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

Mol Cell. 2021 Jan 21;81(2):304-322.e16. doi: 10.1016/j.molcel.2020.11.037. Epub 2020 Dec 22.

Abstract

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.

Keywords: Ebp1; cryo-EM; development; neocortex; neurons; pSILAC/BONCAT mass spectrometry; proteostasis; ribosome; ribosome profiling; selective ribosome profiling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Binding Sites
  • Cell Adhesion Molecules, Neuronal / chemistry
  • Cell Adhesion Molecules, Neuronal / genetics
  • Cell Adhesion Molecules, Neuronal / metabolism
  • Cell Line, Tumor
  • Cryoelectron Microscopy
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Embryo, Mammalian
  • Female
  • Gene Expression Regulation, Developmental*
  • Male
  • Mice
  • Neocortex / cytology
  • Neocortex / growth & development
  • Neocortex / metabolism*
  • Neural Stem Cells / cytology
  • Neural Stem Cells / metabolism
  • Neurogenesis / genetics
  • Neurons / cytology
  • Neurons / metabolism*
  • Primary Cell Culture
  • Protein Binding
  • Protein Biosynthesis*
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Proteostasis / genetics*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Ribosome Subunits, Large, Eukaryotic / genetics*
  • Ribosome Subunits, Large, Eukaryotic / metabolism
  • Ribosome Subunits, Large, Eukaryotic / ultrastructure
  • Signal Recognition Particle / chemistry
  • Signal Recognition Particle / genetics
  • Signal Recognition Particle / metabolism

Substances

  • Cell Adhesion Molecules, Neuronal
  • DNA-Binding Proteins
  • Pa2g4 protein, mouse
  • RNA-Binding Proteins
  • Signal Recognition Particle