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，这种机制已经进化，以防止潜在有毒新生蛋白的损害，并拯救停滞在异常mRNAs上的核糖体。核糖体停滞的原因除了mRNA的异常外，还可以由特定的非最佳密码子对和多碱基的编码序列引起。该研究项目旨在发现导致mRNA序列诱导的核糖体停滞的分子机制，从而控制核糖体相关质量控制(RQC)在这些序列元件上的启动。将遵循三个互补的目标来解决关键问题：(I)停滞诱导序列对核糖体的具体结构影响是什么，(Ii)停滞的核糖体是如何识别的，以及(Iii)RQC反应是如何被触发的。在所有情况下，将使用单粒子低温电子显微镜(CRYO-EM)来获得对这些过程的详细机制洞察。首先，我们将使用已建立的包含已确认的停滞诱导序列的人工mRNA报告系统。我们将对这个系统进行修改，以编码一系列不同的多碱基和串联密码子停滞诱导序列，以分离特定停滞的核糖体。冷冻-EM将被用来揭示停滞的核糖体的特定结构特征，例如肽转移酶中心的潜在扰动。接下来，我们将分离并鉴定一种新的RQC触发因子Hel2 in与上一步中获得的停滞的核糖体形成的复合体。这将通过从酵母细胞中分离或通过体外重组来完成。这一方法有望揭示Hel2为了触发RQC反应而识别特定停滞的核糖体的哪些共同结构特征。最后，我们计划在存在或不存在Hel2的情况下获得与新型RQC触发(RQT)复合体(Slh1-Cue3-Ykr023w)的核糖体。在这里，我们将使用与核糖体-Hel2复合体类似的方法，并使用本地下拉方案来纯化核糖体-RQT复合体，以产生合适的冷冻-EM样品。同时，我们将从原核或真核表达系统(大肠杆菌、酿酒酵母、昆虫或人类细胞培养)中提纯单个组分，并将它们与停滞的核糖体重组。这些结合的方法将使我们能够获得重要的见解，了解核糖体停滞的机制及其通过RQC触发机制的识别。因此，该项目将为多碱基和串联密码子mRNA失速序列引起的失速过程提供详细的机制基础。揭示Hel2是如何与核糖体相互作用的，将有助于揭示它在通过非蛋白酶体泛素化触发RQC中的作用。到目前为止，RQT复合体的后续参与在其机制和功能基础上是完全谜一样的，因此我们的目标是提供第一个关于它在质量控制途径中的结构作用的一瞥。

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.