Biochemical and Genetic Characterization of Ribosome Biogenesis and Functional Diversity

核糖体生物发生和功能多样性的生化和遗传特征

• 批准号: RGPIN-2016-03729
• 负责人: AbouElela, Sherif
• 金额: $ 4.66万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614583
• 关键词: Biochemical; Genetic; Characterization; Ribosome; Biogenesis

Ribosomes are intricate molecular machines that play a central role in translating the genetic code into proteins. In baker’s yeast, assembly of the ribosomal RNA requires a large number of factors and the correct expression of 137 ribosomal protein-coding genes the majority of which are duplicated genes. The events leading to the processing of pre-rRNAs, assembly and export have been extensively studied in vertebrates and yeast, and the overall process is very well understood. However, the mechanism regulating the expression of duplicated ribosomal protein genes and its impact on ribosome biogenesis and function remain unclear. Recently, we discovered that the majority of the ribosomal protein paralogs are not expressed equally and that in most cases they are expressed in a specific ratio required for normal growth. This finding argues against an equal and redundant role for the ribosomal protein genes paralogs. In this proposal, we aim at understanding the mechanism regulating the expression of duplicated genes and its impact on ribosome production and function. Accordingly, we propose two specific aims: 1. Determine the mechanism regulating the expression of duplicated ribosomal protein genes. Under this aim, we will determine how duplicated genes communicate to establish the overall expression of ribosomal proteins. We have recently identified a set of inter-regulated duplicated ribosomal genes, and we will use them as a model for this study. The impact of promoters, intron, transcription termination and cellular ribonucleases on the expression of these paralogs will be monitored and elements that affect inter and intragene regulation will be identified. The outcome of this study will not only explain how the expression of ribosomal proteins is coordinated but will also help us to understand an important component of the general mechanism of gene regulation. 2. Understand the impact of duplicated genes on ribosome biogenesis and function. Under this aim, we will monitor the impact of changing the ratio of ribosomal proteins on ribosome biogenesis, translation and cellular functions. The duplicated genes will be overexpressed from a heterologous promoter, deleted or mutated and the impact on the associated proteins monitored using expression tags. The impact on cell function will be measured using a standard set of phenotypic tests, including temperature sensitivity, drug resistance and use of different carbon sources. The effects of growth conditions on the paralogs expression will also be analyzed in order to identify signals inducing changes in the ribosome composition and function. This project will directly verify the hypothesis of paralogs driven functional diversification of eukaryotic ribosomes and provides a mechanistic frame for how ribosome production is modulated in response to growth conditions.

核糖体 是复杂的分子机器，在翻译 遗传密码转化成蛋白质在面包酵母中，核糖体RNA的组装 需要大量的因子和137个核糖体的正确表达， 蛋白质编码基因，其中大多数是重复基因。的事件 导致前体rRNA的加工、组装和输出， 在脊椎动物和酵母中广泛研究，整个过程非常 很好理解。然而，调控复制的表达的机制， 核糖体蛋白基因及其对核糖体生物发生和功能的影响 仍然不清楚。最近，我们发现大多数核糖体 蛋白质旁系同源物的表达并不相等，在大多数情况下， 以正常生长所需的特定比例表达。这一发现表明， 与核糖体蛋白基因旁系同源物的同等和多余的作用相反。在 本提案旨在了解调节表达的机制 复制基因及其对核糖体产生和功能的影响。因此，委员会认为， 我们提出两个具体目标：确定调节 重复核糖体蛋白基因的表达。在这个目标下，我们会 确定复制的基因如何交流以建立整体表达 核糖体蛋白质。我们最近发现了一套相互调节的 复制的核糖体基因，我们将使用它们作为本研究的模型。的 启动子、内含子、转录终止和细胞 将监测核糖核酸酶对这些旁系同源物表达的影响， 将鉴定影响基因间和基因内调节的元件。的 这项研究的结果不仅解释了核糖体的表达， 蛋白质是协调的，但也将帮助我们了解一个重要的 基因调控的一般机制的组成部分。2.了解 复制基因对核糖体生物发生和功能的影响。根据本 目的是，我们将监测改变核糖体蛋白质的比例对 核糖体生物合成、翻译和细胞功能。复制的基因 将从缺失或突变的异源启动子过表达， 对使用表达标签监测的相关蛋白质的影响。的影响 将使用一组标准的表型测试来测量细胞功能， 包括温度敏感性、耐药性和使用不同的碳 源生长条件对旁系同源物表达的影响也将 以鉴定诱导核糖体变化的信号 组成和功能。这个项目将直接验证假设 旁系同源物驱动真核核糖体功能多样化， 提供了如何调节核糖体产生的机制框架 对生长条件的反应。