Lee S et al. (2014), Upregulation of eIF5B controls cell-cycle arres...

XB-ART-49452

Proc Natl Acad Sci U S A 2014 Oct 14;11141:E4315-22. doi: 10.1073/pnas.1320477111.

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Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages.

Lee S , Truesdell SS , Bukhari SI , Lee JH , LeTonqueze O , Vasudevan S .

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Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met(i)) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.

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Species referenced: Xenopus laevis
Genes referenced: cdk1 dctn6 eif2a eif4g3 eif5b lif mt-tr pou5f3 trna znrd2

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	Fig. 1. eIF5B levels and eIF2α phosphorylation are increased in late immature oocytes (stages V and VI), in which eIF5B promotes translation and maturation. (A) eIF5B and total and phosphorylated eIF2α levels are shown by Western blot analysis comparing 20 oocytes from each stage. In the eIF2α blot the asterisk indicates that the top band is a nonspecific band. (B) Control or eIF5B antisense (AS)-treated stage V immature oocytes were injected with a Firefly luciferase reporter plasmid after 6 h of incubation to measure the effects of eIF5B levels on general translation. In parallel samples, human eIF5B (eIF5B) or vector control mRNA was coinjected with the antisense to observe rescue of the loss of eIF5B function. (C) Western blot of eIF5B and the marker for immaturity, phospho-Cdc2 (p-Cdc2) (5), and its quantitation (given below the blots) from three replicates in oocytes injected with control antisense+GFP (Control), eIF5B antisense+GFP (eIF5B AS), or eIF5B antisense+human eIF5B (eIF5B AS+eIF5B). Phospho-Cdc2 runs variably as multiple bands due to modifications that include multiple-site phosphorylated forms in these extracts to which phosphatase inhibitors were not added (23) and due to a variably observed truncated form that migrates as a lower band and was observed previously (figure S5 in ref. 5). The lack of phosphatase inhibitors in the extracts and the set amount of Cdc2 in these oocytes would limit further increases in phospho-Cdc2 levels upon eIF5B depletion. All bands were quantitated, and the average from multiple experiments is shown below the blots. (D) Control or eIF5B antisense-treated and rescued immature oocytes were scored for germinal vesicle breakdown to measure the effects of eIF5B levels on maturation (Table S1) (5). (E) Ten nanograms of vector control, eIF4G3, or eIF5B mRNAs were injected into immature oocytes; after 6 h, these oocytes were injected with Firefly luciferase reporter plasmid to measure the effects of overexpression of eIF5B or the control translation factor, eIF4G3, on general translation by luciferase analyses. (F) Western blot of eIF5B and the immaturity marker p-Cdc2 and its quantitation from three replicates (shown below the blots), in oocytes with control, eIF4G3, or eIF5B mRNA overexpression (1× = 5 ng; 2× = 10 ng). Multiple bands are observed variably with eIF5B due to dephosphorylation in the absence of phosphatase inhibitors. (G) Control, eIF4G3, or eIF5B overexpression in immature oocytes scored for germinal vesicle breakdown to measure the effects of eIF5B overexpression on maturation (Table S1). Actin (a stable, abundant protein) was used as a loading control. The graphs show the average of three independent experiments; error bars indicate SEM. n.s., not significant. P < 0.05; *P < 0.01.
