Nanopore direct RNA sequencing reveals METTL2A-mediated mC sites in poly(A) RNA.

RNA modifications play critical roles in cellular homeostasis and development by regulating gene expression, RNA metabolism, and translation. Their dysregulation contributes to the development of human diseases, including cancer. 3-methylcytidine (mC) primarily occurs in transfer RNA, where it regulates translation, stem cell pluripotency, and mitochondrial function. mC has also been detected in polyadenylated (poly[A]) RNA by mass spectrometric analysis; however, its transcriptome-wide distribution and functions remain unknown because of its low abundance and technical challenges. Here, we show that , an mC writer, is upregulated and associated with poor prognosis in pancreatic cancer tumors, while also being essential for pancreatic cancer cell proliferation. Using comparative nanopore direct RNA sequencing, we identify potential METTL2A-mediated mC sites in poly(A) RNA. These mC sites are mapped in both messenger RNA and mitochondrial RNA and are enriched in the CC motif and coding sequences. knockdown alters expression of mRNA isoforms, which contains METTL2A-mediated mC sites. Notably, many transcripts with METTL2A-mediated mC sites are upregulated upon knockdown. We reveal the transcriptome-wide presence of mC sites in poly(A) RNA and suggest their potential roles in regulating gene expression.