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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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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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fig5: 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.

Mentions: To measure the impact of the two uORFs on ATF4 translation and its regulation, we sequentially converted their AUG start codon to AUA in the context of the 5′ATF4-GFP reporter (Fig. 5 A). Loss of uORF1 markedly inhibited basal and induced ATF4-GFP translation, whereas loss of uORF2 markedly de-repressed basal ATF4-GFP translation and abolished inducibility (Fig. 5 B). The repressive effect of uORF2 on ATF4 translation is consistent with the highly processive nature of translation and with the lack of a mechanism for translating ribosomes to back track along the mRNA and reinitiate translation at AUGs located 5′ of the stop codon. Therefore, it is likely that all scanning ribosomes initiating translation at uORF2 are precluded from translating ATF4.


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)

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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Related In: Results  -  Collection

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fig5: 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.
Mentions: To measure the impact of the two uORFs on ATF4 translation and its regulation, we sequentially converted their AUG start codon to AUA in the context of the 5′ATF4-GFP reporter (Fig. 5 A). Loss of uORF1 markedly inhibited basal and induced ATF4-GFP translation, whereas loss of uORF2 markedly de-repressed basal ATF4-GFP translation and abolished inducibility (Fig. 5 B). The repressive effect of uORF2 on ATF4 translation is consistent with the highly processive nature of translation and with the lack of a mechanism for translating ribosomes to back track along the mRNA and reinitiate translation at AUGs located 5′ of the stop codon. Therefore, it is likely that all scanning ribosomes initiating translation at uORF2 are precluded from translating ATF4.

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