Ribosomal modifications are associated with mesenchymal fate selection in the neural crest lineage.

Neural crest cells contribute to craniofacial formation by differentiating into skeletogenic mesenchyme and neuro-glial lineages. Using Smart-seq2 single-cell transcriptomics, we show that mesenchymal fate commitment correlates specifically with the expression of rRNA-modifying and ribosome assembly factors, rather than structural ribosomal proteins. Notably, EMG1 and NHP2 introduce key post-transcriptional modifications into 18S rRNA, including m¹acp³ψ at U1248, which requires TSR3 for final maturation. Disrupting NHP2 or TSR3 in vitro and in vivo perturbs cranial neural crest differentiation; post-migratory temporal knockout of Polr1a or Polr1c also causes craniofacial malformations. These findings align with cell type-specific m¹acp³ψ levels during neural crest differentiation. Given the neural crest contribution to neuroblastoma, we analyze patient data to find that elevated ribosomal control and rRNA-modifying proteins predict poorer outcomes. Complementary experiments in neuroblastoma cell lines reveal functional roles for TSR3 and WDR74 in mesenchymal-like tumor states. Together, our results link rRNA modifications and ribosome assembly to fate decisions, suggesting ribosomal heterogeneity shapes both normal development and tumor progression.