Control of cell survival and proliferation by mammalian eukaryotic initiation factor 4B

Abstract

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5' untranslated region (5'UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5'UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.

FIG. 1.

Translation initiation is inhibited in eIF4B-silenced cells. (A) Lysates from eIF4B siRNA-silenced HeLa cells were subjected to Western blotting (WB) for eIF4B and β-actin. (B) Control and eIF4B-silenced cells were labeled with [³⁵S]methionine/cysteine. (C) Polysomes from eIF4B-silenced and control siRNA-treated cells were analyzed on 10 to 50% sucrose density gradients. All procedures are described in Materials and Methods.

FIG. 2.

Translation of mRNAs with structured 5′UTRs is preferentially inhibited in eIF4B-silenced cells. (A to D) Lysates from eIF4B siRNA-silenced HeLa cells were subjected to Western blotting for Cdc25C (A), c-myc (B), ODC (C), and XIAP and Bcl-2 (D) along with eIF4B, GAPDH, and β-actin. (E) Polysomes from eIF4B siRNA-silenced and control siRNA-transfected HeLa cells were fractionated on sucrose density gradients. The effects of eIF4B silencing on the distribution of the indicated mRNAs in gradient fractions are shown.

FIG. 3.

Effects of eIF4B silencing on cell proliferation and survival. (A) Cell counting using Z1 Coulter Counter (Beckman Coulter). (B) Colorimetric analysis using the MTT assay. Values from control cells on day 5 were set at 100%. (C) Cells from parallel cultures were harvested on the third and sixth day posttransfection, equalized for protein content, and subjected to Western blotting for eIF4B and β-actin. (D) HeLa cells were transfected with control or eIF4B-targeting siRNAs and grown for 3 days before cell cycle analysis by flow cytometry was carried out. (E) Control and eIF4B-silenced cells were doubly stained with FITC-annexin V and propidium iodide (PI), and analyzed by flow cytometry 4 days after siRNA transfection. The number in each quadrant represents the percentage of cells in that quadrant. (F) Control and eIF4B-silenced cells were treated with either nocodazole alone or with nocodazole and zVAD and then incubated for an additional 24 h, when the cells were subjected to the MTT assay. Values from nocodazole-treated control cells were set at 100%.

FIG. 4.

eIF4B silencing sensitizes cells to camptothecin-induced apoptosis. (A) The cells were transfected with control or eIF4B-targeting siRNAs. Three days posttransfection, cells were lysed and subjected to Western blotting for cleaved PARP, procaspase 9, eIF4B, and β-actin. (B) eIF4B-silenced and control cells were transfected with either an HA-Bcl-2-expressing vector or empty vector. Cell cycle analysis was carried out using flow cytometry. The percentage of sub-G₁ population is shown in each profile. (C) Control or eIF4B-silenced cells were treated with increasing concentrations of the topoisomerase inhibitor, camptothecin. MTT assay was performed 48 h posttreatment. Values of untreated control and eIF4B-silenced cells were adjusted to 100%.

FIG. 5.

eIF4B silencing results in translational inhibition in a caspase-independent manner. (A) Schematic diagram depicting the apoptotic pathway leading to caspase 3 activation and cleavage of substrates associated with translation initiation. (B) Polysomal profiles of zVAD-treated control and eIF4B-silenced cells. (C) Protein lysates from control and zVAD-treated cells were subjected to Western blotting for cleaved PARP, procaspase 9, eIF4B, and β-actin.