Mapping the m3C epitranscriptome in cancer

About This Grant

Mapping the m3C epitranscriptome in cancer Project Summary RNA modifications are critical regulators of gene expression, with over 170 types identified across RNA molecules. Among these, m6A, m5C, and m1A are quite extensively studied for their roles in mRNA transcription, stability, and translation. In contrast, 3-methylcytidine (m3C) has been predominantly associated with tRNA modification and function, and is required for normal mRNA translation. The potential role of m3C in mRNA remains poorly understood and controversial due to conflicting experimental evidence. Mass spectrometry has suggested the presence of m3C in poly(A)-enriched RNA, but transcriptome-wide studies using chemical cleavage and next-generation sequencing have yet to provide definitive confirmation. Current methods like HAC- seq, ARM-seq, and DM-tRNA-seq are effective for tRNA modifications but inadequate for low-abundance or sub- stoichiometric m3C in mRNA. To address these limitations, this proposal will establish a highly sensitive and precise method (HARP-seq) to map m3C modifications in mRNA at single-nucleotide resolution. By integrating advanced chemical labelling & enrichment strategie, high-throughput sequencing, and bioinformatics analysis, this research will comprehensively characterize the distribution of m3C in mRNA. We will apply the newly developed method to investigate m3C modifications in mRNA across various cancer cell lines, primary tumor samples, and matched normal tissues. These findings will not only resolve current controversies regarding m3C's presence in mRNA but may provide insights into cancer-associated m3C epitranscriptomic changes and advance our comprehension of epitranscriptomic contributions to oncogenesis.