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

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多个蛋白质和6个核糖核酸(RNA)或核糖核酸链。它催化了一种壮观的反应，通过提取和拼接信使RNA(信使RNA)的扩展部分，从人类细胞中几乎每一种蛋白质的蓝图中去除了无关的指令。这一功能至关重要，因为错误拼接会导致各种疾病状态。令人惊讶的是，一个最小的剪接体--只有两个RNA片段--就可以执行催化步骤。然而，这种模型剪接体，或“核酶”，表现出缓慢的速度和较小的产量。这个问题几乎肯定集中在RNA的灵活性和它在没有伴侣蛋白的情况下采用非功能结构的倾向。先前的数据表明，当适当地放置在RNA中时，核苷酸模拟物或具体而言，构象受限的核苷酸(CRN)可以将非功能构象转变为适当的折叠/活性结构。因此，如果核酶样本在CRN替换后经历了显著的速率和产量的提高，这种变化将表明正确折叠的RNA的数量增加。这一提议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，这些核苷酸模拟物被限制在正确折叠的RNA中常见的构象。这一策略可以通过强制剪接体的催化复合体中发现的RNA构象来模拟蛋白质的存在。这项提案详细说明了三个目标，旨在确定CRN在新设计的纯RNA剪接体中的关键位置，本文称为“剪接酶”，以及讲述合理的CRN掺入对RNA结构生物学研究的好处。目的1(建议部分C1)描述了导致新剪接酶构建的思维过程，并讨论了在CRN替换之前该核酶的基础活性的特征。其中包含的初步研究结果表明，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替换的候选位置的结构和生化数据。这里，最近确定的密切相关核酶的结构定义了采用与CRN相同构象的核苷酸。此外，还有数据表明在其他核苷酸位置上也存在这种构型。目的2描述从这些数据推断的剪接酶CRN-替换位置。最后，目标3(提议的C3节)建议使用CRNS和核磁共振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。这一目标假设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。在设计剪接酶和开发CRN作为探针的过程中，本研究试图深入了解剪接催化和剪接体的分子间相互作用所涉及的RNA结构方面，这将为未来各种生物医学和治疗方面的合理操作提供手段。 与公共卫生相关：多达95%的人类基因被剪接体剪接--通常以特定于组织的方式移除无关的遗传信息--通过剪接体。因此，在这个无处不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、痴呆症和失明。因此，通过本文描述的方法表征催化剪接体复合体的基本属性将有助于深入了解影响各种身体系统的条件，并为未来的生物医学和治疗研究奠定基础。

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.