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