From the A. N. Belozersky Institute of
Physico-Chemical Biology, Moscow State University, 119899 Moscow,
Russia and § Department of Microbiology and Immunology,
Morse Institute for Molecular Genetics, State University of New York
Health Science Center at Brooklyn, Brooklyn, New York 11203-2098
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ABSTRACT |
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A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5'-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the ~450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES·eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.
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INTRODUCTION |
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Initiation of protein synthesis in eukaryotes involves the sequential binding of small (40 S) and large (60 S) ribosomal subunits to an mRNA, leading to the assembly of an 80 S initiation complex at the initiation codon (1). The rate-limiting step in this process is recruitment of mRNAs to the 43 S preinitiation complex, which consists of the 40 S ribosomal subunit, methionine-initiator tRNA, and initiation factors, including eIF21 and eIF3. Ribosomal recruitment to most mRNAs requires the m7GpppN cap structure at the 5' end of the mRNA (2), but ribosomal binding to a smaller number of mRNAs is cap- and end-independent, and is instead mediated by an IRES in the 5'-untranslated region (3). One group of IRES elements is exemplified by encephalomyocarditis virus (EMCV) RNA (4). EMCV is a member of the cardiovirus genus of the Picornaviridae family.
Eukaryotic initiation factor eIF4F, which consists of eIF4A, eIF4E, and eIF4G subunits, plays the central role in recruiting mRNAs to 43 S complexes during initiation. The cap structure is recognized by the 24-kDa cap-binding protein eIF4E. eIF4A is an RNA-dependent ATPase/RNA helicase that is thought to unwind cap-proximal regions of the 5'- untranslated region of an mRNA, permitting attachment of the 43 S complex (1, 2). The 154-kDa eIF4G subunit of eIF4F binds to these and other factors, thereby coordinating and enhancing their activities. eIF4E associates with amino acid residues 411-428 of eIF4G, eIF3 binds to the central part of eIF4G (residues 486-886), and eIF4A binds to sites in the central and in the C-terminal thirds of eIF4G (5-7). eIF4G enhances binding both of eIF4E to the cap and of eIF4A to RNA (8, 9). The modular nature of eIF4G supports a model in which it acts as a bridge between the mRNA cap (via eIF4E) and the 40 S subunit (via eIF3, a constituent of the 43 S preinitiation complex) (6). In addition to containing binding sites for these factors, eIF4G contains sequences in its center (7, 10, 11) that are characteristic of RNA binding domains (RBDs) (for review, see Burd and Dreyfuss (12)). A role for this putative RBD in cap-mediated initiation of translation has not been elucidated, but it could contribute to the cap-binding, RNA-binding, and RNA helicase activities of eIF4F (8, 9, 13).
Recently, substantial evidence has implicated eIF4F in cap-independent, IRES-mediated translation initiation of some viral mRNAs (9, 14-17). One function of eIF4F in this process is to enable eIF4A to enter the mRNA-43 S ribosomal preinitiation complex (16). A second function, first identified using EMCV mRNA, is to directly recognize and bind to the IRES (9, 17). This interaction requires the central third of eIF4G, including the putative RBD, and is independent of eIF4A and eIF4E (17). The requirement for eIF4F in EMCV translation can be met by eIF4A and this central RBD-containing domain of eIF4G (9). We have suggested that this specific RNA binding activity of eIF4G may substitute for the cap-binding role of eIF4E in recruiting mRNAs to 40 S subunits (9, 17). In this model, the IRES has an analogous function in the translation process to the 5'-terminal cap; these RNA structures both bind to eIF4F, thereby recruiting 40 S subunits to a specific site on an mRNA. This RNA binding activity of eIF4G could therefore regulate gene expression by facilitating selective translation of cellular IRES-containing mRNAs under conditions when eIF4E is inactive.
Cellular and viral IRESs are large, complex RNAs, and can be assigned to different groups on the basis of sequence and structural similarities. Conserved structural elements may correspond to binding sites for initiation factors that mediate internal ribosomal entry (3, 4). We report here that we have used a combination of chemical and enzymatic protection ("footprinting") assays to map the site in the EMCV IRES that is recognized and bound by eIF4F. This structural element is conserved in the IRESs of several other viruses, including all members of the cardiovirus, aphthovirus, and hepatovirus genera of the Picornaviridae family. The effects of mutations in this structural element on translational activity of these IRESs are consistent with its interaction with eIF4G being a critical step in initiation.
