Functions of mRNA Pseudouridylation

项目总结 多种伪尿苷合成酶(PUS)与人类疾病有关，但其机制 将脓液活性丧失与线粒体肌病、消化障碍、智力障碍联系起来 残疾、对病毒感染的抵抗力、先天性角化不良和各种癌症仍然存在 很大程度上是未知的。在我们目前的知识中，有几个关键的空白 脓液蛋白。尽管PUS蛋白的基本生化活性在催化 尿苷异构化为假尿苷是众所周知的，大多数 人类脓液蛋白是未知的或不完全了解的。我们的长期目标包括 确定所有PUS蛋白的靶标并确定其分子后果 在与疾病相关的细胞环境中用假尿苷修饰特定的RNA。这将是 对于理解脓虚引起的疾病的病因至关重要，并可能揭示 治疗的新靶点。 我们最近的工作(Martinez等人摩尔电池2022)确定沉积了假性尿苷(Ψ 在新生人类Pre-mRNA的数千个位点上共转录 从出生到死亡影响到mRNA代谢的每一步。此续订申请将 严密研究假尿路调节基因表达的生化基础。这个 Ψ的生物效应必须源于U和Ψ之间的化学差异，这直接 影响RNA骨架构象、碱基对的稳定性和蛋白质的结合。我们的 初步数据表明，在实验中存在数百种假尿苷 确定调节RNA结合蛋白(RBPs)和微小RNAs(MiRNAs)的结合部位 在人的HepG2细胞中。Ψ广泛存在于RBP和miRNA结合部位 通过对人工假性DNA的生化研究，证明了 Ψ来确定是否以及在多大程度上将结合mrna-建立一个强大的 这是拟议工作的前提。我们将(1)确定m RNAΨ对功能的影响 与调节的RNA结合蛋白的相互作用，(2)确定假尿苷酸化在 MiRNA介导的基因调控，以及(3)定义受调控的RNA的贡献 假尿路作用于基因表达的调控。 总之，这项拟议的工作将阐明mrna的分子后果。 人细胞中的假尿嘧啶核糖核酸酶及其功能相关的信使核糖核酸 假尿苷合成酶。这将填补一个关键的知识空白，以了解RNA如何 修饰对人类健康控制基因表达至关重要的酶。

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 recent work (Martinez et al. Mol Cell 2022) established that pseudouridine (Ψ) is deposited co-transcriptionally in nascent human pre-mRNA at thousands of sites where it is poised to affect every step of mRNA metabolism from birth to death. This renewal application will rigorously investigate the biochemical basis for mRNA regulation by pseudouridylation. The biological effects of Ψ must originate in chemical differences between U and Ψ, which directly affect RNA backbone conformation, the stability of base pairs, and the binding of proteins. Our preliminary data establish that hundreds of pseudouridines are present within the experimentally determined binding sites of regulatory RNA-binding proteins (RBPs) and micro RNAs (miRNAs) in human HepG2 cells. Widespread occurrence of Ψ in RBP and miRNA binding sites—together with biochemical studies of artificially pseudouridylated RNAs that demonstrate the capacity of Ψ to determine whether, and to what extent, an mRNA will be bound—establish a strong premise for the proposed work. We will (1) Determine impact of mRNA Ψ on functional interactions with regulatory RNA-binding proteins, (2) Identify the roles of pseudouridylation in miRNA-mediated gene regulation, and (3) Define the contributions of regulated RNA pseudouridylation to the control of gene expression. Together, the proposed work will elucidate the molecular consequences of mRNA pseudouridylation in human cells and define functionally relevant mRNA targets of human pseudouridine synthases. This will fill a critical knowledge gap for understanding how RNA modifying enzymes that are essential for human health control gene expression.