The Msi2 RNA binding protein coordinates epithelial regeneration and the inflammatory response to intestinal mucosal injury

项目摘要 肠上皮屏障的破坏发生在许多胃肠道疾病中，包括炎性胃肠道疾病。 肠道疾病、病原体感染、缺血再灌注损伤和放射性肠病。这个屏障 分解启动肠干细胞区室的再生反应， 一种继发于管腔微生物上皮移位的炎症反应。尽管必要性和 由于这两个过程非常接近，人们对它们是如何协调的知之甚少。我们的团队以前 确定了RNA结合蛋白的Msi家族对于激活一种细胞因子是必要的，也是足够的。 兼性肠干细胞（qISC）的静止池，其介导响应于 损伤我们发现Msi2结合了几种控制干细胞循环和代谢的转录物，以及一种抑制干细胞增殖的基因。 一组上皮特异性抗微生物和免疫调节转录物。因此，我假设Msi2 作为兼性干细胞激活的增强剂和免疫抑制剂， 基于细胞类型特异性RNA结合靶标的屏障破坏的应答。 在这个提议中，我将在小鼠中使用MSI2-HyperTRIBE融合蛋白来揭示Msi2 RNA结合 介导肠再生（qISC特异性）和上皮对损伤的炎症反应的靶点 （分泌细胞特异性）。新的HyperTRIBE（通过编辑识别的RNA结合蛋白的靶点）技术 允许在非常小的细胞群体中鉴定蛋白质-RNA相互作用。我会用这个技巧 确定Msi2 RNA结合靶点如何在qISC中响应辐射损伤而变化。我也会使用MSI2- HyperTRIBE小鼠确定Msi2-RNA结合伴侣在分泌谱系上皮细胞中如何变化 （潘氏和杯状细胞）响应假结核耶尔森氏菌感染的免疫攻击。 此外，我将进行体外功能测定，以评估Msi2转录本结合如何影响 再生和免疫相关的候选mRNA。最后，我将测试Msi丢失的功能后果 免疫功能的影响。假结核 Msi 1/2上皮特异性条件性敲除小鼠的胃炎。这些研究将深入了解Msi2 细胞类型特异性功能协调肠损伤的再生和免疫反应 上皮

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.