Grant: Discovery Grants Program - Individual

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.