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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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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.
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fig3: 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.

Mentions: Introduction of a stable stem loop at the 5′ end of ATF4-GFP mRNA markedly diminished reporter translation and blocked all stress inducibility without affecting mRNA levels (Fig. 3, A and B). Furthermore, the 5′ATF4 fragment directed low levels of translation initiation when incorporated as a second cistron downstream of the long luciferase coding region in a bi-cistronic reporter gene (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200408003/DC1). Together, these findings indicate that ribosomes scan the length of the mRNA to reach the ATF4 coding region and that the ATF4 5′ region is unable to direct efficient internal ribosome initiation. Replacement of the SV40 termination and mRNA processing signals of the reporter gene with the endogenous ATF4 mouse genomic fragment did not affect the reporter (Fig. 3 C), suggesting that communication between the 5′ and 3′ ends of the mRNA are unimportant to its translational regulation.


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)

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.
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fig3: 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.
Mentions: Introduction of a stable stem loop at the 5′ end of ATF4-GFP mRNA markedly diminished reporter translation and blocked all stress inducibility without affecting mRNA levels (Fig. 3, A and B). Furthermore, the 5′ATF4 fragment directed low levels of translation initiation when incorporated as a second cistron downstream of the long luciferase coding region in a bi-cistronic reporter gene (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200408003/DC1). Together, these findings indicate that ribosomes scan the length of the mRNA to reach the ATF4 coding region and that the ATF4 5′ region is unable to direct efficient internal ribosome initiation. Replacement of the SV40 termination and mRNA processing signals of the reporter gene with the endogenous ATF4 mouse genomic fragment did not affect the reporter (Fig. 3 C), suggesting that communication between the 5′ and 3′ ends of the mRNA are unimportant to its translational regulation.

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