Role of the SrmB Helicase in Ribosome Assembly investigated by Mass Spectrometry

描述（由申请人提供）：SrmB是一种来自DEAD-盒蛋白家族的RNA解旋酶，具有协助50 S核糖体亚基组装早期步骤的已知功能。已知死亡盒蛋白几乎参与RNA代谢的每一步;然而，它们的具体作用机制在很大程度上是未知的。该项目的目标是通过定量质谱（QMS）研究SrmB在体内协助50 S核糖体亚基组装的机制。首先，我们将使用三个QMS实验（蛋白质库存，脉冲标记，并发现蛋白质组学），以表征核糖体组装中间体的？SrmB大肠杆菌菌株，通过蔗糖梯度超离心分级分离。这些实验将分别报告与完整核糖体内标物相比中间体的核糖体蛋白组成，中间体是否为路径中间体或降解产物，以及它们的核糖体生物合成辅因子组成。我们将通过引入表达WT和突变SrmB的质粒来扰乱系统？SrmB菌株背景，并通过上述QMS实验分析这些的蔗糖梯度级分。其次，我们将WT和突变MBP-SrmB表达质粒导入？SrmB细胞，并用粗裂解物和蔗糖梯度级分的直链淀粉树脂进行MBP标签唐斯，以纯化MBP-SrmB相关的核糖体颗粒。如前所述，将通过QMS分析洗脱级分，这将给出SrmB相关核糖体中间体的表征。第三，我们将研究SrmB和其他核糖体蛋白的关系，通过干扰核糖体蛋白的表达过表达？SrmB E.大肠杆菌细胞的核糖体蛋白S4或S8，其是已知的核糖体蛋白的多顺反子操纵子的自动调节子。这些生长的蔗糖梯度部分将通过QMS进行分析，并且该分析将使我们能够确定特定核糖体蛋白和SrmB的结合之间的功能连接。总之，在本提案的三个目标中提出的实验将从SrmB的角度给出50 S亚基组装的详细图片：也就是说，SrmB结合50 S前体颗粒所需的核糖体蛋白和辅因子，其需要SrmB以与50 S前体颗粒接合，并且所述核糖体蛋白和辅因子独立于SrmB结合并作用于50 S前体颗粒。在包括癌症在内的各种疾病状态中观察到核糖体生物合成的基本过程的失调和DEAD盒蛋白的过表达。这里提出的研究将导致更深入和更详细地了解RNA解旋酶作用于其靶标的分子机制以及核糖体生物合成的复杂过程，这对于设计涉及它们的疾病的有效疗法至关重要。 公共卫生相关性：核糖体组装是细胞中的主要代谢活动之一，并且是所有生物体中细胞生长所必需的;毫不奇怪，大量证据表明核糖体的错误组装是人类疾病的主要原因，包括癌症和其他遗传疾病，如Diamond-Blackfan贫血和严重遗传疾病先天性角化不良。死亡盒蛋白在各种类型的癌细胞中过表达，并且来自相关家族的RNA解旋酶对于引起人类疾病的许多病毒的繁殖是必需的。这里提出的项目将有助于进一步了解SrmB和DEAD盒解旋酶的一般机制，以及核糖体组装的过程，这可用于设计有效的药物和治疗疾病，其中涉及。

DESCRIPTION (provided by applicant): SrmB is an RNA helicase from the DEAD-box family of proteins that has a known function assisting in the early steps of 50S ribosome subunit assembly. DEAD-box proteins are known to be involved in nearly every step of RNA metabolism; however, their specific mechanisms of action are largely unknown. The goal of the proposed project is to investigate the mechanism by which SrmB assists in 50S ribosome subunit assembly in vivo via quantitative mass spectrometry (QMS). First, we will use three QMS experiments (protein inventory, pulse labeling, and discovery proteomics) to characterize ribosome assembly intermediates of the ?SrmB E.coli strain, fractionally separated by sucrose gradient ultracentrifugation. These experiments will report on, respectively, the ribosomal protein composition of the intermediates compared to an intact ribosome internal standard, whether the intermediates are true on path-intermediates or degradation products, and their ribosome biogenesis cofactor composition. We will perturb the system by introducing plasmids expressing WT and mutant SrmB in the ?SrmB strain background and analyze the sucrose gradient fractions of these via the aforementioned QMS experiments. Second, we will introduce WT and mutant MBP-SrmB expression plasmids into ?SrmB cells and perform MBP tag pull downs with amylose resin of both crude lysate and sucrose gradient fractions in order to purify MBP-SrmB-associated ribosomal particles. Elution fractions will be analyzed by QMS, as described before, which will give a characterization of SrmB-associated ribosomal intermediates. Third, we will investigate the relationship between SrmB and other ribosomal proteins by perturbing ribosomal protein expression by overexpression in ?SrmB E. coli cells of ribosomal proteins S4 or S8, which are known autoregulators of polycistrionic operons for ribosomal proteins. Sucrose gradient fractions of these growths will be analyzed by QMS, and this analysis will allow us to determine functional connections between binding of specific ribosomal proteins and SrmB. All together, the experiments proposed in the three aims of this proposal will give a detailed picture of 50S subunit assembly from the standpoint of SrmB: that is, what ribosomal proteins and cofactors are required for SrmB to bind the 50S precursor particle, which require SrmB in order to engage with the 50S precursor particle, and which ribosomal proteins and cofactors bind and act upon the 50S precursor particle independently of SrmB. Both, misregulation of, the fundamental process of ribosome biogenesis and overexpression of DEAD-box proteins are observed in various disease states, including cancer. The studies proposed here will lead to a deeper and more detailed understanding of the molecular mechanism whereby RNA helicases operate on their targets as well as the complex process of ribosome biogenesis, which is paramount for the design of effective therapies for diseases in which they are implicated. PUBLIC HEALTH RELEVANCE: Ribosome assembly is one of the major metabolic activities in cells and is required for cell growth in all organisms; not surprisingly, substantial evidence points to the misassembly of ribosomes as a major cause of human disease, including cancer and other genetic diseases such as Diamond-blackfan anemia and the severe genetic disorder dyskeratosis congenita. DEAD-box proteins are overexpressed in various types of cancer cells and RNA helicases from related families are essential for the propagation of many viruses that cause human diseases. The project proposed here will help further knowledge of the mechanism of SrmB and DEAD-box helicases in general, as well as the process of ribosome assembly, which can be used to design effective drugs and therapies for diseases in which they are implicated.