Sensing of individual stalled 80S ribosomes by Fap1 for non-functional rRNA turnover. Li et al

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself. 20 ribosome profiling (ribo-seq) samples are included in the study. Ribo-seq samples were obtained from (1) total cell lysates (input) collected prior to purification, (2) MS2-purified ribosomes carrying exogenously encoded wild type or mutant rRNA, or (3) co-purified ribosomes from pull-downs of proteins of interest (Mag2, Fap1, or Yil161w). Input samples serve as controls that are compared against purified ribosomes. Each sample has a biological replicate.

细胞可通过感知核糖体碰撞并启动质控通路来应对停滞的核糖体。然而，在不存在核糖体碰撞的情况下，核糖体停滞如何被解决，这一问题至今仍未阐明。
本研究聚焦于解码缺陷型核糖体所表现出的非碰撞性停滞现象，鉴定出Fap1作为停滞传感器，可通过对核糖体蛋白uS3进行多泛素化修饰，触发18S无功能rRNA的降解通路。
核糖体谱分析（ribosome profiling）显示，Fap1在翻译起始位点存在富集，同时也可与正在延伸的单个核糖体结合。
Fap1结合核糖体的冷冻电镜（Cryo-EM）结构解析表明，Fap1可同时在核糖体的mRNA进入与离开通道处探测mRNA，提示其具备mRNA停滞感知活性；此外Fap1可通过空间位阻阻碍经典碰撞型双核糖体的形成。
本研究结果显示，单个停滞的核糖体可作为核糖体功能异常的潜在信号，进而加速核糖体自身的周转过程。
本研究共包含20份核糖体谱分析（ribosome profiling，简称ribo-seq）样本。ribo-seq样本来源分为三类：(1) 纯化前收集的总细胞裂解液（input对照样本）；(2) 经MS2纯化的、携带外源编码野生型或突变型rRNA的核糖体；(3) 通过靶蛋白（Mag2、Fap1或Yil161w）的pull-down（蛋白下拉纯化）实验共纯化得到的核糖体。input对照样本作为对照组，与纯化后的核糖体样本进行比较。每份样本均设置生物学重复。