Characterization of ribosomal stalling sequences and their recognition by the RQT quality control factors

核糖体停滞是质量控制机制的一个标志，它已经进化到防止潜在毒性新生蛋白的损害和拯救在异常mrna上停滞的核糖体。除了mRNA异常外，特定的非最佳密码子对和mRNA的多碱基编码序列也可能导致核糖体停滞。该研究项目旨在发现mRNA序列诱导的核糖体失速的分子机制，并随后控制对这些序列元件启动核糖体相关质量控制（RQC）。将遵循三个互补的目标来解决以下关键问题：(i)诱导停速序列对核糖体的具体结构影响是什么，（ii）如何识别停速核糖体，以及（iii）如何触发RQC反应。在所有情况下，将采用单粒子低温电子显微镜（cryo-EM）来获得这些过程的详细机制。首先，我们将使用一个已建立的人工mRNA报告系统，其中包含已确认的失速诱导序列。我们将修改这一系统，编码一系列不同的多碱基和串联密码子失速诱导序列，以分离特异性失速核糖体。冷冻电镜将用于揭示停滞核糖体的特定结构特征，如肽基转移酶中心的潜在扰动。接下来，我们将分离出一种新的rqc触发因子Hel2，并对其进行结构表征。这将通过从酵母细胞中分离或通过体外重组来完成。该方法有望揭示Hel2识别特异性停滞核糖体的哪些共同结构特征以触发RQC反应。最后，我们计划在Hel2存在或不存在的情况下获得具有新型RQC触发（RQT）复合物（Slh1-Cue3-Ykr023w）的核糖体。在这里，我们将使用与核糖体- hel2复合物类似的方法，并使用天然下拉协议纯化核糖体- rqt复合物，以产生合适的冷冻电镜样品。同时，我们将从原核或真核表达系统（大肠杆菌、酿酒葡萄球菌、昆虫或人类细胞培养）中纯化单个组分，并用停滞的核糖体重建它们。这些结合的方法将使我们对核糖体失速的机制和rqc触发机制的识别获得重要的见解。因此，该项目将为多碱基和串联密码子mRNA延迟序列引起的延迟过程提供详细的机制基础。揭示Hel2如何与核糖体相互作用将揭示其通过非蛋白酶体泛素化触发RQC的作用。到目前为止，RQT复合体的后续作用就其机制和功能基础而言是完全神秘的，因此，我们的目标是提供其在质量控制途径中的结构作用的第一眼。

Ribosomal stalling is a hallmark of quality control mechanisms that have evolved to prevent damage from potentially toxic nascent proteins and rescue of ribosomes stalled on aberrant mRNAs. Besides aberrant mRNA, ribosomal stalling can also be caused by specific non-optimal codon pairs and polybasic encoding sequences of mRNA. The proposed research project aims at discovering the molecular mechanisms, which cause mRNA sequence-induced ribosomal stalling and, subsequently, control initiation of ribosome-associated quality control (RQC) on such sequence elements. Three complementary aims will be followed to address pivotal questions of (i) what the specific structural effects of stalling-inducing sequences on ribosomes are, (ii) how the stalled ribosomes are recognized and (iii) how the RQC response is triggered. In all cases, single-particle cryo-electron microscopy (cryo-EM) will be employed to obtain detailed mechanistic insight into these processes. First, we will use an established artificial mRNA reporter system containing confirmed stalling-inducing sequences. We will modify this system to encode a range of different polybasic and tandem codon stalling-inducing sequences in order to isolate specifically stalled ribosomes. Cryo-EM will be used to reveal specific structural features of stalled ribosomes such as potential perturbations of the peptidyl-transferase center. Next, we will isolate and structurally characterize a novel RQC-triggering factor Hel2 in complex with stalled ribosomes obtained in the previous step. This will be done either by isolation from yeast cells or by in vitro reconstitution. This approach is expected to reveal which common structural features of the specifically stalled ribosomes are recognized by Hel2 in order to trigger the RQC response.Finally, we plan to obtain ribosomes in complex with the novel RQC triggering (RQT) complex (Slh1-Cue3-Ykr023w) in presence or absence of Hel2. Here, we will use a similar approach as for ribosome-Hel2 complexes and use the native pulldown protocol to purify ribosome-RQT complexes to produce suitable cryo-EM samples. In parallel, we will purify the individual components from prokaryotic or eukaryotic expression systems (E. coli, S. cerevisiae, insect or human cell culture) and reconstitute them with stalled ribosomes.These combined approaches will allow us to gain important insights into the mechanisms of ribosomal stalling and its recognition by the RQC-triggering machinery. This project will, thus, provide a detailed mechanistic basis of the stalling process caused by polybasic and tandem codon mRNA stalling sequences. Revealing how Hel2 interacts with the ribosome will shed light on its role in triggering RQC by non-proteasomal ubiquitination. The subsequent engagement of the RQT complex is entirely enigmatic so far with respect to its mechanistic and functional basis, and we therefore aim to provide a first glimpse at its structural role in the quality control pathway.