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Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.

Lu PD, Harding HP, Ron D - J. Cell Biol. (2004)

Bottom Line: In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2.These features are common to regulated translation of GCN4 in yeast.The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

View Article: PubMed Central - PubMed

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

ABSTRACT
Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

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EIF2α phosphorylation is sufficient to up-regulate ATF4 translation. (A) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of Fv2E-PERK expressing wild-type (EIF2AS/S) or mutant (EIF2AA/A) mouse fibroblasts pretreated for 30′ with the indicated concentration of the AP20187 activating ligand. The panels (from top to bottom) show immunoprecipitated radiolabeled endogenous ATF4, all radiolabeled newly synthesized proteins, and immunoblots of phosphorylated and total eIF2α. (B) Polysome profiles of mRNA isolated from untreated mouse fibroblasts and the same cells 60′ after induction of eIF2α phosphorylation by activation of the ligand-inducible Fv2E-PERK eIF2α kinase. Note the accumulation of ribosomal subunits and monosomes in the treated cells. (C) Northern blot analysis of ATF4 and GAPDH mRNA from fractions collected from the gradient shown in B. Note the shift to the right, (heavier) fractions in the peak of the ATF4 mRNA in the treated cells and the shift in opposite direction of the GAPDH peak.
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fig1: EIF2α phosphorylation is sufficient to up-regulate ATF4 translation. (A) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of Fv2E-PERK expressing wild-type (EIF2AS/S) or mutant (EIF2AA/A) mouse fibroblasts pretreated for 30′ with the indicated concentration of the AP20187 activating ligand. The panels (from top to bottom) show immunoprecipitated radiolabeled endogenous ATF4, all radiolabeled newly synthesized proteins, and immunoblots of phosphorylated and total eIF2α. (B) Polysome profiles of mRNA isolated from untreated mouse fibroblasts and the same cells 60′ after induction of eIF2α phosphorylation by activation of the ligand-inducible Fv2E-PERK eIF2α kinase. Note the accumulation of ribosomal subunits and monosomes in the treated cells. (C) Northern blot analysis of ATF4 and GAPDH mRNA from fractions collected from the gradient shown in B. Note the shift to the right, (heavier) fractions in the peak of the ATF4 mRNA in the treated cells and the shift in opposite direction of the GAPDH peak.

Mentions: To determine if eIF2α phosphorylation is sufficient to up-regulate ATF4 translation we made use of a ligand activateable version of PERK. Addition of the AP20187 ligand leads to oligomerization of the chimeric Fv2E-PERK kinase and promotes eIF2α phosphorylation that is uncoupled from ER stress, which normally activates PERK. Incorporation of radiolabeled amino acids into ATF4 increased within minutes of Fv2E-PERK activation and highest levels were observed at relatively low doses of the activator associated with low levels of phosphorylated eIF2α. ATF4 translation was strictly dependent on eIF2α phosphorylation as it was altogether absent from EIF2AS51A mutant mouse fibroblasts lacking the regulatory phosphorylation site on eIF2α (Fig. 1 A).


Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.

Lu PD, Harding HP, Ron D - J. Cell Biol. (2004)

EIF2α phosphorylation is sufficient to up-regulate ATF4 translation. (A) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of Fv2E-PERK expressing wild-type (EIF2AS/S) or mutant (EIF2AA/A) mouse fibroblasts pretreated for 30′ with the indicated concentration of the AP20187 activating ligand. The panels (from top to bottom) show immunoprecipitated radiolabeled endogenous ATF4, all radiolabeled newly synthesized proteins, and immunoblots of phosphorylated and total eIF2α. (B) Polysome profiles of mRNA isolated from untreated mouse fibroblasts and the same cells 60′ after induction of eIF2α phosphorylation by activation of the ligand-inducible Fv2E-PERK eIF2α kinase. Note the accumulation of ribosomal subunits and monosomes in the treated cells. (C) Northern blot analysis of ATF4 and GAPDH mRNA from fractions collected from the gradient shown in B. Note the shift to the right, (heavier) fractions in the peak of the ATF4 mRNA in the treated cells and the shift in opposite direction of the GAPDH peak.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172506&req=5

fig1: EIF2α phosphorylation is sufficient to up-regulate ATF4 translation. (A) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of Fv2E-PERK expressing wild-type (EIF2AS/S) or mutant (EIF2AA/A) mouse fibroblasts pretreated for 30′ with the indicated concentration of the AP20187 activating ligand. The panels (from top to bottom) show immunoprecipitated radiolabeled endogenous ATF4, all radiolabeled newly synthesized proteins, and immunoblots of phosphorylated and total eIF2α. (B) Polysome profiles of mRNA isolated from untreated mouse fibroblasts and the same cells 60′ after induction of eIF2α phosphorylation by activation of the ligand-inducible Fv2E-PERK eIF2α kinase. Note the accumulation of ribosomal subunits and monosomes in the treated cells. (C) Northern blot analysis of ATF4 and GAPDH mRNA from fractions collected from the gradient shown in B. Note the shift to the right, (heavier) fractions in the peak of the ATF4 mRNA in the treated cells and the shift in opposite direction of the GAPDH peak.
Mentions: To determine if eIF2α phosphorylation is sufficient to up-regulate ATF4 translation we made use of a ligand activateable version of PERK. Addition of the AP20187 ligand leads to oligomerization of the chimeric Fv2E-PERK kinase and promotes eIF2α phosphorylation that is uncoupled from ER stress, which normally activates PERK. Incorporation of radiolabeled amino acids into ATF4 increased within minutes of Fv2E-PERK activation and highest levels were observed at relatively low doses of the activator associated with low levels of phosphorylated eIF2α. ATF4 translation was strictly dependent on eIF2α phosphorylation as it was altogether absent from EIF2AS51A mutant mouse fibroblasts lacking the regulatory phosphorylation site on eIF2α (Fig. 1 A).

Bottom Line: In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2.These features are common to regulated translation of GCN4 in yeast.The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

View Article: PubMed Central - PubMed

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

ABSTRACT
Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.

Show MeSH
Related in: MedlinePlus