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. 2008 Apr 21;181(2):293-307.
doi: 10.1083/jcb.200710215.

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

Francisco Ramírez-Valle  1 , Steve Braunstein, Jiri Zavadil, Silvia C Formenti, Robert J Schneider
Affiliations

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

Francisco Ramírez-Valle et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
eIF4GI silencing does not significantly alter global translation rates. MCF10A cells were transfected with the indicated siRNAs. (A) Cells were harvested and protein levels determined by immunoblot analysis (left). Protein synthesis rates for eIF4GI-silenced cells compared with control siRNA-transfected cells (set at 100%, right). (B) Protein synthesis rates in siRNA-transfected cells (left). Extent of silencing determined by immunoblot (right). (C) Protein and phospho-protein levels determined by immunoblot analysis. (D) siRNA transfected cells were treated with 100 nM rapamycin and 10 μM LY294002, or vehicle for 2 h, and protein synthesis rates determined after 2 h of recovery (left). eIF4G1 levels and 4E-BP1 phosphorylation determined (right). (E) Translation elongation was blocked with 200 μM puromycin for 10 min, and protein synthetic rates were measured after treatment or after 30 min of recovery. (F) Translation rates were determined in siRNA silenced cells (top). Efficiency of silencing shown by immunoblot (bottom). Quantification of immunoblots was performed by autoradiogram densitometry using multiple exposures and dilution series. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05.
Figure 2.
Figure 2.
Morphological and proliferative changes in eIF4GI-silenced cells. (A) Immunofluorescence microscopy of cells, performed as described in Materials and methods. Nuclei were stained with propidium iodide and the cytoskeleton with β-actin. Bar, 5 μM. (B) Cell size of control and eIF4GI-depleted cells were analyzed by flow cytometry. X-axis shows cell number, y-axis represents forward scatter (a measure of cell size). Average forward scatter of three independent experiments is shown; P < 0.002 (t test). (C) Proliferation of control (♦), eIF4GII-silenced (▴), and eIF4GI-silenced (▪) cells. (D) Cell cycle distribution of control, eIF4GI-, eIF4GII-, raptor-, and eIF4GI/raptor-silenced cells was determined as indicated in Materials and methods. Data are representative of at least three independent experiments.
Figure 3.
Figure 3.
Metabolic alterations in eIF4GI-depleted cells. (A) MCF10A cells treated with vehicle or 10 μM LY294002 and 100 nM rapamycin (Rapa/Ly) were immunoblotted for the indicated proteins. (B) Control or raptor shRNA-transduced cells were serum/insulin starved and harvested (time 0), or allowed to recover in full medium for the indicated times (minutes, min). Phosphorylation state and total levels of the indicated proteins were determined by immunoblot. (C) As in A, control or raptor shRNA-expressing cells were starved for leucine for 48 h and either frozen or allowed to recover by leucine restitution for 12 h before harvesting. (D) In vitro mTORC1 kinase assays using S6K1 or eIF4GI as substrates. Phospho- and total levels of the indicated proteins were detected by immunoblot. (E) Cells transfected with the specified siRNAs were leucine starved and allowed to recover by leucine restitution. Cell size changes were captured by analysis of forward scatter by flow cytometry showing actual values. (F) Quantitation of cell size changes in control and silenced cells after leucine starvation and recovery, as described in E. (G) Mitochondrial polarization, as measured by the incorporation of the mitochondrial-permeable dye TMRE. Average TMRE loading values of three independent experiments shown; t test, P < 0.005 (right). Quantitation of TMRE incorporation in a panel of siRNA-transfected cells with standard deviations shown (right). (H) ATP levels in control and eIF4GI-silenced cells measured as described in Materials and methods (top). Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.005; **, P < 0.01. Immunoblot analysis of control and silenced cells (bottom).
Figure 4.
Figure 4.
Autophagy induction and reduced cellular proliferation in eIF4GI-silenced cells. (A) 293T cells were transfected with a plasmid encoding GFP-LC3 and treated as shown. Nuclei were stained with propidium iodide (red). Bar, 5 μM. (B) MCF10A cells were treated as indicated and autophagosome formation established by MDC loading. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.05. (C) Endogenous LC3 cleavage in control and eIF4GI-silenced MCF10A cells determined by immunoblot. (D) MCF10A cells expressing AcGFP-LC3 transfected with (a) control (0–5% with dots), (b) eIF4GI (>90% with dots), (c) DAP5 (>90% with dots), (d) eIF4GII (0–5% with dots), (e) eIF4E (<5% with dots) siRNAs, or (f) treated with 200 μM puromycin (0–5% with dots) for 2 h. AcGFP-LC3 clustering analyzed by confocal microscopy. The number of cells with LC3-GFP dots were quantified in five fields of similar numbers of cells, t test (P < 0.001). Bar, 5 μM. (E) Immunoblot analysis showing increased levels of DAP5 in pBMN-DAP5-I-GFP transduced cells compared with vector control. (F) Growth rates on siRNA transfected cells (♦, control; ▴, DAP5; and ▪, eIF4GI). (G) Growth ratios determined from data shown in F.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
Translation of a subset of mRNAs is sensitive to eIF4GI levels. (A) Control and eIF4GI-depleted polysomes and eIF4F components determined by immunoblot analysis. (B) mRNAs collected from polysomal fractions were pooled and compared with total mRNAs by microarray hybridization. Targets were clustered according to their relative change in the polysome fractions compared with total mRNA levels. Four clusters were derived, representing mRNAs whose polysomal association in eIF4GI-depleted cells either decreases without a change in total mRNA (top left) or despite an increase in total mRNA (top right). Clusters of mRNAs whose polysomal association increases either with (bottom right) or without a decrease in total mRNA levels are shown (bottom left). (C) Control or eIF4GI-depleted cells probed for the indicated proteins by immunoblot analysis. (D) Diagram of pRL-HCV IRES-FL reporter mRNAs with mda-7 wild-type or mutant 5′ UTRs (top). Cap-dependent translation of bicistronic constructs in the indicated cells, depicted as mda-7 wild-type or mutant 5′ UTR-directed translation normalized to IRES activity. Error bars represent standard deviation, calculated from at least three independent experiments. Statistical analysis used the t test; *, P < 0.005.
Figure 7.
Figure 7.
eIF4GI in the context of mTORC1 and other eIF4G family members. mTORC1 signaling regulates cellular growth at various levels, including increasing anabolic processes (ribosome biogenesis and increased protein synthesis), and preventing catabolic processes such as autophagy and fatty acid oxidation. mTORC1 controls translation through the well-known factors 4E-BPs and S6K. eIF4GI total and phosphorylated levels increase in response to mTORC1 activity. eIF4GI modulates translation of low abundance, uORF-containing mRNAs such as Skp2, promoting decreased levels of p27 and therefore proliferation, as well as preventing autophagy. DAP5 and eIF4GI are both required to maintain overall protein synthesis rates, and silencing results in autophagy. Whether DAP5 is also controlled by mTOR is not known. Both DAP5 and eIF4GI are thought to participate in cap-independent translation during apoptosis. eIF4GII silencing does not alter global translation rates, and it may instead participate in translational reprogramming during differentiation.
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