eIF3d and eIF4G2 mediate an alternative mechanism of cap-dependent but eIF4E-independent translation initiation

Abstract

Initiation of translation for the majority of eukaryotic mRNAs is mediated by a 5' cap structure to which the eukaryotic initiation factor 4E (eIF4E) binds. Inhibition of the activity of eIF4E by 4EBP-1 does not prevent the translation of a number of cellular capped mRNAs, indicative of the existence of previously unexplored mechanisms for the translation of these capped mRNAs without the requirement of eIF4E. eIF4G2, also known as death-associated protein 5 (DAP5), a homolog of eIFGI that lacks the eIF4E binding domain, utilizes eIF3d (a subunit of eIF3) to promote the translation of a subset of these mRNAs. Using fluorescence anisotropy-based equilibrium binding studies, we provide the first quantitative evidence of the recruitment of eIF3d as well as eIF3d and eIFG2 complexes to a subset of human mRNAs. Our quantitative studies demonstrate the critical role a fully methylated 5' mRNA cap structure plays in the recognition and recruitment of eIF3d, as well as the eIF3d and eIFG2 complex. By using luciferase reporter-based in vitro translation assays, we further show that cap-recognition ability correlates with the efficiency of translation of these mRNAs. Essentially, by preferably utilizing eIF3d and eIFG2, specific mRNA subsets are still able to translate in a cap-dependent manner even when eIF4E is sequestered. Our findings offer new insight into the use of eIF3d and eIF4G2 as an alternative for growth and survival under conditions of cellular stress. This novel mechanism of translation may offer new targets for therapeutic regulation of mRNA translation.

Keywords: 7-methylguanosine cap (m(7)G) cap; eIF3d; eIF4G2; fluorescence anisotropy; hypoxia.

Figure 1

Structure of eIF3d and eIF4E cap binding domains.A, the cap binding domain of eIF3d showing the tunnel for mRNA entry and RNA gate, which are unique to eIF3d but absent in the cap-binding pocket of eIF4E. α helices are colored in purple and β strands in green. Figure was prepared with data in PDB ID code 5K4B using UCSF Chimera software (https://www.cgl.ucsf.edu/chimerax/). B, cap binding pocket of eIF4E, shown in gold, in contact with the m⁷G cap which is represented with a ball and stick model. 4EBP-1 (amino acid residues 36–70) shown in red is bound to eIF4E. Figure was prepared with data in PDB ID code 1WKW using UCSF Chimera software. C, schematic showing the domain architecture of human eIF3d. Human eIF3d consists of an unstructured N-terminal region, as well as a cap binding domain, found between amino acid residues 161 and 527. Within the cap-binding domain is the RNA gate which spans amino acid residues 285 to 299. The amino acid residues that make up the RNA gate are shown above the domain box. 4EBP-1, 4E binding protein 1; eIF, eukaryotic initiation factor; m⁷G, 7-methylguanosine; PDB, Protein Data Bank.

Figure 2

Domain architecture of the human eIF4G protein family. eIF4G2 lacks the poly(A) binding protein (PABP) and eIF4E domains which can be found on eIF4GI and eIF4GII. The colored boxes show the domains of interaction for each eIF4G protein family member with the corresponding factor specified above the domain boxes. Each eIF4G protein family member has three HEAT domains, namely HEAT-1, HEAT-2, and HEAT-3, respectively. The MIF4G domain of eIF4G2 is crucial in mediating its interaction with eIF4A and eIF3. The cleavage sites on eIF4G2 are shown with arrows. G434 is the viral protease 2A cleavage site, whereas D792 is the caspase cleavage site. eIF4E, eukaryotic initiation factor 4E.

Figure 3

Equilibrium-binding titrations of fluorescein-labeled m⁷G capped 5′UTRs with eIF3d.A, cartoon showing the fluorescence anisotropy-based equilibrium binding assay. Equilibrium-binding titrations of fluorescein-labeled m⁷G capped 5′UTRs of mRNAs coding for (B), CD101, (C), ITGAE and (D), ACTB with either eIF3d alone, eIF3d in the presence of GDP, eIF3d in the presence of m⁷GDP, or a complex of eIF3d and eIF4G2 in the presence of m⁷GDP. The normalized anisotropy change for the interaction between the fluorescein-labeled, but uncapped, 5′ UTRs with eIF3d was included as a control. Briefly, 10 nM of fluorescein-labeled capped or uncapped mRNAs were titrated with increasing concentration of protein or protein/protein complex in the titration buffer at 25 °C. The anisotropy at each titration point was measured using excitation and emission wavelengths of 495 nm and 520 nm, respectively. Data points correspond to an average of three independent anisotropy measurements. The curves represent the nonlinear fits that were used to obtain the averages and standard deviations for the corresponding K_d values presented in Table 1. ACTB, β-actin; CD101, cluster of differentiation 101; eIF, eukaryotic initiation factor; ITGAE, integrin subunit alpha E; m⁷GDP, 7-methylguanosine diphosphate.

