Project Summary Breakdown of the intestinal epithelial barrier occurs in many gastrointestinal disorders including inflammatory bowel disease, pathogen infection, ischemia-reperfusion injury and radiation enteropathy. This barrier breakdown initiates a regenerative response from the intestinal stem cell compartment and simultaneously elicits an inflammatory response secondary to epithelial translocation of luminal microbes. Despite the necessity and proximity of these two processes, little is known about how they are coordinated. Our group has previously determined that the Msi family of RNA-binding proteins are both necessary and sufficient for activation of a quiescent pool of facultative intestinal stem cells (qISCs) which mediate intestinal regeneration in response to injury. We found that Msi2 binds several transcripts governing stem cell cycling and metabolism, as well as a group of epithelial-specific anti-microbial and immunomodulatory transcripts. Thus, I hypothesize that Msi2 performs dual functions as an enhancer of facultative stem cell activation and a repressor of the immune response to barrier disruption based on cell type specific RNA binding targets. In this proposal, I will employ a MSI2-HyperTRIBE fusion protein in the mouse to uncover Msi2 RNA binding targets that mediate intestinal regeneration (qISC-specific) and the epithelium’s inflammatory response to injury (secretory cell-specific). The novel HyperTRIBE (targets of RNA binding proteins identified by editing) technology allows for the identification of protein-RNA interactions in very small cell populations. I will use this technique to identify how Msi2 RNA binding targets change in qISCs in response to radiation injury. I will also use MSI2- HyperTRIBE mice to determine how Msi2-RNA binding partners change in secretory lineage epithelial cells (Paneth and goblet cells) in response to immune challenge by Yersinia pseudotuberculosis infection. Furthermore, I will perform in vitro functional assays to assess how Msi2 transcript binding affects translation of regenerative and immune related candidate mRNAs. Lastly, I will test the functional consequences of Msi loss on immune function by performing immune activation assays in response to Y. pseudotuberculosis induced gastritis in Msi1/2 epithelial-specific conditional knockout mice. These studies will provide insight into how Msi2 cell-type specific functions coordinate the regenerative and immune responses to injury in the intestinal epithelium.