cistron 42-fold. These results indicate that the eIF4G 5`-UTR directs internal initiation. The ability to continue synthesis of eIF4G when the cell is unable to carry out normal cap-dependent translation may represent an autoregulatory mechanism or be part of the cellular response to stresses that interrupt cap-dependent translation.
Initiation of the overwhelming majority of eukaryotic mRNAs
proceeds by a cap-dependent mechanism whereby the AUG nearest the
5`-end serves as the initiation codon(1) . Yet other modes of
initiation codon selection are used in special cases, e.g. leaky scanning, termination-reinitiation, and internal
initiation(2) . In the latter case, ribosomes are directed to
internal AUGs by an internal ribosome entry sequence
(IRES)( eIF4G forms a
heterotrimeric complex termed eIF4F together with eIF4E, the
cap-binding protein, and eIF4A, an RNA helicase. The eIF4 group
initiation factors (eIF-4A, eIF-4B, eIF4E, and eIF4G) collectively
catalyze the recognition of the mRNA cap, the unwinding of mRNA
secondary structure, and the binding of mRNA to the 43 S preinitiation
complex(16, 17) . eIF4G contains binding sites for
eIF4E (18, 19) as well as eIF4A and
eIF3(19) , the latter being a component of the 43 S
preinitiation complex. The cleavage of eIF4G by picornaviral proteases
separates the N-terminal one-third of the molecule, which binds eIF4E,
from the C-terminal two-thirds, which binds eIF4A and eIF3, effectively
dissociating the cap recognition function from the RNA helicase and
ribosome binding functions of the eIF4 factors(19) . The
cleaved eIF4G molecule is unable to participate in cap-dependent
initiation but continues to carry out internal initiation of
picornaviral RNAs(2) . The 5`-UTRs of human (20) and
yeast (TIF4631 and TIF4632(21) ) eIF4G mRNA
are unusual in several respects. First, they are considerably longer
(368 for human, 528 for yeast) than the average eukaryotic mRNA
5`-UTR(22) . Second, they contain multiple short, open reading
frames (4 for human, 11 for TIF4631, 4 for TIF4632).
Third, human and yeast TIF4631 mRNAs contain a polypyrimidine
tract of 9-11 nt located 50-53 nt upstream from the
initiation codon. (There is a corresponding U-rich region in TIF4632 mRNA.) All of these features are reminiscent of
picornaviral IRES(5) . We present evidence here that the 5`-UTR
of eIF4G mRNA, like picornaviral IRES, acts as a strong translational
enhancer and can efficiently direct internal initiation in
vivo. The luciferase expression vector pGL2-Control (Promega)
was either used directly (here designated pGL2/LUC) or modified to
contain the 5`-UTR of eIF4G mRNA (pGL2/4G/LUC). cDNA corresponding to
nt 1-357 of human eIF4G mRNA was amplified by PCR from pHFC5 (20) with the primers 5`-CCCAAGCTTTCTAGATGGGGGTCC-3` and
5`-CCCAAGCTTTGATATCCTTTCCTCC-3`, which created a HindIII site
at each end. The product was inserted into the unique HindIII
site of pGL2/LUC. The second, third, and fourth upstream ATG codons
were changed to AAG by PCR mutagenesis of the above PCR product (23) to create pGL2/4G2-4/LUC, and all four upstream ATG
codons were changed to AAG to create pGL2/4G1-4/LUC. For
construction of vectors encoding an RNA hairpin, a 72-bp palindromic
sequence consisting of an inverted repeat of the multiple cloning site
of pBluescript II KS (Stratagene) from the XbaI to EcoRV sites was ligated into the unique StuI site of
pGL2/4G/LUC to produce pGL2/H/4G/LUC. A similar insertion was made in
pGL2/LUC to produce pGL2/H/LUC. Because the StuI site is
itself palindromic, the total length of the palindrome when inserted is
78 bp. For construction of vectors expressing a bicistronic mRNA, a
736-bp fragment containing the CAT coding region, flanked by 5`- and
3`-UTRs of 54 and 23 bp, respectively, was obtained from pCAT-Basic
Vector (Promega) by digestion with BanI and SalI and
blunt end-ligated into the StuI site of pGL2/LUC and
pGL2/4G/LUC to produce pGL2/CAT/LUC and pGL2/CAT/4G/LUC, respectively.
