Ythdf family m6A readers promote retinal ganglion cell fate reprogramming and neurite development.

PURPOSE: A comprehensive understanding of the mechanisms regulating the development of retinal ganglion cells (RGCs) and their neurites carries both theoretical significance and translational implications. The YTHDF family members, comprising Ythdf1, Ythdf2, and Ythdf3, are key readers for N6-methyladenosine (m6A), the most abundant internal modification of mRNA. Mounting evidence has demonstrated essential roles of Ythdf family m6A readers in various biological processes. This study aimed to investigate the roles of Ythdf family m6A readers in RGC development and their neurite outgrowth. METHOD: An in vitro induced RGC (iRGC) system was employed to generate RGC-like neurons. Short hairpin RNAs (shRNAs) were used for gene knockdown expression, coding sequence (CDS)-containing constructs for gene overexpression, immunofluorescence staining for protein expression detection, patch-clamp recording for assessing neuronal electrophysiological properties, and RNA-seq for transcriptome profiling. RESULTS: Knockdown of Ythdf1 and Ythdf3 significantly reduced iRGC reprogramming efficiency and axon length, whereas overexpression of these two m6A readers exerted the opposite effects. Ythdf2 knockdown had no impact on iRGC reprogramming or axonogenesis, while Ythdf2 overexpression promoted axon growth. CONCLUSION: Ythdf1 and Ythdf3 are essential for iRGC fate reprogramming and axon development, whereas Ythdf2 is dispensable for these processes but can promote axon growth when overexpressed. These findings reveal important roles of m6A readers in RGC and their neurite development, which may facilitate future fundamental research and translational applications.