Interactions between eIF4AI and its accessory factors eIF4B and eIF4H

Abstract

Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9-10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30-33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.

FIGURE 1.

Nucleotide dependence of eIF4AI containing-complexes. (A) SDS-PAGE of proteins bound and unbound to streptavidin beads from reaction mixes containing biotinylated 32-nt RNA (1 μM), 4AI (1 μM), indicated nucleotides (1.5 mM), and BSA (indicated by *; 0.1 mg/mL). (MW) Molecular weight markers with sizes indicated on left. (B) EMSA of reactions containing ³²P-labeled 32-nt RNA (30 nM) plus 4AI (250 or 750 nM), full-length 4B (250 or 750 nM), and/or nucleotides (1 mM) as indicated. (C) Same as B, except using 4AI and/or 4H. (D, top) Schematic diagram of full-length 4B domain structure, ΔRRM-4B (amino acids 178–611) and C-term-4B (amino acids 332–611). (Bottom) EMSA as in B and C of reactions containing 4AI, full-length 4B, ΔRRM-4B, and/or C-term-4B as indicated. All reactions contained AMPPNP. (*) 4AI:C-term-4B species. (E) SDS-PAGE of proteins bound and unbound to Ni-coated beads in the presence of 32-nt RNA (1 μM) and/or AMPPNP (1.5 mM) as indicated. (Lanes 1–3) His₆-4AI plus untagged ΔRRM-4B; (lanes 4–6) His₆-4H plus untagged 4AI. All proteins were 1 μM. (MW) Molecular weight markers with sizes indicated on left.

FIGURE 2.

Mapping the eIF4B and eIF4H binding sites on eIF4AI. (A) Helicase assays containing ³²P-labeled duplex RNA±indicated DEAD-box proteins (4AI or 4AIII) and accessory factors (ΔRRM-4B, 4H, or MLN51). After 0, 4, 10, and 15 min incubation at 35°C, samples were quenched with SDS and electrophoresed in a nondenaturing polyacrylamide gel to separate duplex and single-stranded RNAs (indicated at right). (Lane 1) duplex alone incubated at 35°C for 15 min; (lane 2) duplex incubated at 95°C prior to electrophoresis to completely separate the strands. (Δ) Duplex degradation product. (B, top) schematic diagram of 4A3133 chimera. Numbers indicate quadrant boundaries. (Bottom) Same as Figure 1A, except pull-down reactions contained 4AI, 4AIII, the 4A3133 chimera, and/or ΔRRM-4B as indicated. All reactions contained BSA (*) and AMPPNP (1.5 mM). (C) Same as Figure 1, B and C, except that all reactions contained AMPPNP (1.5 mM) and the indicated proteins at 1 μM. (D) Same as C, except reactions contained 4AI and ΔRRM-4B and/or 4H at indicated concentrations and RNA was in excess (200 μM).

FIGURE 3.

Micrococcal nuclease (MNase) footprinting of eIF4AI and eIF4AIII complexes. Denaturing PAGE of RNAs recovered after MNase digestion of reactions containing poly(U)₄₀, AMPPNP and indicated proteins (lanes 3–9). (Lanes 1,2) poly(U)₁₈ and poly(U)₄₀ hydrolysis ladders. (Right panels) Darker exposure of lanes 3 and 5 with densitometry traces.

FIGURE 4.

Preferred RNA binding site sizes of eIF4AI complexes. (A) Representative EMSA of reactions containing ³²P-labeled poly(U) hydrolysis ladder, AMPPNP (1.5 mM), and indicated proteins (1 μM). (Boxes) Gel slices excised for subsequent denaturing analysis. (B) Representative denaturing PAGE of RNAs recovered from slices such as those indicated in A, alongside the hydrolysis ladder used in that experiment (lanes 1,5). (*) Nonshifted bands comigrating with 4AI complexes. (C) Plots of relative band intensities versus RNA fragment length for hydrolysis ladders (gray circles) and indicated complexes in panel B (black circles). Minimum preferred RNA length is indicated by arrow.

FIGURE 5.

The stoichiometry of eIF4AI:eIF4B complexes. (A) EMSA as in Figure 1B,C, except all reactions contained AMPPNP (1.5 mM), 4AI, ΔRRM-4B, and/or 4B at indicated concentrations, and RNA was in excess (200 μM). (B) Same as Figure 1E, except all reactions contained AMPPNP (1.5 mM), the indicated proteins (1 μM), and RNA was in excess (10 μM).