In all cases, constructions were verified by DNA sequencing or
restriction endonuclease analysis. K562, HeLa CCL2, HEL 92.1.7, and
Jurkat cells (ATCC, Rockville, MD) were grown in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Cells
were transfected with 30 µg of each luciferase-expressing plasmid
and 5 µg of pCMV A probe for ribonuclease
protection assays was prepared by digestion of pGL2/LUC with XbaI and EcoRI to produce a 540-bp fragment from the
luciferase coding region. This was inserted into pBluescript II SK
(Stratagene), which had been cut with the same enzymes. Antisense RNA
against luciferase mRNA was produced in vitro with T7
polymerase, and ribonuclease protection assays were performed as
described(27) . Human eIF4G mRNA contains open reading
frames at nt 6-39, 67-88, 90-108, and 165-171,
all upstream of the 4188-nt open reading frame that encodes the
protein(20) . According to the ribosome scanning model, such a
5`-UTR would be expected to down-regulate translation in a manner
highly dependent on the presence of the upstream AUGs. To test this, we
placed a DNA segment encoding the first 357 nt of eIF4G mRNA 29 bp
upstream of the coding region for a luciferase reporter gene, driven by
the SV40 promoter (Fig. 1A). The control vector
(pGL2/LUC) and the eIF4G 5`-UTR-containing vector (pGL2/4G/LUC) were
used to transfect the human chronic myelogenous leukemia cell line
K562. Surprisingly, the 5`-UTR enhanced, rather than repressed,
luciferase expression 7.3-fold (Fig. 1B).
Figure 1:
Expression of luciferase in K562 cells
from vectors encoding wild type and mutated forms of the eIF4G 5`-UTR. A, construction of vectors. A DNA segment encoding the 5`-UTR
of human eIF4G mRNA was inserted into pGL2/LUC as described in the
text. Similar constructions were made in which the second through
fourth ATG triplets of the 5`-UTR, or all four ATG triplets, were
mutated to AAG. Lengths of lines and rectangles are
proportional to DNA lengths except for the inserts, which are expanded
2.8-fold. B, luciferase expression from the four plasmids.
Each of the constructions depicted in A was used to transfect
K562 cells as described in the text. Luciferase expression was measured
after 18 h. Inset, ribonuclease protection analysis of
luciferase mRNA levels in each of the cell lines. A portion of the
autoradiogram is shown. The relative intensities of the bands obtained
by scanning the autoradiogram are: pGL2/LUC, 79380; pGL2/4G/LUC, 76949;
pGL2/4G2-4/LUC, 82980; and pGL2/4G1-4/LUC,
88119.
To
determine whether the upstream open reading frames played a role in
translation driven by the eIF4G 5`-UTR, we mutated the vector so as to
produce mRNAs lacking either the second through fourth AUGs
(pGL2/4G2-4/LUC) or all four upstream AUGs (pGL2/4G1-4/LUC; Fig. 1A). The expression of luciferase, however, was
unaltered (Fig. 1B), indicating that the upstream AUGs
do not play a significant role in the translational enhancement. The
presence of the eIF4G 5`-UTR could conceivably alter steady-state
levels of the luciferase mRNA. Hence, we performed ribonuclease
protection analysis of luciferase mRNA in cells transfected with each
of the four constructs (Fig. 1B, inset). The
finding that mRNA levels were similar in all cell lines, and not
proportional to luciferase expression, indicates that the 5`-UTR acts
at the level of translation rather than transcription, splicing, or
mRNA stability. To determine whether the translational enhancement
was unique to K562 cells, we tested other human cell lines of diverse
origins. HeLa cells, derived from a cervical carcinoma, HEL 92.1.7
cells, an erythroleukemia line derived from malignant peripheral blood,
and Jurkat cells, derived from an acute T cell leukemia, were
transfected with pGL2/LUC and pGL2/4G/LUC (Fig. 2). Even though
the level of luciferase expression was considerably lower in all three
cell lines compared with K562 cells (note scale change), the presence
of the 5`-UTR of eIF4G mRNA stimulated rather than inhibited expression
in each case (2.1-fold in HeLa, 3.4-fold in HEL, and 3.7-fold in
Jurkat).
