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

• 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
• 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' -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

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）：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周转之间的平衡来维持的过程。真核外泌体是细胞的主要3 '5'核糖核酸外切酶，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状结构（Exo 9）。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Rrp 44以及细胞核中的分配性水解核糖核酸外切酶Rrp 6相关。已知Rrp 6活性对于加工结构化底物如核糖体RNA是重要的。Exo 9在调节其催化亚基Rrp 44和Rrp 6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降解组装体中一样。由于这些复合物的结构在结构上是保守的，因此推测真核外泌体通过类似的机制起作用是很诱人的。此外，生物化学证据表明，在体外Exo 11中，Rrp 6活性相对于Rrp 44是主要的。因此，另一个问题涉及这两个催化亚基在Exo 9上的可及性，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9-亚基核心与其催化亚基和结合RNA底物的复合物的结构，为结构如何有助于功能提供机制见解。此外，RNA衰变测定将从动力学上描述核心如何调节Rrp 44和Rrp 6的活性，以及孔如何参与RNA衰变。将纯化缺乏亲和标签的重组酵母外泌体蛋白并用于生化重建。该策略将促进9-、10-和11-亚基外泌体复合物的重构，所述复合物除了提供更易于通过表面熵减少结晶的样品之外，还更好地模拟它们的细胞对应物。