eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide

Elevated upstream ribosome occupancies often associate with reduced translation in CDS. (A) RPFs and mRNA reads for IDH2 showing increased 5′UTR ribosome occupancy coupled with decreased CDS RPFs in L96P versus WT cells. (B) Scatter plot comparing log₂TE_L96P versus log₂TE_WT for CDS of 4242 genes with ≥10 RPF reads and ≥32 mRNA reads (average of four samples, two replicates of WT and two replicates of sui1-L96P). Genes with significant greater than or equal to twofold changes in CDS TE in L96P versus WT (FDR < 0.05) are highlighted by red and dark blue dots, respectively. Among them, genes with significantly elevated RRO ratios in the mutant vs WT (ΔRRO_L96P ≥ 2, FDR < 0.05) are denoted by orange circles. (C) Cumulative frequency distributions of CDS log₂ΔTE_L96P values for all 4242 genes (black line), or for genes with RRO values exceeding 1/32 in either WT (blue line) or L96P cells (red line). P-values are shown indicating the significance of differences between the distributions assigned using the Kolmogorov–Smirnov test. (D) Box-plot analysis of CDS ΔTE_L96P values for the groups of mRNAs described in Figure 1D–F showing significantly increased TEs for 5′UTRs (5′UTR_up), NCC_uORFs (uNCC_up), or AUG_uORFs (uAUG_up). The numbers of mRNAs here are smaller than those in Figure 1E,F because of multiple uORFs residing in the same mRNAs. (E) Box-plot analysis of CDS ΔTE_L96P values for all 4242 mRNAs analyzed in panels B–D, for 592 mRNAs lacking 5′UTR length annotations (NoA), and for eight bins of the 4242 mRNAs divided according to 5′UTR lengths, with the numbers of mRNAs in each bin indicated (n). (F,G) Same box-plot analysis as in E but excluding mRNAs with appreciable RRO values exceeding ∼0.03 in WT (E) or L96P cells (F).