Temporal placement of RNA modifications under 5-fluorouracil treatment in human cell culture

ABSTRACT Recent studies have explored the dynamic changes in RNA modification across various organisms, often in response to stressors like chemical agents or drugs. Among these, the anticancer drug 5-fluorouracil (5-FU) has been evaluated regarding its impact on RNA and DNA. Although different alterations in RNA modification have been associated with 5-FU, a mechanistic insight into the dynamic effects on RNA modification is missing. In this study, we provide a detailed insight into the dynamic modification changes for tRNA and rRNA of HEK293T cells exposed to 5-FU using Nucleic Acid Isotope Labeling coupled Mass Spectrometry (NAIL-MS). Consistent with existing literature, we observed a constant loss of m 5 U in newly transcribed tRNA during 5-FU exposure. In addition to m 5 U, we identified a loss of m 5 Um and an increase of Um within tRNA Lys UUU . While cells recover from a 24-hour 5-FU treatment, we found changed abundances of modifications like an increase of mcm 5 U and m 5 C and a decrease of m 7 G and m 1 A in total tRNA. Moreover, we demonstrate that 5-FU affects various rRNA subtypes, altering both their modification stoichiometry and abundance. Taken together, we use NAIL-MS to deconvolute RNA modification abundance changes caused by drug treatment and we uncover patterns of RNA modification adaptation.