Abstract
Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a series of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide, and targeting of the mRNA for decay (No Go Decay or NGD). Using a reverse genetic screen in yeast, we identify Cue2 as the conserved endonuclease that is recruited to stalled ribosomes to promote NGD. Ribosome profiling and biochemistry provide strong evidence that Cue2 cleaves mRNA within the A site of the colliding ribosome. We demonstrate that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay and Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway. Finally, we show that the Cue2-dependent pathway becomes a major contributor to NGD in cells depleted of factors required for the resolution of stalled ribosome complexes. Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells..
Highlights
Translation is a highly regulated process in which ribosomes must initiate, elongate, and terminate accurately and efficiently to maintain optimal protein levels
Recent work in eukaryotes has revealed that ribosome collisions on problematic mRNAs create a unique interface on the aligned 40S subunits that serves as a substrate for E3 ubiquitin ligases, such as Hel2 and Not4, and the RQC-trigger (RQT) complex, comprised of factors Slh1, Cue2 and Rqt4; together these factors are thought to trigger downstream quality control (Ferrin and Subramaniam, 2017; Garzia et al, 2017; Ikeuchi et al, 2019; Juszkiewicz et al, 2018; Juszkiewicz and Hegde, 2017; Matsuo et al, 2017; Simms et al, 2017b; Sundaramoorthy et al, 2017)
Using ribosome profiling and biochemical assays, we show that Cue2 cleaves mRNAs in the A site of collided ribosomes, and that ribosomes which accumulate at these cleaved sites are rescued by the known ribosome rescue factor Dom34 (Guydosh and Green, 2014; Shoemaker et al, 2010)
Summary
Translation is a highly regulated process in which ribosomes must initiate, elongate, and terminate accurately and efficiently to maintain optimal protein levels. While Xrn1-mediated degradation is thought to be the dominant pathway for most general decay in yeast (Anderson and Parker, 1998), the exosome has been implicated as critical for many degradation events in the cell including those targeting prematurely polyadenylated mRNAs (these mRNAs are usually referred to as NonStop Decay (NSD) targets) (Frischmeyer et al, 2002; Tsuboi et al, 2012; van Hoof et al, 2002) In metazoans, it is less clear what the relative contributions of Xrn and the exosome are to the degradation of normal cellular mRNAs. How the endonucleolytic and canonical exonucleolytic decay pathways coordinate their actions on problematic mRNAs remains unknown. Our final model provides key insights into what happens in cells upon recognition of stalled ribosomes on problematic mRNAs, and reconciles how both endo- and exonucleolytic decay act synergistically to resolve these dead-end translation intermediates
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