The 5'-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway

Abstract

Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5' cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.

Figure 1. Interaction affinities among eIF4 factors

Fluorescence anisotropy of eIF4EFl (•), eIF5-Fl (◇), eIF4A-TAMRA (“eIF4A-Rh”;■) or Fl-ssRNA (◇ ) was measured upon addition of increasing concentrations of (A) eIF4G, (B) eIF4E•eIF4G complex, or (C) eIF4B. K_d values are shown in (D). The K_d for the interaction of eIF4E with eIF4A•eIF4G was not measured, but is deduced from the fact that it must be less than the K_d of eIF4E binding eIF4G in the absence of eIF4A. Error bars are average deviations. For all data with error bars n ≥ 2. See Fig. S1 for SDS-PAGE analysis of proteins used.

Figure 2. Interaction affinities between eIF3 and components of the multifactor and pre-initiation complexes

The affinity of eIF3 for initiation factors and ssRNA was determined by monitoring the fluorescence anisotropy of (A) Fl-ssRNA (K_d = 35 ± 10 nM), (B) eIF1-TAMRA (■, K_d = 115 ± 55 nM), and eIF5-TAMRA (•, K_d = 15 ± 2 nM), and (C) eIF4B-Fl (K_d = 380 nM) as a function of eIF3 concentration. (D): Measured binding affinities. Error bars are average deviations, n ≥ 2.

Figure 3. Factor dependence of stable mRNA recruitment to the 43S pre-initiation complex as determined using the native gel shift assay

The mRNAs are shown in (A). (B) Recruitment of cap-unstructured mRNA; (C) recruitment of cap-RPL41A (black) and cap-DAD4 (striped); (D) recruitment of cap-3 bp (striped) and cap-6 bp (black); and (E) recruitment of capped and uncapped, unstructured mRNA (gray), 3 bp (striped) and RPL41A (black). 30 min points are shown. Error bars are average deviations, n ≥ 2. See Fig. S2 for additional information.

Figure 4. Recruitment of uncapped mRNA to the PIC forms complexes unable to stably locate the start codon in the absence of the full complement of factors

(A) Primer-extension (toeprinting) with uncapped (left) and capped (right) RPL41A mRNA. The first four lanes are dideoxy sequencing lanes. The fifth lane contains 40S ribosomal subunits, eIF1 and eIF1A but no TC. The remaining lanes contain 40S ribosomal subunits, eIF1, eIF1A and TC as well as additional factors as indicated. (B) Toeprinting with uncapped RPL41A mRNA and various fragments of eIF4G. All reactions contain the 43S complex, eIF3, eIF4A and eIF4B. (C) Recruitment of capped RPL41A mRNA to the PIC, monitored using a gel shift assay in the presence of eIF3, eIF4A, eIF4B and the following eIF4G fragments: WT eIF4G and eIF4E (•); 460-952 (○); 301-952 -459 (◇); and 2-300 (◇ ). (D) As in (C), but with uncapped RPL41A mRNA. The presence of eIF4E with the eIF4G fragments did not alter the results for the toeprint or gel shift assays (data not shown).

Figure 5. Kinetics of stable recruitment of mRNA to the 43S pre-initiation complex

The fraction of mRNA recruited to the 43S pre-initiation complex was monitored at the times indicated for: cap-RPL41A (A); cap-DAD4 (B); uncap-3 bp (C); uncap-RPL41A (D); and uncap-DAD4 (E). Structures of mRNAs are shown in Fig. 3A. (A) For cap-RPL41A k_obs were determined with eIF3, eIF4A, eIF4E•eIF4G and eIF4B (hereafter, “all recruitment factors;” black; k_obs = 0.25 ± 0.03 min^-1); and in the absence of the following factors: eIF4E•eIF4G (red; k_obs = 0.012 ± 0.008 min^-1); eIF4B (aqua; k_obs = 0.012 ± 0.01 min^-1); eIF4A (orange; k_obs = 0.009 ± 0.003 min^-1); and eIF3 (purple; no recruitment observed). (B) For cap-DAD4 observed rate constants were determined in the presence of all recruitment factors (black; k_obs = 0.09 ± 0.02 min^-1); and in the absence of the following factors: eIF4E•eIF4G (red; k_obs = 0.02 ± 0.01 min^-1); eIF4B (aqua; k_obs = 0.024 ± 0.03 min^-1); eIF4A (orange; k_obs = 0.03 ± 0.02 min^-1); and eIF3 (purple; no recruitment observed). (C) For uncap-3 bp, observed rate constants were determined in the presence (blue; k_obs = 0.83 ± 0.1 min^-1) and absence (green; k_obs = 0.28 ± 0.05 min^-1) of eIF3. Kinetics were followed for a total of 60 minutes. A shorter period is shown here for clarity. (D) For uncap-RPL41A observed rate constants were determined in the presence of all recruitment factors (black; k_obs = 0.22 ± 0.02 min^-1); and in the absence of the following factors: eIF4E•eIF4G (red; k_obs = 0.24 ± 0.04 min^-1); eIF4B (aqua; k_obs ≥ 0.13 ± 0.06 min^-1); eIF4A (orange; k_obs ≥ 0.3 ± 0.15 min^-1); and eIF3 (purple; no recruitment observed). Kinetics were followed for a total of 500 minutes. A shorter period is shown here for clarity. (E) For uncap-DAD4 observed rate constants were determined in the presence of all recruitment factors (black; k_obs = 1.0 ± 0.3 min^-1); and in the absence of the following factors: eIF4E•eIF4G (red; k_obs ≥ 1.2 ± 0.1 min^-1); eIF4B (aqua; k_obs ≥ 0.8 ± 0.05min^-1); eIF4A (orange; k_obs ≥ 0.9 ± 0.5 min^-1); and eIF3 (purple; k_obs = 0.2 ± 0.05 min-¹). Errors are average deviations, n ≥ 2.

Figure 6. eIF3 interactions with the PIC

(A): Reduction in mobility of the 43S complex in native gels caused by eIF3 binding, monitored using ³⁵S-Met-tRNA_i. (B): In the absence of mRNA, eIF3 enhances TC binding to 40S subunits (with eIF1 and eIF1A) by ~2.5-fold (■). In the presence of the unstructured, model mRNA (•) 43S complex formation is only slightly enhanced by the presence of eIF3 (<1.3 fold). (C): The sequences of mRNAs used in (D). (D): The effect of eIF3 on binding of capped versions of the two mRNAs shown in (C) to the PIC: 3′-AUG mRNA (■); 5′-AUG mRNA (•). Error bars are average deviations, n ≥ 2.

Figure 7. The 5’-5’-triphosphate moiety blocks aberrant mRNA recruitment

(A) Observed rate constants (k_obs) for recruitment to the PIC of mRNAs with various 5′-end structures in the presence of all recruitment factors (black bars) and absence of eIF4E•eIF4G (red bars), eIF4A (orange bars) or eIF4B (aqua bars). Measurements were performed as in Fig. 5. Errors are average deviations, n ≥ 2. (B) Cartoon depicting recruitment of uncapped (top) and capped (bottom) natural mRNAs. Interaction between the cap and the PIC blocks mRNA recruitment and the ability of eIF3 on its own to promote rapid and stable recruitment. Interaction between the cap and the eIF4F complex removes this inhibition and allows productive mRNA binding to the PIC. See Fig. S3 for additional information.