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

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):正常细胞功能的一个必要条件是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。真核外切体是细胞中主要的3‘5’外切核糖核酸酶,在细胞质和细胞核中具有多方面的作用,包括RNA的降解、加工和质量控制。酿酒酵母外显体的非催化核心由9个不同的必需亚基组成,它们可能形成环状结构(Exo9)。这个核心与细胞核和细胞质中的前进性、水解性外核糖核酸酶Rrp44以及细胞核中分布的、水解性外核糖核酸酶RRP6有关。已知RRP6活性对于处理核糖体RNA等结构化底物很重要。Exo9在调节其催化亚基Rrp44和RRP6活性方面的确切作用尚不清楚。该领域的一个悬而未决的问题是,是否所有被外切体靶向的RNA底物都穿过了孔,就像它们在古生菌和细菌的RNA降解组装中一样。因为这些复合体的结构是保守的,所以很容易推测真核外体是通过类似的机制工作的。此外,生化证据表明,在体外,RRP6的活性相对于Rrp44在Exo11中占主导地位。因此,另一个问题与Exo9上这两个催化亚基的可及性以及它们之间的相对位置有关。为了解决这些问题,X射线结晶学将被用于确定9-亚基核心及其催化亚基和结合的RNA底物的结构,为结构如何对功能做出贡献提供机制方面的见解。此外,RNA衰变分析将动态地描述核心如何调节Rrp44和RRP6的活动,以及孔洞如何参与RNA衰变。缺乏亲和力标签的重组酵母外切体蛋白将被提纯并用于生化重组。这一策略将促进9-、10-和11-亚单位外切体复合体的重组,这些复合体除了通过表面熵降低提供更易于结晶的样品外,还能更好地模仿它们的细胞同行。

项目成果

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Elizabeth Victorina Wasmuth其他文献

Elizabeth Victorina Wasmuth的其他文献

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{{ truncateString('Elizabeth Victorina Wasmuth', 18)}}的其他基金

Biochemical, structural and molecular dissection of androgen receptor transcriptional activity
雄激素受体转录活性的生化、结构和分子剖析
  • 批准号:
    10655002
  • 财政年份:
    2022
  • 资助金额:
    $ 4.22万
  • 项目类别:
Biochemical, structural and molecular dissection of androgen receptor transcriptional activity
雄激素受体转录活性的生化、结构和分子剖析
  • 批准号:
    10321278
  • 财政年份:
    2021
  • 资助金额:
    $ 4.22万
  • 项目类别:
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:
    8126951
  • 财政年份:
    2011
  • 资助金额:
    $ 4.22万
  • 项目类别:
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
  • 财政年份:
    2011
  • 资助金额:
    $ 4.22万
  • 项目类别:
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:
    8705540
  • 财政年份:
    2011
  • 资助金额:
    $ 4.22万
  • 项目类别:

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