Role of eIF3a in regulating cell cycle progression

Abstract

Translational control is an essential process in regulation of gene expression, which occurs at the initiation step performed by a number of translation initiation factor complexes. eIF3a (eIF3 p170) is the largest subunit of the eIF3 complex. eIF3a has been suggested to play roles in regulating translation of a subset of mRNAs and in regulating cell cycle progression and cell proliferation. In this study, we examined the expression profile of eIF3a in cell cycle and its role in cell cycle progression. We found that eIF3a expression oscillated with cell cycle and peaked in S phase. Reducing eIF3a expression also reduced cell proliferation rate by elongating cell cycle but did not change the cell cycle distribution. However, eIF3a appears to play an important role in cellular responses to external cell cycle modulators likely by affecting synthesis of target proteins of these modulators.

Fig. 1 –

Effect of serum starvation on expression of eIF3a, eIF3b and eIF3d. NIH3T3 cells were cultured in the presence or absence of serum for 48 h followed by collection of cells for analysis of cell cycle distribution or western blot analysis of the expression of eIF3a, eIF3b and eIF3d with GAPDH as a loading control.

Fig. 2 –

Synchronization study of NIH 3T3 cells. NIH 3T3 cells were synchronized by serum starvation for 48 h and then released by adding fresh complete medium. Cells were harvested at different time point for analysis of cell cycle distribution (A), ³H-thymidine incorporation (B), western blot analysis of eIF3a with actin as a loading control (C), and Real-Time RT-PCR analysis of eIF3a mRNA level (D).

Fig. 3 –

The expression level of eIF3a in isolated cells at G0/G1, S, and G2/M phases. NIH 3T3 (A) and H1299 (B) cells were stained with Hoechst 33342 and sorted to fractionate cells at G0/G1, S and G2/M phases. The collected cells were then analyzed again using propidium iodide staining to confirm their cell cycle distribution followed by western blot analysis of eIF3a expression with actin as a loading control.

Fig. 4 –

The effect of mimosine on eIF3a expression and cell cycle distribution. H1299 cells were treated with mimosine for 8 or 48 h followed by analysis of cell cycle distribution and western blot analysis of eIF3a expression with GAPDH as a loading control.

Fig. 5 –

The effect of reducing eIF3a expression on cell cycle distribution. Two stable H1299 cell clones transfected with antisense cDNA of eIF3a (HAS4 and HAS5) together with vector-transfected clones (HVec) were used to test the expression level of eIF3a using western blot analysis with actin as a loading control (A), growth rate (B), and cell cycle distribution (C) using methods as described in Materials and methods.

Fig. 6 –

Effect of reducing eIF3a expression on cellular response to serum starvation. Stable H1299 clones transfected with vector (HVec) or antisense eIF3a (HAS4 and HAS5) [9] were cultured in normal condition with serum or in the absence of serum for 24 h followed by analysis of cell cycle distribution.

Fig. 7 –

Effect of reducing eIF3a expression on cellular response to hydroxyurea and nocodazole. Stable H1299 clones transfected with vector (HVec) or antisense eIF3a (HAS4 and HAS5) [9] were treated with various concentrations of hydroxyurea (HU) or nocodazole (Noco) for 48 h. Cells were then collected for analysis of cell cycle distribution.