Characterization of a New Model Spliceosomal Ribozyme: Activity and Structure Pro

新模型剪接体核酶的表征：活性和结构 Pro

• 批准号: 8211835
• 负责人: Joshua Alan Boyer
• 金额: $ 5.22万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-03 至 2013-01-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8211835
• 关键词: 3' Splice Site; 5' Splice Site; Adenosine; Adopted; Affect; Base Pairing; Binding; Biochemical; Blindness; Cataloging; Catalogs; Catalysis; Catalytic RNA; Cell Extracts; Complex; Data; Dementia; Disease; Equilibrium; Exons; Foundations; Future; Gel; Genetic; Heterogeneity; Human; Instruction; Introns; Kinetics; Label; Literature; Marriage; Measurement; Mediating; Messenger RNA; Methods; Modeling; Molecular Chaperones; Molecular Conformation; Mutation; NMR Spectroscopy; Nucleotides; Population; Positioning Attribute; Process; Proteins; RNA; RNA Folding; RNA Splicing; Radio; Reaction; Reporting; Research; Resolution; Ribonucleases; Sampling; Simulate; Small Nuclear RNA; Source; Spliced Genes; Spliceosomes; Structural Biologist; Structure; Techniques; Therapeutic; Tissues; U5 small nuclear RNA; analog; base; body system; chemical synthesis; combat; design; experience; flexibility; insight; intermolecular interaction; macromolecule; nucleotide analog; protein complex; public health relevance; structural biology; three dimensional structure; tool

DESCRIPTION (provided by applicant): The spliceosome is a huge complex of macromolecules, including over 200 proteins and 6 ribonucleic acids (RNA) or ribo-nucleotide chains. It catalyzes a spectacular reaction removing extraneous instructions from the blueprints of almost every protein in the human cell by extracting and splicing expanses of messenger RNA (mRNA). This function is vital as mis-splicing results in various disease states. Surprisingly, a minimized spliceosome-only two RNA pieces-can perform the catalytic step. However, this model spliceosome, or "ribozyme," demonstrates a slow rate and small yields. This problem is almost certainly centered in RNA's flexibility and its tendency to adopt non-functional structures in the absence of chaperoning proteins. Previous data suggest nucleotide mimics or specifically, conformationally restricted nucleotides (CRNs), can shift non- functional conformations to properly folded/active structures when appropriately placed within an RNA. Thus, if a sample of ribozymes experience a substantially enhanced rate and yield upon CRN substitution this change would indicate an increase in the number of properly folded RNAs. This proposal aspires to design a new catalytic RNA that better recapitulates splicing's two-step reaction by using these nucleotide mimics that are restricted to conformations common in properly folded RNAs. This strategy may simulate the presence of proteins by enforcing the RNA conformation found in the spliceosome's catalytic complex. This proposal details three aims targeted at identifying key positions for CRN inclusion in a newly designed RNA-only spliceosome, herein termed the "splicezyme," as well as recounting the benefits of rational CRN incorporation for RNA structural biology studies. Aim 1 (proposal section C1) describes the thought processes resulting in the new splicezyme construct, and discusses the characterization of the basal activity of this ribozyme prior to CRN substitution. Initial reported findings contained therein suggest the splicezyme successfully generated the first-step product. Proposal section C2 chronicles the structural and biochemical data used to identify candidate positions for rational CRN substitution. Here, the recently-determined structure of a closely related ribozyme defines nucleotides that adopt the same conformation as CRN. Also, there are data implying this configuration in other nucleotide positions. Aim 2 describes splicezyme CRN-substitution positions extrapolated from these data. Finally, aim 3 (proposal section C3) proposes to characterize the three-dimensional structure of an undetermined portion of the splicezyme using CRNs and NMR spectroscopy, a technique that enables spatial measurements with atomic resolution. This aim hypothesizes that CRNs will reduce misfolded RNA accumulation, a major problem of RNA structural biology. In devising the splicezyme and developing CRNs as probes, this research seeks insight into RNA structural aspects involved in splicing catalysis and the spliceosome's intermolecular interactions, which will provide means for rational manipulation in various future biomedical and therapeutic pursuits. PUBLIC HEALTH RELEVANCE: As many as 95% of all human genes are spliced-have extraneous genetic information removed, often in a tissue-specific manner-by the spliceosome. Thus, malfunctions in this ubiquitous process can produce a variety of disparate disease states that are otherwise unrelated, e.g. hemachromatosis, dementia, and blindness. Therefore, characterizing the fundamental attributes of the catalytic spliceosome complex through methods described here will provide insight into conditions affecting various body systems and introduce a foundation for future biomedical and therapeutic pursuits.

描述（由申请人提供）：剪接体是大分子的巨大复合物，包括200多种蛋白质和6种核糖核酸（RNA）或核糖核苷酸链。它通过提取和拼接膨胀的使者RNA（mRNA）来催化壮观的反应，从人类细胞中几乎每种蛋白质的蓝图中除去了无关指令。此功能至关重要，因为错误切开会导致各种疾病状态。令人惊讶的是，最小化的剪接体仅两个RNA零件可以执行催化步骤。但是，该模型剪接或“核酶”表现出缓慢的速率和较小的产率。这个问题几乎可以肯定地集中在RNA的灵活性以及在没有伴侣蛋白的情况下采用非功能结构的趋势。先前的数据表明核苷酸模拟物或具体的构象受限核苷酸（CRN），当适当地放置在RNA中时，可以将非功能构象转移到正确折叠/活性结构。因此，如果核酶样品的速率大大提高，并且在CRN替代时产量会表明正确折叠的RNA数量增加。该建议渴望设计一种新的催化RNA，该RNA通过使用这些核苷酸模拟物限制在正确折叠的RNA中常见的构象，从而更好地概括了剪接的两步反应。该策略可以通过在剪接体的催化复合物中实施RNA构象来模拟蛋白质的存在。 该提案详细介绍了三个目标，目的是确定在新设计的仅RNA剪接体中CRN包含的关键位置，此处称为“剪接”，并叙述了RNA结构生物学研究的理性CRN掺入的好处。 AIM 1（提案第C1部分）描述了导致新剪接构建的思维过程，并讨论了在CRN取代之前该核酶基础活性的表征。其中包含的最初报告的发现表明，剪接成功生成了第一步产品。提案部分C2记录了用于确定理性CRN替代的候选位置的结构和生化数据。在这里，最近确定的紧密相关核酶的结构定义了采用与CRN相同构象的核苷酸。同样，有数据暗示在其他核苷酸位置上进行这种构型。 AIM 2描述了从这些数据推断的剪接CRN固定位置。最后，AIM 3（提案第C3）提出了使用CRN和NMR光谱法表征剪接的三维结构的三维结构，该技术可以通过原子分辨率进行空间测量。这个目标假设CRN将减少RNA结构生物学的主要问题。在设计剪接和开发CRN作为探针时，这项研究寻求深入了解与剪接催化涉及的RNA结构方面和剪接体的分子间相互作用，这将为各种未来的生物医学和治疗性追求提供理性操纵的手段。 公共卫生相关性：多达95％的人类基因是剪接的无关遗传信息，通常是通过组织特异性的 - 剪接体的方式。因此，在这个无处不在的过程中发生故障可以产生各种不同相关的不同疾病状态，例如血红体病，痴呆和失明。因此，通过此处描述的方法来表征催化剪接体复合物的基本属性，将洞悉影响各种身体系统的条件，并为未来的生物医学和治疗性追求介绍基础。