Exploring N6-Methyladenosine and long transcripts in glioma cells and extracellular vesicles

Glioma represents the most common primary central nervous system (CNS) neoplasm in adults. Previous studies have identified m ⁶ A as a key player in glioma pathogenesis, promoting cell stemness and tumor cell proliferation. However, there is also evidence of a potential anti-cancer role of m ⁶ A. Here, we present a comprehensive overview of the m ⁶ A landscape in a glioma cell line model using Nanopore direct RNA sequencing platform. The study investigates functional and biological impact of targeted knockdown (KD) of key m ⁶ A regulators (reader: IGF2BP2, writer: METTL3, eraser: ALKBH5). Transcriptome wide mapping of m ⁶ A demonstrated enrichment in CDS and 3’UTR regions. Differential patterns of non-DRACH motifs were observed across the KD conditions. Our analysis identified 716 common m ⁶ A sites across all conditions and clusters of KD specific modifications enriched for distinct functional pathways. IGF2BP2 KD was associated with pathways involving protein metabolism and apoptosis regulation. METTL3 KD was linked to cell cycle and stress response regulation and ALKBH5 KD impacted ribosome biogenesis and protein synthesis. Here, for the first time we also report detection of full-length RNA transcripts in extracellular vesicles (EVs) derived RNA from knockdown and control conditions. These findings underscore the unique contributions of each m ⁶ A regulator to glioma cell transcriptomics and suggest potential pathways for elucidating the overall role of m ⁶ A machinery in glioma pathogenesis. Important clinical applications include identification of novel diagnostic, prognostic, and therapeutic m ⁶ A RNA biomarkers.