Nanopore direct RNA sequencing reveals METTL2A-mediated m3C sites in poly(A) RNA
- Shuhei Mitsutomi1,2,
- Anzu Sugawara1,
- Masahide Seki3,
- Yutaka Suzuki3,
- Sotaro Miyao1,
- Haozhe Du1,
- Kenji Takahashi4,5,
- Yusuke Mizukami4,5,
- Kenzui Taniue1,4 and
- Nobuyoshi Akimitsu1
- 1Isotope Science Center, The University of Tokyo, Tokyo, 113-0032, Japan;
- 2Research Institute, National Cancer Center, Tokyo, 104-0045, Japan;
- 3Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 277-8561, Japan;
- 4Division of Gastroenterology, Department of Medicine, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan;
- 5Center for Intractable Diseases and ImmunoGenomics, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan
Abstract
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 (m3C) primarily occurs in transfer RNA, where it regulates translation, stem cell pluripotency, and mitochondrial function. m3C 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 METTL2A, an m3C 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 m3C sites in poly(A) RNA. These m3C sites are mapped in both messenger RNA and mitochondrial RNA and are enriched in the CC motif and coding sequences. METTL2A knockdown alters expression of S100A4 mRNA isoforms, which contains METTL2A-mediated m3C sites. Notably, many transcripts with METTL2A-mediated m3C sites are upregulated upon METTL2A knockdown. We reveal the transcriptome-wide presence of m3C sites in poly(A) RNA and suggest their potential roles in regulating gene expression.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.280269.124.
- Received November 24, 2024.
- Accepted September 26, 2025.
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