Abstract
Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5’-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5’ UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.
Highlights
The endoplasmic reticulum (ER) is a cellular organelle in which secretory and membrane proteins are synthesized, and folded
We had shown that the level of Drosophila ATF4 protein increases in response to ER stress caused by misexpression of mutant Rhosopsin-1[19], which prompted us to examine whether the mechanism of ATF4 induction upon ER stress is conserved in Drosophila
To better understand how ATF4 expression is regulated during normal development as well as in ER stress, we developed an in vivo ATF4 reporter modified from ATF4 luciferase reporter
Summary
The endoplasmic reticulum (ER) is a cellular organelle in which secretory and membrane proteins are synthesized, and folded. One of those signaling arms is mediated by PKR-like ER kinase (PERK) [7] This transmembrane kinase is activated upon excessive stress in the ER, and activates a downstream transcription factor, ATF4, a member of the leucine-zipper ATF/CREB family of DNA binding proteins [8]. ATF40s transcriptional targets include those involved in antioxidant stress response, amino acid transportation and biosynthesis Both PERK and GCN2 directly phosphorylate and inactivate eukaryotic initiation factor 2α (eIF2α) [10]. When PERK or GCN2 are activated, the decrease of functional eIF2 complex makes the AUG start codon recognition less efficient This allows the scanning ribosomes to occasionally bypass the final uORF and to give the ATF4 main ORF an opportunity to be recognized for translation [11]. As ATF4 is known to have causal effects in the progression of various neurodegenerative or metabolic diseases, this tool may facilitate the study of ATF4 function in Drosophila models of human diseases
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