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

通过核糖体将mRNA翻译成蛋白质是一个中心过程，该过程受到错误和环境影响。因此，由于许多原因，这一过程可能会减慢或停止，导致停滞的核糖体与尾随的核糖体碰撞。这些核糖体碰撞被识别，以便被解决，并作为有问题的翻译，elevening应激反应和触发质量控制途径的代理。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。这种复合物在酵母中由三个亚基组成，在人类中由多达四个亚基组成（hRQT/ASC-1复合物），具有大的Ski 2样解旋酶Slh 1（人类中的ASCC 3）。Slh 1/ASCC 3含有两个ATP酶盒，提供核糖体分裂所需的中心解旋酶活性。在通过失速感应E3连接酶Hel 2（人类中的ZNF 598）对小核糖体亚基进行泛素修饰后，RQT被募集到失速的核糖体。我们最近在酵母系统中使用生物化学测定和冷冻EM，发现RQT与小的40 S核糖体亚基结合，需要新出现的mRNA来重塑，从而使碰撞的核糖体不稳定并分裂。然而，RQT裂解活性的确切分子机制，例如，其激活机制和单个潜在解旋酶盒的贡献尚不清楚。关于人类RQT复合物（hRQT）的整体结构、其与停滞的核糖体的相互作用及其在碰撞解离中的功能模式，知之甚少。因此，我们建议在酵母和人类系统中研究RQT驱动核糖体裂解的分子机制。主要使用体外重建方法和测定，我们将分析Slh 1的单个N-和C-末端ATP酶盒的RNA解旋酶活性。此外，我们将使用cryo-EM，通过采用不同的mRNA捕获条件，直接观察酵母核糖体上的mRNA结合RQT。在人类系统中，我们将通过冷冻-EM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功能。此外，我们建议使用体外重建试验和hRQT富集条件下的体内拔出可视化hRQT结合碰撞的人核糖体，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。这些结果也可能有助于更好地理解病理学的潜在机制，例如由功能失调的hRQT系统引起的某些类型的癌症。

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.