Structure and Function of Kinase Family Receptors Regulating Translation

项目摘要 这项建议研究了两个相关的受体的结构和功能机制 蛋白激酶家族，核糖核酸酶L(AIMS 1，2)和GCN2(AIMS 3)。这些受体是普遍表达的 并调节蛋白质合成(翻译)以减轻细胞压力。核糖核酸酶L感觉 由双链RNA(DsRNA)引起的应激，而GCN2检测到由营养限制引起的应激。 哺乳动物细胞对dsRNA的存在高度敏感。DsRNA分子通常很少见， 但由于富含dsRNA的内源性重复序列的激增，在病毒感染的细胞和癌细胞中广泛存在。 元素，这使得研究dsRNA反应至关重要。然而，我们对 DsRNA-核糖核酸酶L途径。我们在这个提案的目标1和2中的目标是理解分子 全面调控核糖核酸酶的机制和广泛刻画翻译的新机制 由本实验室发现的核糖核酸酶L调控。这项研究具有生物医学意义，因为核糖核酸酶L，在 除了具有抗癌和抗病毒作用外，它还是一种抗脂肪受体，可能对治疗 以及预防传染病、肿瘤和代谢性疾病。在目标1中，我们将阐明 核糖核酸酶L通过获得核糖核酸酶L的潜伏期结构对其进行调控。我们的假设是，核糖核酸酶L 形成一个明确的潜在结构，限制不可控的核糖核酸酶L信号，以保护健康组织。我们 将使用低温电子显微镜(CRYO-EM)的结构分析来验证这一假设。在目标2中，我们将使用 细胞生物学、生物化学和我们小组开发的一种创新的RNA-SEQ方法来建立 核糖核酸酶的翻译调控机制2019年，我们发表了我们的发现，核糖核酸酶L执行 作为控制细胞内蛋白质合成的一种策略，主动翻译的mRNAs的核内裂解。 这项研究中的初步数据为这种核糖核酸酶的衰退提供了进一步的见解，并表明核糖核酸酶L展示了 对信使核糖核酸编码区的明显偏好，使我们产生了核糖核酸酶L的作用是 再加上翻译。我们将在我们的调查中彻底检验这一假设。 在目标3中，我们将工作扩展到调节全球翻译的相关受体GCN2。GCN2是一种 在人类基因组中，应激蛋白激酶位于核糖核酸酶L的近端。GCN2是 对蛋白质平衡、记忆功能和癌症新陈代谢很重要。GCN2是饥饿的感应器 一旦结合了未带电的tRNA，就会抑制全球蛋白质的合成，以缓解营养不足。这个 由于缺乏GCN2的结构信息，GCN2的激活机制尚不清楚 调节的tRNA敏感结构域。我们将使用低温电子显微镜来确定传感器结构域的结构。 我们的研究将提供新的知识，为建立一个全面的机制做出贡献 理解应激蛋白对翻译的调节作用。我们的工作也将贡献一种新的方法，LRtcB RNA-seq，作为研究哺乳动物信使核糖核酸衰变的工具。

Project Summary This proposal investigates structural and functional mechanisms of two related receptors from the family of protein kinases, RNase L (Aims 1, 2) and Gcn2 (Aim 3). These receptors are ubiquitously expressed in human tissues and regulate protein synthesis (translation) to mitigate cellular stresses. RNase L senses stress caused by double-stranded RNA (dsRNA), whereas Gcn2 detects stress caused by nutrient limitation. Mammalian cells are highly sensitive to the presence of dsRNA. DsRNA molecules are normally rare, but are widespread in virus-infected cells and in cancer cells due to upsurge of dsRNA-rich endogenous repeat elements, which makes studying dsRNA responses critically important. Yet, we have a poor understanding of the dsRNA-RNase L pathway. Our goal in Aims 1 and 2 of this proposal is to understand the molecular mechanism regulating RNase L generally and to extensively characterize the novel mechanism of translational regulation by RNase L, discovered by our laboratory. This work is biomedically significant because RNase L, in addition to having anticancer and antiviral roles, is an antilipogenic receptor that could be important for treating and preventing infectious, neoplastic, and metabolic illnesses. In Aim 1 we will elucidate the mechanism of RNase L regulation by obtaining the structure of RNase L in its latent state. Our hypothesis is that RNase L forms a defined latent structure that restricts uncontrollable RNase L signaling to protect healthy tissues. We will test this hypothesis using structural analysis by cryo electron microscopy (cryo-EM). In Aim 2, we will use cell biology, biochemistry and an innovative RNA-seq approach developed in our group to establish the mechanism of translation regulation by RNase L. In 2019, we published our discovery that RNase L performs endonucleolytic cleavage of actively translating mRNAs as a strategy to control cell-wide protein synthesis. Preliminary data in this proposal provide further insights into this mRNA decay and show that RNase L exhibits a distinctive preference for mRNA coding regions, leading us to the hypothesis that the action of RNase L is coupled to translation. We will test this hypothesis thoroughly in our investigation. In Aim 3, we extend our work to a related receptor regulating global translation, Gcn2. Gcn2 is a mechanistically poorly understood stress kinase located proximally to RNase L in the human kinome. Gcn2 is important for proteostasis, memory function, and cancer metabolism. Gcn2 serves as a sensor of starvation that, upon binding uncharged tRNAs, inhibits global protein synthesis to mitigate nutrient deficits. The mechanism of Gcn2 activation is poorly understood due to the absence of structural information about its regulatory tRNA-sensing domain. We will employ cryo-EM to determine the structure of the sensor domain. Our research will provide new knowledge and contribute to building a comprehensive mechanistic understanding of stress kinases regulating translation. Our work will also contribute a new method, LRtcB RNA-seq, as a tool for studying mammalian mRNA decay.