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

描述（由申请人提供）：smp是一种来自DEAD-box蛋白家族的RNA解旋酶，在50S核糖体亚基组装的早期步骤中具有已知的功能。DEAD-box蛋白几乎参与了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smp在体内协助50S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，脉冲标记和发现蛋白质组学）来表征？SrmB大肠杆菌菌株，用蔗糖梯度超离心分离。这些实验将分别报告中间体的核糖体蛋白质组成与完整核糖体内标准的比较，中间体是否在路径中间体或降解产物上是真实的，以及它们的核糖体生物发生辅助因子组成。我们将通过引入表达WT和突变型smp的质粒来扰乱该系统。SrmB菌株背景，并通过上述QMS实验分析其蔗糖梯度分数。其次，我们将WT和突变mbp - smp表达质粒引入？用粗裂解物和蔗糖梯度组分的直链淀粉树脂进行MBP标签拉下，以纯化MBP-SrmB相关核糖体颗粒。如前所述，洗脱部分将通过QMS进行分析，这将给出srmb相关核糖体中间体的表征。第三，我们将研究smp与其他核糖体蛋白之间的关系，通过在？含有核糖体蛋白S4或S8的smp大肠杆菌细胞，它们是已知的核糖体蛋白多顺壳子操纵子的自身调节因子。这些生长的蔗糖梯度部分将通过QMS进行分析，这一分析将使我们能够确定特定核糖体蛋白与smp结合之间的功能联系。综上所述，本提案的三个目标中提出的实验将从smp的角度详细描述50S亚基组装：即smp结合50S前体颗粒需要哪些核糖体蛋白和辅因子，哪些核糖体蛋白和辅因子需要与50S前体颗粒结合，哪些核糖体蛋白和辅因子独立于smp结合并作用于50S前体颗粒。在包括癌症在内的各种疾病状态中都观察到核糖体生物发生的基本过程调控不当和DEAD-box蛋白的过度表达。这里提出的研究将导致对RNA解旋酶作用于其靶点的分子机制以及核糖体生物发生的复杂过程的更深入和更详细的理解，这对于设计有效治疗与它们有关的疾病至关重要。

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.