Abstract The Fragile X Messenger Ribonucleoprotein (FMRP) is a selective RNA-binding protein that localizes to the cytoplasm and the nucleus. The loss of FMRP results in Fragile X Syndrome (FXS), an Autism Spectrum Disorder. FMRP interacts with ribosomes and regulates the translation of mRNAs essential for neuronal development and synaptic plasticity. However, the biochemical nature of this translation regulation is unknown. Here we report that a key feature of FMRP-mediated translation regulation during neuronal differentiation is modulating the 2’O-methylation of ribosomal RNA. 2’O-methylation, facilitated by C/D box snoRNAs in the nucleus, is a major epitranscriptome mark on rRNA, essential for ribosome assembly and function. We found that FMRP influences a distinct rRNA 2’O-Methylation pattern across neuronal differentiation. We show that in H9 ESCs, FMRP interacts with a selected set of C/D box snoRNA in the nucleus resulting in the generation of ribosomes with a distinct pattern of rRNA 2’O-Methylation. This epitranscriptome pattern on rRNA undergoes a significant change during the differentiation of ESCs to neuronal precursors and cortical neurons. ESCs display maximum hypomethylated residues on rRNA, which is eventually reduced in neuronal precursors and post-mitotic cortical neurons and this is correlated to the change in global protein synthesis among the states of differentiation. Importantly, this gradual change in the 2’O-methylation pattern during neuronal differentiation is altered in the absence of FMRP, which could impact neuronal development and contribute to dysregulated protein synthesis observed in Fragile X Syndrome. This also suggests the need for diversity in functional ribosomes during the early stages of development.

Altering rRNA 2’O-methylation pattern during neuronal differentiation is regulated by FMRP