Epitranscriptomic regulation by m6A RNA methylation after stroke

中风后 m6A RNA 甲基化的表观转录组调控

• 批准号: 10604801
• 负责人: Raghu VEMUGANTI
• 金额: $ 61.81万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10604801
• 关键词: Acute; Adenine; Adult; Anxiety; Apoptosis; Apoptotic; Binding; Binding Sites; Biological Assay; Brain; Brain Injuries; Brain Ischemia; Cerebrum; Chemicals; Chronic; Cognitive; Complex; Data; Dedications; Down-Regulation; Gene Expression; Genes; Goals; Hypermethylation; Industry; Infarction; Inflammation; Ischemia; Ischemic Brain Injury; JUN gene; Luciferases; Mediating; Mediator; Mental Depression; Messenger RNA; Methylation; Modification; Motor; Mus; NADP; Neurodegenerative Disorders; Neurons; Pathogenesis; Phenocopy; Plasmids; Post-Transcriptional RNA Processing; Proto-Oncogene Proteins c-jun; RNA; RNA Degradation; RNA Splicing; RNA methylation; Reader; Recovery of Function; Regulation; Reporter; Rodent; Role; Signal Transduction; Specificity; Testing; Therapeutic; Transcript; Transcription Initiation Site; Translations; base; central nervous system injury; demethylation; efficacy testing; epitranscriptomics; functional group; functional improvement; gray matter; improved; methyl group; motor recovery; mutant; nucleobase; overexpression; post stroke; prevent; promoter; protein expression; sex; stroke therapy; therapeutic evaluation; transcription factor; transcriptome; vector; white matter damage

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.

许多官能团将共价连接到RNA上，以增强细胞转录组的功能。 特别是，甲基修饰N6-甲基趋化胺（M6A）被认为是在 成人大脑。 M6a凭借其潜水员和背景特定的角色在MRNA加工中，在进展中隐含 急性中枢神经系统损伤和慢性神经退行性疾病。 由于尚未评估M6A在中风后大脑中的作用，我们将检验假设“ M6A高甲基化 是缺血后脑损伤的主要调节剂。” 初步研究表明，瞬时局灶性缺血显着增加了成人的M6A甲基化 小鼠大脑。我们还观察到M6A作者复合物是没有改变的，但是富含脑部的M6A橡皮擦FTO 在冲程后大脑中被下调。初步研究表明，用FTO AAV9进行治疗 载体增加了大脑FTO的表达，并防止了中风后大脑中的M6a高甲基化。这 表明FTO下调而不是增加甲基化，是造成观察到的持续的原因 中风后M6A羟基甲基化。 FTO AAV9也减少了次要脑损伤，并改善了电动机 两种遭受局灶性缺血的性别小鼠的认知功能恢复。我们进一步观察到 用NADPH治疗的势后治疗会激活FTO和脱甲基甲基化的M6A标记的RNA。 NADPH也受到了保护 中风后大脑。基于此，AIM 1将评估中风后大脑是否可以通过诱导的FTO保护 符合许多楼梯标准的NADPH。 在缺血性大脑中，所有122个RNA高甲基甲基甲基化，C-Jun RNA拥有最高数量 19个真正的M6A图案。 C-Jun是凋亡基因表达和FTO过表达的介体 脱甲基化的C-Jun mRNA，并防止其在冲程后大脑中的翻译。基于此，AIM 2将测试 如果C-JUN HYPER-M6A甲基化介导了中风后凋亡和随后的脑损伤。 M6A读取器YTHDF1的结合促进了M6A标记的转录本的翻译。显示的初步数据 在局灶性之后，YTHDF1表达的诱导和M6a-甲基化的C-Jun与YTHDF1的结合增加 缺血。我们进一步观察到，FTO的过表达下降了M6A-甲基化C-JUN的抽象 并减少了其与YTHDF1的结合。 ythdf1 - / - 小鼠在 缺血。这表明M6A甲基化增加和YTHDF1结合对于翻译至关重要 C-Jun。基于此，AIM 3将测试M6A读取器YTHDF1是否在缺血后脑损伤中起作用。 总体目标是了解中风后M6A甲基化的机制和治疗潜力。