Figure 9. Disruption of Ded1 NTD interactions with eIF4A or eIF4E alter the kinetics of 48S assembly in the reconstituted system for a synthetic mRNA with Cap-proximal SL.

(A–B) Kinetics of 48S PIC assembly was analyzed in reactions containing reconstituted 43S PICs, a radiolabeled capped reporter mRNA containing a cap-proximal SL of predicted ΔG of −8.1kcal/mol located five nt from the 5’end (depicted schematically), and different concentrations of mutant or WT Ded1 protein. Formation of 48S PICs was detected using a native gel mobility shift assay and measured as a function of time, allowing determination of observed rates at each Ded1 concentration. The maximum rates (k_max) (A) and Ded1 concentration at the half-maximal rate (K_1/2) (B) were determined from three replicate sets of assays and the individual values (black points) and mean values (bar heights) are plotted for experiments containing no Ded1, WT full-length Ded1 (FL Ded1), Ded1 lacking N-terminal residues 2–116 (ded1ΔNTD), or FL Ded1 harboring the indicated NTD substitutions that impair binding to eIF4A (green bars) or eIF4E (orange bars). (C) Model to account for the greater requirements for the Ded1-NTD interactions with eIF4A and eIF4E in stimulating translation of mRNAs with strong secondary structures versus relatively unstructured 5’UTRs. Ded1 interactions with each of the subunits of the eIF4F complex lowers the concentration of Ded1 required for its recruitment to the capped 5’ ends of all mRNAs, where it can unwind structures that impede PIC attachment or scanning to the start codon. Structured mRNAs (right) require a more stable association between Ded1 and eIF4F for maximum Ded1 recruitment and, hence, are relatively more dependent on having both eIF4A and eIF4E contacts with the Ded1 NTD intact. Unwinding stable SL structures for the latter additionally requires the enhancement of Ded1 unwinding activity conferred by its interaction with eIF4A (red arrows on the right) to achieve maximum acceleration of PIC attachment or scanning. (D) Schematic summary of the relative importance of interactions of the Ded1 NTD with eIF4A and eIF4E and the Ded1 CTD with eIF4G, in stimulating Ded1 recruitment to mRNA and its RNA helicase activity. (i) In WT cells, Ded1’s multiple interactions with the subunits of eIF4F enhance recruitment of Ded1 in complex with eIF4F to the m⁷G cap to form a stable activated mRNP, which can subsequently recruit the 43S PIC and efficiently scan the 5’UTR to locate the AUG codon (not depicted). Eliminating the Ded1 CTD and its direct contact to eIF4G (ii, red Δ), confers a modest reduction in Ded1 function, whereas greater reductions are conferred by substitution mutations in the Ded1 NTD (red asterisks) that impair Ded1 interaction with eIF4E (iii) or eIF4A (iv). Even greater decreases in Ded1 function are seen on combining each of the Ded1 NTD substitutions with deletion of the Ded1 CTD (v–vi).

Figure 9—figure supplement 1. Effects of Ded1 NTD substitutions that impair eIF4A or eIF4E binding on 48S PIC assembly on RPL41A mRNA in the yeast reconstituted system.

(A–B) The analysis was conducted exactly as described in Figure 9A–B for the same panel of Ded1 proteins, except using RPL41A mRNA. (C) Summary of effects of Ded1 NTD substitutions on k_max (in units of min⁻¹) and K_1/2 (in nM) for CP-8.1 mRNA determined from the data in Figure 9A–B, and for RPL41A mRNA determined from the data in panels A-B above, as shown for three replicate sets of assays.