During protein synthesis, GTPase elongation factor-1α (eEF1A) enables delivery of the appropriate aminoacyl-tRNA (aa-tRNA) to the A site of the ribosome. Ternatin-4 is a cyclic peptide that blocks translation by interfering with eEF1A-mediated release of aa-tRNA. Quality-control pathways are normally activated to restore translation, but the exact mechanisms for resolving ternatin-4-induced stalling are not clear. Oltion et al. found that ternatin-4 induced degradation of eEF1A through the ubiquitin–proteasome system, with a CRISPRi screen revealing RNF14 and RNF25 as the relevant E3 ligases involved in this process. Knockdown of either RNF14 or RNF25 was sufficient to block ternatin-4-mediated degradation of eEF1A. To identify the direct targets of ubiquitination by RNF14 and RNF25, the authors performed SILAC-based proteomics, revealing ubiquitination sites in eEF1A, as well as in ribosomal proteins such as RSP27A and the ribosomal collision sensor GCN1. Follow-up studies showed that RNF14 directly ubiqiutinated eEF1A at K385 in cells but required direct interaction with GCN1; a GCN1 mutant unable to bind to RNF14 blocked ternatin-4-induced degradation of eEF1A. Interestingly, although RNF25 was genetically required for eEF1A degradation, RNF25 did not directly ubiquitinate eEF1A and instead ubiquitinated RSP27A at K107 to enable RNF14-mediated ubiquitination of eEF1A. Although further studies are needed to tease out the direct biochemical connections, the findings from Oltion et al. reveal a unique E3 network for resolving ternatin-4-mediated ribosomal stalling.
Original reference: Cell 186, 346–362 (2023)
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