Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation - PubMed (original) (raw)

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

I B Lomakin et al. Mol Cell Biol. 2000 Aug.

Abstract

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.

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Figures

FIG. 1

Schematic representation of eIF4GI. PABP, eIF4E, eIF4A, eIF3, EMCV IRES, and Mnk1 binding regions are shown.

FIG. 2

Specificity of interaction between eIF4GI mutants and the EMCV IRES. A toeprint analysis of binary-complex formation on the EMCV IRES with eIF4GI deletion mutants (A) and eIF4GI insertion-substitution mutants (B) was performed as described in Materials and Methods. The full-length cDNA extension product is marked E, the position of the stop site due to binding of eIF4G is indicated at C786, and a stop site detected on EMCV RNA irrespective of the presence or absence of eIF4GI that was used as an internal standard for quantitation is marked N. Reference lanes T, C, G, and A depict the EMCV cDNA sequence.

FIG. 3

Interaction of eIF4GI(697–1076) mut1 with β-globin RNA as assayed by an electrophoretic mobility shift assay. The positions of free RNA and of the RNA-eIF4GI complex are indicated.

FIG. 4

Primer extension analysis of 48S initiation complexes assembled on EMCV RNA using translation mix (eIF1, eIF1A, eIF2, eIF3, eIF4A, eIF4B, initiator tRNA, and 40S subunits) (lanes 3 to 7 and 9 to 21) with eIF4F (lanes 2 and 10) or eIF4GI mutants (lanes 3 to 7 and 11 to 21) as indicated. The full-length cDNA extension product is marked E, the position of the stop site due to binding of eIF4GI is indicated at C786, and cDNA products labelled AUG826 and AUG834 terminated at stop sites 15 to 17 nt downstream of the stated initiation codon. Reference lanes T, C, G, and A depict the EMCV cDNA sequence.

FIG. 5

Influence of eIF4A on the interaction of eIF4GI mutants with the EMCV IRES. Toeprint analysis of ribonucleoprotein complex formation on the EMCV IRES with eIF4GI deletion mutants (A) and eIF4GI insertion-substitution mutants (B) in the presence and absence of eIF4A, as indicated, was performed. The position of the stop site due to binding of eIF4GI is indicated at C786; for greater clarity, only this part of each gel is shown. These bands were quantitated by PhosphorImager analysis and normalized as described in Materials and Methods. Values are shown schematically relative to the intensity of the C786 band in the absence of factors, which was arbitrarily assigned a value of 1; gray and black bars represent values obtained in the absence and presence of eIF4A, respectively.

FIG. 6

Interaction between eIF4A and eIF4G determined by the electrophoretic mobility shift assay, showing the specific supershift of the β-globin mRNA–eIF4GI complex in the presence of eIF4A. The positions of free RNA, the RNA-eIF4GI complex, and the RNA-eIF4GI-eIF4A complex are indicated.

FIG. 7

Interaction of insertion and substitution mutant eIF4GI(697–1076) polypeptides with immobilized eIF4A in a direct binding assay, as described in Materials and Methods. eIF4A was visualized by Coomassie blue staining, and eIF4GI polypeptides were detected by Western blotting with anti-T7 tag antibodies.

FIG. 8

Interaction of eIF3 with immobilized eIF4GI deletion mutant polypeptides in a direct-binding assay, as described in Materials and Methods. The eIF3 p170 subunit is indicated on the left and was visualized by Western blotting with a specific monoclonal antibody.

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Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation - PubMed (original) (raw)