N6-Methyladenosine Methylome in Duchenne Muscular Dystrophy

项目摘要/摘要 Duchenne肌营养不良症(DMD)是一种致命性的X连锁肌肉退行性疾病。患有DMD的儿童 在他们生命的第一个十年内被限制在轮椅上，并在第三个十年内死亡。没有有效的方法 治疗这种毁灭性疾病的方法。DMD病理生理学中的关键生肌过程 分子水平尚不完全清楚，阻碍了DMD治疗方法的发展。为了填满这个 知识鸿沟，我们一直在调查一种新的转录后基因调控模式， 被称为表位转录组学，在DMD的发病机制中发挥作用。墓志翻译是一个动态的过程， 通过可逆的mRNAs化学修饰来调节RNA的稳定性、剪接和翻译。N6- 甲基腺苷(M6A)是最丰富的表位转录标志。M6A标志由 甲基转移酶样3(METTL3)和甲基转移酶样14(METTL14)，并被脂肪块和 肥胖相关基因(FTO)和烷基化修复同源基因5(ALKBH5)。选择性地选择特定的阅读器蛋白 识别m6A修饰的mRNAs并控制它们的命运。我们最近的发现显示，M6A mRNA甲基化 调节DMD的肌肉发生，M6A写入器、擦除器和M6A标记的水平受到严格调节 在成肌细胞分化、肌肉再生和DMD的过程中。这些研究表明，M6A的mRNA. 甲基化在DMD的病理生理学中起着关键作用。在这里，我们建议获得更深层次的机械论见解 转化为DMD中M6A基因的甲基化。我们将执行转录组范围的分析，以映射m6A-修饰 一口井的骨骼肌干细胞(MuSCs)、原代成肌细胞和骨骼肌中的mRNAs(甲基组) 建立DMD的MDX小鼠模型和相应的健康小鼠模型。我们还将绘制M6A甲基组 在DMD患者来源和健康的成肌细胞中，将我们的结论扩展到更具临床相关性的模型 系统。我们将机械地表征选定的m6A靶标mRNA的优先子集，这两个之前 与DMD相关的基因，以及该小说，以确定它们与DMD的肌肉退行性变之间的联系。我们的建议 研究将通过揭示DMD中转录组范围的m6A靶向mRNAs产生重大影响，并提供 对这一新机制的失调引起的DMD病理生理学的洞察。

PROJECT SUMMARY/ABSTRACT Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease. Children with DMD become restricted to wheelchairs within the first decade of their lives and die within the third. There is no effective treatment available for this devastating disease. Key myogenic processes in DMD pathophysiology at the molecular level are not completely understood, hindering the development of therapies for DMD. To fill this knowledge gap, we have been investigating whether an emerging mode of post-transcriptional gene regulation, known as epitranscriptomics, plays a role in DMD pathogenesis. Epitranscriptomics is a dynamic process that regulates RNA stability, splicing, and translation by reversible chemical modifications of mRNAs. N6- methyladenosine (m6A) is the most abundant epitranscriptomic mark. The m6A marks are installed by methyltransferase like 3 (METTL3) and methyltransferase like 14 (METTL14) and erased by fat mass and obesity-associated (FTO) and alkylation repair homolog 5 (ALKBH5). Specific reader proteins selectively recognize m6A-modified mRNAs and control their fates. Our recent findings revealed that m6A mRNA methylation regulates myogenesis in DMD and that levels of the m6A writers, erasers, and m6A marks are tightly regulated during myoblast differentiation, muscle regeneration, and in DMD. These studies suggest that m6A mRNA methylation plays a pivotal role in DMD pathophysiology. Here, we propose to gain a deeper mechanistic insight into m6A mRNA methylation in DMD. We will perform a transcriptome-wide analysis to map m6A-modified mRNAs (methylome) in skeletal muscle stem cells (MuSCs), primary myoblasts, and skeletal muscle of a well- established mdx mouse model of DMD and its counterpart healthy mice. We will also map the m6A methylome in DMD patient-derived and healthy myoblasts to extend our conclusions to a more clinically relevant model system. We will mechanistically characterize a select subset of prioritized m6A target mRNAs, both previously associated with DMD, as well as the novel, to establish their link to muscle degeneration in DMD. Our proposed research will make a significant impact by revealing transcriptome-wide m6A target mRNAs in DMD and provide insights into DMD pathophysiology incurred by the dysregulation of this novel mechanism.