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MATERIALS AND METHODS |
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Plasmids-- Plasmids have been described (9, 17, 20, 21). pTE17 contains EMCV nt 378-1155 downstream of a T7 promoter. pTE8 and pTE10 are identical to pTE17 except for deletions of EMCV nt 485-647 and nt 701-763, respectively. pET28(His6-eIF4G457-1396) (9) was renamed pET28(His6-eIF4G457-1404), reflecting corrections to the sequence of eIF4G (7).
In Vitro Transcription-- Plasmids pTE8, pTE10, and pTE17 were linearized by digestion with PstI. Transcription with T7 RNA polymerase and subsequent purification of RNA were done as described previously (17).
Purification of Factors-- Native eIF4F and recombinant eIF4A, eIF4B, and eIF4G457-1404 and recombinant eIF4A were purified as described elsewhere (9, 17). The trans-dominant eIF4A R362Q mutant (22) was a kind gift of N. Sonenberg (McGill University, Montreal).
Assembly of RNA-Protein Complexes-- RNP complexes were formed by incubating initiation factors and EMCV IRES transcripts for 5 min at 30 °C in buffer (100 mM potassium acetate, 2 mM magnesium acetate, 2 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol). Reactions contained 1 µg of EMCV RNA (4 pmol) and 1 µg of eIF4G457-1404 (10 pmol), 3 µg of eIF4F (13 pmol), 1 µg of eIF4A (22 pmol), 1.5 µg (21 pmol) of eIF4B, and ATP (1 mM), as indicated in the text, in a volume of 20 µl.
Chemical and Enzymatic Footprinting-- RNP complexes were probed with RNase V1, DMS, and CMCT as described previously (23). Cleaved or modified sites were identified by primer extension, done using avian myeloblastosis virus reverse transcriptase and the primers 5'-CTCAAAAGTGAGAGAGTGCGC-3' (complementary to nt 884-864), 5'-CGCTTGAGGAGAGCCAT-3' (complementary to nt 669-653), and 5'-GGGGTTCCGCTGCC-3' (complementary to nt 539-526), as appropriate.
Toeprint Analysis of RNP Complexes-- Toeprint analysis of EMCV mRNA·eIF4G457-1404 complexes was done as described previously (9, 17) using the primer 5'-GTCAATAACTCCTCTGG-3' (complementary to EMCV nt 957-974).
UV Cross-linking-- UV cross-linking of RNP complexes consisting of the EMCV IRES and recombinant eIFs 4A, 4B, and 4G457-1404 was done as described previously (9), except that were indicated, DMS was included in reactions at the same concentration as used in probing experiments (23).
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RESULTS |
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eIF4G Binds to the Oligo(A) Loop between the J and K Domains of the EMCV IRES-- The structure of the EMCV IRES is shown schematically in Fig. 1. As we have previously shown, eIF4F bound to this IRES arrests primer extension at C786 (9, 17). Although we could not exclude that the target site for eIF4F is a complex structure formed by more than one of the principal domains of the IRES, the simplest and most likely possibility was that eIF4G binds to the J-K domain. The results of chemical and enzymatic footprinting presented here are wholly consistent with this hypothesis.
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Specificity of Interaction of eIF4G with the Oligo(A) Loop Is
Determined by Its Structural Context--
Specificity of interaction
of eIF4G with the EMCV IRES can be inferred from our recent reports (9,
17), in which we found that binding of eIF4G457-1404 to
the EMCV 701-763 deletion mutant and to an AAAAAU775
UA deletion-insertion mutant did not arrest primer extension at
C786. These results are consistent with a loss of affinity
of eIF4G for the mutant IRESs. The minimal activity of these two
mutants in translation initiation strongly supports the functional
importance of the interaction of eIF4G with the IRES. To investigate
this issue further, footprinting was done on the
701-763 mutant in the presence and absence of eIF4G457-1404. The oligo(A)
loop between J and K domains that is bound by eIF4G in the wild type
IRES is intact in this mutant (Fig.
