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-BOX蛋白家族的RNA解旋酶，在50S核糖体亚单位组装的早期步骤中具有已知的功能。已知DEAD-box蛋白几乎参与了RNA新陈代谢的每一步；然而，它们的具体作用机制在很大程度上还不清楚。该项目的目标是通过定量质谱学(QMS)研究SrmB在体内协助50S核糖体亚单位组装的机制。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)来表征通过蔗糖梯度超速离心法分离的？srmB大肠杆菌株的核糖体组装中间产物。这些实验将分别报告中间体与完整的核糖体内部标准相比的核糖体蛋白质组成，中间体在路径中间体或降解产物上是否正确，以及它们的核糖体生物发生辅因子组成。我们将通过在？srmB菌株背景中引入表达WT和突变体srmB的质粒来扰动系统，并通过上述QMS实验分析这些基因的蔗糖梯度组分。其次，我们将WT和突变型MBP-SrmB表达载体导入SrmB细胞，并用粗裂解产物和蔗糖梯度组分的直链淀粉树脂进行MBP标签的下拉，以纯化MBP-SrmB相关的核糖体颗粒。洗脱组分将通过QMS进行分析，如前所述，这将给出与SrmB相关的核糖体中间体的特征。第三，我们将通过在大肠杆菌中过度表达核糖体蛋白S4或S8来干扰核糖体蛋白的表达，从而研究SmB与其他核糖体蛋白之间的关系。S4或S8是已知的核糖体蛋白多环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SrmB结合之间的功能联系。总之，本提案的三个目标中提出的实验将从SrmB的角度给出50S亚基组装的详细图景：即，哪些核糖体蛋白和辅因子需要SrmB与50S前体颗粒结合，需要SrmB才能与50S前体颗粒结合，以及哪些核糖体蛋白和辅因子独立于SrmB结合和作用于50S前体颗粒。核糖体生物发生的基本过程的调控错误和DEAD-BOX蛋白的过度表达在包括癌症在内的各种疾病状态中都被观察到。本文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，这对于设计有效的治疗它们所涉及的疾病是至关重要的。 与公共卫生相关：核糖体组装是细胞中的主要代谢活动之一，是所有生物体细胞生长所必需的；毫不奇怪，大量证据表明，核糖体组装错误是人类疾病的主要原因，包括癌症和其他遗传性疾病，如钻石-布莱克凡贫血和严重的遗传性疾病先天性角化不良。DEAD-box蛋白在各种类型的癌细胞中过表达，来自相关家族的RNA解旋酶是许多导致人类疾病的病毒传播所必需的。该项目将有助于进一步了解srmB和DEAD-box解旋酶的一般机制，以及核糖体组装过程，该过程可用于设计有效的药物和治疗与其相关的疾病。

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.