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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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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.
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fig4: 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.

Mentions: To determine if the scanning ribosomes initiate translation at the two uORFs we fused these to GFP and transfected the reporters into the Fv2E-PERK(+) CHO cells. Pulse labeling and immunoprecipitation showed high levels of translation initiation at both uORF1 and uORF2 under basal conditions (Fig. 4). Translation initiation at these uORFs was 10–20-fold higher than at ATF4. But unlike ATF4, which is translationally induced by eIF2α(P), both uORF1 and uORF2 were repressed by these conditions.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: 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.
Mentions: To determine if the scanning ribosomes initiate translation at the two uORFs we fused these to GFP and transfected the reporters into the Fv2E-PERK(+) CHO cells. Pulse labeling and immunoprecipitation showed high levels of translation initiation at both uORF1 and uORF2 under basal conditions (Fig. 4). Translation initiation at these uORFs was 10–20-fold higher than at ATF4. But unlike ATF4, which is translationally induced by eIF2α(P), both uORF1 and uORF2 were repressed by these conditions.

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