RQT complex dissociates ribosomes collided on endogenous RQC substrate SDD1

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of collided di- and preferentially tri-ribosomes. Distinct trisome architecture was visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.核糖体相关质量控制（Ribosome-associated quality control, RQC）是真核细胞中在翻译停滞时激活的拯救通路。碰撞核糖体首先被识别，随后发生解离，新生肽段也随之被降解。然而，内源性RQC诱导序列以及泛素依赖的核糖体解离的分子机制仍有待进一步阐明。本研究从酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）中鉴定出SDD1 mRNA作为内源性RQC底物，并通过突变分析与冷冻电镜（cryo-EM）实验揭示了其依赖于mRNA与新生肽段的翻译停滞机制。SDD1 mRNA的体外翻译可重构碰撞二聚核糖体与优先靶向三聚核糖体的Hel2依赖型多泛素化反应。冷冻电镜可视化了独特的三聚核糖体结构，该结构为Hel2相较于二聚核糖体实现更高效的识别提供了结构基础。随后，RQC触发复合物（RQC trigger, RQT）的Slh1解旋酶亚基以ATP依赖的方式优先解离首个停滞的多泛素化核糖体。综上，本研究为RQC及其在维持细胞蛋白质稳态中的生理功能提供了关键的机制性见解。