A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development

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.

Modulation of the Proteostasis Machinery to Overcome Stress Caused by Diminished Levels of t6A‐Modified tRNAs in Drosophila

“…The last one overlaps Atf4 coding sequence in a different reading frame, therefore inhibiting its translation in the unstressed cells where upon ER stress, the main reading frame is translated [22]. In the reporter, Atf4 5’UTR is fused to dsRed ORF, then in stressed cells dsRed will be translated [36]. In control animals, no dsRed signal was detected (Figure 4A); however, when tcs2 (Figure 4B) or TCTC subunits were silenced (Figure 4C–E), a signal was detected, indicating that the UPR is active via the PERK/ATF4 pathway, confirming and extending our previous results for tcs3 and tcs5 [21].…”

“…Stocks were obtained from the Bloomington Drosophila Stock Center (BDSC) and the Vienna Drosophila Resource Center (VDRC) [25]. The Atf4 5’UTR::dsRed reporter is described in reference [36] and Xpb1::GFP is described in reference [37]. …”

The Drosophila metallopeptidase superdeath decouples apoptosis from the activation of the ER stress response

“…Tunicamycin inhibits N-linked glycosylation in the ER, inducing a massive ER stress response through the UPR (29). This fivehour treatment is sufficient to significantly activate the UPR in the larvae, leading to developmental delay and lethality (15,30). We found that Ubi-superdeathi larvae are significantly more resistant to tunicamycin-induced lethality as compared to Ubi-control larvae ( Fig 5B, Table S1).…”

The integrated stress response regulates BMP signaling through effects on translation

“…crc is a bZIP transcription factor sharing sequence and functional homology with mammalian ATF4 (Hewes et al, 2000, Kang et al, 2015. In order to confirm activation of the ISR, we generated an antibody able to detect endogenous crc by western blot (Fig 2C and Supplementary Fig S2).…”