N⁶-methyladenosine (m6A), a prevalent internal methylation pattern in mammalian mRNA, plays a pivotal role in various aspects of RNA biology. m6A modification has been implicated in the regulation of hematopoiesis and hematologic malignancies, particularly in the context of Acute Myeloid Leukemia (AML). However, the comprehensive understanding of m6A distribution and stoichiometry at a single-base resolution, along with its interaction with other RNA features, remains limited. Existing methodologies for m6A mapping face challenges in providing single-nucleotide resolution, creating a gap in our understanding of m6A variation within transcripts and its interconnectedness with other post-transcriptional regulatory mechanisms. To address these limitations, we employed nanopore direct RNA sequencing on the myeloid leukemia cell line MOLM13, obtaining a comprehensive and transcriptome-wide profiling of m6A stoichiometry and polyA tail lengths at the single-molecule level. Our study focused on the impact on the modifications and correlation with other RNA features when the m6A writer METTL3 is depleted, demonstrating its role in post-transcriptional regulations and their interplay within full-length mRNA molecules in AML cells. METTL3 deletion led to alterations in m6A levels, polyA tail lengths, and transcript expression patterns. Overall, this study provides a foundation for further investigations into the epitranscriptomic landscape of AML and opens avenues for targeted therapeutic interventions.
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