Assembly of the eukaryotic small ribosomal subunit

真核小核糖体亚基的组装

• 批准号: 10612107
• 负责人: Sebastian Klinge
• 金额: $ 33.9万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-20 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612107
• 关键词: Address; Biochemical; Biogenesis; Biology; Cell Nucleolus; Cell Separation; Cells; Chemicals; Code; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cytoplasm; Defect; Disease; Elements; Engineering; Enzymes; Event; Genes; Genetic; Genetic Diseases; Genetic Transcription; Goals; Growth; Hematological Disease; Human; Human Genome; Laboratories; Light; Link; Location; Mass Spectrum Analysis; Messenger RNA; Metabolism; Molecular; Molecular Conformation; Molecular Machines; Nuclear; Nuclear Export; Pathway interactions; Peptides; Physical condensation; Positioning Attribute; Process; Proliferating; Protein Biosynthesis; Public Health; Quality Control; RNA; RNA Degradation; RNA Helicase; RNA Processing; Regulation; Research; Resolution; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Series; Structure; Techniques; Transact; Yeasts; exosome; genome editing; human disease; innovation; insight; novel strategies; particle; programs; ribosomopathy; structural biology; yeast genetics

Project Summary Ribosomes are molecular machines composed of ribosomal RNAs and up to 80 ribosomal proteins. These large assemblies catalyze protein synthesis in all cells. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled with the help of more than 200 non-ribosomal factors as a series of molecular snapshots of assembly intermediates. Combining genetic, biochemical and mass spectrometry approaches with cryo-EM is an essential step to engineer, trap, isolate and determine atomic-resolution molecular snapshots of transient assembly intermediates of ribosomal subunits. Eukaryotic ribosome assembly can be subdivided into four stages, co-transcriptional assembly events and initial maturation of small and large ribosomal subunit precursors in the nucleolus, nuclear maturation of pre-40S and pre-60S particles, nuclear export, and cytoplasmic maturation. While late events in eukaryotic ribosome assembly are relatively well characterized, the early assembly of ribosomal subunits in the nucleolus is still poorly understood. Especially the mechanisms responsible for early human small ribosomal subunit assembly have remained elusive due to a biomolecular condensate that retains these early particles in human nucleoli. This proposal describes new approaches to define the molecular mechanisms that govern nucleolar maturation of the eukaryotic small ribosomal subunit. My laboratory has developed new human genome editing and biochemical approaches that now enable us to efficiently tag and isolate early nucleolar assembly intermediates of the human small ribosomal subunit. The synergistic use of these approaches has allowed us to overcome previously intractable biochemical hurdles, thereby enabling the detailed study of essential early assembly intermediates of the small ribosomal subunit. Insights from these studies will shed light onto both the mechanisms that are employed during eukaryotic ribosome assembly to coordinate key processing events as well as how defects in eukaryotic ribosome assembly can result in human blood disorders, which are collectively termed ribosomopathies.

项目摘要 核糖体是由核糖体RNA和多达80种核糖体蛋白组成的分子机器。 这些大的组装体催化所有细胞中的蛋白质合成。该项目的长期目标是 了解真核生物核糖体是如何在200多个非核糖体的帮助下组装的。 作为组装中间体的一系列分子快照的因素。结合基因，生化 和质谱方法与冷冻EM是一个必不可少的步骤，工程，陷阱，隔离， 确定核糖体瞬时组装中间体的原子分辨率分子快照 亚单位。 真核生物核糖体组装可分为四个阶段，共转录组装事件 以及核仁、核内核糖体大小亚基前体的初始成熟 前40S和前60S颗粒的成熟、核输出和细胞质成熟。而晚期 真核生物核糖体组装中的事件相对较好地表征， 对核仁中的核糖体亚基仍然知之甚少。尤其是那些 对于早期人类小核糖体亚基组装仍然难以捉摸，由于生物分子 在人类核仁中保留这些早期粒子的凝聚物。 这一建议描述了新的方法来确定的分子机制，管理核仁 真核小核糖体亚基的成熟。我的实验室开发出了新的人类基因组 编辑和生物化学方法，现在使我们能够有效地标记和分离早期核仁 人核糖体小亚基的组装中间体。这些方法的协同使用 使我们能够克服以前难以克服的生物化学障碍，从而使详细的 核糖体小亚基早期组装中间体的研究。 从这些研究的见解将阐明这两种机制，采用期间 真核生物核糖体组装以协调关键加工事件以及真核生物中的缺陷如何 核糖体组装可导致人类血液疾病，这些疾病统称为 核糖体病