Functional Characterization of the Pseudouridine Synthase PUS10

项目摘要 炎症性肠病（IBD）是胃肠道的慢性炎症性疾病，影响肠道的功能。 全球数百万人。大多数患有IBD的患者表现出慢性活动性疾病， 症状，代表了一个明确的未满足的需要，以促进我们对IBD病理学的理解，并确定新的 治疗目标多个全基因组关联研究已鉴定假尿苷合酶10 （PUS10）作为IBD的易感基因。PUS10编码一种酶，负责安装修饰的 核苷酸假尿苷（pseudouridine，简称pDR）在人类tRNA的一个子集，并可能靶向mRNA以及。人士 IBD相关的PUS10等位基因表达降低，提示PUS10依赖性的转录因子表达的缺失可能与IBD的发病有关。 其靶RNA中的一种或多种是其在IBD中的作用的基础。然而，PUS10- 依赖性假尿苷化是未知的。值得注意的是，我的初步分析表明，失去PUS10 活性启动上皮向间质转化（EMT），这是一种促纤维化细胞过程， 在IBD中失调，并且已知有助于其他慢性炎性疾病的发病机制。我 这项工作旨在阐明PUS10依赖性假尿苷化的分子功能，并确定其在以下方面的作用： 急救小组启动。 它对编码和非编码RNA都有深远的影响，影响RNA的加工、衰变和降解。 翻译--为PUS10调节基因表达建立了广泛的潜力。我将开始描述 PUS10的作用通过全面绘制和定量PUS10依赖性转录组， 使用增强的DNA谱分析方法，以确保鉴定可能的疾病相关底物（目的1）。 随着PUS10靶位点的确定，我将系统地分析PUS10依赖性转录因子对tRNA的影响， 功能和基因表达，使用具有时间控制PUS10活性的工程化细胞系， 区分tRNA和mRNA假尿苷酸化的直接作用（目的2）。然后我会把这些 IBD相关细胞生理学的分子扰动-直接鉴定EMT的精确方面 受PUS10的假尿苷化活性调节（Aim 3）。 完成拟议的工作将大大推进我们对IBD的认识， 危险基因PUS10。这些研究将全面确定PUS10的分子后果- 依赖性假尿苷化，并描绘其在EMT的调节作用-一个细胞过程中的显着 与IBD病理学的临床相关性。

PROJECT SUMMARY Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract affecting millions of individuals worldwide. The majority of patients afflicted with IBD exhibit chronically active disease symptoms, representing a clear unmet need to advance our understanding of IBD pathology and identify new therapeutic targets. Multiple genome-wide association studies have identified pseudouridine synthase 10 (PUS10) as a susceptibility gene for IBD. PUS10 encodes an enzyme responsible for installing the modified nucleotide pseudouridine (Ψ) in a subset of human tRNAs and is likely to target mRNA as well. Individuals with IBD-associated PUS10 alleles exhibit decreased expression, suggesting that loss of PUS10-dependent Ψ in one or more of its target RNAs underlies its role in IBD. However, the functional consequences of PUS10- dependent pseudouridylation are unknown. Notably, my preliminary analysis demonstrates that loss of PUS10 activity initiates an epithelial-to-mesenchymal transition (EMT), a pro-fibrotic cellular process that is dysregulated in IBD and known to contribute to the pathogenesis of other chronic inflammatory disorders. My work seeks to elucidate the molecular function of PUS10-dependent pseudouridylation and pinpoint its roles in EMT initiation. Ψ has profound impacts in both coding and non-coding RNA, affecting RNA processing, decay, and translation—establishing a broad potential for PUS10 to regulate gene expression. I will begin to characterize the role of PUS10 by comprehensively mapping and quantifying PUS10-dependent Ψ transcriptome-wide, using augmented Ψ profiling methods to ensure possible disease-relevant substrates are identified (Aim 1). With PUS10 target sites defined, I will systematically profile the effects of PUS10-dependent Ψ on tRNA function and gene expression, using engineered cell lines with temporal control of PUS10 activity to discriminate between the direct effects of tRNA and mRNA pseudouridylation (Aim 2). I will then relate these molecular perturbations to IBD-relevant cellular physiology—identifying the precise aspects of EMT directly regulated by PUS10’s pseudouridylation activity (Aim 3). Completion of the proposed work will significantly advance our understanding of the poorly characterized IBD risk gene PUS10. These studies will comprehensively determine the molecular consequences of PUS10- dependent pseudouridylation and delineate its role in the regulation of EMT—a cellular process of significant clinical relevance to IBD pathology.