m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs [RNA-seq and DART-seq]

The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a regulator of mRNA localization in neurons. Transcriptome-wide analysis of mRNA localization in neurons following genetic loss of m6A reveals hundreds of transcripts that exhibit altered subcellular localization. Additionally, using a reporter system, we show that mutation of specific m6A sites in select neuronal transcripts diminishes their localization to neurites. Single molecule fluorescent in situ hybridization experiments further confirm our findings and identify the m6A reader proteins YTHDF2 and YTHDF3 as mediators of this effect. Our findings reveal a novel function for m6A in controlling mRNA localization in neurons and enable a better understanding of the mechanisms through which m6A influences gene expression in the brain. Overall design: RNA abundance measurement and m6A sites profiling in the soma and neurite compartments of wildtype and Mettl3-/- hippocampal neurons. Primary neurons were grown on microporous membrane and treated with KCl for 2 hours. Total RNA was extracted from the top (soma) and bottom (neurite) of the membrane and used for RNA-seq or in vitro DART-seq library preparation in duplicates. For DART-seq, Mettl3-/- samples are used as control for identification of m6A sites in WT samples.