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

项目摘要 许多胃肠道疾病，包括炎症性疾病，都会出现肠上皮屏障的破坏。 肠道疾病、病原体感染、缺血再灌注损伤和放射性肠病。这道屏障 破裂启动来自肠道干细胞隔室的再生反应，同时引起 腔内微生物上皮移位引起的继发性炎症反应。尽管有必要和 由于这两个过程接近，人们对它们是如何协调的知之甚少。我们小组之前已经 确定MSI家族的RNA结合蛋白对于激活一种 静止的兼性肠干细胞(QISCs)池，介导肠道再生 受伤。我们发现MSI2结合了几个调控干细胞周期和新陈代谢的转录本，以及一个 一组上皮细胞特异的抗微生物和免疫调节转录本。因此，我假设MSI2 作为兼性干细胞激活的增强剂和免疫的抑制者起双重作用 根据细胞类型特定的RNA结合靶点对屏障破坏的反应。 在这项提议中，我将在小鼠中使用MSI2-HyperTRIBE融合蛋白来揭示MSI2 RNA结合 介导肠道再生的靶点(qISC特异性)和上皮对损伤的炎症反应 (特定于分泌细胞)。新型编辑识别RNA结合蛋白靶标技术 允许在非常小的细胞群体中识别蛋白质-RNA相互作用。我将使用这个技巧来 确定qISCs中MSI2 RNA结合靶点是如何响应辐射损伤的。我还将使用MSI2- HyperTRIBE小鼠确定MSI2-RNA结合伙伴在分泌谱系上皮细胞中的变化 (Paneth细胞和杯状细胞)，以响应假结核耶尔森菌感染的免疫挑战。 此外，我将进行体外功能分析，以评估MSI2转录结合如何影响翻译 再生和免疫相关候选基因。最后，我将测试MSI丢失的功能后果 假结核杆菌诱导的免疫激活试验对免疫功能的影响 Msi1/2上皮特异性条件性基因敲除小鼠的胃炎。这些研究将提供对MSI2如何 细胞类型的特定功能协调肠道损伤的再生和免疫反应 上皮组织。

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.