Abstract
Upon exposure to environmental stress, phosphorylation of the α subunit of eIF2 (eIF2α-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2α-P is that encoding the transcription factor CHOP (DDIT3/GADD153). Enhanced levels of CHOP promote cell death when ISR signaling is insufficient to restore cell homeostasis. Preferential translation of CHOP mRNA occurs by a mechanism involving ribosome bypass of an inhibitory upstream ORF (uORF) situated in the 5'-leader of the CHOP mRNA. In this study, we used biochemical and genetic approaches to define the inhibitory features of the CHOP uORF and the biological consequences of loss of the CHOP uORF on CHOP expression during stress. We discovered that specific sequences within the CHOP uORF serve to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP coding sequence. As a consequence, deletion of the CHOP uORF substantially increases the levels and modifies the pattern of induction of CHOP expression in the ISR. Enhanced CHOP expression leads to increased expression of key CHOP target genes, culminating in increased cell death in response to stress.
Highlights
In response to a variety of environmental stresses, protein synthesis is modulated to facilitate reprogramming of gene expression to ameliorate stress damage
The integrated stress response (ISR) features many preferentially translated genes, including those encoding transcription factors ATF4 (CREB2) and CHOP (DDIT3/GADD153), which serve to reprogram the transcriptome to respond to cellular stress, and GADD34 (PPP1R15A), which interacts with the catalytic subunit of protein phosphatase 1 to target eIF2␣-P for dephosphorylation and restore protein synthesis [3,4,5,6]
Using biochemical and genetic approaches, we show that specific sequences within the upstream ORFs (uORFs) in the CHOP mRNA serve to stall translation elongation, which culminates in lowered reinitiation of translation at the CHOP coding sequences (CDS)
Summary
Preferential translation of CHOP and GADD34 are suggested to occur via a “bypass mechanism,” in which a single inhibitory uORF is bypassed during eIF2␣-P by a process involving, in part, a less than optimal start codon context [4, 5]. Genome-wide analyses of changes in translation in response to eIF2␣-P recently indicated that ϳ40% of mammalian mRNAs that contain uORFs are distributed among those transcripts whose translation are either enhanced, repressed, or resistant to eIF2␣-P [14]. This finding suggests that there are specific properties of each uORF in mRNAs that determine the mechanism by which translation initiation at the CDS is regulated and that uORFs can serve to either activate or repress downstream translation. This study illuminates key features of uORFs that direct preferential mRNA translation in the ISR and the roles that these translational control mechanisms play in restoring cell homeostasis in response to environmental stresses
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