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eIF4GI links nutrient sensing by mTOR to cell proliferation and inhibition of autophagy.

Ramírez-Valle F, Braunstein S, Zavadil J, Formenti SC, Schneider RJ - J. Cell Biol. (2008)

Bottom Line: Depletion or overexpression of other eIF4G family members did not recapitulate these results.The majority of mRNAs that were translationally impaired with eIF4GI depletion were excluded from polyribosomes due to the presence of multiple upstream open reading frames and low mRNA abundance.These results suggest that the high levels of eIF4GI observed in many breast cancers might act to specifically increase proliferation, prevent autophagy, and release tumor cells from control by nutrient sensing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Translation initiation factors have complex functions in cells that are not yet understood. We show that depletion of initiation factor eIF4GI only modestly reduces overall protein synthesis in cells, but phenocopies nutrient starvation or inhibition of protein kinase mTOR, a key nutrient sensor. eIF4GI depletion impairs cell proliferation, bioenergetics, and mitochondrial activity, thereby promoting autophagy. Translation of mRNAs involved in cell growth, proliferation, and bioenergetics were selectively inhibited by reduction of eIF4GI, as was the mRNA encoding Skp2 that inhibits p27, whereas catabolic pathway factors were increased. Depletion or overexpression of other eIF4G family members did not recapitulate these results. The majority of mRNAs that were translationally impaired with eIF4GI depletion were excluded from polyribosomes due to the presence of multiple upstream open reading frames and low mRNA abundance. These results suggest that the high levels of eIF4GI observed in many breast cancers might act to specifically increase proliferation, prevent autophagy, and release tumor cells from control by nutrient sensing.

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eIF4GI depletion blocks Skp2 mRNA translation. (A) Immunoblot of the indicated proteins in cells transfected with specific siRNAs. (B) Control or eIF4GI-depleted cells treated with proteasome inhibitor and proteins detected by immunoblot. (C) Total Skp2 mRNA levels in eIF4GI-silenced cells, determined by qRT-PCR analysis. (D) qRT-PCR analysis of Skp2 and Hsp27 mRNA in polysomes with eIF4GI depletion. (E) Immunoprecipitation of 35S-Met labeled lysates with the indicated antibodies. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05.
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fig5: eIF4GI depletion blocks Skp2 mRNA translation. (A) Immunoblot of the indicated proteins in cells transfected with specific siRNAs. (B) Control or eIF4GI-depleted cells treated with proteasome inhibitor and proteins detected by immunoblot. (C) Total Skp2 mRNA levels in eIF4GI-silenced cells, determined by qRT-PCR analysis. (D) qRT-PCR analysis of Skp2 and Hsp27 mRNA in polysomes with eIF4GI depletion. (E) Immunoprecipitation of 35S-Met labeled lysates with the indicated antibodies. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05.

Mentions: Because eIF4GI silencing results in increased G1 checkpoint accumulation, we investigated the effect on Skp2 mRNA translation. Skp2 promotes ubiquitin-dependent proteasome degradation of cell cycle inhibitor p27 (Pagano, 2004). eIF4GI silencing strongly increased levels of p27 protein, consistent with loss of Skp2 expression (Fig. 5 A). Inhibition of the proteasome with MG132 only minimally increased Skp2 levels in eIF4GI-silenced cells, suggesting that eIF4GI silencing primarily inhibits Skp2 mRNA translation (Fig. 5 B), also supported by decreased protein synthesis of Skp2 without alteration of mRNA levels (Fig. 5, C and E). In contrast, DAP5 depletion increased p27 levels without altering Skp2 levels (Fig. 5 E). Thus, despite similar effects on growth, eIF4GI and DAP5 perform distinct functions (Lee and McCormick, 2006). This result is consistent with qRT-PCR analysis of total and polysome-bound Skp2 mRNA (Fig. 5, C and D), a significant fraction of which shifts to lighter polysomes with eIF4GI depletion. In contrast, Hsp27 mRNA shifts to heavier polysomes with eIF4GI depletion, consistent with the array analysis (described later) and increased protein levels (Fig. 5 D). These results show that eIF4GI silencing impairs cell proliferation, in part, by reducing the translation of Skp2 mRNA and thereby increasing the abundance of p27 protein.


eIF4GI links nutrient sensing by mTOR to cell proliferation and inhibition of autophagy.

