Epitranscriptomic regulation by m6A RNA methylation after stroke

中风后 m6A RNA 甲基化的表观转录组调控

基本信息

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
项目类别：
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

项目摘要

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.

许多功能基团与 RNA 共价连接，以增强细胞转录组的功能。特别地，甲基修饰N6-甲基腺苷(m6A)被认为是该结构中的第五个核碱基。 m6A 在 mRNA 加工中具有多种且特定于环境的作用，因此参与了这一进展。急性中枢神经系统损伤和慢性神经退行性疾病。由于 m6A 在中风后大脑中的作用尚未评估，我们将检验“m6A 高甲基化”这一假设是缺血后脑损伤的主要调节剂。” 初步研究表明，成人短暂性局灶性缺血显着增加 m6A 甲基化我们还观察到 m6A writer 复合体没有改变，但大脑中富含 m6A 擦除器 FTO。初步研究表明，FTO AAV9 治疗后大脑中的表达下调。载体增加了中风后大脑中 FTO 的表达并防止 m6A 高甲基化。表明 FTO 下调，而不是甲基化增加，才是观察到的持续的原因中风后 FTO AAV9 的 m6A 高甲基化也减少了继发性脑损伤，并改善了运动功能。我们进一步观察到，局灶性缺血的雌雄小鼠的认知功能恢复情况。中风后使用 NADPH 治疗可激活 FTO 并使 m6A 标记的 RNA 去甲基化，NADPH 也受到保护。基于此，目标 1 将评估是否可以通过诱导 FTO 来保护中风后大脑。 NADPH 遵循许多 STAIR 标准。在缺血脑中甲基化超过 5 倍的所有 122 个 RNA 中，c-Jun RNA 的数量最多 19 个 bonafide m6A 基序的 c-Jun 是凋亡基因表达和 FTO 过度表达的介质。基于此，Aim 2 将测试 c-Jun mRNA 的去甲基化并阻止其在中风后大脑中的翻译。 c-Jun 高 m6A 甲基化是否介导中风后细胞凋亡和随后的脑损伤。初步数据显示，m6A 阅读器 YTHDF1 的结合可促进 m6A 标记转录本的翻译。诱导 YTHDF1 表达并增加 m6A 甲基化 c-Jun 与 YTHDF1 的结合我们进一步观察到 FTO 过度表达降低了 m6A 甲基化 c-Jun 的丰度。并减少了与 YTHDF1-/- 的结合，YTHDF1-/- 小鼠在给药后并未表现出 c-Jun 蛋白表达的诱导。这表明 m6A 甲基化和 YTHDF1 结合的增加对于翻译至关重要。基于此，Aim 3 将测试 m6A 阅读器 YTHDF1 是否在缺血后脑损伤中发挥作用。总体目标是了解中风后 m6A 甲基化的机制和治疗潜力。