Functions of mRNA Pseudouridylation

项目摘要 多种假尿苷合酶（PUS）与人类疾病有关，但 将PUS活性丧失与线粒体肌病，消化系统疾病， 智力残疾、抵抗病毒感染、先天性角化不良和各种癌症 仍然在很大程度上未知。我们目前对生物多样性的了解存在一些关键差距 PUS蛋白的功能。虽然PUS蛋白在催化细胞凋亡中的基本生化活性 尿苷到假尿苷的异构化是很好理解的， 大多数人PUS蛋白是未知的或不完全已知的。我们的长期目标包括 鉴定所有PUS蛋白的靶点，并确定PUS蛋白的分子结果。 在疾病相关的细胞环境中用假尿苷修饰特异性RNA。这将 对于了解由PUS缺乏引起的疾病的病因至关重要，并可能揭示 新的治疗靶点。 我们以前的研究表明，假尿苷是一种普遍的修饰新生 前信使RNA。这些结果以及体外定量研究表明， 假尿苷可以影响RNA-蛋白质和RNA-RNA相互作用，导致我们的中枢神经系统 假设前体mRNA假尿苷化在以下水平控制人类基因表达 前mRNA剪接。为了支持这一假设，我们已经证明，一个前， mRNA修饰假尿苷合酶PUS 1引起前mRNA的广泛变化 在人类细胞中进行剪接，有超过3，000个PUS 1敏感的选择性剪接事件 鉴定我们的具体目标是：（1）确定剪接相关的前mRNA靶点， 主要的前mRNA假尿苷化酶：PUS 1、PUS 7和RPUSD 4;和（2） 阐明假尿苷敏感剪接的分子机制。 拟议的工作将提供关键的深入了解的分子功能 pseudouridines在pre-mRNAs和可能揭示真核基因调控的新模式。通过 建立前mRNA作为一个广泛的新类底物的PUS酶，我们的工作 暗示有缺陷的剪接是一个合理的，但未充分研究的机制， 许多人类疾病的活动。

PROJECT SUMMARY Multiple pseudouridine synthases (PUS) are implicated in human disease, but the mechanisms that connect loss of PUS activity to mitochondrial myopathy, digestive disorders, intellectual disability, resistance to viral infection, dyskeratosis congenita, and diverse cancers remain largely unknown. There are several critical gaps in our current knowledge of the functions of PUS proteins. Although the basic biochemical activity of PUS proteins in catalyzing the isomerization of uridine to pseudouridine is well understood, the specific RNA targets of most human PUS proteins are unknown or incompletely known. Our long-term goals include identifying the targets of all PUS proteins and determining the molecular consequences of modification of specific RNAs with pseudouridine in disease-relevant cellular contexts. This will be critical for understanding the etiology of diseases caused by PUS deficiency and may reveal new therapeutic targets for treatment. Our previous studies revealed that pseudouridine is a prevalent modification of nascent pre-messenger RNA. These results, together with quantitative in vitro studies showing that pseudouridine can affect both RNA-protein and RNA-RNA interactions, lead to our central hypothesis that pre-mRNA pseudouridylation controls human gene expression at the level of pre-mRNA splicing. In support of this hypothesis, we have demonstrated that loss of one pre- mRNA modifying pseudouridine synthase, PUS1, causes widespread changes to pre-mRNA splicing in human cells, with more than 3,000 PUS1-sensitive alternative splicing events identified. Our Specific Aims are to (1) Define the splicing-relevant pre-mRNA targets of the predominant pre-mRNA pseudouridylating enzymes: PUS1, PUS7, and RPUSD4; and (2) Elucidate the molecular mechanisms of pseudouridine-sensitive splicing. The proposed work will provide key insight into the molecular functions of pseudouridines in pre-mRNAs and may reveal novel modes of eukaryotic gene regulation. By establishing pre-mRNAs as a broad new class of substrates for PUS enzymes, our work implicates defective splicing as a plausible but understudied mechanism connecting loss of PUS activity to numerous human diseases.