Kinetic Dissection of the RNA Chaperone Protein CYT-19

RNA 伴侣蛋白 CYT-19 的动力学解剖

• 批准号: 6889603
• 负责人: Rick Russell
• 金额: $ 29.56万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6889603
• 关键词: RNA splicing; Tetrahymena; chemical kinetics; intermolecular interaction; molecular chaperones; oligonucleotides; protein folding; protein structure function; ribozymes; sedimentation equilibrium; site directed mutagenesis; stoichiometry; thermodynamics

DESCRIPTION (provided by applicant): The proposed research is a dissection of the targeting and action of the RNA chaperone CYT-19 protein as it facilitates folding of a group I RNA. Although RNA chaperones were proposed long ago, the first demonstration that any protein functions naturally to chaperone RNA folding was in 2002, when Lambowitz and colleagues showed that the Neurospora crassa CYT-19 protein functions by accelerating folding of several group I introns. These introns also require the splicing factor CYT-18, additionally suggesting that CYT-19 may be targeted to RNAs by CYT-18. CYT-19 is a DExD/H-box protein, which are present in all organisms and involved in virtually every process that involves structured RNA, including essential processes like ribosome biogenesis and pre-mRNA splicing, as well the replication of viruses including HCV. DExD/H-box proteins are generally thought to use ATP binding and hydrolysis to mediate structural rearrangements of RNA, facilitating folding to a functional structure or transitions between functional structures. However, little is known about their mechanisms of action or about what governs their specificity for RNA or RNA-protein substrates, largely because of the complexity of many of their targets. The system above provides a unique opportunity for biochemical dissection because it is sufficiently simple that it can be readily manipulated, yet sufficiently complete that it captures a physiological action of a DExD/H-box protein. However, folding of Neurospora group I introns is not well characterized and the best-studied intron populates multiple misfolded forms, inhibiting a deep biochemical dissection. This proposal therefore focuses instead on the group I RNA from Tetrahymena and its derivative that binds CYT-18. The Tetrahymena RNA is extensively characterized and folds to a discrete misfolded form whose re-folding to the native state is also accelerated by CYT-19. Specific aims are to 1) Use kinetics approaches to probe the molecular action of CYT-19 in re-folding the Tetrahymena ribozyme, comparing the reaction to nonspecific unwinding of duplex RNA and testing specific models for the mechanism of ribozyme re-folding; 2) Examine determinants and mechanisms of specificity conferred by the CYT-18 protein; 3) Use sedimentation equilibrium to probe the self-association behavior of CYT-19 in solution and bound to RNA, then use this information to probe the multimeric state of CYT-19 as it acts; 4) Explore an unexpected activity of CYT-19, dissociation of an oligonucleotide substrate from the ribozyme. These experiments are intended to provide novel and fundamental understanding of how a DExD/H-box protein acts as an RNA chaperone and how it is targeted. Results here are expected to guide models for the action and targeting of DExD/H box proteins involved in all aspects of RNA metabolism and function.

描述（由申请人提供）： 拟议的研究是对 RNA 伴侣 CYT-19 蛋白的靶向和作用的剖析，因为它促进 I 组 RNA 的折叠。尽管RNA伴侣很早以前就被提出，但任何蛋白质对伴侣RNA折叠自然发挥作用的第一次证明是在2002年，当时Lambowitz及其同事证明粗糙脉孢菌CYT-19蛋白质通过加速几个I组内含子的折叠来发挥作用。这些内含子还需要剪接因子 CYT-18，另外表明 CYT-19 可能被 CYT-18 靶向 RNA。 CYT-19 是一种 DExD/H-box 蛋白，存在于所有生物体中，几乎参与涉及结构化 RNA 的每个过程，包括核糖体生物发生和前 mRNA 剪接等基本过程，以及包括 HCV 在内的病毒的复制。 DExD/H-box 蛋白通常被认为利用 ATP 结合和水解来介导 RNA 的结构重排，促进折叠成功能结构或功能结构之间的转换。然而，人们对它们的作用机制或控制它们对 RNA 或 RNA-蛋白质底物特异性的因素知之甚少，这主要是因为它们的许多靶标非常复杂。上述系统为生化解剖提供了独特的机会，因为它足够简单，可以轻松操作，但又足够完整，可以捕获 DExD/H-box 蛋白的生理作用。然而，脉孢菌 I 组内含子的折叠尚未得到很好的表征，并且研究最充分的内含子填充了多种错误折叠形式，抑制了深层生化解剖。因此，该提案重点关注来自四膜虫的 I 组 RNA 及其结合 CYT-18 的衍生物。四膜虫 RNA 被广泛表征并折叠成离散的错误折叠形式，CYT-19 也加速其重新折叠至天然状态。具体目标是 1) 使用动力学方法探测 CYT-19 在四膜虫核酶重折叠中的分子作用，比较双链体 RNA 的非特异性解旋反应并测试核酶重折叠机制的特定模型； 2) 检查CYT-18蛋白赋予的特异性的决定因素和机制； 3) 使用沉降平衡来探测溶液中 CYT-19 与 RNA 结合的自缔合行为，然后使用该信息来探测 CYT-19 作用时的多聚体状态； 4) 探索 CYT-19 的意外活性，即寡核苷酸底物与核酶的解离。这些实验旨在提供关于 DExD/H-box 蛋白如何充当 RNA 伴侣及其如何靶向的新颖且基本的理解。这里的结果有望指导参与 RNA 代谢和功能各个方面的 DExD/H 盒蛋白的作用和靶向模型。