Long-read transcriptomic profiling of a glioma cell line following m6A regulator perturbations

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification and is enriched in the central nervous system (CNS), yet its role in glioma remains incompletely defined. Using long-read direct RNA sequencing, we mapped transcriptome-wide m6A modifications in a single glioma cell line following targeted knockdown of the m6A reader IGF2BP2, writer METTL3, and eraser ALKBH5. Across perturbations, the global architecture of m6A, including transcript class, positional enrichment, and site multiplicity, was largely preserved, while differential methylation was weakly coupled to gene expression. In contrast, m6A regulator perturbation coincided with widespread isoform switching and with changes in untranslated regions, coding potential, and predicted transcript fate, largely independent of bulk gene expression changes. Public glioma datasets further supported the relevance of isoform-specific changes in glioma. Together, these findings highlight isoform-level transcript variation associated with m6A regulator perturbation and support the use of long-read, isoform-resolved approaches to study RNA regulatory states in glioma. • Long-read RNA sequencing was used to profile m6A in glioma cells. • Global m6A architecture remained broadly preserved across conditions. • m6A regulator perturbation coincided with widespread isoform changes. • External glioma datasets supported selected regulator and isoform patterns.