The regulation of Xrp1 expression by uORFs and main ORF sequences and its function in Drosophila disease models

The Integrated Stress Response (ISR) mediates cellular adaptation to endoplasmic reticulum (ER) stress, amino acid deprivation, and mitochondrial dysfunction. The ISR regulates gene expression in part by preferentially translating the transcription factor ATF4, a process regulated by upstream open reading frames (uORFs) in its 5' leader. In Drosophila, Xrp1 is another transcription factor induced during the ISR, but the precise underlying mechanism remains unclear. Here, we report that Xrp1 induction in response to ER stress is regulated by both its uORFs and the main ORF sequence. Xrp1 has seven splice isoforms, and the two predominant transcripts expressed in eye imaginal discs contain uORFs. Expressing the ER stress-imposing ninaEG69D transgene in this tissue induced Xrp1 expression without significantly changing the Xrp1 splice isoform composition. The uORF-containing 5' leaders, particularly the AUG codon of the second uORF, inhibited DsRed expression when placed upstream of the reporter. Unlike ATF4, the uORF-containing 5' leader alone was insufficient to mediate the main ORF induction, but Xrp1 induction occurred in ninaEG69D-expressing discs when Xrp1's 5' leader and the main ORF sequence were both present. Functionally, Xrp1 was required to maintain the integrity of Drosophila photoreceptors exposed to constant light. In a different disease model, parkin mutants activated Xrp1 target gene expression in specific tissues and Xrp1 loss enhanced the viability of parkin mutant flies during adult eclosion. These results provide molecular and pathological insights into Xrp1 regulation and function in disease models.