Ramírez-Valle F, Braunstein S, Zavadil J, Formenti SC, Schneider RJ - J. Cell Biol. (2008)

eIF4GI depletion blocks Skp2 mRNA translation. (A) Immunoblot of the indicated proteins in cells transfected with specific siRNAs. (B) Control or eIF4GI-depleted cells treated with proteasome inhibitor and proteins detected by immunoblot. (C) Total Skp2 mRNA levels in eIF4GI-silenced cells, determined by qRT-PCR analysis. (D) qRT-PCR analysis of Skp2 and Hsp27 mRNA in polysomes with eIF4GI depletion. (E) Immunoprecipitation of 35S-Met labeled lysates with the indicated antibodies. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05.
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fig5: eIF4GI depletion blocks Skp2 mRNA translation. (A) Immunoblot of the indicated proteins in cells transfected with specific siRNAs. (B) Control or eIF4GI-depleted cells treated with proteasome inhibitor and proteins detected by immunoblot. (C) Total Skp2 mRNA levels in eIF4GI-silenced cells, determined by qRT-PCR analysis. (D) qRT-PCR analysis of Skp2 and Hsp27 mRNA in polysomes with eIF4GI depletion. (E) Immunoprecipitation of 35S-Met labeled lysates with the indicated antibodies. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05.
Mentions: Because eIF4GI silencing results in increased G1 checkpoint accumulation, we investigated the effect on Skp2 mRNA translation. Skp2 promotes ubiquitin-dependent proteasome degradation of cell cycle inhibitor p27 (Pagano, 2004). eIF4GI silencing strongly increased levels of p27 protein, consistent with loss of Skp2 expression (Fig. 5 A). Inhibition of the proteasome with MG132 only minimally increased Skp2 levels in eIF4GI-silenced cells, suggesting that eIF4GI silencing primarily inhibits Skp2 mRNA translation (Fig. 5 B), also supported by decreased protein synthesis of Skp2 without alteration of mRNA levels (Fig. 5, C and E). In contrast, DAP5 depletion increased p27 levels without altering Skp2 levels (Fig. 5 E). Thus, despite similar effects on growth, eIF4GI and DAP5 perform distinct functions (Lee and McCormick, 2006). This result is consistent with qRT-PCR analysis of total and polysome-bound Skp2 mRNA (Fig. 5, C and D), a significant fraction of which shifts to lighter polysomes with eIF4GI depletion. In contrast, Hsp27 mRNA shifts to heavier polysomes with eIF4GI depletion, consistent with the array analysis (described later) and increased protein levels (Fig. 5 D). These results show that eIF4GI silencing impairs cell proliferation, in part, by reducing the translation of Skp2 mRNA and thereby increasing the abundance of p27 protein.

Bottom Line: Depletion or overexpression of other eIF4G family members did not recapitulate these results.The majority of mRNAs that were translationally impaired with eIF4GI depletion were excluded from polyribosomes due to the presence of multiple upstream open reading frames and low mRNA abundance.These results suggest that the high levels of eIF4GI observed in many breast cancers might act to specifically increase proliferation, prevent autophagy, and release tumor cells from control by nutrient sensing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Translation initiation factors have complex functions in cells that are not yet understood. We show that depletion of initiation factor eIF4GI only modestly reduces overall protein synthesis in cells, but phenocopies nutrient starvation or inhibition of protein kinase mTOR, a key nutrient sensor. eIF4GI depletion impairs cell proliferation, bioenergetics, and mitochondrial activity, thereby promoting autophagy. Translation of mRNAs involved in cell growth, proliferation, and bioenergetics were selectively inhibited by reduction of eIF4GI, as was the mRNA encoding Skp2 that inhibits p27, whereas catabolic pathway factors were increased. Depletion or overexpression of other eIF4G family members did not recapitulate these results. The majority of mRNAs that were translationally impaired with eIF4GI depletion were excluded from polyribosomes due to the presence of multiple upstream open reading frames and low mRNA abundance. These results suggest that the high levels of eIF4GI observed in many breast cancers might act to specifically increase proliferation, prevent autophagy, and release tumor cells from control by nutrient sensing.

Show MeSH
Related in: MedlinePlus