	Fig. 2. eIF5B levels and eIF2α phosphorylation are increased in ES cells in which eIF5B depletion decreases general translation. (A) Western blot showing eIF5B levels and eIF2α phosphorylation in mouse ES cells [+LIF (ESC)] compared with differentiating EBs in the absence of LIF (24). Oct4 is an ES cell marker. Actin was used as a loading control. (B) Western blot of stem cells transfected with the luciferase reporter plasmid pTRIPZ control shRNA vector or with eIF5B-specific shRNA. shRNA was induced with doxycycline for 3 d in ES cells or for 3 d after differentiation into EBs (11 d −LIF). Pooled EBs and ES cells were checked for depletion. (C) Luciferase reporter assay normalized to total nucleic acid levels of ES cells (+LIF) and EBs (11 d −LIF) transfected with pTRIPZ control or eIF5B shRNA. Error bars indicate SEMs from three independent experiments. LIF, leukemia inhibitory factor. *P < 0.05.
	Fig. 3. eIF5B levels and eIF2α phosphorylation are transiently increased in THP1 cells serum-starved for 1 d in which eIF5B depletion promotes earlier G0 arrest. (A) Western blot analysis of eIF5B levels over days of serum starvation (SS1D, SS2D, SS4D, SS7D) compared with serum-grown cells (S+). (B, Left) Western blot analysis of eIF2α phosphorylation and eIF2α levels in THP1 cells that had been serum-starved for 1 d (SS1D) compared with serum-grown cells (S+). p27/KIP1 (p27) marks cell-cycle arrest. (Right) The phosphorylated form and total eIF2α levels from three experiments were quantitated. (C) By using a doxycycline-inducible lentiviral vector (which expresses eIF5B-specific shRNA, eIF5B shRNA, as in Fig. 2) that is stably transduced into THP1 cells, eIF5B was knocked down by 63–67% as observed by Western analysis. Actin was used as a loading control. (D) eIF5B shRNA was induced with doxycycline in serum-containing medium for 3 d; then one half of the cells were shifted to serum-free medium. Cell counts were plotted relative to day 0 cell counts to measure cell proliferation over 3 d. (Left) eIF5B depletion had no effect on proliferation rates in serum-grown cells. (Right) Upon serum starvation, G0 arrest was observed earlier in eIF5B-depleted cells than in undepleted cells. (E) MTS assay (Promega) to measure cell growth after 3 d in serum-grown and serum-starved eIF5B-depleted and control cells. (F, Left) Western blot analysis of eIF5B, p27, and p21CIP1 (p21) in eIF5B-depleted and control cells after 1 d of serum starvation. (Right) Quantitation from three experiments. Tubulin was used as a loading control. Graphs show the average of three independent experiments; error bars indicate SEM. P < 0.05; *P < 0.01.
	Fig. 4. eIF5B depletion decreases translation in serum-starved cells. (A) 35S-methionine incorporation into newly synthesized protein without (Control) and with (eIF5B shRNA) eIF5B depletion in THP1 cells either grown in serum-containing medium (S+) or serum-starved for 1 d (SS1D). (Upper Left) Equal amounts of extracts, separated by SDS/PAGE, were exposed on the phosphorimager and normalized to Tubulin, a stable, abundant protein, for loading by Western analysis (Lower Left). (Right) 35S-methionine incorporation was quantitated. The graph shows the average of three independent experiments. Error bars indicate SEM. *P < 0.01. (B) Luciferase reporter assay in cells that had been serum-starved for 1 d with and without eIF5B depletion. The graph shows the average of five independent experiments. Error bars indicate SEM; P < 0.03.

