Isoform-level profiling of m⁶A epitranscriptomic signatures in human brain

The RNA modification N6-methyladenosine (m⁶A) is highly abundant in human brain and implicated in neurological disorders. Profiling m⁶A within RNA isoforms is a critical step toward understanding the complex mechanisms that underpin brain function and disease; however, we lack an isoform-level atlas of m⁶A sites in the brain. We applied Oxford Nanopore direct RNA sequencing (DRS) to three postmortem human brain regions-prefrontal cortex, caudate nucleus, and cerebellum-to simultaneously investigate the transcriptome and epitranscriptome at the isoform level. We identified 57,000 m⁶A sites within 15,000 isoforms, revealing both isoform- and brain region-specific patterning of m⁶A modifications. The prefrontal cortex exhibited a distinctive profile of specifically modified isoforms enriched in excitatory neurons and had the highest proportion of unannotated m⁶A sites. A population of isoforms were hypermodified and associated with excitatory neurons in all brain regions. Our results demonstrate the utility of isoform-level profiling of RNA modifications and provide insights into brain region specificity with implications for development and disease.