Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development. [Display omitted] •Near-atomic resolution structure of translating ribosomes in the nervous system•Ebp1 binds the 60S peptide tunnel exit during active protein synthesis•Ebp1 regulates start codon initiation and N-terminal peptide elongation•Ebp1 is abundant in early-born neocortex neurons and regulates their morphology Kraushar et al. visualize protein synthesis in the developing mouse brain at near-atomic resolution. Ebp1 binds the 60S tunnel exit to regulate translation initiation and N-terminal peptide elongation proteome-wide. Ebp1 is particularly abundant in early-born neocortex neural stem cells and regulates neuronal morphology, impacting cell adhesion molecule synthesis.