Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity

Abstract

Elevated eukaryotic initiation factor 4E (eIF4E) levels frequently occur in a variety of human cancers. Overexpression of eIF4E promotes cellular transformation by selectively increasing the translation of proliferative and prosurvival mRNAs. These mRNAs possess highly structured 5'-UTRs that impede ribosome recruitment and scanning, yet the mechanism for how eIF4E abundance elevates their translation is not easily explained by its cap-binding activity. Here, we show that eIF4E possesses an unexpected second function in translation initiation by strongly stimulating eukaryotic initiation factor 4A (eIF4A) helicase activity. Importantly, we demonstrate that this activity promotes mRNA restructuring in a manner that is independent of its cap-binding function. To explain these findings, we show that the eIF4E-binding site in eukaryotic initiation factor 4G (eIF4G) functions as an autoinhibitory domain to modulate its ability to stimulate eIF4A helicase activity. Binding of eIF4E counteracts this autoinhibition, enabling eIF4G to stimulate eIF4A helicase activity. Finally, we have successfully separated the two functions of eIF4E to show that its helicase promoting activity increases the rate of translation by a mechanism that is distinct from its cap-binding function. Based on our results, we propose that maintaining a connection between eIF4E and eIF4G throughout scanning provides a plausible mechanism to explain how eIF4E abundance selectively stimulates the translation of highly structured proliferation and tumor-promoting mRNAs.

Keywords: ATPase; DEAD-box; protein synthesis.

Fig. 1.

eIF4E stimulates eIF4A helicase activity. (A) Unwinding assay: a Cy3-labeled reporter RNA is annealed to an uncapped RNA loading strand with a 20-nt overhang. A BHQ-labeled RNA is annealed to the loading strand to quench the reporter fluorescence. ATP-dependent helicase activity dissociates the reporter, resulting in increased Cy3 fluorescence. A DNA capture strand prevents reannealing. Each assay contains 50 nM RNA substrate, 2 mM ATP-Mg, and 1 µM each protein component unless otherwise stated (Materials and Methods). (B) Representative unwinding time course of helicase reactions containing eIF4A, eIF4B, and eIF4G_682–1105 (black) or eIF4G in the absence (blue) or presence of eIF4E (magenta). A cartoon depicts human eIF4G domains. (C) Initial rate of duplex unwinding (fraction per minute) for eIF4A, eIF4B, and varied concentrations of eIF4G in the absence or presence of eIF4E. Data are fit to the Hill equation as described in SI Materials and Methods. The K_d,app and maximum initial rates of unwinding (i.e., “A”) are means of three independent experiments ± SEM.

Fig. 2.

eIF4E stimulation of eIF4F helicase activity is eIF4A-dependent and cap-independent. (A) Initial rates of duplex unwinding for helicase reactions containing 1 µM eIF4A, eIF4B, eIF4E, and eIF4G in the absence or presence of 0.3% DMSO (final concentration) ± 3 µM hippuristanol. (B) Initial rates of duplex unwinding for helicase reactions containing 1 µM eIF4A, eIF4B, eIF4E, and 0.5 µM eIF4G in the absence or presence of m⁷GTP (1 µM or 20 µM). Data are presented as means of three independent experiments ± SEM.

Fig. 3.

The eIF4E-binding domain in eIF4G regulates eIF4A activity. Initial rates of duplex unwinding (fraction per minute) for each reaction containing 1 µM eIF4A and eIF4B are plotted vs. increasing concentrations of eIF4G_557–1600 (A) or eIF4G_557–1137 (B) in the absence or presence of 1 µM eIF4E. Data are fit to the Hill equation (SI Materials and Methods). The K_d,app and maximum initial rates of unwinding (i.e., “A”) are shown as means of three independent experiments ± SEM. Cartoons are shown to depict human eIF4G domains in the constructs used. (C) Initial rates of duplex unwinding for different truncations of eIF4G ± eIF4E. Each reaction contains 1 µM eIF4G, eIF4A, eIF4B, and eIF4E.

Fig. 4.

eIF4E stimulates translation independent of cap-binding. (A) Schematic of the luciferase translation assay: the boxB RNA element recruits λ-eIF4G_557–1600 to the reporter RNA construct. The mRNA contains two additional stem loops between the boxB hairpin and the luciferase reporter gene as described in Materials and Methods. An inhibitory hairpin is located upstream of the boxB element to prevent any 5′ end-dependent ribosome loading. The binding of λ-eIF4G_557–1600 enables eIF4F and eIF4B to unwind any secondary structure so that the 43S preinitiation complex (gray) can be recruited to the mRNA independent of cap-binding. (B) Bar graph of relative luciferase translation rates measured for 30 min at 30 °C for reactions containing 250 nM RNA and 1 µM λ-eIF4G_557–1600 with or without 2 µM eIF4E, 10 µM 4E-BP1, and 20 µM m⁷GTP. Each reaction is normalized to basal nonspecific levels of luciferase translation in the absence of λ-eIF4G_557–1600.

Fig. 5.

Proposed mechanism by which eIF4E enhances translation of structured mRNAs. In the absence of eIF4E, the eIF4E-binding domain maintains a conformation of eIF4G that possesses low eIF4A helicase stimulating activity. Upon eIF4E binding, a conformation of eIF4G is induced that possesses a high eIF4A helicase stimulating activity.