EXPLORING THE GLIOMA M6A RNA EPITRANSCRIPTOME WITH DIRECT RNA SEQUENCING

Abstract AIMS Despite aggressive treatment, glioblastoma patients suffer from an invariably poor prognosis. Whilst substan- tial progress has been made in understanding the role of DNA methylation in brain tumour pathology, methy- lation of RNA has received comparatively little attention, despite being a key driver of cellular differentiation and neurodevelopment. N6-methyladenosine (m6A) is the most abundant RNA modification and is, crucially, reversible. The immense intra-tumoral heterogeneity and phenotypic plasticity of glioblastoma are major barri- ers to successful treatment, and dynamic m6A methylation may mediate such behaviour, by flexibly influencing transcriptional networks via the regulation of mRNA metabolism. METHODS Nanopore sequencing technology uniquely enables direct sequencing of RNA, and therefore detection of RNA modifications within their sequence context. We present nanopore direct RNA sequencing data from four in- house glioma-derived cell lines. We validate this approach using a novel drug inhibitor of the major m6A methyl- transferase, METTL3. RESULTS We define transcriptome-wide RNA methylome profiles for four cell lines and identify common candidate path- ways which exhibit methylation. We confirm robust reduction in m6A marks following treatment with a novel METTL3 inhibitor. We further compare the transcriptional landscape of wild-type tumours against these treated samples, demonstrating differential expression of biological processes linked to RNA metabolism. CONCLUSION We demonstrate that nanopore direct RNA sequencing can resolve m6A modifications at single-base resolu- tion, preserving m6A stoichiometry. We validate this approach with a known inhibitor of METTL3, and further identify differentially expressed pathways in response to such inhibition. We show that glioma cell lines demon- strate extensive methylation throughout the transcriptome, and exhibit a degree of inter-tumoral heterogeneity of these m6A signatures.