Asc1, Hel2, and Slh1 couple translation arrest to nascent chain degradation

Sitron, Cole S.; Park, Joseph Hun; Brandman, Onn

doi:10.1261/rna.060897.117

Cited by 117 publications

(155 citation statements)

References 50 publications

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“…4D compared to 4A): 21 nt RPFs accumulate on the 2 nd through 5 th CGA codons and 28 nt RPFs accumulate 30 nts behind the lead ribosome; we also see modest accumulation of RPFs downstream of the stalling sequence as previously reported (Fig. 4D) (Sitron, Park, & Brandman, 2017). SLH1 deletion does not stabilize 16 nt RPFs, and therefore, likely does not act on cleaved Cue2-substrates.…”

Section: Ribosome Profiling Provides High-resolution View Of Cue2 Clesupporting

confidence: 82%

The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay

D’Orazio

Sinha

et al. 2019

Preprint

118

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Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a sequence of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide via the Ribosome-mediated Quality control Complex (RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Previous studies provide strong evidence for the existence of an endonuclease involved in the process of NGD though the identity of the endonuclease and the extent to which it contributes to mRNA decay remain unknown. 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. Finally, we show that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay. Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway, but becomes a major contributor in cells depleted of factors required for the resolution of stalled ribosome complexes (the RQT factors including Slh1). Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells. One Sentence Summary:Cue2 is the endonuclease that cleaves mRNA at ribosome stall sites.

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Section: Ribosome Profiling Provides High-resolution View Of Cue2 Clesupporting

confidence: 82%

The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay

D’Orazio

Sinha

et al. 2019

Preprint

118

View full text Add to dashboard Cite

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“…Moreover, initiating the RQC pathway requires an additional protein complex, the so-called RQC-trigger (RQT) complex (Matsuo et al, 2017). It is composed of the RNA helicase family protein Slh1/ Rqt2, the ubiquitin-binding protein Cue3/Rqt3 and yKR023W/Rqt4 (Matsuo et al, 2017;Sitron et al, 2017). The RQT complex was found to associate with the ribosome and Hel2, and the ubiquitinbinding activity of Cue3/Rqt3 and ATPase activity of Slh1/Rqt2 were shown to be crucial to trigger RQC (Matsuo et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways

et al. 2019

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Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no‐go decay) and the nascent polypeptide (RQC: ribosome‐associated quality control). RQC requires Hel2‐dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2‐dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di‐ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo‐EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2‐mediated cleavages upstream of the disome, governed by initial Not4‐mediated monoubiquitination of eS7 and followed by Hel2‐mediated K63‐linked polyubiquitination. We propose that Hel2‐mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC‐uncoupled NGD outside the disome (NGDRQC−) can occur in a Not4‐dependent manner.

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“…In yeast the equivalent E3 ligase HEL2 was shown to be important for stalling (Letzring et al, 2013; Saito et al, 2015). Recent studies using ribosomal profiling have, however, argued that HEL2 is not required for stalling but instead in its absence mRNA reporters are stabilized (Sitron et al, 2017). Here, nonetheless, deletion of HEL2 results in increased ribosome occupancy (> twofold) beyond the stall site.…”

Section: Introductionmentioning

confidence: 99%

Ribosome Collision Is Critical for Quality Control during No-Go Decay

2017

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SUMMARY No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively our data suggests that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off target effects.

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Asc1, Hel2, and Slh1 couple translation arrest to nascent chain degradation

Cited by 117 publications

References 50 publications

The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay

The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay

Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways

Ribosome Collision Is Critical for Quality Control during No-Go Decay

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