Synaptic N⁶-methyladenosine (m⁶A) epitranscriptome reveals functional partitioning of localized transcripts

A localized transcriptome at the synapse facilitates synapse-, stimulus- and transcript-specific local protein synthesis in response to neuronal activity. While enzyme-mediated mRNA modifications are known to regulate cellular mRNA turnover, the role of these modifications in regulating synaptic RNA has not been studied. We established low-input m⁶A-sequencing of synaptosomal RNA to determine the chemically modified local transcriptome in healthy adult mouse forebrains and identified 4,469 selectively enriched m⁶A sites in 2,921 genes as the synaptic m⁶A epitranscriptome (SME). The SME is functionally enriched in synthesis and modulation of tripartite synapses and in pathways implicated in neurodevelopmental and neuropsychiatric diseases. Interrupting m⁶A-mediated regulation via knockdown of readers in hippocampal neurons altered expression of SME member Apc, resulting in synaptic dysfunction including immature spine morphology and dampened excitatory synaptic transmission concomitant with decreased clusters of postsynaptic density-95 (PSD-95) and decreased surface expression of AMPA receptor subunit GluA1. Our findings indicate that chemical modifications of synaptic mRNAs critically contribute to synaptic function.