Functional Characterization of the Pseudouridine Synthase PUS10

项目总结 炎症性肠病是胃肠道的慢性炎症性疾病，影响 全世界数以百万计的人。大多数患有IBD的患者表现出慢性活动性疾病。 症状，代表着一种明显的未得到满足的需求，以促进我们对IBD病理的理解并识别新的 治疗靶点。多项全基因组关联研究已确定假性尿苷合成酶10 (PUS10)是IBD的易感基因。PUS10编码一种酶，负责安装经过修饰的 核苷酸假尿苷(Ψ)在人类tRNA的一个子集中，也可能以mR NA为靶标。具有以下特征的个人 与IBD相关的PUS10等位基因表达降低，表明依赖于PUS10的Ψ在 它的一个或多个靶RNA是其在IBD中作用的基础。然而，PUS10的功能后果是- 依从性假性尿路尚不清楚。值得注意的是，我的初步分析表明，10美元的损失 活动启动上皮向间充质转化(EMT)，这是一个促纤维化的细胞过程，是 在IBD中调节失调，已知与其他慢性炎症性疾病的发病机制有关。我的 这项工作旨在阐明PUS10依赖的假尿苷酸化的分子功能，并精确定位其在 EMT启动。 Ψ对编码和非编码的核糖核酸都有深远的影响，影响核糖核酸的处理、衰变和 翻译--为PUS10调控基因表达奠定了广阔的潜力。我将开始描述 PUS10通过全面定位和量化依赖于PUS10的Ψ在整个转录组中的作用， 使用增强型Ψ分析方法确保确定可能与疾病相关的底物(目标1)。 在定义了PUS10靶点之后，我将系统地描述依赖PUS10的Ψ对tRNA的影响 功能和基因表达，使用具有时间控制的PUS10活性的工程细胞系来 区分tRNA和信使核糖核酸假尿酸的直接作用(目标2)。然后，我将讲述这些 IBD相关细胞生理学的分子扰动--直接识别EMT的精确方面 受PUS10‘S假尿酸活性调节(目标3)。 拟议工作的完成将大大促进我们对特征不佳的IBD的了解 风险基因PUS10。这些研究将全面确定PUS10的分子后果- 依赖性假尿路反应及其在EMT调节中的作用--这是一个有意义的细胞过程 与IBD病理的临床相关性。

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.