Purpose: Introduction Glioblastoma carries an invariably poor prognosis despite multimodal therapy. While DNA methylation has transformed the molecular classification of brain tumours, RNA methylation remains relatively unexplored, despite its central role in neurodevelopment and cell-state regulation. N6-methyladenosine (m6A) is the most abundant internal RNA modification and influences nuclear export, splicing, translation, and RNA stability. Because m6A is dynamically regulated, it may enable cells to rapidly remodel transcriptional programmes in response to microenvironmental cues. Objectives Glioblastoma exhibits marked intra-tumoral heterogeneity and phenotypic plasticity, promoting recurrence after treatment. We hypothesise that dynamic m6A regulation contributes to these processes by modulating RNA metabolism. Our aim is to map m6A-modified transcripts and pathways in glioma to identify mechanisms that may underlie therapy resistance.

Methods: Nanopore long-read sequencing enables direct interrogation of native RNA and the detection of RNA modifications within their sequence context. Using this technology, we profiled the m6A methylome of in-house glioma-derived cell lines to identify modification-enriched genes and pathways. We extended these analyses to patient samples, comparing direct RNA sequencing data from 6 astrocytomas, 4 glioblastomas, and 3 normal brain tissues. Results We generated high-resolution RNA methylome maps from glioma tissues and identified pathways displaying differential m6A methylation between tumour and normal brain. Comparative analysis of astrocytoma and glioblastoma revealed distinct transcriptional signatures and differential isoform usage associated with m6A status.