5-Fluorouridine leaves ribosomal RNA scars discovered by nanopore direct RNA sequencing

5-Fluorouracil (5-FU) is an anticancer drug that is metabolically activated to disrupt the functions of DNA and RNA. It remains a mystery how 5-FU in cells impacts the ribosomal RNA modification landscape at nucleotide resolution. Nanopore direct RNA sequencing was used to monitor rRNA modifications in E. coli and human cells exposed to 5-fluorouridine (5-FrU). Sequencing synthetic RNA standards identified that 5-FrU is nearly indistinguishable from U by ionic current and is miscalled as Ψ by the Ψ-aware base-calling model at a low but above-background rate (7.5% on average), resulting in Ψ losses being modestly underestimated by the sequencing method. In E. coli, 5-FrU slowed growth and decreased rRNA modification, most strongly at Ψ residues and also at m5U, Um, m4Cm, and m62A sites. At a subset of 23S Ψ sites, 5-FrU produced ionic current populations distinct from controls comprised of the parent nucleotide U or the substrate Ψ, found in the wild-type E. coli sample; these new signals suggest the cellular pseudouridine synthases attempted catalysis at this site but could not complete the isomerization reaction. In human HEK293T cells, 5-FrU decreased modification levels broadly across Ψ, Nm, m5C, and m6A in the 18S, 28S, and 5.8S rRNAs, with variation from position to position. Additionally, increased global base-calling errors were found, which are consistent with 5-FrU interference in RNA base calling and the drug-induced cellular RNA base damage. These data show that fluoropyrimidine exposure leaves site-specific scars in rRNA modification patterns that nanopore sequencing can detect directly.