Kinetic Dissection of the RNA Chaperone Protein CYT-19

描述(由申请人提供)： 这项拟议的研究是对RNA伴侣蛋白CyT-19的靶向和作用的剖析，因为它促进了I组RNA的折叠。虽然RNA伴侣很久以前就被提出了，但第一次证明任何蛋白质都具有天然的伴侣RNA折叠功能是在2002年，当时兰博维茨和他的同事们证明了粗糙脉孢菌的细胞色素T-19蛋白是通过加速几个I组内含子的折叠来发挥功能的。这些内含子还需要剪接因子Cyt-18，这另外表明Cyt-19可能通过Cyt-18靶向RNA。Cyt-19是一种DExD/H-box蛋白，存在于所有生物体中，几乎参与了所有涉及结构RNA的过程，包括核糖体生物发生和前mRNA剪接等必要过程，以及包括丙型肝炎病毒在内的病毒的复制。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知之甚少，也不知道是什么决定了它们对RNA或RNA-蛋白质底物的专一性，这主要是因为它们的许多靶标都很复杂。上面的系统为生化解剖提供了一个独特的机会，因为它足够简单，易于操作，但又足够完整，可以捕捉到DExD/H-box蛋白的生理作用。然而，脉孢霉I组内含子的折叠并不是很好的特征，研究最好的内含子填充了多种错误折叠的形式，阻碍了深入的生化解剖。因此，这项建议将重点放在四膜虫及其与细胞色素T-18结合的衍生物的I族RNA上。四膜虫RNA具有广泛的特征，并折叠成离散的错误折叠形式，其重新折叠到自然状态也被细胞色素T-19加速。具体目标是1)利用动力学方法探测CyT-19在四膜虫核酶重折叠中的分子作用，比较双链RNA非特异性解离的反应和测试核酶重折叠机制的特定模型；2)检测CyT-18蛋白质所决定的决定因素和特异性的机制；3)利用沉淀法检测CyT-19在溶液中并与RNA结合的自结合行为，然后利用这些信息探测CyT-19在作用过程中的多聚体状态；4)探索Cyt-19出人意料的活性，使寡核苷酸底物从核酶上解离。这些实验旨在提供对DExD/H-box蛋白如何作为RNA伴侣以及它是如何被靶向的新颖和基本的理解。这一结果有望指导DExD/H box蛋白在RNA代谢和功能的各个方面的作用和靶向模型。

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.