4EHP and NELF-E regulate factors required for ATF4 expression in the Drosophila fat body

Cells adapt to proteostatic and metabolic stresses, in part, by triggering the phosphorylation of eIF2alpha and the subsequent synthesis of ATF4. eIF2alpha phosphorylation facilitates translational induction of ATF4 by regulating reinitiation of the scanning ribosomes at the ATF4 mRNA's 5' leader. In addition to eIF2alpha, ATF4 induction requires other regulators that remain poorly understood. Here, we report an ATF4 regulatory network consisting of eIF4E-Homologous Protein (4EHP), NELF-E, the 40S ribosome, and eIF3 subunits. Specifically, 4EHP was required for ATF4 signaling in the Drosophila larval fat body and in disease models associated with abnormal ATF4 signaling. 4EHP's mRNA cap-binding domain specifically interacted with NELF-E mRNA, which encodes a regulator of pol II-mediated transcription. Both NELF-E and 4EHP were required for the expression of ATF4 and enzymes that mediate the serine-one-carbon pathway. Knockdown of NELF-E also reduced several subunits of the 40S ribosome and the eIF3 translation initiation factor complex. Reduction of Rps12 and eif3h suppressed physiological ATF4 induction. These results uncover a previously unrecognized ATF4 regulatory network that impacts stress adaptation and pathological outcomes. Overall design: We knocked down 4EHP in the Drosophila fat body using the dcg-Gal4 driver that drived 4EHP RNAi (VDRC #38399). NELF-E was knocked down by the expression of NELF-E RNAi (VDRC #21009) with the dcg-Gal4 driver. To identify 4EHP binding mRNAs, we expressed 4EHP fused to ADAR in the fat body (using dcg-Gal4) and compared the extent of mRNA editing with a negative control 4EHP-ADAR that has an impaired mRNA cap-binding domain. The fat bodies were dissected from late third instar male larvae and the RNAs were extracted using Trizol. PolyA mRNA was sequenced through Novaseq 6000.