Mechanisms of RNA binding and remodeling proteins

摘要RNA的操作需要RNA结合蛋白和atp依赖的分子马达蛋白的作用，这些蛋白被认为是RNA序列中转运、重塑和解开二级结构的分子马达蛋白。这些分子马达中有许多是DEAD-box或密切相关的蛋白质。正链RNA病毒，如丙型肝炎病毒（HCV）需要这些蛋白质的活性来进行病毒复制。确定这些蛋白质的分子机制对我们理解RNA代谢具有重要意义，并将促进我们对RNA病毒如何复制的理解。本项目的目的是确定丙型肝炎病毒RNA结合蛋白和RNA重塑酶的RNA重塑机制。非结构蛋白3 （NS3）是一种RNA重塑酶（或解旋酶），是丙型肝炎病毒复制所必需的。我们提出了一种新的RNA重塑模型，即低聚酶的多个亚基一起工作来熔化RNA中的二级结构。我们的模型预测NS3以缓慢的动力学步骤参与双链的缓慢解扭，随后以依赖atp的方式进行快速易位。我们将通过结合生物物理、生化和生物学实验来测试这个模型。我们将通过使用蛋白质足迹和质谱结合来确定蛋白质-蛋白质相互作用的特定位点。不形成寡聚结构的NS3的截短形式将被检查，以确定蛋白质-蛋白质相互作用在RNA重塑中发挥的特定作用。我们的工作已经确定NS5A是一种与自身和NS3相互作用的RNA结合蛋白。NS5A的结构代表了核酸识别的一个新领域，我们有望揭示该蛋白与RNA结合的结构/功能关系。蛋白-蛋白相互作用的重要性将通过制备二聚化受阻的NS5A变异体来确定，然后测试这些变异体的RNA结合活性和在细胞中支持HCV复制的能力。NS3和NS5A之间的相互作用将使用新的生化和生物学方法进行详细研究。在目标1中，我们将验证NS3对RNA重塑的假设。在目的2中，我们将确定NS5A二聚化在RNA结合中的作用。在目标3中，我们将检验NS5A作为NS3解旋酶活性的加工因子的假设。确定HCV RNA结合和重塑蛋白的机制将揭示破坏HCV复制途径的新分子方法。因此，了解蛋白质结合和操纵RNA的分子机制具有重要的生物学和医学意义。

DESCRIPTION (provided by applicant): Mechanisms of RNA binding and remodeling proteins Abstract Manipulation of RNA requires the action of RNA binding proteins and ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the Hepatitis C virus (HCV) require the activity of these proteins for viral replication. Determination of the molecular mechanisms of these proteins is of fundamental importance to our understanding of RNA metabolism in general and will advance our understanding of how RNA virus' are replicated. The goal of this project is to determine the mechanism of RNA remodeling by an RNA binding protein and an RNA remodeling enzyme from the Hepatitis C virus. Non-structural protein 3 (NS3) is an RNA remodeling enzyme (or helicase), that is necessary for HCV replication. We propose a new model for RNA remodeling by this enzyme whereby multiple subunits of an oligomeric enzyme work together to melt out secondary structures in RNA. Our model predicts that NS3 participates in slow untwisting of the duplex in slow kinetic step, followed by rapid translocation in an ATP-dependent manner. We will test this model by using a combination of biophysical, biochemical, and biological experiments. We will identify the specific sites of protein-protein interactions by using protein footprinting coupled with mass spectrometry. Truncated forms of NS3 that do not form oligomeric structures will be examined to determine the specific roles that protein-protein interactions play in RNA remodeling. Our work has identified NS5A as an RNA binding protein that interacts with itself and with NS3. The structure of NS5A represents a new fold in nucleic acid recognition, and we are poised to uncover the structure/function relationship for RNA binding by this protein. The importance of protein-protein interactions will be determined by preparing variants of NS5A that are impeded in dimerization, followed by testing of those variants for RNA binding activity and for support of HCV replication in cells. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will test our hypothesis for RNA remodeling by NS3. In aim 2, we will determine the role of dimerization of NS5A in RNA binding. In aim 3, the we will test the hypothesis that NS5A serves as a processivity factor for NS3 helicase activity. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of biological and medical significance. PUBLIC HEALTH RELEVANCE: Mechanisms of RNA binding and remodeling proteins Narrative: Proteins that bind and manipulate RNA are of fundamental importance to many biological processes including translation, transcription, and gene regulation. RNA binding protiens also play key roles in viruses such as the Hepatitis C Virus, which infects almost 3 % of the world's population. Over 75% of HCV infections never resolve, resulting in persistent virus infection that can lead to liver fibrosis and, progressively, to severe and fatal diseases, including liver cirrhosis and liver cancer. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of general biological and medical significance.