Lu Lu, Avijite Kumer Sarkar, Lan Dao, Y. Liu, Chunlong Ma, Phyo Han Thwin, Xuyao Chang, George Yoshida, Annie Li, Cenjing Wang, Crace Westerkamp, Lauren Schmitt, Maag Chelsey, Monzon Stephanie, Yu Zhao, Yaping Liu, Xiong Wang, Ling‐Qiang Zhu, Dan Liu, Jason Tchieu, Makoto Miyakoshi, Haining Zhu, Christina Groß, Ernest Pedapati, Nathan Salomonis, Craig Erickson, Ziyuan Guo
Abstract Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and the subsequent loss of the RNA-binding protein FMRP. N6-methyladenosine (m 6 A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP binds and regulates the stability of m 6 A-containing transcripts. However, how FMRP deficiency impacts transcriptome-wide m 6 A modifications in FXS remains unknown. To address this, we generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from healthy individuals and FXS patients. Electrophysiology recordings revealed synaptic and neuronal network defects in FXS iPSC-derived neurons. Transcriptome-wide analysis revealed striking m 6 A hypermethylation predominantly affecting synapse-associated transcripts. Mechanistically, we demonstrated that FMRP deficiency drives the aberrant translational upregulation of core m 6 A writers, a causal relationship definitively validated using CGG-corrected isogenic control lines. Targeted genetic knockdown of the m 6 A writer METTL3 successfully rescued synaptic phenotypes in FXS neurons, whereas its overexpression in control neurons phenocopied these synaptic defects, confirming the causal role of m 6 A dysregulation in FXS pathology. Notably, pharmacological intervention with the METTL3 inhibitor STM-2457 normalized methylation on synapse-associated transcripts and restored synaptic transmission in FXS neurons. Together, our findings uncover an FMRP-dependent epitranscriptomic mechanism contributing to FXS pathogenesis and suggest a promising avenue for m 6 A-targeted therapies.