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: 8061049
• 负责人: Joshua Alan Boyer
• 金额: $ 4.84万
• 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/8061049
• 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; 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（申请人提供）：剪接体是一个巨大的大分子复合体，包括200多种蛋白质和6种核糖核酸（RNA）或核糖核苷酸链。它催化了一个惊人的反应，通过提取和剪接信使RNA （mRNA）的扩展，从人类细胞中几乎所有蛋白质的蓝图中去除多余的指令。当错误剪接导致各种疾病状态时，这个功能是至关重要的。令人惊讶的是，一个最小化的剪接体——只有两个RNA片段——可以执行催化步骤。然而，这种模式剪接体，或“核酶”，显示出缓慢的速率和小产量。这个问题几乎可以肯定地集中在RNA的灵活性和它在没有陪伴蛋白的情况下采用非功能性结构的倾向上。先前的数据表明，核苷酸模拟物，特别是构象限制核苷酸（crn），当适当地放置在RNA中时，可以将非功能构象转变为正确折叠/活性结构。因此，如果核糖酶样品在CRN替换后的速率和产率显著提高，这种变化将表明正确折叠rna的数量增加。本研究旨在设计一种新的催化RNA，通过使用这些核苷酸模拟物来更好地概括剪接的两步反应，这些核苷酸模拟物仅限于正确折叠RNA中常见的构象。这种策略可以通过加强剪接体催化复合体中发现的RNA构象来模拟蛋白质的存在。该提案详细介绍了三个目标，旨在确定新设计的仅RNA剪接体（此处称为“剪接酶”）中CRN包含的关键位置，并叙述了合理的CRN掺入RNA结构生物学研究的好处。目标1（提案部分C1）描述了导致新剪接酶结构的思维过程，并讨论了在CRN取代之前该核酶的基础活性的表征。其中包含的初步报告结果表明，剪接酶成功地产生了第一步产物。提案部分C2记录了用于确定合理CRN替代的候选位置的结构和生化数据。在这里，最近确定的一种密切相关的核酶的结构定义了采用与CRN相同构象的核苷酸。此外，也有数据表明在其他核苷酸位置也有这种构型。目的2描述了从这些数据推断出的剪接酶crn取代位置。最后，目标3（提案部分C3）建议使用crn和核磁共振波谱（一种能够进行原子分辨率空间测量的技术）表征剪接酶未确定部分的三维结构。这一目的假设crn将减少错误折叠的RNA积累，这是RNA结构生物学的一个主要问题。在设计剪接酶和开发crn作为探针的过程中，本研究旨在深入了解剪接催化和剪接体分子间相互作用的RNA结构方面，这将为未来各种生物医学和治疗追求的合理操作提供手段。