Ribonuclease function of APE1

生命系统中最基本的活动之一是将其遗传信息 (DNA) 表达为蛋白质，蛋白质是执行维持有机体生存的大部分生物功能的分子机器。 DNA 复制成蛋白质分两步进行：首先，DNA 复制成 mRNA（信使核糖核酸；化学性质与 DNA 相似）；其次，mRNA被用作模板来制造特定的蛋白质。 因此，对于给定基因，蛋白质的量取决于 mRNA 的量。 由于 mRNA 丰度的变化而导致的许多蛋白质的过量或短缺可能会导致疾病，例如癌症。 mRNA 的丰度高度依赖于其降解速度。 因此，了解mRNA降解机制的工作原理，对于提高我们的分子生物学基础科学知识，以及揭示一些人类疾病发病机制的信息至关重要。2009年，我们实验室偶然发现脱嘌呤/脱嘧啶核酸内切酶1（APE1），一种众所周知的DNA修复酶，具有体外降解特定mRNA的能力，并能控制细胞内特定mRNA的表达。 自从我们的开创性发现以来，至少有三种类似的酶被发现位于 DNA 和 RNA 代谢的联系中。 对于包括 APE1 在内的许多酶，其核糖核酸酶活性（切割和降解 RNA 的能力）的生物学意义仍然未知。我提出的研究的主要目标是了解内源形式以及最近发现的 APE1 分泌形式的核糖核酸酶功能。 我们将生成两种类型的细胞系统，它们被设计为携带野生型 APE1 及其变体，以帮助我们检验我们的假设。 我们将确定核糖核酸酶活性与野生型 APE1 及其变体对细胞活力的影响之间是否存在相关性（目标 I）。 使用 RNA-seq 技术，我们的目标是确定 APE1 核糖核酸酶活性的细胞 RNA 靶标（目标 I）。 我们将确定分泌的野生型 APE1 及其最常见的变体 D148E 是否具有核糖核酸酶活性，以及​​这是否对细胞活力和细胞死亡有任何影响（目标 II）。 我们将研究 APE1 控制肿瘤发生中重要的 RNA 结合蛋白的机制（目标 III）。这项研究有望深入了解 APE1 在哺乳动物细胞中的核糖核酸酶功能，这将有助于拓宽我们对基因表达调控以及 DNA 修复和 RNA 代谢过程如何联系的理解。

One of the most fundamental activities in living systems is to express their genetic information (DNA) into proteins, the molecular machines that carry out most of the biological functions that keep an organism alive. The copying of DNA into proteins occurs in two steps: first, DNA is copied into mRNA (messenger ribonucleic acid; chemically similar to DNA); second, mRNA is used as a template to make specific proteins. For a given gene, the amount of protein is therefore dependent on the amount of mRNA. An excess or shortage of many proteins due to changes in the abundance of mRNA can lead to diseases, such as cancer. The abundance of mRNAs is highly dependent on the rate of its degradation. Therefore, an understanding of how the mRNA degradation machinery works is critical for increasing our basic scientific knowledge of molecular biology, as well as for revealing information on the pathogenesis of some human diseases.In 2009 our lab fortuitously discovered that apurinic/apyrimidinic endonuclease 1 (APE1), an enzyme well-known to play a role in DNA repair, has an ability to degrade specific mRNAs in vitro and can control the expression of specific mRNA in cells. Since our pioneering discovery at least three similar enzymes have been discovered to be at the nexus of DNA and RNA metabolism. For many of these enzymes, including APE1, the biological significance of their enzymatic ribonuclease activity (ability to cleave and degrade RNA) is still unknown.The major goal of my proposed research is to understand the ribonuclease function of the endogenous form as well as the recently discovered secreted form of APE1. We will generate two types of cell systems engineered to carry the wild type of APE1 and its variants to help us test our hypotheses. We will determine whether there is correlation between the ribonuclease activity and impact on cell viability by wild-type APE1 and its variants (Aim I). Using a technology known as RNA-seq, we aim to determine the cellular RNA targets of the ribonuclease activity of APE1 (Aim I). We will determine whether the secreted wild-type APE1 and its most common variant D148E have ribonuclease activity and if this has any effect on cell viability and cell death (Aim II). We will investigate the mechanism whereby APE1 controls an important RNA-binding protein involved in tumorigenesis (Aim III).The proposed research is expected to provide insights into the ribonuclease function of APE1 in mammalian cells, which will help broaden our understanding of the regulation of gene expression and how the processes of DNA repair and RNA metabolism may be linked.