真核核糖体组装过程的结构和功能研究

Ribosome biogenesis is a highly complicated and coordinated process that includes the assembly of four rRNAs and about 80 ribosomal proteins into subunit precursors and the transportation of precursors across nuclear membrane. The fidelity and efficiency of ribosome assembly in vivo is not only closely related to the function of the cells, but also associated with many human diseases. For examples, a collection of genetic diseases, termed as ribosomopathy, including SDS, DBA, 5q-syndrome, DKC, CHH and TCS, are caused by mutations in genes involved in the maturation and assembly of ribosomal subunits. Especially, 90% of cases of SDS patients are caused by mutations of a ribosomal maturation factor SBDS, which catalyzes the late-stage maturation of the 60S subunit in cytoplasm. In animal cells, the process of the assembly and maturation of ribosomal subunits is monitored by the MDM2-p53 pathway, and defects in the process leads to increased risk of many types of cancers. The structural and functional study of ribosome assembly in eukaryotic cells is not only of fundamental importance to the general biology, particularly in the field of protein translation. More importantly, mechanistic elucidation of the roles of disease-associated ribosomal maturation factors could have intermediate impact, as to the understanding of the pathogenesis in related diseases, as well as the clues and possible drug targets for therapeutic interventions. This proposal aims to study the functional and structural aspects of a number of ribosomal maturations factors in the model system of yeast, using the cryo-EM single particle method as a major tool. The two specific objectives are: (1) functional elucidation of several maturation factors, by determining the structures of ribosomal subunits bound with maturation factors, with a focus on disease related factors, including Sdo1 (the yeast homolog of SBDS), Efl1, Lsg1; (2) depiction of the in vivo assembly pathway of the ribosomal subunits, by using isolated in vivo assembly precursors with TAP (tandem affinity purification)-labelled assembly factors, such as Rrp5, Alb1, Nog1, Nog2 and Rsa4.

真核核糖体的组装成熟是一个高度复杂和协同的过程，包括4个rRNA和大约80个核糖体蛋白跨核膜转运及组装。核糖体的正确组装与否关系到细胞功能的执行以及生物体的生理状态。许多人类遗传疾病与核糖体组装成熟相关，如SDS，DBA， DKC，CHH和TCS等一大类统称为ribosomopathy的疾病。本项目拟采用生物化学和冷冻电镜相结合的方法，以酵母为模型，从两个方面研究真核核糖体的组装：（A）核糖体成熟因子，特别是与疾病相关的几种因子，例如Sdo1、Lsg1、Efl1等在组装中的分子功能；（B）以体内纯化的核糖体亚基组装中间体为材料，阐释与一些重要的成熟因子（Nog1、Nog2、Alb1、Rrp5等）相关的核糖体亚基体内组装的分子途径（assembly pathway）。这些核糖体组装机制的研究不仅具有重要的基础科学价值，还可能揭示相关疾病的发生机理，为临床治疗提供线索。

真核核糖体的组装成熟是一个高度复杂和协同的过程，包括4个rRNA和大约80个核糖体蛋白跨核膜转运及组装。核糖体的正确组装与否关系到细胞功能的执行以及生物体的生理状态。本项目采用生物化学和冷冻电镜相结合的方法，以酵母为模型，对酵母核糖体60S亚基组装、多种核糖体组装因子进行了研究，分析了核糖体亚基组装过程中相关因子的结构和功能。重要研究成果包括1）酵母细胞核内的pre-60S组装前体的结构解析（Nature,2016）以及2）细胞质晚期的pre-60S组装前体（NSMB，2017）的结构研究。这些工作在真核核糖体组装过程的结构和功能研究领域做出了重要的原创贡献，阐明了系列科学问题。