Biochemical and Genetic Characterization of Ribosome Biogenesis and Functional Diversity

核糖体是一种复杂的分子机器，在将遗传密码转化为蛋白质方面发挥着核心作用。在面包酵母中，核糖体RNA的组装需要许多因素和137个核糖体蛋白编码基因的正确表达，其中大部分是重复基因。导致前rRNA加工和组装的事件已经被广泛研究。然而，调控核糖体蛋白基因表达的机制以及基因复制对核糖体生物发生和功能的影响尚不清楚。通过对酵母中每个核糖体蛋白基因的系统缺失和分析，我们发现在正常条件下不需要的次要拷贝对胁迫下的生长至关重要。值得注意的是，这项由NSERC资助的工作发现了新的调节层，通过RNA剪接和RNA稳定性的变化来调节核糖体的组成和翻译。这些发现反对核糖体蛋白基因的等同和多余的作用。在这项研究中，我们旨在了解复制基因表达的调控机制及其对核糖体和细胞功能的影响。因此，我们提出了两个具体的目标：1.确定复制核糖体蛋白基因差异表达的调控机制；2.评估复制基因对核糖体生物发生和功能的贡献。在第一个目标下，我们将确定复制的基因如何沟通，以建立核糖体蛋白的整体表达，以及它们的表达比例在不同的生长条件下是如何调节的。我们最近发现了一组相互调控的复制核糖体基因，它们在应激状态下调节细胞生长并促进细胞分裂，我们将使用它们作为本研究的模型。将研究启动子、内含子、转录终止和细胞核糖核酸酶对这些准基因表达的影响，并确定它们促进细胞生长和对应激反应的机制。这项研究的结果不仅将解释核糖体蛋白的表达是如何协调的，还将有助于我们理解细胞应激反应的新方面。在第二个目标下，我们将监测改变核糖体蛋白比例对核糖体生物发生、翻译和细胞功能的影响。复制的基因将从异源启动子过表达、缺失或突变，并通过一套完整的转录和表型分析来确定对核糖体生物发生、基因表达、翻译和细胞表型的影响。这一研究计划将直接验证真核细胞核糖体功能多样化的假说，并为核糖体生产的调节如何影响细胞的功能和生长提供一个机制框架。

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 many 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 and assembly have been extensively studied. However, the mechanism regulating the expression of ribosomal protein genes and the impact of gene duplication on ribosome biogenesis and function remain unclear. Through systematic deletion and analysis of each ribosomal protein gene in yeast we discovered that the minor copies, which are not required for growth under normal condition, become critical for growth under stress. Remarkably, this work funded by NSERC uncovered new regulatory layers that modulate ribosome composition and translation through changes in RNA splicing and RNA stability. These findings argue against an equal and redundant role for the ribosomal protein genes paralogs. In this research program, we aim at understanding the mechanism regulating the expression of duplicated genes and its impact on ribosome and cell function. Accordingly, we propose two specific aims: 1. Determine the mechanism regulating the differential expression of duplicated ribosomal protein genes and 2. Evaluate the contribution of duplicated genes to ribosome biogenesis and function. Under the first aim, we will determine how duplicated genes communicate to establish the overall expression of ribosomal proteins and how their expression ratio is modulated under different growth conditions. We have recently identified a set of inter-regulated duplicated ribosomal genes that regulate cell growth under stress and promote cell division, and we will use them as a model for this study. The impact of promoters, introns, transcription termination and cellular ribonucleases on the expression of these paralogs will be examined and the mechanisms by which they contribute to cell growth and response to stress identified. The outcome of this study will not only explain how the expression of ribosomal proteins is coordinated but will also help us understand new aspects of cell stress response. Under the second 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 ribosome biogenesis, gene expression, translation and cellular phenotype determined using an integrated set of transcriptomic and phenotypic assays. This research program will directly verify the hypothesis of paralogs driven functional diversification of eukaryotic ribosomes and provides a mechanistic frame for how the modulation of ribosome production may influence cell function and growth.