Characterizing dynamics and functions of RNA modifications in ribosome biogenesis

描述(申请人提供)：RNA修饰在生物学中普遍存在。在核糖体中，它们调节其结构和功能，调节抗生素耐药性，并与人类疾病有关。最近rRNA修饰的鉴定和结构定位的成功促进了核糖体生物发生过程中RNA修饰的机制研究。虽然以前的大部分工作都集中在体外修饰酶的结构和特异性的表征上，但单个rRNA修饰如何整合到复杂的多步骤生物发生过程中仍不清楚。这项拟议的研究将建立现代质谱学方法和稳定同位素标记用于rRNA修饰的定量分析，并应用这一技术来破译在大肠杆菌核糖体组装过程中rRNA修饰的时间和调节。为了将RNA修饰置于现有的核糖体组装框架中，我们将利用细菌遗传学，通过系统地耗尽单个核糖体蛋白、生物发生因子和修饰酶，在组装途径的不同点引入扰动。然后，将应用质谱学来表征累积的组装中间体中的蛋白质组成和rRNA修饰，以揭示单个rRNA修饰和其他组装步骤之间的关系模式。通过为大肠杆菌中整个rRNA修饰途径提供新的机制见解，这些研究为在高等生物体中的工作奠定了基础，从长远来看，这将有助于理解rRNA修饰步骤在生物发生中的具体作用及其与疾病的相关性。

DESCRIPTION (provided by applicant): RNA modifications are ubiquitous in biology. In ribosome, they regulate its structure and functions, modulate antibiotic drug resistance, and have been implicated in human diseases. Recent successes in identification and structural localization of rRNA modifications facilitate mechanistic studies of RNA modifications during the ribosome biogenesis. While most of the prior efforts have been focused on characterization of structure and specificity of modification enzymes in vitro, how individual rRNA modifications are integrated in the complex multistep biogenesis process remains unclear. The proposed study will establish the utility of a modern mass spectrometry methodology and stable isotope labeling for quantitative analysis of rRNA modifications, and apply this technique to decipher the timing and regulation of rRNA modifications during ribosome assembly in E. coli. To place RNA modifications into the existing framework of ribosome assembly we will use bacteria genetics to introduce perturbations at different points on the assembly pathway via systematic depletions of individual ribosomal proteins, biogenesis factors and modification enzymes. Then, mass spectrometry will be applied to characterize protein composition and rRNA modifications in the accumulated assembly intermediates to reveal patterns of relationships between individual rRNA modifications and other assembly steps. By providing new mechanistic insights into the entire rRNA modification pathway in E. coli, these studies set a stage for work in higher organisms which in the long term will help to understand the specific roles of rRNA modification steps in biogenesis and their relevance to diseases.