The role of EXOSC8 during mouse embryonic stem cells differentiation into neural precursor cells

Abstract

The role of EXOSC8 during mouse embryonic stem cells differentiation into neural precursor cells. Understanding the interplay between transcriptional and translational regulation is essential for deciphering stem cell differentiation. Here, we present a comprehensive study investigating the role of EXOSC8, a subunit of the core of the RNA exosome complex (EC), in mouse embryonic stem cell (mESC) differentiation toward neural precursor cells (NPC). By employing a controlled embryoid body (EB) formation protocol using diamond-bottom well plates and optimized cell seeding, we achieved synchronized neural induction. Transcriptional profiling confirmed progressive downregulation of pluripotency genes and upregulation of neuroectodermal markers across differentiation stages. We established and validated stable mESC lines expressing Ribo-STAMP and DOX-inducible shRNA constructs targeting EXOSC8, achieving 88% protein depletion. Integration of Ribo-STAMP with bulk and single-cell RNA-sequencing revealed dynamic shifts in both transcriptome and translatome, including genes regulated exclusively at the translational level. EXOSC8 knockdown led to altered expression of genes involved in protein ubiquitination and mRNA surveillance, affirming the RNA exosome’s role in post-transcriptional control. Through immunofluorescence and subcellular fractionation, we observed stage-specific relocalization of EXOSC8, shifting from a balanced nuclear-cytoplasmic distribution in mESCs to predominant cytoplasmic enrichment during intermediate stages, and partial nuclear return in neural precursors. Co-immunoprecipitation and mass spectrometry revealed a differentiation-stage-specific EXOSC8 interactome, transitioning from nucleolar and ribosome biogenesis factors to chromatin regulators, mRNA transporters, and finally to cytoskeletal proteins involved in axon development. These findings suggest that EXOSC8 localization is functionally programmed, enabling context-specific interactions that support lineage progression and neuronal differentiation. Finally, single-cell transcriptome and translatome analysis uncovered heterogeneity and revealed dynamic regulatory changes during differentiation under EXOSC8 perturbation. Collectively, this study establishes a robust experimental framework to dissect transcriptional, translational, and spatial regulation in stem cell fate commitment and highlights the RNA exosome complex as a modular RNA regulator during development.