NONU-1 encodes a conserved endonuclease required for mRNA translation surveillance

Abstract

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. However, the nuclease(s) responsible remain unknown. Here we identify NONU-1 as a novel endoribonuclease 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 and with properties similar to the unknown endonuclease; and (3) the domain architecture and catalytic residues of NONU-1 are conserved throughout metazoans and eukaryotes, respectively. We extend our results in C. elegans to homologous factors in S. cerevisiae, showing conservation of function of the NONU-1 protein across billions of years of evolution.Our work establishes the identity of a previously unknown factor critical to translation surveillance and will inform mechanistic studies at the intersection of translation and mRNA decay. 1 5 10 15 20 25 2 30 35 40 45 50 that organism. NONU-1 contains a conserved IF3-C fold domain previously implicated in processing RNA. Our results identify a critical new component of the translation surveillance machinery in two model organisms and suggest why this factor has been recalcitrant to discovery in S. cerevisiae. RESULTS nonu-1 encodes a novel factor required for Nonstop mRNA DecayWe previously developed a phenotypic reporter in C. elegans that allowed us to identify Nonstop mRNA Decay factors via reverse and forward genetics ( Figure 1A, (Arribere and Fire 2018)).Briefly, the reporter was constructed using the unc-54 locus as expression and function of this gene has been extensively studied (Brenner 1974;Epstein et al. 1974;Dibb et al. 1985Dibb et al. , 1989Moerman et al. 1982;Bejsovec and Anderson 1988;Anderson and Brenner 1984) and unc-54 has been used in previous suppressor screens (Hodgkin et al. 1989). To construct a Nonstop reporter at the unc-54 locus we first integrated a GFP at the C-terminus of UNC-54 by CRISPR/Cas9. We also removed all stop codons from the 3'UTR and integrated a ribosomal skipping T2A sequence between the C-terminal GFP and the remainder of the 3'UTR. The T2A sequence is a viral-derived peptide that cotranslationally releases the upstream protein and allows UNC-54::GFP to escape Nonstop protein decay (so-called "Ribosome Quality Control", (Bengtson and Joazeiro 2010;Shao et al. 2013;Shen et al. 2015)). We hereafter refer to the unc-54::gfp::t2a::nonstop reporter as unc-54 (Nonstop). Animals with the unc-54 (Nonstop) reporter deficient in Nonstop mRNA Decay exhibit derepression of the locus, as evidenced by increased GFP fluorescence, mRNA expression, and egg laying (unc-54 encodes a muscle myosin required in the vulva for egg laying) ( Figure 1B, (Arribere and Fire 2018)).Although our initial screen successfully identified C. elegans' skih-2 and ttc-37 (homologs of S. cerevisiae SKI2 and SKI3, respectively), ...

No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

“…It has been recently reported that Cue2 is the endonuclease that cleaves NGD/NSD targeted RNAs 36,37 . Its action has been proposed to occur in the ribosome A site and therefore deviates significantly from our observations.…”

Extraction of mRNA from Stalled Ribosomes by the Ski Complex

“…Another important question is whether the mRNA-extracting function of the Ski complex could play a role in the RQT-mediated or any other surveillance and quality control mechanism. Notably, nonstop suppression in a C. elegans skih-2 nonu-1 double mutant was greater than in either single mutant, suggesting that the activities of NONU-1 and the Ski complex may not be strongly interdependent (Glover et al, 2019).…”

“…Although endonucleolytic cleavage of mRNA upstream of the stall site is an established hallmark of No-Go decay (Doma and Parker, 2006), the nature of this process has remained obscure. Recent studies showed that ribosomal ubiquitination acts as a signal for recruitment to the stall site of an endonuclease, identified as Cue2 in Saccharomyces cerevisiae (D'Orazio et al, 2019) and NONU-1 in Caenorhabditis elegans (Glover et al, 2019). Cleavage is reported to occur in the A site of collided ribosomes (D'Orazio et al, 2019).…”

Cellular RNA surveillance in health and disease

“…In either case, the ribosome stalls after translating to the end of the mRNA. As in no-go decay, removal of the arrested ribosome involves endonucleolytic cleavage of the mRNA and ribosome rescue by PELO and HBS1 (25), and a preliminary report has identified Cue2/N4BP2 as the required endonuclease (29).…”