Figure 4

Equilibrium-binding titrations of fluorescein-labeled m⁷G and ApppG capped 5′UTRs with the eIF3d and eIF4G2 complex. Briefly, 10 nM of fluorescein-labeled m⁷G or ApppG capped 5′ UTRs of (A) CD101, (B) ITGAE, and (C) ACTB coding mRNAs were titrated with increasing concentration of protein/protein complex in the titration buffer at 25 °C. Saturating amounts of 5 μM eIF3d and 5 μM eIF4G2 protein/protein complex in the syringe were injected automatically into the cuvette containing the mRNA over a time course of 30 min. The anisotropy at each titration point was measured using excitation and emission wavelengths of 495 nm and 520 nm, respectively. Data points correspond to an average of three independent anisotropy measurements, and the curves represent the nonlinear fits that were used to obtain the averages and standard deviations for the corresponding K_d values presented in Table 1. ACTB, β-actin; eIF, eukaryotic initiation factor; CD101, cluster of differentiation 101; ITGAE, integrin subunit alpha E; m⁷G, 7-methylguanosine.

Figure 5

Comparison of fluorescein labeled m⁷G capped or uncapped 5′UTRs binding with eIF4E or a complex of eIF4E and 4EBP-1. Equilibrium-binding titrations of fluorescein-labeled m⁷G capped 5′UTRs of mRNAs coding for (A) CD101, (B) ITGAE, and (C) ACTB with either eIF4E and 4EBP-1 complex, or eIF4E alone are shown. The normalized anisotropy changes for the interaction between the fluorescein-labeled, but uncapped, 5′UTRs with the eIF4E and 4EBP-1 complex, or eIF4E alone were included as internal controls to allow comparisons of differing batches of RRL. Briefly, 10 nM of fluorescein-labeled capped or uncapped mRNAs were titrated with increasing concentration of protein or protein/protein complex in the titration buffer at 25 °C. The anisotropy at each titration point was measured using excitation and emission wavelengths of 495 nm and 520 nm, respectively. Data points correspond to an average of three independent anisotropy measurements. The curves represent the nonlinear fits that were used to obtain the averages and standard deviations for the corresponding K_d values presented in Table 1. 4EBP-1, 4E binding protein 1; ACTB, β-actin; CD101, cluster of differentiation 101; eIF4E, eukaryotic initiation factor 4E; ITGAE, integrin subunit alpha E; m⁷G, 7-methylguanosine; RRL, rabbit reticulocyte lysate.

Figure 6

Effect of eIF4G2 on the cap-binding activity of eIF4E. Equilibrium-binding titrations of fluorescein-labeled m⁷G and ApppG capped 5′UTRs of mRNAs coding for (A), CD101, (B), ITGAE, and (C) ACTB with either eIF4E and eIF4G2 protein/protein mixtures, or eIF4G2 alone. Briefly, 10 nM of fluorescein-labeled m⁷G or ApppG capped mRNAs were titrated with increasing concentration of protein or protein/protein mixtures in the titration buffer at 25 °C. The anisotropy at each titration point was measured using excitation and emission wavelengths of 495 nm and 520 nm, respectively. Data points correspond to an average of three independent anisotropy measurements, and the curves represent the nonlinear fits that were used to obtain the averages and standard deviations for the corresponding K_d values presented in Table 1. ACTB, β-actin; CD101, cluster of differentiation 101; eIF4E, eukaryotic initiation factor 4E; ITGAE, integrin subunit alpha E; m⁷G, 7-methylguanosine.

Figure 7

Translational efficiencies of m⁷GpppA and ApppG capped-UTR-Luc mRNAs.A, schematic showing the m⁷GpppA and ApppG reporter constructs. Translational outputs of m⁷GpppA and ApppG capped 5′ UTR-luc-mRNAs (B) CD101, (C) ITGAE, and (D), ACTB. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired student's t test; ∗∗∗, p < 0.001. ACTB, β-actin; CD101, cluster of differentiation 101; ITGAE, integrin subunit alpha E; m⁷G, 7-methylguanosine.

Figure 8

Effect of increasing concentrations of eIF3d and eIF4G2 on the translational yields of m⁷GpppA capped transcripts. Panels (A–C) represent the CD101, ITGAE, and ACTB 5′ UTR encoding mRNA luciferase constructs, respectively. Luciferase activity was normalized to a control eIF3d dependent mRNA, CD101. Luciferase activity measured in RRL for CD101 with the addition of the 125 nM eIF3d/eIF4G2 protein mixture was set at 100% and used as an internal positive control. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired Student's t test; ∗∗∗, p < 0.001. ACTB, β-actin; CD101, cluster of differentiation 101; eIF4E, eukaryotic initiation factor 4E; ITGAE, integrin subunit alpha E; m⁷G, 7-methylguanosine; RRL, rabbit reticulocyte lysate.

Figure 9

Effect of increasing concentrations of eIF4E on the translational outputs of m⁷GpppA cappedtranscripts . Panels (A–C), represent the 5′ UTR luc-mRNAs encoding CD101, ITGAE, and ACTB, respectively. Luciferase activity was normalized to a control eIF4E dependent mRNA, ACTB. Luciferase activity measured in RRL for ACTB with the addition of 125 nM eIF4E was set at 100% and used as an internal positive control. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired student's t test; ∗p < 0.033, ∗∗p = 0.002; ∗∗∗p < 0.001. 4E; ITGAE, integrin subunit alpha E; ACTB, β-actin; CD101, cluster of differentiation 101; eIF4E, eukaryotic initiation factor m⁷G, 7-methylguanosine; RRL, rabbit reticulocyte lysate.

Supplementary Figure 1

Revised supplementary Figure 2

Revised supplementary Figure 3