Physical and functional probing of DEAD-box proteins as general RNA chaperones

作为一般 RNA 伴侣的 DEAD-box 蛋白的物理和功能探测

• 批准号: 7737923
• 负责人: Rick Russell
• 金额: $ 32.34万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737923
• 关键词: Adopted; Affect; Binding; Biological Assay; Boxing; C-terminal; Catalytic RNA; Cell physiology; Colon; Complex; Disease; Docking; Elements; Fluorescence Microscopy; Foundations; Free Energy; Funding; Goals; Hepatitis C virus; Human; Hydroxyl Radical; In Vitro; Introns; Knowledge; Life; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Metabolism; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Neurospora crassa; Oligonucleotides; Pathway interactions; Physiological; Population; Process; Proteins; RNA; RNA Splicing; RNA Stability; RNA-Binding Proteins; Reaction; Relative (related person); Rest; Role; Solutions; Specificity; Structure; Suggestion; System; Tail; Testing; Tetrahymena; Tetrahymena thermophila; Translations; Viral; Virus Replication; Work; base; conformer; human disease; insight; mRNA Precursor; mutant; overexpression; physical property; preference; protein function; protein transport; public health relevance; single molecule; three dimensional structure; yeast protein

DESCRIPTION (provided by applicant): DExD/H-box proteins are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. These proteins are thought to use energy from ATP to facilitate RNA conformational changes and folding transitions, but relatively little is known on a molecular level about how they manipulate RNA structure. While many of the RNA and RNA-protein targets of DExD/H-box proteins are large, complex, and difficult to study in vitro, it was shown in 2002 that the Neurospora crassa CYT-19 protein functions in folding of several mitochondrial group I introns. Further work indicated that CYT-19 can also interact productively with group II introns, indicating that it possesses general RNA chaperone activity, and the related yeast protein Mss116p was shown to function similarly. The goal of this project has been to use the well-defined, tractable system of CYT-19 and group I RNAs to dissect the mechanisms of RNA chaperone activity by DExD/H-box proteins. In addition to increasing knowledge of essential cellular processes, this work has implications for diseases, as DExD/H-box proteins are required for replication of viruses including HCV, and overexpression of human DExD/H-box proteins is linked to colon and prostate cancer. Progress during the current funding period led to a general model for RNA chaperone activity, around which this proposal is centered. First, CYT-19 can disrupt RNA structure indiscriminately, unfolding both the native state and a long-lived misfolded conformer of a non-cognate group I intron with efficiencies that depend on the relative stabilities of the RNA species but not on any specific structural features. However, its activity is not fully indiscriminate; it acts preferentially on structured RNAs by forming an additional 'tethering' interaction, and it unwinds a short helix of a group I intron (P1) only when P1 does not 'dock' into tertiary contacts with the rest of the RNA. These results suggest that CYT-19 may disrupt preferentially misfolded RNAs that cannot pack correctly. The goals of the current proposal are to delve further into the physical basis of RNA chaperone activity by DExD/H-box proteins, to probe for specificity using CYT-19 and its physiological substrates, and to test hypotheses on the mechanisms and implications of tethering and inhibition by tertiary contacts. Specific Aims are: 1) to use an oligonucleotide displacement assay and directed hydroxyl radical footprinting to probe pathways of RNA chaperone activity on group I introns; 2) to determine whether CYT-19 acts on its cognate group I introns with specificity that is absent with a non-cognate RNA; 3) to probe further the inhibition by tertiary contact formation, including testing the hypothesis that the inhibition is a general feature of chaperone activity by determining whether it is present for different structural elements and within a different group I intron; and 4) to test the hypotheses that the tethering interaction is formed by a highly basic 'tail' of CYT-19 and related DExD/H-box proteins, and that this interaction can remain intact during local unwinding. Results are expected to guide models for DExD/H-box proteins in all aspects of RNA metabolism. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how RNA chaperone proteins assist RNAs as they fold to specific structures and exchange between structures. These proteins are required for viral replication and are linked to human cancer, so understanding how they function is important for understanding and ultimately treating human disease.

描述（由申请人提供）：几乎每个由结构化RNA进行的过程所需的DEXD/H-box蛋白，从MRNA剪接和翻译到蛋白质和RNA的细胞内运输。这些蛋白质被认为使用来自ATP的能量来促进RNA构象变化和折叠过渡，但是在分子水平上，人们对它们如何操纵RNA结构的了解相对较少。虽然DEXD/H-box蛋白的许多RNA和RNA蛋白靶标是大型，复杂且难以在体外研究的，但2002年表明，神经孢子Crassa cyt-19蛋白在折叠几个线粒体I组INTRON的折叠中的功能。进一步的工作表明，CYT-19还可以与II组内含子有效地相互作用，表明它具有一般的RNA伴侣活性，并且相关的酵母蛋白MSS116P的功能相似。该项目的目的是使用CYT-19和I组RNA的定义明确的，可拖动的系统来通过DEXD/H-box蛋白来剖析RNA伴侣活性的机制。除了对基本细胞过程的知识越来越多，这项工作还对疾病具有影响，因为DEXD/H-box蛋白需要复制包括HCV在内的病毒，并且人dexD/H-box蛋白的过表达与结肠和前列腺癌有关。在当前资金期间的进展导致了RNA伴侣活动的一般模型，该提案围绕着该建议。首先，Cyt-19可以不加区分地破坏RNA结构，从而展现了本地状态和长期存在的非认知基团I内含子的错误折叠构象体，其效率取决于RNA物种的相对稳定性，但不取决于任何特定的结构特征。但是，它的活性并非完全不可分割。它通过形成额外的“绑定”相互作用来优先在结构化的RNA上起作用，并且仅当P1不将P1“ dock”与其余RNA接触到第三次接触时，它才能放弃I组内含子（P1）的短螺旋。这些结果表明，Cyt-19可能会优先破坏无法正确包装的RNA。当前建议的目标是通过DEXD/H-box蛋白进一步研究RNA伴侣活性的物理基础，以使用CYT-19及其生理底物探测特异性，并对第三键接触的绑带和抑制作用的机制和含义进行假设。具体目的是：1）使用寡核苷酸位移测定法和定向羟基自由基足迹以探测I组内含子上RNA伴侣活性的途径； 2）确定Cyt-19是否具有具有非认知RNA的特异性的同源I组内含子； 3）进一步探测第三接触形成的抑制作用，包括测试抑制是通过确定是否存在于不同的结构元素和不同组I内含子内的抑制作用的假设。 4）为了测试束缚相互作用的假设是由Cyt-19和相关DEXD/H-box蛋白的高度碱性“尾巴”形成的，并且在局部放松期间，这种相互作用可以保持完整。预期在RNA代谢的各个方面，将指导DEXD/H-box蛋白的模型。 公共卫生相关性：该项目的目的是了解RNA伴侣蛋白如何帮助RNA折叠成特定的结构和结构之间的交换。这些蛋白质是病毒复制所必需的，并且与人类癌症有关，因此了解它们的作用对于理解和最终治疗人类疾病至关重要。