Biochemical and Genetic Characterization of Ribosome Biogenesis and Functional Diversity

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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.