Molecular mechanism of recognizing individual stalled ribosomes by the E3 ligases Mag2 and Fap1

由于许多原因，核糖体对mRNA的翻译可能会在翻译的所有阶段减慢甚至停止。这些停滞的核糖体需要被识别，以引发应激反应，降解有缺陷的翻译组分和产物，并触发下游质量控制途径。几项生化和结构研究表明，与停滞的核糖体碰撞的尾随核糖体提供了一个容易识别的替代物，即，核糖体碰撞，这被证明是细菌和真核生物的关键信号。然而，停滞的起始核糖体和在低起始效率的mRNA上停滞的核糖体不太可能导致碰撞，并且需要特异性识别单个停滞的80 S核糖体。在这里，类似于碰撞识别，E3泛素连接酶，如人类细胞中的RNF 10或酵母中的Mag 2和Fap 1，最近被发现在识别这些个体停滞的核糖体中发挥核心作用，然后通过特定核糖体蛋白的泛素化标记用于下游过程。然而，这些识别和修饰活性的基础分子机制尚不清楚。因此，本项目的总体目标是阐明酵母E3连接酶Mag 2和Fap 1及其人类同源物RNP 10和NFX 1启动质量控制途径的分子机制。具体来说，我们建议解决以下主要问题：一个缓慢的核糖体的功能是由Mag 2识别，执行uS 3单泛素化和什么是识别和多泛素化的个人80 S单体由Fap 1及其辅因子的结构基础？这些停滞的80 S核糖体是如何回收的？RNF 10驱动停滞的人类核糖体的识别和泛素化的机制是什么？Fap 1同源物NFX 1如何与人类核糖体相互作用？我们建议使用冷冻电子显微镜（cryo-EM），以提供Mag 2结合和Fap 1相互作用的核糖体复合物，无论是在体外重建或亲和纯化的酵母细胞的结构。类似地，我们建议使用cryo-EM的结构分析来表征RNF 10和NFX 1与停滞核糖体的相互作用模式。我们希望提供这些复合物的动态行为的见解，从而阐明它们如何在真核细胞中作为传感器的个别停滞的核糖体。这将允许从分子水平上理解这些中心和普遍保守的质量控制过程。

For numerous reasons the translation of mRNA by ribosomes can slow down or even stall during all phases of translation. These stalled ribosomes need to be recognized in order to elicit stress responses, degrade the faulty translation components and products, and trigger downstream quality control pathways. Several biochemical and structural studies revealed that trailing ribosomes that collide with the stalled ribosome provide a readily recognizable proxy, i.e., ribosomal collisions, which turned out to be a key signal in bacteria and eukaryotes. However, stalled initiating ribosomes and ribosomes stalling on mRNAs with low initiation efficiency are unlikely to result in collisions and require the specific recognition of individual stalled 80S ribosomes. Here, similar to collision recognition, E3 ubiquitin ligases, such as RNF10 in human cells or Mag2 and Fap1 in yeast, were recently discovered to play a central role in the recognition of such individual stalled ribosomes which are then tagged by ubiquitination of specific ribosomal proteins for downstream processes. However, the underlaying molecular mechanisms of these recognition and modification activities are not known. Therefore, the overall goal of this project is to elucidate the molecular mechanism of quality control pathway initiation by yeast E3 ligases Mag2 and Fap1 and their human homologs RNP10 and NFX1. Specifically, we suggest to address the following main questions: What features of a slow ribosome are recognized by Mag2 to perform uS3 mono-ubiquitination and what is the structural basis for recognition and poly-ubiquitination of individual 80S monosomes by Fap1 and its cofactors? How are these individual stalled 80S ribosomes recycled? What is the mechanism of RNF10 driven recognition and ubiquitination of stalled human ribosomes and how does the Fap1 homolog NFX1 interact with the human ribosome? We suggest to use cryo-electron microscopy (cryo-EM) to provide structures of Mag2-bound and Fap1-interacting ribosomal complexes which are either reconstituted in vitro or affinity purified from yeast cells. Analogously, we suggest using structural analysis by cryo-EM to characterize the mode of interaction of RNF10 and NFX1 with stalled ribosomes. We expect to provide insights into the dynamic behavior of these complexes thereby elucidating how they function in eukaryotic cells as sensors for individual stalled ribosomes. This will allow for a molecular understanding of these central and universally conserved quality control processes.