Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer.

Post-transcriptional modifications on mRNA are crucial for mRNA fate and function. The current lack of a comprehensive method for high-coverage and sensitive quantitative analysis of mRNA modifications significantly limits the discovery of new mRNA modifications and understanding mRNA modifications' occurrence, dynamics and function. Here, a highly sensitive, high-throughput and robust LC-MS/MS-based technique, mRQuant, was developed to simultaneously detect and quantify 84 modified ribonucleosides in cellular mRNA. Using mRQuant, we quantified 32-34 modified ribonucleosides across several human cancer and non-cancer cell lines and uncovered cancer- and cancer type-specific signatures. Analyses of cisplatin- and paclitaxel-treated HeLa cells and drug-resistant variants revealed several drug resistance-associated modifications. Among them, m¹A exhibited significant differences across multiple cell types and between cancerous and non-cancerous cells. Knocking down mRNA m¹A writer or eraser protein resulted in altered cell viability, cell cycle and apoptosis in HeLa cells, suggesting a role of mRNA m¹A in cancer. Transcriptomic and proteomic analyses further revealed the molecular mechanisms underlying the phenotypic variation.