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. 2007 Aug;27(16):5619-29.
doi: 10.1128/MCB.00035-07. Epub 2007 Jun 11.

Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway

Céline Chauvin  1 , Samia Salhi, Olivier Jean-Jean
Affiliations

Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway

Céline Chauvin et al. Mol Cell Biol. 2007 Aug.

Abstract

Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity.

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Figures

FIG. 1.
FIG. 1.
Expression of eRF3a during the cell cycle. HCT116 cells were synchronized in G1/S with thymidine treatment for 24 h, released, and harvested at time zero (T0), 4 h (T4), 8 h (T8), and 14 h (T14) after release. HCT116 cells were synchronized in mitosis with nocodazole treatment for 14 h, shaken off, released, and harvested at time zero (N0) and 4 h (N4) after release. (A) Fluorescence-activated cell sorting (FACS) analysis. Event count (y axis) versus DNA content (x axis) values are shown. (B) Western blotting using anti-eRF3a, anti-eRF1, or anti-α-tubulin (α-tub) antibodies as indicated.
FIG. 2.
FIG. 2.
eRF3a depletion alters cell cycle progression and cell morphology. HCT116 cells were electroporated with either empty pSuper vector (Sup) as a control or plasmid expressing si-3a1 targeting eRF3a mRNA (3a1) and analyzed 3 days after electroporation. (A) FACS analysis. Event count (y axis) versus DNA content (x axis) values are shown. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are indicated below the graph. (B) The percentages of cells in G1, S, and G2/M were determined for nine independent electroporation experiments and are expressed as means and standard deviations. The significance of the 3a1 sample values with respect to the Sup basal value was determined by a two-tailed t test for G1, S, and G2/M cells. The P values were less than 0.0001 for the three cell types. (C) Western blot analysis using anti-eRF3a, anti-eRF1, or anti-α-tubulin (α-tub) antibodies as indicated on the left. (D) Phase-contrast microscopy images.
FIG. 3.
FIG. 3.
eRF3a-depleted cells are arrested in the G1 phase of the cell cycle. HCT116 cells electroporated with either pSuper vector (Sup, upper panels), or plasmid expressing si-3a1 (3a1, lower panels) were synchronized in mitosis by nocodazole treatment for 14 h. Mitotic cells were shaken off, released for the time indicated above each panel, and analyzed by flow cytometry. Event count (y axis) versus DNA content (x axis) values are shown.
FIG. 4.
FIG. 4.
eRF3a-depleted cells are arrested in late G1 phase. HCT116 cells were electroporated with either pSuper vector (Sup) or plasmid expressing si-3a1 (3a1) and analyzed 3 days after electroporation by Western blotting using anti-eRF3a, anti-cyclin D1, anti-cyclin E, anti-cyclin A, and anti-α-tubulin (α-tub) antibodies as indicated on the left.
FIG. 5.
FIG. 5.
eRF1 depletion and suppressor tRNA expression do not induce G1 arrest. HCT116 cells were electroporated with either pSuper vector (Sup); plasmids expressing si-3a1 targeting eRF3a mRNA (3a1); plasmids expressing si-1Y targeting eRF1 mRNA (1Y); or the plasmid ptRNAam, ptRNAoc, or ptRNAop, expressing amber (Am), ochre (Oc), or opal (Op) suppressor tRNA, respectively, and analyzed 3 days after electroporation. (A) FACS analysis. Event count (y axis) versus DNA content (x axis) values are shown. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are indicated below the graph. (B) Phase-contrast microscopy images. (C) Western blot analyses of eRF3a, eRF1, and α-tubulin (α-tub).
FIG. 6.
FIG. 6.
eRF3a GTP-bound form is required to restore cell cycle progression in eRF3a-depleted cells. HCT116 cells were coelectroporated with either pSuper vector (Sup) or plasmid expressing si-3a1 (3a1) and with either the empty expression vector pBK-CMV (pBK) or plasmids overexpressing either human eRF3a (3a), human eRF3a mutated in its GTPase domain (3aNN), human eRF3b (3b), or a truncated form of human eRF3a containing only the C-terminal domain (HC). Thirty hours after electroporation, cells were synchronized in mitosis by nocodazole treatment, shaken off, and released for 16 h. (A) Flow cytometry analysis of cells electroporated with the plasmids indicated in abbreviation above each panel. Relative event count (y axis) versus DNA content (x axis) values are shown. Cells with DNA content values lower than 2N were excluded from the count. Percentages of cells in G1 (2N DNA content) and G2/M (4N DNA content) are indicated in the lower panel. (B) Western blot analysis of eRF3a and α-tubulin (α-tub) in each cell sample as indicated below. (C) Western blot analysis of eRF3a-depleted cells overexpressing eRF3b (3a1-3b) or not (3a1-pBK) using anti-eRF3b and anti-α-tubulin (α-tub) antibodies as indicated on the left. (D) Western blot analysis of eRF3a-depleted cells overexpressing N-terminally truncated eRF3a (3a1-HC) or not (3a1-pBK) using anti-eRF3a-C antibodies as indicated above. Entire eRF3a and N-terminally truncated eRF3a (HC) proteins are indicated by arrows on the right.
FIG. 7.
FIG. 7.
eRF3a depletion causes a decrease in the rate of amino acid incorporation into proteins. HCT116 cells electroporated with either pSuper vector, plasmid expressing si-3a1 targeting eRF3a mRNA, or plasmid expressing si-1Y targeting eRF1 mRNA were labeled 3 days after electroporation for 0 to 6 h with a mixture of [35S]methionine and [35S]cysteine. Total proteins (30 μg) were spotted on Whatman 3MM filter paper and TCA precipitated. (A) Radioactivity (counts per minute [cpm]) was determined by scintillation counting. Results are shown for pSuper vector (filled circles), plasmid expressing si-3a1 (filled squares), and plasmid expressing si-1Y (filled triangles). (B) Western blot analysis of the radiolabeled cells expressing pSuper vector (Sup), si-3a1 (3a1), or si-1Y (1Y) using anti-eRF3a, anti-eRF1, or anti-α-tubulin (α-tub) antibodies as indicated on the left.
FIG. 8.
FIG. 8.
eRF3a depletion causes a loss of polysomes. Cytoplasmic extracts of HCT116 cells electroporated with either pSuper vector (Sup) or plasmid expressing si-3a1 (3a1) were fractionated by 15 to 50% sucrose gradient centrifugation as described in Materials and Methods. The absorbance profile of the gradient is shown (sedimentation was from left to right), and the collected fractions are indicated on the x axis.
FIG. 9.
FIG. 9.
eRF3a depletion induces the hypophosphorylation of mTOR targets. Cytoplasmic extracts of HCT116 cells from two independent electroporation experiments with either pSuper vector (Sup) or plasmid expressing si-3a1 (3a1) were analyzed by Western blotting using antibodies directed against eRF3a, S6K1, phospho-S6K1 Thr389 (P-S6K1), ribosomal protein S6 and phospho-S6 Ser240/244 (P-S6), 4E-BP1 and phospho-4E-BP1 Thr70 (P-4E-BP1), phospho-Akt Ser473 (P-Akt), phospho-Erk1/2 Ser202 and Tyr204 (P-Erk1/2), eIF4E, and α-tubulin (α-tub), which served as a loading control.
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