Reversible mRNA methylation in oligodendrocyte development and CNS myelination

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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.