Thymidylate synthase RIP-seq from DNA-repair-deficient HCT116 cells treated with RTX or 5FdUR [RIP-Seq]

Inhibition of TS disrupts the dUTP:dTTP balance, leading to uracil misincorporation, futile base excision repair cycles, DNA strand breaks, and ultimately cell death. Beyond its catalytic role, TS also binds RNA, autoregulating its own translation and interacting with transcripts such as p53 and c-myc, thereby linking TS activity to broader post-transcriptional regulatory networks.

To better understand how TS inhibitory drugs influence this network, we performed TS-coupled RNA-coimmunoprecipitation (TS-RIP) and sequencing from mismatch repair-deficient, UNG-inhibited HCT116 colon cancer cells treated with 5FdUR or RTX. TS-RIP revealed RNA-binding targets of TS, in addition to the known p53 and TS mRNAs. Our findings underscore the multifaceted impact of TS inhibition, linking enzymatic disruption to RNA-level regulation and revealing drug-specific differences in cellular responses.