Requirement of RNA binding of mammalian eukaryotic translation initiation factor 4GI (eIF4GI) for efficient interaction of eIF4E with the mRNA cap

Abstract

Eukaryotic mRNAs possess a 5'-terminal cap structure (cap), m(7)GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.

FIG. 1.

Recombinant human eIF4GI proteins. (A) Diagram of eIF4GI constructs. PABP-, eIF4E-, eIF4A-, and eIF3-binding sites and the RNA-binding region are indicated. (B) Purified murine eIF4E and human eIF4GI fragments (2 μg) were analyzed by SDS-12% PAGE followed by Coomassie blue staining. The amino acid numbers of eIF4GI fragments are indicated above the lanes. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.

FIG. 2.

Functional analysis of recombinant eIF4GI fragments. Rabbit reticulocyte lysates treated with rhinovirus 2A^pro (+) or control buffer (−) were supplemented with recombinant eIF4GI fragments (200 nM) as indicated. Capped poly(A)⁺ luciferase mRNA (1 μg/ml) was added to the extract, and in vitro translation and luciferase assays were performed as described in Materials and Methods. The amino acid numbers of eIF4GI fragments are indicated below the graph. RLU, relative light units.

FIG. 3.

The interaction of recombinant eIF4GI fragments with β-globin RNA as assayed by an EMSA. [³²P]UTP-labeled β-globin RNA was incubated with recombinant eIF4GI fragments. In lane 1, no protein was added. eIF4GI fragments were added at the indicated concentrations. The positions of free RNA and the RNA-eIF4GI complex are shown to the right of the gel.

FIG. 4.

Recombinant eIF4GI fragments containing the RNA-binding region enhance the association between eIF4E and the cap structure. (A) Dose-dependent interaction of eIF4E with the cap structure. Cap-labeled luciferase poly(A)⁺ RNA was incubated with the amounts (nanomolar concentrations) of recombinant eIF4E indicated in the figure. eIF4E was cross-linked to the RNA as described in Materials and Methods. m⁷GDP was added (+) or not added (−) as indicated. (B) Quantitative analysis of the eIF4E bands in panel A. The intensities of the bands were measured using NIH ImageJ. The value obtained for the band in lane 1 was set at 1 (bar 1). (C) Recombinant full-length eIF4GI fragment enhances the interaction between the cap structure and eIF4E. Cap-labeled RNA was incubated with recombinant eIF4E and eIF4GI(84-1599). Increasing amounts of eIF4E were incubated with eIF4GI (20 nM) at the molar ratios indicated in the figure. (D) Quantitative analysis of the enhancement of eIF4E cross-linking to the cap in panel C. The band intensities in lanes 1, 5, and 9 were set at 1 (gray bars). (E) Recombinant eIF4GI fragments containing the RNA-binding region enhance the interaction between the cap structure and eIF4E. Cap-labeled RNA was incubated with recombinant eIF4E and eIF4GI fragments at a molar ratio of 1:1. The eIF4GI fragments are indicated above the figure. (F) Quantitative analysis of eIF4E bands in panel E. The band intensity in lane 1 was set at 1. The data shown in panels A, C, and E are representative of three experiments. Quantitative data with means plus standard deviations (error bars) are shown in panels B, D, and F.

FIG. 5.

Analysis of recombinant La RRM1-fused eIF4GI fragments. (A) Schematic representations of La RRM1-fused eIF4GI fragments. The positions of the eIF4E-binding site on eIF4GI and the RRM1 of the La autoantigen are indicated above the fragments. (B) Recombinant La RRM1-fused eIF4GI fragments and the full-length La(1-408) were analyzed by SDS-12% PAGE and Coomassie blue staining. Lane 1, eIF4GI(197-674)La(1-202); lane 2, eIF4GI(602-674)La(1-202); lane 3, La(1-408). The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.

FIG. 6.

La RRM1 confers on the N-terminal region of eIF4GI the ability to stimulate the interaction between the cap structure and eIF4E. (A) Cap-labeled RNA and eIF4E were incubated with recombinant eIF4GI fragments as indicated in the figure at a molar ratio of eIF4E to eIF4GI of 1:1. Proteins were cross-linked and analyzed as described in Materials and Methods. m⁷GDP was added (+) or not added (−) as indicated. (B) Quantitative analysis of eIF4E bands in panel A. The band intensity in lane 1 was set at 1. (C) Dose-dependent interaction between the cap structure and eIF4E by the addition of increasing concentrations of eIF4GI and La RRM1-fused N-terminal eIF4GI fragments. Cap-labeled RNA and eIF4E were incubated with recombinant eIF4GI fragment and La RRM1-fused N-terminal eIF4GI fragments, respectively, at the molar ratios of eIF4E to eIF4GI indicated in the figure. (D) Quantitative analysis of eIF4E bands in panel C. The band intensity of lane 1 in panel C was set at 1. The data shown in panels A and C are representative of three experiments. Quantitative data with means and standard deviations (error bars) for these experiments are shown in panels B and D. (E) RNA-binding ability and the interaction between the cap structure and eIF4E of La RRM1-fused N-terminal eIF4GI fragments. The RNA-binding ability of the three fragments and the ability of the three fragments to enhance the cap-eIF4E interaction are shown (+++, strong; ++, moderate, −, none). (F) Model of the stimulatory effect of La RRM1 on the interaction between the cap structure and eIF4E.

FIG. 7.

Model of the mechanism by which eIF4GI stimulates the interaction between the cap structure and eIF4E. Binding of eIF4GI to the mRNA body brings eIF4E to the vicinity of the cap structure and consequently stabilizes the interaction between the cap and eIF4E. The thickness of the double-headed arrow represents the strength of the interaction. eIF4GI further enhances this interaction by interacting with PABP.