Isoform-level profiling of m 6 A epitranscriptomic signatures in human brain

The RNA modification N6-methyladenosine (m 6 A) is highly abundant in human brain and implicated in neurological disorders. Profiling m 6 A 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 m 6 A 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 m 6 A sites within 15,000 isoforms, revealing both isoform- and brain region–specific patterning of m 6 A modifications. The prefrontal cortex exhibited a distinctive profile of specifically modified isoforms enriched in excitatory neurons and had the highest proportion of unannotated m 6 A 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.