Reversible mRNA methylation in oligodendrocyte development and CNS myelination

摘要 髓鞘对高等脊椎动物的正常神经系统功能是必不可少的，最近的数据表明 髓鞘重塑对运动学习至关重要。此外，中枢神经系统髓鞘和少突胶质细胞 负责其合成的是一系列神经疾病的靶点，包括遗传(例如， 脑白质营养不良)和获得性(如多发性硬化症)疾病。因此，至关重要的是，我们必须 全面了解调控少突胶质细胞发育的途径和机制 和髓鞘形成。在这里，我们建议探索少突胶质细胞发育的表观遗传调控， 功能和对环境变化的反应。组蛋白脱乙酰酶对染色质的重塑 甲基化和非编码RNA的基因沉默是表观遗传机制，已经 证明在中枢神经系统髓鞘形成过程中起着关键作用。在这里描述的研究中，可逆的作用 将检测少突胶质细胞系细胞中RNA的甲基化情况。最近，N6-甲基腺苷 (M6A)被证明是第一个可逆RNA甲基化的例子。蛋白质“作家”、“橡皮”和 这个RNA标记的“读取器”已经被发现，强烈地表明这些动态的RNA修饰 发挥监管作用。读者已经被证明影响稳定性、翻译、剪接和细胞内 含有m6A的信使核糖核酸的定位，因此这种修饰是快速微调基因的理想位置 表情。我们建议采用遗传方法来确定RNA甲基化是否会影响 少突胶质细胞谱系细胞的发育和功能。多蛋白复合体催化m6A甲基化 真核基因的表达。甲基转移酶样物(METTL)3和14，它们在m6A编写器中形成异二聚体， 已被证明是该复合体的酶成分，并具有基因抑制作用 导致含有m6A的信使核糖核酸显著减少。尽管Mettl14基因缺失的小鼠表现为胚胎 致命性，我们有携带Mettl14基因的等位基因的小鼠，我们将在这些研究中使用它。这个 Mettl14条件突变小鼠将与一些不同的Cre驱动系结合使用，以测试 可逆RNA甲基化在少突胶质细胞发育和发育中起关键调节作用的假设 功能。此外，还将对少突胶质细胞系细胞表达的甲基化RNA转录本进行分析。 使用含有m6A的RNA下拉方法与RNA测序相结合。在多大程度上 M6A标记改变了特定mRNAs的稳定性、剪接、翻译和细胞内转运 少突胶质细胞也将被确定。此外，Mettl14基因将在少突胶质细胞中失活 在成年小鼠的血统细胞中检测甲基化RNA在维持 少突胶质细胞的功能，以及这些细胞对脱髓鞘和炎症的反应。这些 动物也将允许我们开始探索可逆的RNA甲基化在运动中所起的潜在作用 学习。综上所述，本提案中描述的研究将为深入了解 少突胶质细胞系细胞中M6A RNA甲基化。

Abstract Myelin is essential for normal nervous system function in higher vertebrates, and recent data suggest that myelin remodeling is critical for motor learning. Moreover, CNS myelin sheath and the oligodendrocytes responsible for its synthesis are the targets of a number of neurological conditions, including genetic (e.g. leukodystrophies) and acquired (e.g. multiple sclerosis) disorders. Therefore, it is critically important that we gain a complete understanding of the pathways and mechanisms that regulate oligodendrocyte development and myelin formation. Here, we propose to explore the epigenetic regulation of oligodendrocyte development, function and response to environmental changes. Chromatin remodeling by histone deacetylases, DNA methylation and gene silencing by non-coding RNAs are epigenetic mechanisms that have already been shown to play a critical role in CNS myelination. In the studies described here the role that the reversible methylation of RNA plays in oligodendrocyte lineage cells will be examined. Recently, N6-methyladenosine (m6A) was shown to be the first example of reversible RNA methylation. Protein “writers”, “erasers” and “readers” of this RNA mark have been discovered, strongly suggesting that these dynamic RNA modifications play a regulatory role. Readers have been shown to influence the stability, translation, splicing and intracellular localization of m6A-containing mRNA, such that this modification is ideally positioned to rapidly fine-tune gene expression. We propose to take a genetic approach to determine if RNA methylation influences oligodendrocyte lineage cell development and function. A multiprotein complex catalyzes the m6A methylation of eukaryotic mRNA. Methyltransferase like (METTL) 3 and 14, which form a heterodimer in the m6A writer, have been shown to be the enzymatic components of this complex, with the genetic inhibition of either resulting in a substantial reduction of m6A-containing mRNA. Although Mettl14 null mice display embryonic lethality, we have mice that carry a floxed allele of the Mettl14 gene that we will use in these studies. The Mettl14 conditional mutant mice will be used in combination with a number of distinct Cre driver lines to test the hypothesis that reversible RNA methylation plays a crucial regulatory role in oligodendrocyte development and function. In addition, the methylated RNA transcripts expressed by oligodendrocyte lineage cells will be profiled using an m6A-containing RNA pull-down approach in combination with RNA-sequencing. The degree to which the m6A marks alters the stability, splicing, translation and intracellular transport of specific mRNAs in oligodendrocytes will also be determined. Moreover, the Mettl14 gene will be inactivated in oligodendrocyte lineage cells in adult mice to examine the requirement of methylated RNA in the maintenance of oligodendrocyte function, as well as the response of these cells to demyelination and inflammation. These animals will also allow us to begin to explore the potential role that reversible RNA methylation plays in motor learning. Together, the studies described in this proposal will provide considerable insight into the function of m6A RNA methylation in oligodendrocyte lineage cells.