Biophysical studies of H/ACA snoRNAs and psi in rRNA

该项目的长期目标是在分子水平上了解前核糖体RNA(pre-rRNA)转录后修饰过程的关键方面，包括修饰酶识别分子特异性RNA的基础和RNA分子内修饰的功能基础。这项研究集中在真核生物rRNA中的伪尿苷修饰位点以及引入它们的小核仁核糖核蛋白颗粒(SnoRNPs)。该项目将探索snoRNP蛋白与其RNA配体的相互作用，并以生化方法和高分辨率核磁共振为主要工具，研究snoRNA介导的在真核大亚基rRNA功能重要区域选择假尿苷酸位点的热力学和结构效应。具体地说，将讨论以下主题：(1)snoRNA-rRNA复合物的结构和热力学研究；(2)真核大亚基rRNA未修饰和伪尿嘧啶修饰形式的比较结构和热力学研究。这些研究结果直接关系到理解蛋白质-RNA相互作用的特异性和亲和力，以及RNA分子用来调整局部构象和稳定性的机制。这些研究补充了对snoRNPs催化的假尿苷酸化反应的遗传学研究，并共同为理解最常见和系统发育保守的RNA碱基修饰的功能作用提供了一条途径。在这些研究中开发的方法将作为结构生物学家和化学生物学家的工具。

The long-term goal of this project is to understand key aspects of the post-transcriptional modification process of pre-ribosomal RNA (pre-rRNA) at the molecular level, including the basis of molecule specific RNA recognition by modification enzymes and the functional basis for modifications within RNA molecules. This research focuses on pseudouridine modification sites within eukaryotic rRNA and the small nucleolar ribonucleoprotein particles (snoRNPs) that introduce them. The project will explore the interaction of snoRNP proteins with their RNA ligand, and examine the thermodynamic and structural effects of snoRNA-mediated selection of pseudouridylation sites in functionally important regions of the eukaryotic large subunit rRNA using biochemical methods and high resolution NMR spectroscopy as the principal tools. Specifically, the following topics will be addressed: (1) structural and thermodynamic studies of snoRNA-rRNA complexes and (2) comparative structural and thermodynamic studies of the unmodified and pseudouridyl-modified forms of the eukaryotic large subunit rRNA. The results of these studies are directly relevant to understanding the specificity and affinity of protein-RNA interactions and the mechanisms used by RNA molecules to tweak local conformation and stability. These investigations complement genetic studies of the pseudouridylation reactions catalyzed by the snoRNPs and together provide a route to understanding the functional roles of the most common and phylogenetically conserved RNA base modification. The methods developed during these studies will serve as tools for structural biologists and chemical biologists.