Abstract 2754: Long-read direct RNA sequencing identifies m3C modification sites in poly(A) RNAs from pancreatic cancer cells

Abstract Covalent modifications provide a precise and effective mechanism to regulate the biogenesis, stability, and function of biological molecules, including DNA, RNA, proteins, and lipids. More than 300 distinct chemical modifications have been identified in both coding and non-coding RNAs, forming a diverse RNA modification landscape known as the epitranscriptome. RNA modifications are mediated by three classes of proteins: writers, which add modifications; erasers, which remove them; and readers, which recognize and interpret modified RNAs. RNA modifications play critical roles in cell homeostasis and cell developmental processes by regulating gene expression, RNA metabolism, and translation into proteins. Dysregulation of RNA modifications has been linked to the pathogenesis of various human diseases, including cancer. Recent studies has shown that 3-methylcytidine (m3C) is primarily located at position 32 on the anticodon loop of certain transfer RNAs (tRNAs). This modification is catalyzed by m3C writers, such as METTL2A, METTL2B, METTL6, and METTL8. These writers and their cofactors have been associated with human diseases. m3C has also been detected in polyadenylated RNA (poly(A) RNA) at levels approximately 1000-fold lower than in tRNA through LC-MS/MS analysis. However, its transcriptome-wide distribution and functions remain unknown due to its low abundance and the absence of commercially available anti- m3C antibodies suitable for immunoprecipitation. Here, we demonstrated that METTL2A expression is upregulated in pancreatic tumor tissues and that high METTL2A levels are associated with poor prognosis in pancreatic cancer patients. To further investigate the role of METTL2A, we performed comparative direct RNA sequencing on the pancreatic cancer cell line AsPC-1 to identify potential METTL2A-mediated m3C sites in poly(A) RNA. These m3C sites were enriched in the CC motif and predominantly located in the coding sequences of mRNA, mitochondrial mRNA, and mitochondrial ribosomal RNA. Moreover, METTL2A downregulated m3C RNAs and altered the balance between specific isoforms containing METTL2A-mediated m3C sites by promoting exon inclusion and shifting isoform expression, underscoring a potential role of m3C in poly(A) RNA in regulating gene expression. In conclusion, our approach may contribute to uncovering the functions and characteristics of previously unknown RNA modifications regulated by various factors. Citation Format: Kenzui Taniue, Shuhei Mitsutomi, Anzu Sugawara, Kenji Takahashi, Yusuke Mizukami, Nobuyoshi Akimitsu. Long-read direct RNA sequencing identifies m3C modification sites in poly(A) RNAs from pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2754.