Epitranscriptomic Control of Local Gene Expression in Neural Stem Cells

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Abstract During brain development, neurons are generated from neural stem cells, also called radial glial cells (RGCs). These cells exhibit a unique morphology with a long basal process that extends to the pia to form endfeet. Basal processes serve critical roles as scaffolds for neuronal migration and can also influence neurogenesis. Despite their importance for neurodevelopment, our understanding of molecular regulation within these basal radial glial structures remains poor. Our group recently discovered that RGC endfeet contain a specific transcriptome which can be locally translated. This suggests that local transcriptomic regulation is important for controlling gene expression in RGCs. However, our understanding of post-transcriptional mechanisms that regulate mRNAs within RGCs is very limited. Recently, methylation of adenosine residues in RNA (m6A) has emerged as a pervasive feature of the transcriptome which plays important roles in the regulation of gene expression. m6A is particularly abundant within the brain, and recent studies have shown that dynamic methylation enables cells to fine-tune the expression of subsets of the transcriptome. Moreover, the m6A methyltransferase, METTL3, is essential for promoting differentiation of stem cells, including RGCs. Our preliminary data indicate that m6A is present in the local transcriptome of RGC endfeet, suggesting the intriguing but untested possibility that mRNA methylation controls sub-cellular events in this important stem cell population. This proposal will test the novel hypothesis that RGC subcellular compartments contain distinct repertoires of methylated mRNAs and that mRNA modifications contribute to local gene expression regulation in the developing brain. We will first employ novel methods developed by our group for RGC endfeet isolation coupled with global m6A mapping strategies to identify the local methylome in RGCs. We will determine the transcripts whose localization to endfeet is dependent upon m6A and test the impact of RNA methylation upon local translation. We will additionally test the hypothesis that FMRP influences endfeet localization by binding m6A. Discoverying how FMRP targets RNAs in RGCs is important given that FMRP mutation influences cortical development and causes Fragile X syndrome. Collectively, these studies will provide the first identification of m6A-containing mRNAs in RGC endfeet and will uncover the transcripts for which local expression in RGCs is m6A- dependent. This work will provide a foundation for future studies designed to investigate the consequences of local RGC mRNA regulation on neural stem cell function and brain development.