Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172506&req=5

fig2: 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.

Mentions: The ATF4 mRNA has two conserved uORFs implicated in translational regulation (Harding et al., 2000a). The 5′ uORF1 has three codons whereas the 3′ uORF2 is longer and extends into the ATF4 coding region. To establish a system for studying the impact of cis acting elements on ATF4 translation, we inserted the 5′ end of the ATF4 mRNA into a GFP reporter gene fusing the coding regions of ATF4 and GFP immediately 3′ of the termination codon of uORF2 (Fig. 2 A). A constitutive cytomegalovirus promoter drove transcription of this 5′ATF4-GFP reporter and SV40 viral signals specified termination and 3′ mRNA processing.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: The ATF4 mRNA has two conserved uORFs implicated in translational regulation (Harding et al., 2000a). The 5′ uORF1 has three codons whereas the 3′ uORF2 is longer and extends into the ATF4 coding region. To establish a system for studying the impact of cis acting elements on ATF4 translation, we inserted the 5′ end of the ATF4 mRNA into a GFP reporter gene fusing the coding regions of ATF4 and GFP immediately 3′ of the termination codon of uORF2 (Fig. 2 A). A constitutive cytomegalovirus promoter drove transcription of this 5′ATF4-GFP reporter and SV40 viral signals specified termination and 3′ mRNA processing.

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