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	Fig. 1. eIF5B levels and eIF2α phosphorylation are increased in late immature oocytes (stages V and VI), in which eIF5B promotes translation and maturation. (A) eIF5B and total and phosphorylated eIF2α levels are shown by Western blot analysis comparing 20 oocytes from each stage. In the eIF2α blot the asterisk indicates that the top band is a nonspecific band. (B) Control or eIF5B antisense (AS)-treated stage V immature oocytes were injected with a Firefly luciferase reporter plasmid after 6 h of incubation to measure the effects of eIF5B levels on general translation. In parallel samples, human eIF5B (eIF5B) or vector control mRNA was coinjected with the antisense to observe rescue of the loss of eIF5B function. (C) Western blot of eIF5B and the marker for immaturity, phospho-Cdc2 (p-Cdc2) (5), and its quantitation (given below the blots) from three replicates in oocytes injected with control antisense+GFP (Control), eIF5B antisense+GFP (eIF5B AS), or eIF5B antisense+human eIF5B (eIF5B AS+eIF5B). Phospho-Cdc2 runs variably as multiple bands due to modifications that include multiple-site phosphorylated forms in these extracts to which phosphatase inhibitors were not added (23) and due to a variably observed truncated form that migrates as a lower band and was observed previously (figure S5 in ref. 5). The lack of phosphatase inhibitors in the extracts and the set amount of Cdc2 in these oocytes would limit further increases in phospho-Cdc2 levels upon eIF5B depletion. All bands were quantitated, and the average from multiple experiments is shown below the blots. (D) Control or eIF5B antisense-treated and rescued immature oocytes were scored for germinal vesicle breakdown to measure the effects of eIF5B levels on maturation (Table S1) (5). (E) Ten nanograms of vector control, eIF4G3, or eIF5B mRNAs were injected into immature oocytes; after 6 h, these oocytes were injected with Firefly luciferase reporter plasmid to measure the effects of overexpression of eIF5B or the control translation factor, eIF4G3, on general translation by luciferase analyses. (F) Western blot of eIF5B and the immaturity marker p-Cdc2 and its quantitation from three replicates (shown below the blots), in oocytes with control, eIF4G3, or eIF5B mRNA overexpression (1× = 5 ng; 2× = 10 ng). Multiple bands are observed variably with eIF5B due to dephosphorylation in the absence of phosphatase inhibitors. (G) Control, eIF4G3, or eIF5B overexpression in immature oocytes scored for germinal vesicle breakdown to measure the effects of eIF5B overexpression on maturation (Table S1). Actin (a stable, abundant protein) was used as a loading control. The graphs show the average of three independent experiments; error bars indicate SEM. n.s., not significant. P < 0.05; *P < 0.01.
	Fig. 2. eIF5B levels and eIF2α phosphorylation are increased in ES cells in which eIF5B depletion decreases general translation. (A) Western blot showing eIF5B levels and eIF2α phosphorylation in mouse ES cells [+LIF (ESC)] compared with differentiating EBs in the absence of LIF (24). Oct4 is an ES cell marker. Actin was used as a loading control. (B) Western blot of stem cells transfected with the luciferase reporter plasmid pTRIPZ control shRNA vector or with eIF5B-specific shRNA. shRNA was induced with doxycycline for 3 d in ES cells or for 3 d after differentiation into EBs (11 d −LIF). Pooled EBs and ES cells were checked for depletion. (C) Luciferase reporter assay normalized to total nucleic acid levels of ES cells (+LIF) and EBs (11 d −LIF) transfected with pTRIPZ control or eIF5B shRNA. Error bars indicate SEMs from three independent experiments. LIF, leukemia inhibitory factor. *P < 0.05.
	Fig. 3. eIF5B levels and eIF2α phosphorylation are transiently increased in THP1 cells serum-starved for 1 d in which eIF5B depletion promotes earlier G0 arrest. (A) Western blot analysis of eIF5B levels over days of serum starvation (SS1D, SS2D, SS4D, SS7D) compared with serum-grown cells (S+). (B, Left) Western blot analysis of eIF2α phosphorylation and eIF2α levels in THP1 cells that had been serum-starved for 1 d (SS1D) compared with serum-grown cells (S+). p27/KIP1 (p27) marks cell-cycle arrest. (Right) The phosphorylated form and total eIF2α levels from three experiments were quantitated. (C) By using a doxycycline-inducible lentiviral vector (which expresses eIF5B-specific shRNA, eIF5B shRNA, as in Fig. 2) that is stably transduced into THP1 cells, eIF5B was knocked down by 63–67% as observed by Western analysis. Actin was used as a loading control. (D) eIF5B shRNA was induced with doxycycline in serum-containing medium for 3 d; then one half of the cells were shifted to serum-free medium. Cell counts were plotted relative to day 0 cell counts to measure cell proliferation over 3 d. (Left) eIF5B depletion had no effect on proliferation rates in serum-grown cells. (Right) Upon serum starvation, G0 arrest was observed earlier in eIF5B-depleted cells than in undepleted cells. (E) MTS assay (Promega) to measure cell growth after 3 d in serum-grown and serum-starved eIF5B-depleted and control cells. (F, Left) Western blot analysis of eIF5B, p27, and p21CIP1 (p21) in eIF5B-depleted and control cells after 1 d of serum starvation. (Right) Quantitation from three experiments. Tubulin was used as a loading control. Graphs show the average of three independent experiments; error bars indicate SEM. P < 0.05; *P < 0.01.
	Fig. 4. eIF5B depletion decreases translation in serum-starved cells. (A) 35S-methionine incorporation into newly synthesized protein without (Control) and with (eIF5B shRNA) eIF5B depletion in THP1 cells either grown in serum-containing medium (S+) or serum-starved for 1 d (SS1D). (Upper Left) Equal amounts of extracts, separated by SDS/PAGE, were exposed on the phosphorimager and normalized to Tubulin, a stable, abundant protein, for loading by Western analysis (Lower Left). (Right) 35S-methionine incorporation was quantitated. The graph shows the average of three independent experiments. Error bars indicate SEM. *P < 0.01. (B) Luciferase reporter assay in cells that had been serum-starved for 1 d with and without eIF5B depletion. The graph shows the average of five independent experiments. Error bars indicate SEM; P < 0.03.