Kinetic Dissection of the RNA Chaperone Protein CYT-19

描述（由申请人提供）： 拟议的研究是解剖RNA伴侣CYT-19蛋白的靶向和作用，因为它有助于I组RNA的折叠。虽然RNA分子伴侣很早以前就被提出，但第一次证明任何蛋白质都能自然地伴侣RNA折叠是在2002年，当时Lambowitz和同事们发现粗糙脉孢菌CYT-19蛋白通过加速几个I组内含子的折叠来发挥作用。这些内含子也需要剪接因子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，几乎参与所有涉及结构化RNA的过程，包括核糖体生物合成和前mRNA剪接等基本过程，以及包括HCV在内的病毒复制。DExD/H-box蛋白通常被认为使用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-蛋白质底物的特异性的因素知之甚少，这主要是因为它们的许多靶点很复杂。上述系统为生化解剖提供了独特的机会，因为它足够简单，可以容易地操作，但又足够完整，可以捕获DExD/H盒蛋白的生理作用。然而，链孢菌组I内含子的折叠没有得到很好的表征，并且研究最多的内含子存在多种错误折叠形式，抑制了深层的生化解剖。因此，该提议将重点放在来自四膜虫的I组RNA及其结合CYT-18的衍生物上。四膜虫RNA被广泛表征，并折叠成离散的错误折叠形式，其再折叠成天然状态也被CYT-19加速。具体目标是：1）利用动力学方法探索CYT-19在四膜虫核酶重折叠中的分子作用，比较双链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）研究CYT-18蛋白赋予的特异性决定因素和机制; 3）利用沉降平衡来探测CYT-19在溶液中与RNA结合的自缔合行为，然后利用该信息来探测CYT-19在其作用时的多聚体状态; 4）探索CYT-19的意想不到的活性，从核酶解离寡核苷酸底物。这些实验旨在提供对DExD/H-box蛋白如何作为RNA伴侣以及如何靶向的新的和基本的理解。这里的结果预计将指导模型的行动和靶向DExD/H盒蛋白参与RNA代谢和功能的各个方面。

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.