Collided ribosomes are rescued by the endonuclease Rae1 and trans- translation
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Ribosome stalling is a major problem in all domains of life. When a ribosome stalls, trailing ribosomes may catch up to and collide with the stalled ribosome, depleting protein synthesis capacity. Here, we describe a novel pathway used by Gram-positive bacteria to rescue ribosome collisions. We used the ATPase defective ABCF protein YdiF(EQ 2 ) to induce ribosome stalling and collisions in Bacillus subtilis . Ribosome profiling (Ribo-seq) of YdiF(EQ 2 )-expressing cells revealed that collided ribosomes are enriched for tmRNA, a functional RNA involved in trans -translation. We confirmed that tmRNA tagging activity is globally increased upon expression of any ATPase defective ABCF as well as in cells treated with the collision-inducing antibiotic erythromycin, suggesting this is a generalizable mechanism to rescue stalled and collided ribosomes. The global increase in tmRNA tagging that occurred in response to both erythromycin and YdiF(EQ2) induced collisions was dependent on the Rae1 endonuclease. Loss of trans -translation in cells experiencing widespread ribosome collisions leads to a severe fitness defect, consistent with the importance of this pathway in rescuing ribosomes stalled on truncated mRNAs that result from ribosome collisions. Altogether, our work supports a model in which Rae1 cleaves mRNA on collided ribosomes, thereby generating a truncated mRNA substrate for trans -translation and leading to rescue and recycling of the collided ribosomes. We term this mechanism C ollision- A ssociated R ae1-induced trans- Translation (CART). CART broadens the repertoire of tools that bacteria use to manage ribosome collisions.
Significance
Prolonged ribosome stalling leads to ribosome collisions, which are rescued by specialized factors. While ribosome collisions have been extensively studied in eukaryotes, our understanding of collision rescue in bacteria is in its infancy. Data described here are the first to directly show that tmRNA mediates rescue of collided ribosomes in a Gram-positive bacterium, Bacillus subtilis . This pathway is analogous to what occurs in model organisms such as Escherichia coli and Saccharomyces cerevisiae , but relies on an unrelated nuclease, Rae1. Since B. subtilis and E. coli are on opposite ends of the bacterial phylogenetic tree, and since Rae1 is broadly conserved in bacteria, our findings suggest that mRNA cleavage arose convergently in distantly related bacteria as a strategy to rescue ribosome collisions. Convergent evolution of these pathways highlights the importance of rescuing collided ribosomes in all organisms. Moreover, insights into ribosome rescue in E. coli and B. subtilis can guide studies of ribosome rescue in bacteria with intermediary phylogenetic relatedness to these two model organisms.
