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Cellular translation surveillance rescues ribosomes that stall on problematic mRNAs. During translation surveillance, endonucleolytic cleavage of the problematic mRNA is a critical step in rescuing stalled ribosomes. Here we identify NONU-1 as a factor required for translation surveillance pathways including no-go and nonstop mRNA decay. We show that (1) NONU-1 reduces nonstop and no-go mRNA levels; (2) NONU-1 contains an Smr RNase domain required for mRNA decay; (3) the domain architecture and catalytic residues of NONU-1 are conserved throughout metazoans and eukaryotes, respectively; and (4) NONU-1 is required for the formation of mRNA cleavage fragments in the vicinity of stalled ribosomes. We extend our results in C.elegans to homologous factors in S.cerevisiae, showing the evolutionarily conserved function of NONU-1. Our work establishes the identity of a factor critical to translation surveillance and will inform mechanistic studies at the intersection of translation and mRNA decay.

SUMMARY Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2′, 3′-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5′ tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair., Graphical Abstract, In Brief Hughes et al. demonstrate that a bacterial RNA repair operon, containing the RtcB RNA ligase and the RtcA RNA cyclase, is regulated by binding of 5′ tRNA halves ending in 2′, 3′-cyclic phosphate to the RtcR transcriptional activator. These studies show how tRNA fragments can regulate bacterial gene expression.