Defining the biological roles of the 2',3'-cyclic phosphatases Angel1 and Angel2

摘要 保持细胞转录组的保真度对细胞的活力和生物功能至关重要。因此， 为了检测和降解异常的RNA转录物，细胞有许多机制。功能障碍 在这些机制中会在微观水平上导致细胞功能的生理问题，如果不加以检查， 会导致影响整个生物体的疾病状态。因此，我们理解这些是至关重要的。 核糖核酸质量控制的机制。近年来，RNA修饰已成为关键的调节因子 核糖核酸的功能。这些修饰之一是在编码和非编码RNA中都发现的3‘端修饰： 2‘，3’-环磷酸盐。2‘，3’-环磷酸参与了许多重要的生物途径； 然而，尽管这些环状磷酸盐中间体在RNA加工和质量中的重要性 控制，这种修饰的新陈代谢仍然知之甚少。最近，有两个事态发展 现在可以让我们了解2‘，3’-环磷酸盐的新陈代谢： Angel1和Angel2蛋白作为环磷酸酶的鉴定及2‘，3’-环的形成 基于磷酸的连接实验，它利用tRNA连接酶并使其能够识别被修饰的RNA 带有2‘，3’-环磷酸盐。而关于天使一号S和天使二号S活动的新信息为 环磷酸酶是令人兴奋的，但关于它们所起的生物学作用的信息仍然很少。 在2‘，3’-环磷酸盐代谢中。我在Lykke-Andersen实验室的研究试图通过 利用基于tRNA连接酶的捕获终止于2‘，3’-环磷酸的RNA。具体而言 目的1、明确Angel1‘S在核糖体相关质量控制途径中的生物学作用。上一首 Lykke-Andersen实验室进行的研究表明，Angel1与RQC和RQC的成员有关 EIF4E及其催化活性是RQC底物mRNAs衰变的限速因子。因此，我会 确定在RQC Angel1中作为环磷酸酶的作用位置，并定义其重要性 与eIF4E的相互作用和作为eIF4E抑制剂的潜在作用。此外，通过我的第二个目标，我将确定 Angel2在RNA加工中的生物学作用。先前的研究表明，Angel2的过度表达 导致tRNA连接酶底物的积累。因此，通过这个特定的目标，我将探索天使2的S 作为tRNA连接酶潜在抑制物的活性。我将再次利用tRNA连接酶为基础 捕获实验验证Angel2在tRNA连接酶途径中的靶点。通过识别和定义生物 Angel1和Angel2环磷酸酶的作用，这项研究将致力于 解开以前研究不足的2‘，3’-环磷酸盐在调节细胞的 转录组。

Abstract Maintaining the fidelity of the cell’s transcriptome is critical to a cell’s viability and biological function. As such, there are many mechanisms the cell has in order to detect and degrade aberrant RNA transcripts. Dysfunction in these mechanisms can lead to physiological issues in the cell’s function on a micro-level and if unchecked, can lead to disease states affecting the whole organism. Therefore, it is critical that we understand these mechanisms of RNA quality control. In recent years, RNA modifications have emerged as key regulators of RNA functions. One of these modifications is a 3’ end modification found in both coding and non-coding RNAs: the 2’,3’-cyclic phosphate. The 2’,3’-cyclic phosphate is involved in many important biological pathways; however, despite the importance of these cyclic phosphate intermediates in RNA processing and quality control, the metabolism of this modification is still poorly understood. Recently, two developments have emerged that now allow us to approach an understanding of the metabolism of the 2’,3’-cyclic phosphate: identification of two proteins, Angel1 and Angel2, as cyclic phosphatases and the development of a 2’,3’-cyclic phosphate-based ligation assay which utilizes tRNA ligase and makes it possible to identify RNAs modified with a 2’,3’-cyclic phosphate. While the new information regarding both Angel1’s and Angel2’s activities as cyclic phosphatases is exciting, there is still a paucity of information regarding the biological role that each play in 2’,3’-cyclic phosphate metabolism. My research in the Lykke-Andersen lab seeks to resolve these issues by taking advantage of the tRNA-ligase based capture of RNAs terminating in 2’,3’-cyclic phosphate. In specific aim 1, I will define Angel1’s biological role in the ribosome-associated quality control pathway (RQC). Previous studies conducted by the Lykke-Andersen lab reveal that Angel1 associates with members of the RQC and eIF4E and its catalytic activity acts as a rate-limiting factor for decay of RQC substrate mRNAs. Thus, I will identify where in the RQC Angel1 is acting as a cyclic phosphatase as well as define the importance of its interaction with and potential action as an inhibitor of eIF4E. Additionally, through my second aim I will identify the biological role of Angel2 in RNA processing. Previous studies have shown that overexpression of Angel2 causes accumulation of tRNA ligase substrates. Thus, through this specific aim, I will probe for Angel2’s activity as a potential inhibitor of tRNA ligase. I will once again take advantage of the tRNA ligase-based capture assay to verify targets of Angel2 in the tRNA ligase pathway. By identifying and defining the biological roles of the Angel1 and Angel2 cyclic phosphatase, the research proposed in this fellowship will work to unravel the previously understudied role that the 2’,3’-cyclic phosphate has in regulating the cell’s transcriptome.