Structural and Biochemical Characterization of the S. cerevisiae RNA Exosome

描述（由申请人提供）：正常细胞功能的一个要求是 RNA 稳态，这是一个通过转录和 RNA 周转之间的平衡维持的过程。真核外泌体是细胞主要的 3'5' 外切核糖核酸酶，具有多方面的作用，包括细胞质和细胞核中的 RNA 降解、加工和质量控制。酿酒酵母外泌体的非催化核心由 9 个不同且必需的亚基组成，这些亚基可能形成环形结构 (Exo9)。该核心与细胞核和细胞质中的进行性水解核糖核酸外切酶 Rrp44 相关，并与细胞核中的分配性水解核糖核酸外切酶 Rrp6 相关。已知 Rrp6 活性对于处理结构化底物（例如核糖体 RNA）非常重要。 Exo9 在调节其催化亚基 Rrp44 和 Rrp6 活性中的确切作用尚不清楚。该领域的一个突出问题是，外泌体靶向的所有 RNA 底物是否都穿过孔，就像它们在来自古细菌和细菌的 RNA 降解组件中一样。由于这些复合物的结构在结构上是保守的，因此很容易推测真核外泌体通过类似的机制发挥作用。此外，生化证据表明，在体外 Exo11 中，Rrp6 活性相对于 Rrp44 而言占主导地位。因此，另一个问题涉及 Exo9 上这两个催化亚基的可及性，以及它们相对于彼此的位置。为了解决这些问题，X射线晶体学将用于确定与其催化亚基和结合的RNA底物复合的9亚基核心的结构，从而提供结构如何影响功能的机制见解。此外，RNA 衰变测定将在动力学上描述核心如何调节 Rrp44 和 Rrp6 的活性，以及​​孔如何（如果有的话）参与 RNA 衰变。缺乏亲和标签的重组酵母外泌体蛋白将被纯化并用于生化重建。该策略将促进 9、10 和 11 亚基外泌体复合物的重建，从而更好地模拟其细胞对应物，此外还通过表面熵减少提供更适合结晶的样品。 公共健康相关性：RNA 转录和衰变之间的平衡对于正常的细胞功能至关重要。 RNA 外泌体参与 RNA 加工、成熟、降解和质量控制。外泌体介导的过程的破坏与癌症和自身免疫性疾病等疾病有关。该项目将研究 RNA 外泌体调节这些关键活动的机制。

DESCRIPTION (provided by applicant): A requirement for proper cellular function is RNA homeostasis, a process maintained through a balance between transcription and RNA turnover. The eukaryotic exosome is the cell's major 3'5' exoribonuclease, with multi-faceted roles that include RNA degradation, processing, and quality control in the cytoplasm and nucleus. The non-catalytic core of the Saccharomyces Cerevisiae exosome is composed of 9 distinct and essential subunits that likely form a ring-shaped structure (Exo9). This core is associated with a processive, hydrolytic exoribonuclease, Rrp44 in the nucleus and cytoplasm, and with the distributive, hydrolytic exoribonuclease, Rrp6 in the nucleus. Rrp6 activities are known to be important for processing structured substrates such as ribosomal RNA. The precise role of Exo9 in modulating the activities of its catalytic subunits, Rrp44 and Rrp6 is unknown. An outstanding question in the field is whether all RNA substrates targeted by the exosome are threaded through the pore, as they are in RNA degradation assemblies from archaea and bacteria. Because the architecture of these complexes is structurally conserved, it is tempting to speculate that the eukaryotic exosome operates through a similar mechanism. Additionally, biochemical evidence shows that Rrp6 activity is predominant relative to Rrp44 in Exo11 in vitro. Therefore, another question pertains to the accessibility of these two catalytic subunits on Exo9, and their positions relative to each other. To address these questions, X-ray crystallography will be used for structure determination of the 9-subunit core in complex with its catalytic subunits and a bound RNA substrate, providing mechanistic insight to how architecture contributes to function. Furthermore, RNA decay assays will kinetically describe how the core modulates the activities of Rrp44 and Rrp6, and how, if at all, the pore is involved in RNA decay. Recombinant yeast exosome proteins that lack affinity tags will be purified and used for biochemical reconstitution. This strategy will facilitate reconstitution of the 9-, 10-, and 11-subunit exosome complexes that better mimic their cellular counterparts in addition to providing samples more amenable to crystallization through surface entropy reduction. PUBLIC HEALTH RELEVANCE: The balance between RNA transcription and decay is essential for proper cellular function. The RNA exosome is involved in RNA processing, maturation, degradation, and quality control. Disruption of exosome-mediated processes are associated with diseases such as cancer and autoimmune disorders. This project will address the mechanism by which the RNA exosome regulates these key activities.