RNF25 is activated as a response to amino acid starvation-induced ribosome collisions in competition with GCN2

Surveillance of mRNA translation relies on a suite of ribosome-associated quality control pathways. Recently, a novel pathway induced by trapping of translation factors in the ribosomal A-site has been described, involving ubiquitination of RPS27A/eS31 by the human E3 ubiquitin ligase RNF25. Here, we show that not only ribosome-stalling by low doses of translation inhibitors, but also amino acid starvation induces RPS27A/eS31 ubiquitination, identifying a natural trigger of RNF25 activation. Even under optimal growth conditions, RNF25 senses and resolves transient ribosome stalls. RPS27A/eS31 ubiquitination specifically depends on the ribosome collision sensor GCN1, a known cofactor of GCN2 involved in the integrated stress response. RNF25 and GCN2 both possess a GCN1-binding RWD domain, indicating a competitive relationship, with GCN2 acting as a negative regulator of RNF25 activation. Although both RNF25 and GCN2 respond to amino acid starvation, RPS27A/eS31 ubiquitination by RNF25 is not required for GCN2 activation, showing that both act in independent pathways. We propose that the RNF25 pathway acts as a first line of defence to resolve ribosome collisions, outcompeted by GCN2 binding to GCN1 under acute stress.