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

描述(申请人提供)：DEAD-box蛋白是超家族2 RNA解旋酶，几乎是结构RNA执行的每一个过程所必需的，从前mRNA剪接和翻译到蛋白质和RNA的细胞内运输。它们也是复制包括HIV-1和丙型肝炎病毒在内的病毒所必需的，过度表达与结肠癌和前列腺癌有关。这些蛋白质利用ATP结合和水解产生的能量来促进RNA的构象变化和折叠过渡，但我们对它们如何操纵RNA结构的分子知识有限。来自机械学研究的见解对于深入理解基本的生物过程以及理解和最终治疗重要的病毒疾病和癌症至关重要。我们重点研究了真菌Dead-box蛋白Cyt-19和Mss 116p，它们在线粒体I组和II组自剪接内含子的折叠中起着通用RNA伴侣的作用。这些系统对于机理研究来说是强大的，因为RNA相对简单和容易处理，并且它们的催化活性为自然态的形成提供了可靠和灵敏的读数。在1-5年的资助中(2004-2009)，我们使用了来自嗜热四膜虫的一个研究充分的I族内含子来证明CyT-19作为一个真正的伴侣发挥功能，促进了长时间错误折叠的构象的折叠，而不是在下游的催化步骤中发挥作用。我们发现，Cyt-19非特异性地破坏结构，没有区分天然和错误折叠的构象，这种活性有利于天然RNA的积累，因为它比错误折叠的构象更稳定。自从提交了一份获得ARRA两年资助(2009-2011年)的续签申请以来，我们取得了进一步的进展。单分子荧光和快速动力学直接表明，细胞色素T-19能被三级结构强烈抑制，只有在螺旋自发地从内含子核心的三级接触中‘脱离’后，才能解开一个短螺旋。快速动力学实验也支持一个模型，在该模型中，CyT-19的C末端‘尾’与结构RNA接触，并拴住解旋酶核心以破坏附近的结构，一系列速率测量表明，CyT-19在一个依赖于ATP的构象变化的单个周期中完全解开短的RNA螺旋。综上所述，这项工作导致了一个通过DEAD-box蛋白质进行RNA伴侣活性的通用模型，其中蛋白质通过拴系定位到结构RNA上，并且它们非过程地破坏二级结构的暴露元件，使这些片段有机会形成新的接触。在这里，我们建议在两个重要方向上测试和扩展该模型。在目标1和2中，我们将使用简单的双链底物和大量的实验方法来探索ATPase循环是如何与RNA解离相耦合的，以及拴系在多大程度上限制了解旋酶核心的位置和取向。在目标3和4中，我们将使用物理和化学方法跟踪依赖CyT-19和Mss 116p的第一组和第二组内含子在体内的折叠，测试特定的假设，并探索我们的一般伴侣活性模型是否描述了这些蛋白质对其同源RNA折叠的影响。 与公共卫生相关：该项目的目标是了解RNA伴侣蛋白在RNA折叠到特定结构以及结构之间的交换时如何帮助RNA。这些蛋白质是病毒复制所必需的，并与人类癌症有关，因此了解它们的功能对于理解和最终治疗人类疾病非常重要。

DESCRIPTION (provided by applicant): DEAD-box proteins are superfamily 2 RNA helicases that are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. They are also required for replication of viruses including HIV-1 and HCV and overexpression is linked to colon and prostate cancer. These proteins use energy from ATP binding and hydrolysis to facilitate RNA conformational changes and folding transitions, but we have limited molecular knowledge about how they manipulate RNA structure. Insights from mechanistic studies will be critical for deep understanding of fundamental biological processes and for understanding and ultimately treatment of important viral diseases and cancer. We have focused on the fungal DEAD-box proteins CYT-19 and Mss116p, which function as general RNA chaperones in folding of mitochondrial group I and group II self-splicing introns. These systems are powerful for mechanistic studies because the RNAs are relatively simple and tractable, and their catalytic activity provides a robust and sensitive readout for formation of the native state. In years 1-5 of funding (2004-2009), we used a well-studied group I intron from Tetrahymena thermophila to show that CYT-19 functions as a true chaperone, facilitating refolding of a long-lived misfolded conformation without functioning in the downstream catalytic steps. We found that CYT-19 disrupts structure non-specifically, without distinguishing native from misfolded conformations, and this activity favors accumulation of the native RNA because it is much more stable than the misfolded conformation. Since submitting a renewal that received two years of ARRA funding (2009-2011), we made further advances. Single molecule fluorescence and rapid kinetics showed directly that CYT-19 can be strongly inhibited by tertiary structure, unwinding a short helix only after the helix spontaneously 'undocks' from tertiary contacts with the intron core. Rapid kinetics experiments also supported a model in which a C-terminal 'tail' of CYT-19 contacts structured RNA and tethers the helicase core to disrupt nearby structure, and a series of rate measurements indicated that CYT-19 completely unwinds short RNA helices in a single cycle of ATP- dependent conformational changes. Together, the work leads to a general model for RNA chaperone activity by DEAD-box proteins in which the proteins are localized to structured RNAs by tethering, and they disrupt exposed elements of secondary structure non-processively to allow these segments an opportunity to form new contacts. Here we propose to test and extend this model in two important directions. In Aims 1 and 2, we will use simple duplex substrates and an arsenal of experimental approaches to probe how the ATPase cycle is coupled to RNA unwinding and to what extent tethering constrains the position and orientation of the helicase core. In Aims 3 and 4 we will use physical and chemical approaches to follow folding of group I and group II introns that rely on CYT-19 and Mss116p in vivo, testing specific hypotheses and probing whether our model for general chaperone activity describes the effects of these proteins on folding of their cognate RNAs. 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.