3A). The A residues in this
loop are totally accessible to DMS attack and are therefore still
exposed and unpaired (Fig. 3B). Nevertheless, they were not
protected at all by eIF4G457-1404 from DMS modification
(Fig. 3B).
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Structural Elements Upstream of the J-K Domain Do Not Affect
Binding of eIF4G to the EMCV IRES--
As noted above, eIF4G made no
specific contact with any part of the IRES other than the J-K domain.
Specifically, no protection from chemical or enzymatic modification was
detected in the H, I, or L domains (data not shown). The H domain and
adjacent upstream residues are bound by PTB, an auxiliary factor in
EMCV IRES function (23, 24). However, we could not rule out the
possibility that these domains indirectly affect the interaction of
eIF4G with the J-K domain. Significantly, no functional role has been
ascribed to the large central domain I in various picornaviruses,
although mutational analysis has shown that it is important for IRES
activity (19, 21, 25-29). We used toeprinting to investigate whether deletion of the upper conserved part of domain I in the EMCV
485-647 IRES mutant affected binding of eIF4G457-1404.
This deletion did not change either the position or intensity of the
toeprint at C786 with respect to the full-length cDNA
(Fig. 4). This result indicates that the
upper conserved part of domain I does not contribute to the affinity of
the eIF4G-EMCV IRES interaction.
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The Presence of eIFs 4A and 4B Does Not Change the Pattern of Protection of the EMCV IRES by eIF4G457-1404-- EMCV IRES-mediated translation initiation requires ATP, eIF4A, and eIF4G, and is augmented by eIF4B (9, 16, 17). In UV cross-linking experiments done using radiolabeled EMCV IRES transcripts, eIF4G457-1404 strongly enhanced radiolabeling of eIF4A and eIF4B, but eIF4G457-1404 itself did not become strongly labeled (Fig. 5, lanes 1-6). These results are wholly consistent with our previous data (9) and suggest that these three factors form a complex on the EMCV IRES. UV cross-linking of different combinations of these factors to this IRES was not altered in the presence of DMS (Fig. 5, lanes 1-6 and 7-12). We therefore used DMS and RNase V1 in footprinting experiments to determine whether eIFs 4A, 4B, and 4G457-1404 bound to a specific site on the IRES or altered its conformation in any way.
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DISCUSSION |
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We recently found that eIF4G directly recognizes the EMCV IRES and that this interaction is important in recruiting the IRES to ribosomes (9, 17). The results of the footprinting experiments reported here show that the eIF4G binding site consists of an oligo(A) loop and three adjacent helices at the junction of the J and K domain of this IRES (Fig. 7). The bases of the oligo(A) loop are bound directly by eIF4G.
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A similar structural element comprising an oligo(A) loop at the
junction of three helices occurs at an identical position in the IRESes
of many other picornaviruses. They include all cardioviruses, all
aphthoviruses, echovirus 22, equine rhinoviruses 1 and 2, and hepatitis
A virus (3, 4, 18, 19, 35, 36). The conservation of A residues in the
loop of this motif is especially remarkable. Indeed, a single
nucleotide substitution (A772C) in the loop strongly impairs EMCV IRES
activity (37). The majority of phenotypic reversions of this mutation
occurred by restoration of a purine (preferably A) residue in the
mutated position (38). In addition to the loop, mutations in adjacent
helices (such as deletion of nt 727-730 in the J2 helix) (37) also
significantly impair IRES activity. A more extensive deletion
(701-760) in this domain totally inactived the IRES (21). The
oligo(A) loop in this mutant is still in a single-stranded conformation
and is completely accessible to DMS modification (21) but its
structural context is quite different from wild type (Fig.
3A). We found that this mutant IRES is unable to bind eIF4G,
a result that emphasizes the importance of the structural context
surrounding the oligo(A) loop for recognition by eIF4G. The
conformation of the eIF4G binding site may be affected by other
RNA-binding factors. We have previously suggested that the active
conformation of the EMCV IRES which enables it to bind to essential
factors such as eIF4G is stabilized by PTB (23). PTB binds to sites at
the 5' and 3' borders of the EMCV IRES, including the apical K2 hairpin
of the J-K domain. Recent studies that have shown that the dependence
of the EMCV IRES on PTB for activity was significantly increased
following insertion of a single A residue into the oligo(A) loop
(AAAAA770-774) (39) are consistent with this proposal.
