An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes

  • Cell. 2023 Jan 5;S0092-8674(22)01538-0. doi: 10.1016/j.cell.2022.12.025.
Keely Oltion  1 ,  Jordan D Carelli  1 ,  Tangpo Yang  2 ,  Stephanie K See  1 ,  Hao-Yuan Wang  2 ,  Martin Kampmann  3 ,  Jack Taunton  4
Affiliations
  • 1. Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 3. Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
  • 4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: [email protected].
PMID: 36638793 DOI: 10.1016/j.cell.2022.12.025
Abstract

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 Ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

Keywords
CRISPRi screen; E3 ligase; K6-linked ubiquitin; RWD domain; diGly proteomics; natural product; protein synthesis; ribosomal ubiquitination; ribosome quality control; translation factor.
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