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An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.

ABSTRACT: Cellular stress such as endoplasmic reticulum stress, hypoxia, and viral infection activates an integrated stress response, which includes the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit overall protein synthesis. Paradoxically, this leads to translation of a subset of mRNAs, like transcription factor ATF4, which in turn induces transcription of downstream stress-induced genes such as growth arrest DNA-inducible gene 34 (GADD34). GADD34 interacts with protein phosphatase 1 to dephosphorylate eIF2alpha, resulting in a negative feedback loop to recover protein synthesis and allow translation of stress-induced transcripts. Here, we show that GADD34 is not only transcriptionally induced but also translationally regulated to ensure maximal expression during eIF2alpha phosphorylation. GADD34 mRNAs are preferentially associated with polysomes during eIF2alpha phosphorylation, which is mediated by its 5'-untranslated region (5'UTR). The human GADD34 5'UTR contains two non-overlapping upstream open reading frames (uORFs), whereas the mouse version contains two overlapping and out of frame uORFs. Using 5'UTR GADD34 reporter constructs, we show that the downstream uORF mediates repression of basal translation and directs translation during eIF2alpha phosphorylation. Furthermore, we show that the upstream uORF is poorly translated and that a proportion of scanning ribosomes bypasses the upstream uORF to recognize the downstream uORF. These findings suggest that GADD34 translation is regulated by a unique 5'UTR uORF mechanism to ensure proper GADD34 expression during eIF2alpha phosphorylation. This mechanism may serve as a model for understanding how other 5'UTR uORF-containing mRNAs are regulated during cellular stress.

SUBMITTER: Lee YY

PROVIDER: S-EPMC2652341 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.

Lee Yun-Young YY Cevallos Randal C RC Jan Eric E

The Journal of biological chemistry 20090108 11

Cellular stress such as endoplasmic reticulum stress, hypoxia, and viral infection activates an integrated stress response, which includes the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit overall protein synthesis. Paradoxically, this leads to translation of a subset of mRNAs, like transcription factor ATF4, which in turn induces transcription of downstream stress-induced genes such as growth arrest DNA-inducible gene 34 (GADD34). GADD34 interacts with protei ...[more]

PMID: 19131336

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Project description:BACKGROUND:Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. RESULTS:Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. CONCLUSIONS:Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Project description:Copper is essential to most living beings but also highly toxic and as such is an important player at the host-pathogen interface. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. Known Cu export and import systems are under transcriptional control, whereas posttranscriptional regulatory mechanisms are yet to be characterized. We identified a three-gene operon, bp2923-bfrG-bp2921, downregulated by copper and notably encoding a TonB-dependent transporter in Bordetella pertussis. We show here that the protein encoded by the first gene, which is a member of the DUF2946 protein family, represents a new type of upstream Open Reading Frame (uORF) involved in posttranscriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologs of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has thus unveiled a new mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator. IMPORTANCE Copper is a transition metal necessary for living beings but also extremely toxic. Bacteria thus tightly control its homeostasis with transcriptional regulators. In this work, we have identified in the whooping cough agent Bordetella pertussis a new control mechanism mediated by a small protein called CruR, for copper-responsive upstream regulator. While being translated by the ribosome CruR is able to perceive intracellular copper, which shuts down the transcription of downstream genes of the same operon, coding for a copper uptake system. This mechanism limits the import of copper in conditions where it is abundant for the bacterium. This is the first report of "posttranscriptional regulation" in response to copper. Homologs of CruR genes head many operons harboring copper-related genes in various bacteria, and therefore the regulatory function unveiled here is likely a general property of this new protein family.

Dataset Information

An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.

Publications

An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.

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