Metabolic Salvage of Epigenetically Modified Pyrimidine Nucleosides

Abstract

Nucleoside salvage, an energy-efficient metabolic alternative to the de novo synthesis pathway in cells, presents a mechanism in which chemically modified RNA nucleosides can be metabolized and potentially incorporated into nascent nucleic acid strands. Alterations of RNA modifications can cause severe disruptions to protein synthesis and are implicated in numerous disease phenotypes. In particular, the rate-limiting pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2) displays potential for substrate promiscuity towards modified pyrimidines. This study aimed to investigate the physiological role of UCK2 in cancer cell metabolism and the effects of the kinase’s mediation of chemically and epigenetically modified nucleoside salvage in cells. The growth of a developed UCK2 knockout cell line in HeLa was assessed through cell viability assays in various metabolic conditions to understand the enzyme’s role in cell proliferation. Through the generation of survival curves for various modified pyrimidines in WT and UCK2 KO cells and LC-QQQ-MS quantitation of total RNA, we probed the structure-activity relationships of these potential modified nucleoside substrates and characterized the role of UCK2 in their metabolism. We find that UCK2 is vital for cell growth amidst diverse metabolic demands and that UCK2 expression mediates the toxicity of many modified pyrimidines of diverse chemical properties. Moreover, our study provides evidence for the role of UCK2 in the salvage and incorporation of modified nucleosides into RNA. These results provide valuable information about the role of pyrimidine salvage in modified nucleoside toxicity and cellular metabolism while raising new mechanistic questions.