真核核糖体的组装是一个高度复杂且精密调控的过程，包括80种核糖体蛋白的正确合成与有序结合，以及4种rRNAs的正确转录、修饰、折叠与加工。此外，有超过200种组装因子参与调控，然而大部分的结构及功能还不明确。有研究表明人类中某些组装因子的突变会导致核糖体合成的失调进而引起一系列核糖体相关的人类疾病。真核核糖体的组装过程较为保守，酵母与人类核糖体的大部分组装因子具有较高同源性。对酵母核糖体组装过程的研究有助于我们了解人类核糖体的组装过程，进而为核糖体相关的人类疾病提供有效的治疗方案。因此本项目以酵母组装因子Sda1为目标蛋白，以5S RNP的旋转过程为主要研究对象，将蛋白晶体学与单颗粒冷冻电镜技术结合，旨在阐释Sda1蛋白在核糖体成熟过程中的具体分子作用机制，揭示5S RNP的旋转机制及Sda1与核糖体大亚基作用时期的核糖体组装过程，进而深入阐明酵母细胞核内较晚时期核糖体大亚基的组装过程。

Ribosome biogenesis in eukaryotes is a highly complex and extremely sophisticated process involving assembly of ribosomal proteins and transcription, modification, folding and processing of ribosomal RNAs. This process is regulated by more than 200 assembly factors. Loss-of-function mutations of certain assembly factors in human lead to dyregulation of ribosome biogenesis and may result in several human diseases referred to as ribosomopathies. However, the structure and function of most assembly factors remain unknown. Therefore, understanding the mechanism of ribosome biogenesis in yeast will provide a framework to insight into how human ribosomes are synthesized and help us find an effective treatment for those human diseases. Based on our previously solved cryo-EM structures of Nog2-TAP pre-60S ribosomes, we will focus on the rotation of 5S RNP by affinity purification of nucleoplasmic pre-60S ribosomes from Saccharomyces cerevisiae using the epitope-tagged Sda1 protein as a bait. With the help of protein crystallography and single-particle cryo-EM, we can not only interpret how Sda1 protein functions in ribosome biogenesis and the molecular mechanism of 5S RNP maturation, but also reveal the dynamic assembly process of 60S subunits during the interaction with Sda1.