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（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。它催化了一个壮观的反应，通过提取和剪接大量的信使RNA（mRNA），从人类细胞中几乎每一种蛋白质的蓝图中去除无关的指令。这种功能至关重要，因为错误剪接导致各种疾病状态。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，这种模型剪接体或“核酶”表现出缓慢的速率和小的产量。这个问题几乎可以肯定是集中在RNA的灵活性和它的倾向，采取非功能性的结构，在没有伴侣蛋白。先前的数据表明，核苷酸模拟物或具体地，构象限制核苷酸（CRN）当适当地放置在RNA内时，可以将非功能性构象转变为适当折叠/活性结构。因此，如果核酶样品在CRN取代后经历显著增强的速率和产率，则这种变化将表明正确折叠的RNA的数量增加。该提案旨在设计一种新的催化RNA，通过使用这些核苷酸模拟物更好地再现剪接的两步反应，这些核苷酸模拟物仅限于正确折叠的RNA中常见的构象。这种策略可以通过加强剪接体催化复合物中发现的RNA构象来模拟蛋白质的存在。 该提案详细介绍了三个目标，目标是确定CRN包含在新设计的仅RNA剪接体中的关键位置，本文称为“剪接酶”，以及叙述合理的CRN掺入RNA结构生物学研究的好处。目的1（提案部分C1）描述了新的剪接酶构建的思想过程，并讨论了CRN取代前这种核酶的基本活性的表征。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。提案第C2节记录了用于确定合理CRN取代的候选位置的结构和生化数据。在这里，最近确定的结构密切相关的核酶定义的核苷酸，采用相同的构象作为CRN。此外，有数据表明在其他核苷酸位置存在这种构型。目的2描述剪接酶CRN取代位置外推从这些数据。最后，目标3（提案部分C3）提出使用CRN和NMR光谱来表征剪接酶的未确定部分的三维结构，这是一种能够以原子分辨率进行空间测量的技术。这个目标假设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。在设计的剪接酶和开发CRN作为探针，本研究旨在深入了解RNA的结构方面参与剪接催化和剪接体的分子间相互作用，这将提供合理的操作在未来的各种生物医学和治疗的追求。 公共卫生关系：多达95%的人类基因都是通过剪接体剪接的，剪接体通常以组织特异性的方式去除无关的遗传信息。因此，这个普遍存在的过程中的故障可以产生各种不同的疾病状态，否则是无关的，例如血色病，痴呆和失明。因此，通过本文描述的方法表征催化剪接体复合物的基本属性将提供对影响各种身体系统的条件的洞察，并为未来的生物医学和治疗追求奠定基础。