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

定义 2,3-环状磷酸酶 Angel1 和 Angel2 的生物学作用

• 批准号: 10752449
• 负责人: Megan Elizabeth Dowdle
• 金额: $ 7.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2026-07-16
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752449
• 关键词: Affect; Biochemical; Biological; Biological Assay; Biological Process; Cell Survival; Cell physiology; Cells; Code; DNA; Development; Disease; Ensure; Eukaryota; Family; Fellowship; Functional disorder; Gene Silencing; Genes; Goals; Ligation; Mediating; Messenger RNA; Metabolism; Modification; Pathway interactions; Peptide Initiation Factors; Periodicity; Phosphoric Monoester Hydrolases; Physiological; Play; Process; Proteins; Quality Control; RNA; RNA Processing; RNA Splicing; RNA Stability; Regulation; Reporter; Repression; Research; Ribosomes; Role; Site; Transcript; Transfer RNA; Translating; Translations; U6 small nuclear RNA; Untranslated RNA; Whole Organism; Work; XBP1 gene; inhibitor; inorganic phosphate; knock-down; member; overexpression; prevent; repaired; response; tRNA Ligase; transcriptome

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.

抽象的 维持细胞转录组的保真度对于细胞的活力和生物功能至关重要。 细胞有许多机制来检测和降解异常的 RNA 转录本。 这些机制可能会导致细胞功能在微观层面出现生理问题，如果不加以控制， 可能导致影响整个有机体的疾病状态，因此，我们了解这些至关重要。 近年来，RNA 修饰已成为 RNA 质量控制的关键调节因子。 RNA 功能之一是在编码和非编码 RNA 中发现的 3' 末端修饰： 2',3'-环磷酸盐 2',3'-环磷酸盐参与许多重要的生物途径； 然而，尽管这些环状磷酸中间体在 RNA 加工和质量中很重要 控制，这种修饰的代谢仍然知之甚少，最近有两个进展。 现在，我们可以了解 2',3'-环磷酸盐的代谢： 鉴定出两种蛋白质 Angel1 和 Angel2 为环状磷酸酶，并开发了 2',3'-环状磷酸酶 基于磷酸盐的连接测定，利用 tRNA 连接酶，可以识别修饰的 RNA 具有 2',3'-环磷酸酯，而有关 Angel1 和 Angel2 活性的新信息为 环磷酸酶令人兴奋，但有关每种酶所发挥的生物学作用的信息仍然很少 我在 Lykke-Andersen 实验室的研究旨在通过 2',3'-环磷酸盐代谢来解决这些问题。 利用基于 tRNA 连接酶的以 2',3'-环磷酸盐终止的 RNA 捕获。 目标 1，我将定义 Angel1 在核糖体相关质量控制途径 (RQC) 中的生物学作用。 Lykke-Andersen 实验室进行的研究表明，Angel1 与 RQC 成员有联系，并且 eIF4E 及其催化活性充当 RQC 底物 mRNA 衰变的限速因素。 确定 RQC Angel1 中的何处充当环磷酸酶并定义其重要性 此外，通过我的第二个目标，我将确定与 eIF4E 的相互作用以及作为 eIF4E 抑制剂的潜在作用。 先前的研究表明Angel2在RNA加工中的生物学作用。 导致tRNA连接酶底物的积累，因此，通过这个具体目标，我将探究Angel2的情况。 作为 tRNA 连接酶的潜在抑制剂，我将再次利用基于 tRNA 连接酶的优势。 通过识别和定义生物学来验证 Angel2 在 tRNA 连接酶途径中的靶点。 Angel1 和 Angel2 环磷酸酶的作用，本奖学金提出的研究将致力于 揭示了 2',3'-环磷酸在调节细胞中的先前未被充分研究的作用 转录组。