Single-cell and spatial epitranscriptomics of m⁶A: Technologies, regulatory mechanisms, and biological insights

Transcription serves as the primary mRNA blueprint for gene expression; however, transcription function is further regulated by co-transcriptional and post-transcriptional mechanisms, including N6-methyladenosine (m⁶A), the most widespread internal mRNA modification. The mammalian m⁶A landscape is primarily established by the METTL3-METTL14 writer complex, with METTL3 serving as its catalytic subunit, while accessory writer components, erasers, and readers contribute to the regulation and interpretation of m⁶A marks. Conventional bulk sequencing averages m⁶A profiles across large cell populations, masking cell- and tissue-specific heterogeneity in m⁶A deposition and interpretation. Studies show that the functional outcome of METTL3-mediated m⁶A deposition depends on the co-expression of the reader and eraser proteins in the same cell, and that these vary across cell types, developmental stages, and tissue compartments. Recent single-cell and spatial epitranscriptomic technologies are beginning to resolve this context-dependent regulation, revealing that m⁶A can promote mRNA decay, translation, stabilization, or altered splicing depending on cellular context. In this review, we discuss emerging technologies for single-cell and spatially resolved m⁶A profiling, using METTL3 as a conceptual framework for understanding how m⁶A deposition is linked to transcriptional state and subsequently decoded by cell-specific regulatory machinery. We further discuss their applications, technological challenges, multi-omics integration, and clinical potential in development, immunity, neuronal biology, and cancer.