The organization of the eIF4G binding site on the EMCV IRES is
consistent with the general paradigm for the interaction of RBDs with
RNA. Their binding normally involves sequence nonspecific electrostatic
interactions with the sugar-phosphate backbone of base-paired or
stacked RNA, followed by base-specific interaction with apical or
internal RNA loops (32, 33). Bound RNA remains exposed on the -sheet
RNA-binding surface of the RBD and is potentially accessible for
interaction with other RNA-binding proteins (12, 33). Our results
suggest that eIFs 4A and 4B may bind to the binary (eIF4G·IRES)
complex in this way. UV cross-linking of eIF4A and eIF4B to the EMCV
IRES is enhanced most significantly by eIF4G when all three factors are
present together (9) (Fig. 6). UV cross-linking of eIF4B to the related
foot-and-mouth disease virus IRES also requires cytosolic co-factor(s),
which we suggest are eIFs 4A and 4G, and involves only the J-K domain
(40). Moreover, eIF4A and the EMCV IRES both bind to the same central
domain of eIF4G (7, 9). However, the observation that eIFs 4A and 4B
did not result in additional protection of binary (eIF4G·IRES) complexes from chemical or enzymatic modification suggests that their
interaction with the IRES is transient. During initiation, it may be
stabilized by other components of the translation apparatus such as
constituents of the 43 S complex.
In this study we found that addition of eIF4A, eIF4B, or both to the binary (eIF4G·IRES) complex did not alter the susceptibility of any part of the IRES to chemical/enzymatic modification, including the hairpin that constitutes domain L between the eIF4G binding site and the initiation codon. This has important implications for the role of eIF4A in recruitment and attachment of a 43 S complex to the EMCV IRES, which therefore does not involve unwinding of mRNA in a classical helicase reaction, but may instead involve rearrangement and accommodation of the IRES in the mRNA-binding cleft of the 40 S subunit. This process of accommodation may require the concerted action of all components needed to form the 48 S preinitiation complex. This model for the activity of the eIF4A subunit of eIF4F may also apply to attachment of 43 S complexes to mRNA in the cap-dependent mode of translation initiation.
Taken together, our results show that eIF4G is an essential factor in EMCV IRES-mediated initiation (17) and suggest that it plays a dual role in this process. One role is selection of EMCV mRNA by specific interaction with the IRES, in a manner analogous to the selection of capped mRNAs by the cap-binding protein eIF4E (9, 17). This results in recruitment of mRNAs to ribosomes by virtue of this interaction and the interaction of eIF4G with eIF3 (6). A second role for eIF4G in IRES-mediated initiation is to recruit eIFs 4A and 4B to the IRES, possibly as a prelude to accommodation of the initiation codon and flanking regions in the mRNA-binding cleft of the 43 S complex.
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ACKNOWLEDGEMENTS |
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We thank Dmitri Kolkevich, Yakov Alexeev, Alexey Kuzubov, and Rodney Romain for technical assistance. We gratefully acknowledge Nahum Sonenberg for providing the trans-dominant eIF4A-mutant. We thank A. Kaminski and R. Jackson for communicating their results prior to publication.
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FOOTNOTES |
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* This work was supported by grants from the National Science Foundation (to C. U. T. H.), from the Russian Foundation for Basic Investigations (to I. N. S.), and from the Howard Hughes Medical Institute (to C. U. T. H. and I. N. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: c/oDr. C. U. T. Hellen. Tel.: 718-270-1034; Fax: 718-270-2656; E-mail: shatsky{at}ins.genebee.msu.su.
1 The abbreviations used are: eIF, eukaryotic initiation factor; EMCV, encephalomyocarditis virus; PTB, pyrimidine tract binding protein; IRES, internal ribosomal entry site; RBD, RNA binding domain; DMS, dimethylsulfate; CMCT, 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodiimide metho-p-toluenesulfonate; nt, nucleotide.
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REFERENCES |
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