Epitranscriptomic regulation by m6A RNA methylation after stroke

University Of Wisconsin-Madison, Madison WI

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Abstract

Many functional groups attach covalently to RNAs to enhance the functionality of the cellular transcriptome. Particularly, the methyl group modification N6-methyladenosine (m6A) is regarded as the fifth nucleobase in the adult brain. With its diverse and context-specific roles in mRNA processing, m6A is implicated in the progression of acute CNS injuries and chronic neurodegenerative diseases. As the role of m6A in post-stroke brain is not yet evaluated, we will test the hypothesis “m6A hypermethylation is a major modulator of post-ischemic brain damage.” Preliminary studies showed that transient focal ischemia significantly increases the m6A methylation in adult mouse brain. We also observed that the m6A writer complex is unaltered, but the brain-enriched m6A eraser FTO was down-regulated in the post-stroke brain. Preliminary studies showed that treatment with an FTO AAV9 vector increased cerebral FTO expression and prevented m6A hypermethylation in the post-stroke brain. This indicates that FTO downregulation, rather than increased methylation, is responsible for the observed sustained m6A-hypermethylation after stroke. FTO AAV9 also decreased secondary brain damage, and improved motor and cognitive functional recovery in mice of both sexes subjected to focal ischemia. We further observed that post-stroke treatment with NADPH activates FTO and demethylates m6A-tagged RNAs. NADPH also protected post-stroke brain. Based on this, Aim 1 will evaluate if post-stroke brain can be protected by inducing FTO with NADPH following many STAIR criteria. Of all the 122 RNAs hypermethylated by >5-fold in the ischemic brain, c-Jun RNA harbors the highest number of 19 bonafide m6A motifs. c-Jun is a mediator of apoptotic gene expression, and FTO overexpression demethylated c-Jun mRNA and prevented its translation in the post-stroke brain. Based on this, Aim 2 will test if c-Jun hyper-m6A methylation mediates post-stroke apoptosis and the ensuing brain damage. Binding of m6A reader YTHDF1 promotes translation of m6A-tagged transcripts. Preliminary data showed induction of YTHDF1 expression and increased binding of m6A-methylated c-Jun to YTHDF1 following focal ischemia. We further observed that FTO overexpression decreased the abundance of m6A-methylated c-Jun and reduced its binding to YTHDF1. YTHDF1-/- mice didn’t show induction of c-Jun protein expression after ischemia. This indicates that both increased m6A methylation and YTHDF1 binding are essential for translation of c-Jun. Based on this, Aim 3 will test if m6A reader YTHDF1 plays a role in post-ischemic brain damage. The overall goal is to understand the mechanisms and therapeutic potential of m6A methylation after stroke.