Increased competition for eIF4 factors or 40S ribosomal subunits disfavors translation preinitiation complex assembly on poorly translated mRNAs with high G/A nucleotide ratios in 5’ leaders [Rec-Seq]

We tested the RCM using the yeast reconstituted initiation system by measuring PIC assembly on all native mRNAs in parallel (Rec-Seq) at different 40S subunit concentrations. We found broad agreement with the RCM, as mRNAs showing the largest decreases in PIC assembly on reducing [40S] tend to be the least efficient at the lowest [40S]/[mRNA] ratio tested and to display low translational efficiencies (TEs) in wild-type yeast.

Conclusions in line with the RCM also emerged from Rec-Seq conducted in the absence of mRNA-recruitment factors eIF4E–eIF4G or eIF4B, and by ribosome profiling analysis of a tif3Δ mutant lacking eIF4B, wherein mRNAs least efficient in PIC assembly or translation in the absence of factor tend to show the greatest impairment when factor is withdrawn and to exhibit low TEs in yeast. Remarkably, occurrence of G nucleotides in 5′UTRs is associated with low PIC assembly and TE in vivo and with heightened sensitivity to limiting 40S and initiation factors.