Distinct ribosome states trigger diverse mRNA quality control pathways

Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully-designed reporter mRNAs to perform genetic screens and functional assays in S. cerevisiae. We characterize the roles of Hel2 and Syh1 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 acts as the primary link to mRNA decay in NGD. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new mechanistic insight into the role of Syh1 in NGD and define the molecular triggers that determine how distinct pathways target mRNAs for degradation in yeast.关键衔接蛋白可将真核生物中特定类别异常mRNA的翻译过程与mRNA降解相偶联。非最优密码子的翻译速率缓慢会引发密码子最优性介导的降解（codon-optimality-mediated decay, COMD）；而核糖体在停滞位点的长时间停留则会触发停滞降解（no-go decay, NGD）。介导上述两类过程的降解因子的具体身份，以及它们响应翻译应激的分子机制，仍是尚未阐明的研究方向。我们借助精心设计的报告基因mRNA，在酿酒酵母（S. cerevisiae）中开展遗传筛选与功能测定实验。我们对Hel2与Syh1在NGD报告基因mRNA上协调翻译抑制与mRNA降解的作用进行了系统表征，发现Syh1是NGD通路中连接mRNA降解的核心纽带。值得注意的是，我们发现上述NGD相关因子并不参与富含非最优密码子的mRNA的降解过程。此外，我们证实此前被报道参与COMD过程的关键因子Not5，仅对NGD靶向mRNA的降解具有轻微的促进作用。最后，我们通过核糖体谱分析（ribosome profiling）揭示了与每一种报告基因mRNA相关的独特核糖体状态，这些状态可合理解释NGD与COMD因子对这些报告基因mRNA降解的调控作用。综上，本研究为Syh1在NGD通路中的功能提供了全新的机制视角，并阐明了酿酒酵母中决定不同降解通路靶向mRNA的分子触发因素。