Structural and Biochemical Characterization of the S. cerevisiae RNA Exosome

描述（由申请人提供）：正常细胞功能的要求是RNA稳态，这是一个通过转录和RNA周转之间的平衡来维持的过程。真核外泌体是细胞中主要的3‘5’外核糖核酸酶，在细胞质和细胞核中具有多方面的作用，包括RNA降解、加工和质量控制。Saccharomyces Cerevisiae外泌体的非催化核心由9个不同的基本亚基组成，可能形成环状结构（Exo9）。该核心与核和细胞质中的进程性水解外核糖核酸酶Rrp44以及核中的分布性水解外核糖核酸酶Rrp6有关。众所周知，Rrp6的活性对于处理结构底物（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，是否所有被外泌体靶向的RNA底物都穿过孔，就像它们在古细菌和细菌的RNA降解组装中一样。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生物的外泌体通过类似的机制起作用。此外，生化证据表明，在体外Exo11中，相对于Rrp44， Rrp6的活性占主导地位。因此，另一个问题涉及到这两个催化亚基在Exo9上的可及性，以及它们彼此之间的相对位置。为了解决这些问题，x射线晶体学将用于确定9亚基核心及其催化亚基和结合的RNA底物的结构，为结构如何促进功能提供机制见解。此外，RNA衰变分析将动态地描述核如何调节Rrp44和Rrp6的活性，以及如果有的话，孔如何参与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组。这种策略将促进9-、10-和11-亚基外泌体复合物的重组，更好地模仿它们的细胞对应物，除了提供更适合通过表面熵降低结晶的样品外。

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.