Fig 2: DAP5 and eIF3d are both required for translation of DAP5-dependent mRNAs. a Overexpression of 4E-BP1 by stable cDNA transformation in MDA-MB-231 cells silenced with siRNAs: non-silencing (NSi), DAP5, eIF4GI, eIF3d, DAP5 + eIF4GI, and DAP5 + eIF3d. b Cap chromatography of cell lysates with or without 4E-BP1 cDNA overexpression. Representative immunoblots show almost total sequestration of eIF4E by overexpressed 4E-BP1. c Representative immunoblots of MDA-MB-231 cells silenced with NSi or DAP5 siRNAs as in (a) probed for proteins whose mRNAs were found to be reduced in polyribosomes by DAP5 silencing (Table 1). Lamin A, AUF1, and GAPDH served as DAP5-independent controls. Results are representative of three independent experiments

Fig 3: DAP5 interacts with eIF3d to drive selective translation independently of eIF4E and eIF4GI. a Distribution of mRNAs encoding c-JUN, ACTB, MMP1, and CDK12 in 48S ribosome-mRNA complexes in 293T cell in vitro mRNA programmed translation extracts (see Supplementary Fig. 5 flow chart for methodology). In vitro translation extracts lacking endogenous mRNAs were programmed with in vitro-synthesized capped and polyadenylated mRNAs. 48S and 60S ribosome subunit distribution profiles from in vitro translation extracts were plotted by relative absorbance at 254 nm against elution fractions. Non-programmed extracts served as controls. b Quantitative RT–PCR analysis of the in vitro translation fractions shown in (a) expressed as relative mRNA abundance as a fraction of total recovered mRNA. c Immunoblot analysis of initiation factors in isolated 48S ribosome-mRNA translation complexes fractionated by sucrose gradients formed with mRNAs encoding c-JUN, ACTB, MMP1, or CDK12. Total protein is a control from non-programmed 293T cell in vitro translation extracts. Ribosomal protein S19 (rpS19) is a loading control (n = 3)

Fig 4: Detailed analysis of initiation codons identified by TISCA. (A–C) Read aggregation plots for GTI-seq as well as for Sel-TCP-seq of eIF3D-V5 for EIF2AK1 (A), LMNB2 (B), and SAFB (C) mRNAs. Red and blue circles show GTI-seq peaks and 40S decreasing points, respectively; black inverted triangles indicate TIS positions identified by TISCA; red and black vertical lines denote novel TIS positions identified by TISCA and canonical TIS positions, respectively; and white inverted triangles with blue vertical lines indicate TIS positions identified only by GTI-seq. (D) Motif analysis for surrounding sequences of AUG, CUG, GUG, UUG and ACG initiation codons identified by TISCA.

Fig 5: eIF2 dependency of translation initiation at near-cognate codons. (A) Immunoblot analysis of eIF2A and eIF2D in HEK293T cells deficient in eIF2A (eIF2A KO), eIF2D (eIF2D KO), or both eIF2A and eIF2D (DKO) as well as in WT cells. Two independent clones of each type were examined, and HSP90 was analyzed as a loading control. (B) Fold change in RNA abundance for eIF2α mRNA in HEK293T cells transfected with siRNAs specific for eIF2α (eIF2α KD #1 or #2) relative to HEK293T cells transfected with a control siRNA. padj, adjusted P value. (C) Immunoblot analysis of eIF2α in HEK293T cells transfected with siRNAs as in (B). Lanes 1 and 2 correspond to biological replicates. (D) Immunoblot analysis of ATF4 as well as of phosphorylated (p-) and total forms of eIF2α in WT HEK293T cells treated with 40 μM arsenite for the indicated times. (E) Numbers of ORFs showing a significant change (adjusted P value of <0.05) in translation efficiency (TE) in eIF2A KO, eIF2D KO, and DKO cells, in eIF2α KD cells, as well as in arsenite-treated WT HEK293T cells compared with control cells. For eIF2A KO, eIF2D KO, DKO and eIF2α KD cells, ORFs not commonly up-regulated or down-regulated in two clones or with two different siRNAs were removed. (F) GSEA plots for ORFs initiated at near-cognate codons whose translation efficiency was down-regulated by arsenite treatment. Translation of these ORFs is compared between eIF2α KD cells and control HEK293T cells. NES, normalized enrichment score. (G, J) Read aggregation plots for GTI-seq and Sel-TCP-seq of eIF3D-V5 for NPM1 (G) and HNRNPA2B1 (J) mRNAs. Red and blue circles show GTI-seq peaks and 40S decreasing points, respectively; black inverted triangles indicate TISs identified by TISCA; and red and black vertical lines denote novel TISs identified by TISCA and canonical TISs, respectively. (H, K) Read aggregation plots of Ribo-seq for NPM1 (H) and HNRNPA2B1 (K) mRNAs in HEK293T cells transfected with control or eIF2α siRNAs. The average of two replicates is shown. (I, L) Fold change in translation efficiency for mORF and the extended ORF of NPM1 (I) and HNRNPA2B1 (L) as analyzed by RiboDiff in eIF2A KO, eIF2D KO, DKO and eIF2α KD cells as well as in arsenite-treated WT cells compared with control cells.