Figure 2:
Effect of the eIF4G 5`-UTR on luciferase
expression in various cell lines. HeLa CCL2, HEL 92.1.7, and Jurkat
cells were grown and transfected with pGL2/LUC and pGL2/4G/LUC as
described in the text. Luciferase and
The possibility that expression of luciferase from these
constructs is due to leaky scanning or termination-reinitiation is
unlikely for several reasons. Some of the upstream AUG triplets in the
5`-UTR of eIF4G mRNA have good sequence contexts for
initiation(20) ; the second AUG in the 5`-UTR is out of frame
with the luciferase coding region; and there was no significant change
in expression when these AUGs were altered (Fig. 1B).
We tested directly for an internal entry mechanism by two different
approaches. First, we placed a palindromic sequence 45 bp upstream of
the luciferase coding region (pGL2/H/LUC; see Fig. 3A).
The free energy of formation of the transcribed 78-nt RNA hairpin was
calculated to be -78.9 kcal/mol using the computer program OLIGO
(National Biosciences), considerably more stable than the -50
kcal/mol previously shown to be sufficient for preventing movement of
the scanning small ribosomal subunit(28) . The hairpin reduced
luciferase expression to 2.5% of the control vector containing no
palindrome (pGL2/H/LUC versus pGL2/LUC; Fig. 3B). However, when sequences encoding the 5`-UTR
of eIF4G mRNA were placed between the palindrome and the luciferase
cistron, expression was stimulated 119-fold (pGL2/H/4G/LUC versus pGL2/H/LUC). The presence of the palindrome in the eIF4G
5`-UTR-containing vectors caused only a slight drop (26%) in luciferase
expression (pGL2/4G/LUC versus pGL2/H/4G/LUC).
Figure 3:
Effect of an RNA hairpin on in vivo expression of luciferase. A, construction of vectors. A
DNA palindrome encoding an RNA hairpin (H) with free energy of
formation of -78.9 kcal/mol was inserted upstream of the
luciferase cistron in two different constructs, using the procedures
described in the text. B, K562 cells were transfected with the
plasmids shown in A, and luciferase expression was measured as
described in the text.
Bicistronic
mRNAs have been effectively used in vivo to demonstrate the
existence of IRES in both picornaviral (3, 29, 30) and non-picornaviral (9, 10) RNAs. As most ribosomes fail to continue
through the intercistronic region, the relative translational
efficiency of the downstream cistron is greatly reduced unless preceded
by an IRES. As a second approach to test for internal initiation, we
inserted DNA encoding the eIF4G 5`-UTR into the 68-bp spacer separating
CAT and luciferase cistrons (Fig. 4A). Expression of
the downstream cistron was stimulated 42-fold compared with the
bicistronic construct containing the spacer alone (Fig. 4C). CAT was synthesized in cells containing both
constructs (Fig. 4B), although the presence of the
eIF4G 5`-UTR depressed its expression. This has also been observed for in vivo expression driven by both BiP and poliovirus IRES (9) and is thought to represent competition between
cap-dependent and IRES-dependent translation of bicistronic mRNA.
Figure 4:
Expression of CAT and luciferase from
bicistronic mRNAs in K562 cells. A, construction of vectors.
Vectors encoding bicistronic mRNAs containing CAT and luciferase
cistrons were constructed as described in the text. In one case, a DNA
segment encoding the 5`-UTR of eIF4G mRNA was inserted between them. B, expression of CAT activity. K562 cells were transfected
with the plasmids shown in A, and CAT activity was measured as
described in the text. The autoradiogram obtained from a thin-layer
chromatogram is shown, with positions of the origin (O),
chloramphenicol (C), acetylchloramphenicol (AC; two
isomers), and diacetylchloramphenicol (DAC) indicated. C, expression of luciferase activity in the same transfected
cells.
The foregoing results suggest that eIF4G mRNA is translated in
vivo by a cap-independent mechanism involving internal initiation.
