226C Poster - 01. Cell Stress and cell death
Saturday April 09, 1:30 PM - 3:30 PM
An in vivo Drosophila screen to identify new regulators of ATF4 signaling
Author: Kristoffer Walsh
Affiliation: NYU Vilcek Institute, New York, NY
Keywords: h. other (Integrated Stress Response); h. translational regulation
In response to various physiological or external stresses, eukaryotic cells activate the Integrated Stress Response (ISR) to regulate protein synthesis and gene expression. ISR signaling begins when stress-activated kinases phosphorylate the translational initiation factor eIF2a, inhibiting general mRNA translation. Under these conditions, a small number of specially-regulated mRNAs – including that of transcription factor ATF4 – are translationally upregulated to activate a stress-induced gene expression program.
Our lab has previously demonstrated that cap-dependent translation inhibitor 4EBP is induced by ATF4 in the Drosophila fat body through the amino acid deprivation-activated ISR kinase GCN2. The 4EBP intron contains ATF4-binding sites that not only respond to environmental stress but also show physiological ATF4 activity during the development of larval fat body tissue. We developed a Drosophila-based ATF4 reporter that relies upon the 4EBP-intron ATF4-binding sites to drive the expression of dsRed, which we designated 4EBP-dsRed.
Using 4EBP-dsRed, I performed an in vivo RNAi screen to find new regulators of ATF4 in the Drosophila larval fat body. Among over 200 RNAi lines, 20 showed a decrease in the 4EBP-dsRed signal relative to wild-type larvae, implicating them as candidate positive regulators of ATF4. We have proceeded to validate these candidates through confocal microscopy and qPCR of endogenous 4EBP. By performing additional experiments in Drosophila tissue and cell culture, we aim to determine how these candidates regulate ATF4 and the ISR.
ISR dysregulation has been implicated in various diseases, including neurodegenerative disorders such as Charcot-Marie-Tooth disease. Identifying new positive regulators of ATF4 could provide a greater understanding of the ISR, potentially aiding the development of new drugs and other therapeutics.