The transcriptional and splicing landscape of intestinal organoids undergoing nutrient starvation or endoplasmic reticulum stress

Jessica Tsalikis, Qun Pan, Ivan Tattoli, Charles Maisonneuve, Benjamin J. Blencowe, Dana J. Philpott, Stephen E. Girardin

The intestinal epithelium plays a critical role in nutrient absorption and innate immune defense. Recent studies showed that metabolic stress pathways, in particular the integrated stress response (ISR), control intestinal epithelial cell fate and function. Here, we used RNA-seq to analyze the global transcript level and alternative splicing responses of primary murine enteroids undergoing two distinct ISR-triggering stresses, endoplasmic reticulum (ER) stress and nutrient starvation. Our results reveal the core transcript level response to ISR-associated stress in murine enteroids, which includes induction of stress transcription factors, as well as genes associated with chemotaxis and inflammation. We also identified the transcript expression signatures that are unique to each ISR stress. Among these, we observed that ER stress and nutrient starvation had opposite effects on intestinal stem cell (ISC) transcriptional reprogramming. In agreement, ER stress decreased EdU incorporation, a marker of cell proliferation, in primary murine enteroids, while nutrient starvation had an opposite effect. We also analyzed the impact of these cellular stresses on mRNA splicing regulation. Splicing events commonly regulated by both stresses affected genes regulating splicing and were associated with nonsense-mediated decay (NMD), suggesting that splicing is modulated by an auto-regulatory feedback loop during stress. In addition, we also identified a number of genes displaying stress-specific splicing regulation. We suggest that functional gene expression diversity may arise during stress by the coordination of alternative splicing and alternative translation, and that this diversity might contribute to the cellular response to stress. Together, these results provide novel understanding of the importance of metabolic stress pathways in the intestinal epithelium. Specifically, the importance of cellular stresses in the regulation of immune and defense function, metabolism, proliferation and ISC activity in the intestinal epithelium is highlighted. Furthermore, this work highlights an under-appreciated role played by alternative splicing in shaping the response to stress and reveals a potential mechanism for gene regulation involving coupling of AS and alternative translation start sites.