Reversible mRNA methylation in oligodendrocyte development and CNS myelination

摘要 髓鞘是高等脊椎动物正常神经系统功能所必需的，最近的数据表明， 髓磷脂重塑对于运动学习至关重要。此外，CNS髓鞘和少突胶质细胞 负责其合成的是许多神经病症的靶点，包括遗传（例如， 脑白质营养不良）和获得性（例如多发性硬化）病症。因此，我们必须 获得调节少突胶质细胞发育的途径和机制的完整理解 和髓磷脂形成。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。组蛋白去乙酰化酶介导的染色质重塑 非编码RNA的甲基化和基因沉默是表观遗传机制， 在中枢神经系统髓鞘形成中起关键作用。在这里描述的研究中， 将检查少突胶质细胞谱系细胞中RNA的甲基化。最近，N6-甲基腺苷 (m6A)被证明是可逆RNA甲基化的第一个例子。蛋白质“作家”，“橡皮擦”和 已经发现了这种RNA标记的“读者”，强烈表明这些动态RNA修饰 发挥监管作用。已经显示出阅读器影响稳定性、翻译、剪接和细胞内的表达。 定位含有m6 A的mRNA，使得这种修饰理想地定位于快速微调基因 表情我们建议采用遗传学方法来确定RNA甲基化是否影响 少突胶质细胞谱系细胞发育和功能。多蛋白复合物催化m6 A甲基化 真核生物的mRNA。甲基转移酶样（L）3和14，在m6 A writer中形成异二聚体， 已被证明是这种复合物的酶成分，具有遗传抑制作用， 导致含m6 A的mRNA的显著减少。虽然Mettl 14基因敲除小鼠显示胚胎 致死性，我们有携带Mettl 14基因的floxed等位基因的小鼠，我们将在这些研究中使用。的 Mettl 14条件突变小鼠将与许多不同的Cre驱动系组合使用，以测试Mettl 14条件突变小鼠的基因表达。 假设可逆的RNA甲基化在少突胶质细胞发育中起着至关重要的调节作用， 功能此外，将分析由少突胶质细胞谱系细胞表达的甲基化RNA转录物 使用含有m6 A的RNA下拉方法结合RNA测序。的程度 m6 A标记改变了细胞中特定mRNA的稳定性、剪接、翻译和细胞内转运， 还将测定少突胶质细胞。此外，Mettl 14基因在少突胶质细胞中失活， 谱系细胞在成年小鼠中，以检查甲基化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 动物也将使我们开始探索可逆的RNA甲基化在运动中的潜在作用。 学习总之，本提案中所述的研究将为深入了解 少突胶质细胞系细胞中的m6 A 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.