5-Fluorouracil Treatment Represses Pseudouridine-Containing miRNA Export into Extracellular Vesicles (RNA-seq)

5-Fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. We examined the impact of 5-FU on the expression and export of small RNAs into small extracellular vesicles (sEVs) and regulation of post-transcriptional small RNA modifications (PTxMs). EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular small RNAs provide yet another layer of gene regulation. We found that treatment of colorectal cancer (CRC) cells with 5-FU represses sEV export of miRNA and snRNA-derived RNAs, but promotes export of snoRNA-derived RNAs. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and sEV small RNA profiles. In contrast, 5-FU exposure led to increased levels of cellular small RNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modification, and mislocalization of sEV small RNAs.