Structural and Biochemical Characterization of the S. cerevisiae RNA Exosome

酿酒酵母 RNA 外泌体的结构和生化特征

基本信息

批准号：
8520346
负责人：
Elizabeth Victorina Wasmuth
金额：
$ 4.22万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-11 至 2015-08-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8520346
关键词：
5&apos -exoribonuclease Address Affinity Archaea Architecture Autoimmune Diseases Bacteria Biochemical Biological Assay Catalysis Catalytic Domain Cell Nucleus Cell physiology Cells Complex Crystallization Crystallography Cues Cytoplasm Disease Entropy Environment Equilibrium Eukaryota Exoribonucleases Functional RNA Gene Expression Genetic Transcription Homeostasis In Vitro Label Malignant Neoplasms Mediating Messenger RNA Molecular Morphologic artifacts Nature Nuclear Pathway interactions Positioning Attribute Process Proteins Quality Control RNA RNA Binding RNA Decay RNA Degradation RNA Processing Recombinants Relative (related person)Resolution Ribosomal RNA Role Route Saccharomyces cerevisiae Sampling Shapes Small RNA Structure Substrate Specificity Surface System Time Transcript Transcription Process Translations X-Ray Crystallography Yeasts base genetic analysis insight milligram particle reconstitution response scaffold

项目摘要

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.

描述（由申请人提供）：适当的细胞功能的一个要求是 RNA 稳态，这是通过转录和 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。