117 Transcript-resolved mapping of the m6A RNA modification landscape in glioma using nanopore direct RNA sequencing

Abstract

Glioblastoma (GBM) remains uniformly fatal despite multimodal therapy. While DNA methylation profiling has transformed brain tumour classification, post-transcriptional RNA modifications remain comparatively underexplored. N6-methyladenosine (m6A) is the most abundant internal RNA modification and regulates splicing, nuclear export, translation and RNA stability. As a dynamic and reversible mark, m6A may enable glioma cells to rapidly remodel transcriptional networks in response to microenvironmental and therapeutic stress. We performed direct RNA sequencing using Oxford Nanopore Technologies to enable transcript-resolved detection of m6A within native RNA molecules. We analysed glioma-derived cell lines and primary tissue, including three low-grade IDH-mutant astrocytomas, three high-grade IDH-mutant astrocytomas, three glioblastomas and three representative normal brain samples. Modification-aware basecalling and transcript-level modelling were used to infer m6A status at DRACH consensus motifs. Differential methylation and isoform usage were analysed across tumour types and compared with normal brain. We generated transcriptome-wide m6A profiles from glioma tissues and cell lines, establishing reproducible modification signatures across samples. Comparative analysis identified candidate transcripts exhibiting altered m6A signal between tumour and normal brain. Differential modification patterns were observed across tumour types, alongside differences in isoform usage. Integration of modification calls with transcript structure highlights candidate genes in which m6A may contribute to transcript diversity in glioma. Direct RNA long-read sequencing enables transcript-resolved profiling of the glioma m6A landscape. Our data demonstrate tumour-associated variation in RNA modification patterns and support further investigation into the relationship between m6A regulation, RNA metabolism and glioma biology.