The presence of multiple upstream open reading frames would normally
diminish mRNA translation severely(31) , but the 5`-UTR of
eIF4G mRNA actually stimulates translation ( Fig. 1and Fig. 2). An upstream RNA hairpin drastically inhibits
translation, presumably by stopping the scanning small ribosomal
subunit(28) , but insertion of the 5`-UTR of eIF4G rescues
translation (Fig. 3). Finally, the dramatic increase in
translation of the downstream cistron of a bicistronic mRNA (Fig. 4) is difficult to explain without invoking internal
initiation. Yet it should be noted that all of the experiments
described here involve stimulation of a reporter gene and not eIF4G
itself. Although there seems to be a clear rationale for the use of
internal initiation by viruses, the advantage of this mechanism of
initiation for the cellular mRNAs encoding BiP(9) , Antennapedia(10) , and basic fibroblast growth factor (11) is more difficult to understand. In the case of eIF4G,
which is essential for cap-dependent translation but not for internal
initiation, at least in its intact form, there are some intriguing
possibilities why its mRNA would utilize internal initiation. First,
this may represent a normal homeostatic mechanism for the maintenance
of intracellular levels of eIF4G. If eIF4G levels decreased, the
capacity of the cell to carry out cap-dependent initiation would also
decrease. The requirement of eIF4G for internal initiation may be less
than that for cap-dependent initiation, although this is yet to be
demonstrated. If so, the ribosomes and other components of the
translational machinery would then become more available for increased
translation of mRNAs that can utilize a cap-independent route. This
would in turn increase eIF4G levels, completing an autoregulatory loop. Second, there are physiological events that suddenly switch the
cell's predominant mode of initiation from cap-dependent to
cap-independent(32) . The best understood of these is infection
by entero-, rhino-, and aphthoviruses, in which cap-dependent
translation decreases drastically while cap-independent translation
persists. If eIF4G synthesis continues during infection, it may provide
a host defense mechanism whereby viral takeover would be slowed, or
recovery from infection accelerated, by replenishment of intact eIF4G
in order to restore cap-dependent initiation. An alternative view is
that the virus would actually take advantage of the continued synthesis
of eIF4G during infection. Since internal initiation from the IRES of
entero- and rhinoviruses is stimulated by eIF4G
cleavage(13, 14, 15) , presumably through the
generation of cleavage products, continued synthesis of eIF4G may
provide more cleavage products to aid in viral translation.
Distinguishing between these possibilities will require additional
experimentation. Another physiological situation in which the
predominant mode of translation can switch from cap-dependent to
cap-independent is heat shock (reviewed in (33) ). In a variety
of cell types and species, heat shock causes the synthesis of non-HSP
proteins to decrease rapidly, but the synthesis of HSPs remains the
same or increases. The change in translational specificity does not
require transcription of HSP genes or the appearance of new HSPs in the
cytosol. The defect produced by heat shock is preserved in cell lysates
and can be corrected in both Ehrlich (34) and Drosophila(35) cell lysates by a complex of eIF4E and eIF4G. Cells
with very low levels of both factors do not recover from heat
shock(36) . Notably, BiP, which is produced by internal
initiation (9) and continues to be translated in
poliovirus-infected cells(37) , shares many properties with
HSPs. It has partial sequence homology with HSP70 and becomes elevated
during a variety of stress-inducing conditions. The finding that the
5`-UTR of eIF4G mRNA directs internal initiation suggests that eIF4G
may also continue to be synthesized after heat shock and may accelerate
recovery.
)(3, 4) . Internal initiation has
been demonstrated by both in vivo and in vitro experimentation for picornaviruses, including various members of
entero-, rhino-, cardio-, and aphthovirus groups (5) , certain
other viruses(6, 7, 8) , and a few non-viral,
cellular mRNAs(9, 10, 11) . For
Picornaviridae, the rationale for a cap-independent route is evident:
entero-, rhino-, and aphthoviruses encode a protease that cleaves
translation initiation factor eIF4G. (
)This action severely
inhibits cap-dependent translation (12, 13) but
permits IRES-driven internal initiation to continue, actually
stimulating it in the case of entero- and
rhinoviruses(13, 14, 15) .
(Clontech Laboratories, Inc.), the latter
being a control plasmid that encodes
-galactosidase.
Electroporation was performed in triplicate using a Gene Pulser
(Bio-Rad) set at
ohms, 960 microfarads, and 0.22 kV. The number
of cells electroporated was 8
10
for K562, 4
10
for HeLa, and 6
10
for HEL
and Jurkat. Cells were cultured for an additional 18 h and collected,
and aliquots of 1
10
cells were assayed for
luciferase activity (24) using a Monolight 2010 luminometer
(Analytical Luminescence Laboratory) and
-galactosidase
activity(25) . Variations in transfection efficiency were
corrected by normalizing luciferase activity in each sample with
-galactosidase activity. CAT activity was measured in aliquots of
4.5
10
cells using D-threo-[1,2-
C]chloramphenicol
(ICN) as described previously(26) .
-galactosidase were measured
in triplicate, the luciferase activity being normalized separately for
each cell type.