Fig 2: uORFs largely determine the eIF4G2-dependence in 293T cells. The reporter mRNAs were transfected to control cells and those depleted of eIF4G2 along with reference β-globin reporter mRNA coding for Nluc (n ≥ 5). The data are presented as ratios of normalized reporter expression in cells with eIF4G2 knocked down to control cells. (A) The uAUG codons of the APAF1, Maf1, Stard7, AKT2, SMAD1 and UCP2 reporters were mutated to the UAG stop codons to eliminate the uORFs (see panel C) and the effect of eIF4G2 knockdown by siRNA #1 was assayed in 293T cells. (B) Upstream AUG codons were introduced into the 5′ UTRs of TFGβ1 and HSPA2 so that the created uORFs were of the same size and approximately of the same position relative to the main AUG as the Stard7 uORF (see panel C). Then the effect of eIF4G2 knockdown by siRNA #1 was assayed in 293T cells. Statistical significance was determined by Mann–Whitney U test (P < 0.001 in both cases). (C) Schematic in-scale representation of the reporters assayed in panels A, B, D and E. The 5′ UTRs and the start of Fluc CDS are depicted. Arrows indicate uORFs, crosses display the positions of the uAUGs that were substituted for the stop codons in the corresponding reporters. Panels D and E are similar to panels A and B, respectively, except siRNA #2 was used for the eIF4G2 depletion in 293T cells. (F) mRNAs bearing uORF(s), which regulate translation under certain conditions, were assayed for eIF4G2-dependence in 293T cells. Only AKT2, SMAD1 and UCP2 reporter translation was significantly affected by the eIF4G2 knockdown.

Fig 3: eIF4G2 promotes eIF4E-dependent translation. (A) Control or eIF4G2 knocked down 293T cells were treated with either vehicle (DMSO) or 1 μM PP242 to activate eIF4E-BPs. Then the cells were transfected with the indicated mRNA reporters along with β-globin reference mRNA coding for Fluc or Nluc, as an appropriate internal reference (n ≥ 5). The data are presented as ratios of normalized reporter expression in cells treated with 1 μM PP242 to that in vehicle-treated cells. The dotted line at 1, therefore, corresponds to a behaviour identical to that of the reference β-globin reporter. The average ∼2.8-fold drop in the reference β-globin mRNA translation is shown by the dashed line, which therefore delineates a totally PP242-unresponsive translation. (B) Data from panel A were replotted to show if the mTOR inactivation alters the response to eIF4G2 knockdown (siRNA #1). The statistical significance is determined by the Mann–Whitney U test. The exact P-values for HIV1, TGFβ1 and HSPA2 are 0.009, 0.0016 and 0.0012, respectively. (C) Western blotting analysis of control and eIF4G2 knocked down 293T cells treated with either 1 μM PP242 or vehicle (DMSO). The PP242 treatment resulted in the disappearance of Thr37/Thr46 phosphorylation of eIF4E-BP1, which is a result of the mTOR inhibition.

Fig 4: Validation of the eIF4G2 translational targets. (A) Results of in vitro transcribed m7G-capped and polyadenylated reporters' transfection into 293T cells treated with either control or anti-eIF4G2 siRNAs. Unlike the other figures, this one incorporates data from all relevant experiments. All reporters were co-transfected with the reference β-globin reporter mRNA coding for Fluc or Nluc, as appropriate. The data are presented as ratios of normalized reporter expression in cells with eIF4G2 knocked down to control cells. For all eIF4G2 targets, β-globin and CDK1 reporters the number of replicates exceeds 20. For the non-targets n ≥ 5. (B) Western blot analysis of the eIF4G2 knockdown in 293T cells, GAPDH as a loading control. (C) Results of mRNA transfections in Huh7 cells (similar to panel A). (D) Western blot analysis of the eIF4G2 knockdown in Huh7 cells, GAPDH as a loading control.

Fig 5: eIF4G2 promotes ribosomal scanning inside and/or downstream of the uORFs in 293T cells. The reporter mRNAs were transfected to control cells and those depleted of eIF4G2 along with reference β-globin reporter mRNA coding for Nluc (n ≥ 5). The data are presented as ratios of normalized reporter expression in cells with eIF4G2 knocked down to control cells. (A) The uORFs of APAF1, Maf1, Stard7 and UCP2 reporters were fused to the Fluc sequence (see panel C) and the involvement of eIF4G2 in uORF translation was assayed in 293T cells. siRNA #1 was used for the eIF4G2 depletion. (B) The stop codons of Maf1, Stard7 and UCP2 uORFs were mutated so that the extended uORFs became overlapped with the Fluc CDS (see panel C) to eliminate a possibility of translation reinitiation. The eIF4G2-dependence of these reporters was assayed in 293T cells treated either with control or anti-eIF4G2 siRNA #1. (C) Schematic in-scale representation of the reporters assayed in panels A, B, D and E. The 5′ UTRs and the start of Fluc CDS are depicted. Arrows indicate the wild type and extended uORFs in the 5′ UTRs of assayed reporters. Panels D and E are similar to panels A and B, respectively, except siRNA #2 was used for the eIF4G2 depletion in 293T cells.