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. 2004 Oct 11;167(1):27-33.
doi: 10.1083/jcb.200408003.

"V体育ios版" Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response

Phoebe D Lu  1 , Heather P Harding, David Ron
Affiliations

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

Phoebe D Lu et al. J Cell Biol. .

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 VSports手机版. 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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Figures

Figure 1.
Figure 1.
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 (EIF2A S/S) or mutant (EIF2A A/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.
Figure 2.
Figure 2.
The 5′ end of the ATF4 mRNA mediates translational regulation of the gene. (A) Organization of the 5′ end of the mouse ATF4 mRNA, the derivative 5′ATF4-GFP reporter and the parental GFP reporter. (B) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of wild-type CHO cells or cells stably overexpressing the COOH terminus of GADD34 (which blocks eIF2α phosphorylation) transfected with the indicated reporter plasmids. The cells were treated with the endoplasmic reticulum stress-inducing agent thapsigargin (TG) for the indicated time and the encoded GFP fusion proteins (top) and endogenous ATF4 (bottom) were immunoprecipitated with specific antibodies. (C) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of CHO cells transfected with the ATF4-GFP reporter and pretreated for 30 min with the indicated concentration of arsenite (ARS), the electrophile methyl-methanesulfonate (MMS), histidinol (HIS, which activates GCN2), thapsigargin (TG), and the encoded GFP fusion proteins (top), the NPTII, encoded by a different gene on the same plasmid (second panel) and endogenous ATF4 (third panel) were immunoprecipitated with specific antisera. The GFP/NPTII ratio provides an estimate of the translational inducibility of the reporter in the treated cells. Immunoblots of phosphorylated (eIF2α-P) and total eIF2α from parallel lysates are shown in the bottom panels. (D) Autoradiogram of radiolabeled proteins from Fv2E-PERK expressing CHO cells treated with the indicated concentration of the activating AP20187 ligand for 30′ before and during the 20′ labeling pulse. The cells were transfected with the ATF4 reporter (5′ATF4-GFP), a cricket paralysis virus internal ribosome entry site reporter (IGR IRE5-YFP) or a GFP translational reporter derived from the 5′ end of mouse CD36 gene (mCD36-GFP). GFP, endogenous ATF4 and NPTII were immunoprecipitated as in C with specific antisera.
Figure 3.
Figure 3.
ATF4 mRNA translation requires ribosomes scanning. (A) Predicted structure of the mRNA expressed by the reporter genes. Depicted is the stem loop introduced at the 5′ end of the mRNA and replacement of vector derived viral termination and poly-adenylation sequences (thin line) with ATF4 derived termination sequences (white rectangle). (B) Autoradiogram of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated CHO cells expressing Fv2E-PERK. The cells were transfected with ATF4-GFP reporters with or without the stable stem-loop structure at the 5′ end of the mRNA. Radiolabeled GFP, NPTII, and endogenous ATF4 were immunoprecipitated with a specific antisera. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. The ratio of the labeled GFP to RNA signal in each lane is provided. (C) As in B, cells were transfected with ATF4.GFP reporters with a viral 3′ termination sequences (5′ATF4.GFP), or the genomic ATF4 3′ termination sequences (5′3′ATF4.GFP) or the parental GFP reporter.
Figure 4.
Figure 4.
Both ATF4 uORFs are translated under basal conditions. (A) Predicted structure of the mRNA expressed by the reporter genes used in these experiments. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK(+) CHO cells transfected with the ATF4-GFP reporter, or reporters fusing uORF1 or uORF2 to GFP. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes. The low basal expression of NPTII in cells transfected with the uORF1-GFP is a reproducible if unexplained finding.
Figure 5.
Figure 5.
Interplay of repressive and stimulatory uORFs regulates ATF4 translation. (A) mRNA expressed by the reporter genes used in these experiments. Where indicated, the initiating AUG codons of the uORFs were converted to AUA and the stop codons between the colinear uORF1 and uORF2 converted to UCG and UAC, respectively. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK(+) CHO cells transfected with the 5′ATF4-GFP reporter, or reporters with a mutation in the start codon of uORF1, uORF2 or both uORFs, or the parental GFP reporter. Cytoplasmic RNA from a parallel sample of untreated transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. (C) As in B except cells were transfected with reporters in which the stop codons between the two uORFs were converted to Ser or Tyr codons.
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References

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