Epitranscriptomic regulation by m6A RNA methylation after stroke

许多官能团共价连接到RNA以增强细胞转录组的功能。 特别地，甲基修饰N6-甲基腺苷（m6 A）被认为是腺苷酸中的第五个核碱基。 成人大脑由于其在mRNA加工中的多样性和背景特异性作用，m6 A参与了 急性中枢神经系统损伤和慢性神经退行性疾病。 由于m6 A在卒中后大脑中的作用尚未得到评估，我们将检验“m6 A高甲基化”的假设。 是缺血后脑损伤的主要调节剂。” 初步研究表明，短暂局灶性脑缺血显著增加了成年人m6 A甲基化， 老鼠的大脑我们还观察到，m6 A书写复合体没有改变，但大脑丰富的m6 A擦除器FTO 在中风后的大脑中被下调。初步研究显示，用FTO AAV 9 载体增加脑FTO表达并防止中风后脑中m6 A高甲基化。这 表明FTO下调，而不是甲基化增加，是观察到的持续性 中风后m6 A高甲基化FTO AAV 9还减少了继发性脑损伤，并改善了运动神经功能。 和认知功能恢复。我们进一步观察到， 中风后用NADPH治疗激活FTO并使m6 A标记的RNA去甲基化。NADPH也保护 中风后的大脑基于此，目标1将评估是否可以通过用以下物质诱导FTO来保护中风后大脑： NADPH遵循许多STAIR标准。 在缺血性脑中所有122种RNA高甲基化>5倍，c-Jun RNA的数量最多， 19个真正的m6 A基序c-Jun是凋亡基因表达和FTO过表达的介导者 去甲基化c-Jun mRNA并阻止其在中风后大脑中的翻译。基于此，Aim 2将测试 如果c-Jun高m6 A甲基化介导中风后细胞凋亡和随后的脑损伤。 m6 A阅读器YTHDF 1的结合促进m6 A标记的转录物的翻译。初步数据显示 诱导YTHDF 1表达和增加m6 A-甲基化c-Jun与YTHDF 1的结合 缺血我们进一步观察到FTO过表达降低了m6 A-甲基化c-Jun的丰度， 并降低其与YTHDF 1的结合。YTHDF 1-/-小鼠在注射后未显示c-Jun蛋白表达的诱导。 缺血这表明增加的m6 A甲基化和YTHDF 1结合对于翻译是必需的。 基于此，Aim 3将测试m6 A读取器YTHDF 1是否在缺血后脑损伤中起作用。 总体目标是了解卒中后m6 A甲基化的机制和治疗潜力。

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.