Structural and functional analysis of the ribosomal quality control trigger complex RQT

核糖体质量控制触发复合物 RQT 的结构和功能分析

• 批准号: 512515806
• 负责人: Professor Dr. Roland Beckmann
• 金额: --
• 依托单位: Genzentrum - Laboratorium für molekulare Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/512515806?language=en
• 关键词: Structural; functional; analysis; ribosomal; quality

The translation of mRNA into proteins by ribosomes is a central process which is subject to errors and environmental impact. Thus, for numerous reasons this process can slow down or come to a halt resulting in stalled ribosomes that collide with trailing ones. These ribosomal collisions are recognized in order to be resolved and to serve as a proxy for problematic translation which elicits stress responses and triggers quality control pathways. A key step for collision clearance and a prerequisite to trigger ribosome associated quality control (RQC) is the dissociation of the stalled ribosomes into subunits which is facilitated by the ribosome associated quality control trigger complex RQT. This complex consists in yeast of three and in humans of up to four subunits (hRQT/ASC-1 complex) with a large Ski2-like helicase Slh1 (ASCC3 in humans). Slh1/ASCC3 contains two ATPase cassettes providing the central helicase activity required for ribosomal splitting. Upon ubiquitin modification of the small ribosomal subunit by the stall sensing E3 ligase Hel2 (ZNF598 in humans), RQT is recruited to the stalled ribosomes. Using biochemical assays and cryo-EM in the yeast system, we recently discovered that RQT binds to the small 40S ribosomal subunit and requires the emerging mRNA to remodel and thereby destabilize and split the collided ribosomes. Yet, the exact molecular mechanism of the splitting activity of RQT, e.g., its activation mechanism and the contribution of the individual potential helicase cassettes is not clear yet. Even less is known about the overall architecture of the human RQT complex (hRQT), its interaction with stalled ribosomes and its mode of function in collision dissociation. Therefore, we propose to investigate the molecular mechanism of RQT driven ribosomal splitting in the yeast and the human system. Using mainly in vitro reconstitution approaches and assays, we will analyze the RNA helicase activity of the individual N- and C-terminal ATPase cassettes of Slh1. In addition, we will use cryo-EM in order to directly visualize mRNA-bound RQT on the yeast ribosome by employing different mRNA trapping conditions. In the human system we will directly visualize by cryo-EM the trimeric and tetrameric hRQT in order to gain insights in its overall architecture and potential autoregulatory features. Moreover, we suggest to use in vitro reconstitution assays and in vivo pullouts under hRQT-enriching conditions to visualize hRQT bound to collided human ribosomes, representing distinct intermediate states of the splitting process. Together, this will provide us with biochemical and structural information required to understand the molecular mechanisms employed by RQT/hRQT for the clearance of collided ribosomes and trigger of quality control pathways. The results may also be of value to better understand the underlying mechanisms of pathologies such as certain types of cancer that are caused by a dysfunctional hRQT system.

核糖体将mRNA翻译成蛋白质是一个受错误和环境影响的核心过程。因此，由于种种原因，这一过程可能会减慢或停止，导致核糖体停滞不前，与尾随的核糖体相撞。这些核糖体碰撞被识别是为了解决，并作为问题翻译的代理，引发应激反应和触发质量控制途径。碰撞清除的关键步骤和触发核糖体相关质量控制（RQC）的先决条件是停滞的核糖体解离成亚基，这是由核糖体相关质量控制触发复合物RQT促进的。该复合体在酵母中有3个亚基，在人类中有多达4个亚基（hRQT/ASC-1复合体），具有一个大的ski2样解旋酶Slh1（人类中的ASCC3）。Slh1/ASCC3包含两个atp酶盒，提供核糖体分裂所需的中央解旋酶活性。通过失速感应E3连接酶Hel2（人类中的ZNF598）对小核糖体亚基进行泛素修饰，RQT被招募到失速核糖体中。利用酵母系统中的生化分析和冷冻电镜，我们最近发现RQT与小40S核糖体亚基结合，并需要新出现的mRNA进行重塑，从而破坏和分裂碰撞的核糖体。然而，RQT分裂活性的确切分子机制，如其激活机制和单个潜在解旋酶盒的贡献尚不清楚。人类RQT复合体（hRQT）的整体结构、其与停滞核糖体的相互作用及其在碰撞解离中的功能模式所知甚少。因此，我们建议在酵母和人体系统中研究RQT驱动核糖体分裂的分子机制。主要采用体外重组方法和检测，我们将分析Slh1的单个N端和c端atp酶盒的RNA解旋酶活性。此外，我们将使用冷冻电镜，通过采用不同的mRNA捕获条件，直接观察酵母核糖体上mRNA结合的RQT。在人体系统中，我们将通过低温电镜直接可视化三聚体和四聚体hRQT，以便深入了解其整体结构和潜在的自动调节特征。此外，我们建议在hRQT富集条件下使用体外重构分析和体内提取来观察hRQT与碰撞的人核糖体结合，代表分裂过程的不同中间状态。总之，这将为我们提供了解RQT/hRQT清除碰撞核糖体和触发质量控制途径的分子机制所需的生化和结构信息。这些结果对于更好地理解由hRQT系统功能失调引起的某些类型的癌症等病理的潜在机制也有价值。