This proposal outlines a plan to study the dynamics of ribosome assembly in yeast, and the relationship between ribosome biogenesis and growth and cell cycle control. The synthesis of ribosomes in eukaryotes requires >200 assembly factors to direct the ribosomal RNA processing and ordered assembly of >100 ribosomal proteins. While the framework for rRNA processing is well established, there is no coherent mechanism for the dynamics of biogenesis factor binding and release or association of the ribosomal proteins. Furthermore, the control of the ribosome biogenesis program is poorly understood in two key respects. First, the mechanism by which synthesis of the ribosomal proteins in the cytoplasm is coordinated with the dynamics for ribosome assembly in the nucleus is poorly understood. Second, although there is ample evidence for the association between defects in ribosome biogenesis and defects in cell growth and division, the mechanism by which ribosome biogenesis is monitored to regulate growth and progression through the cell cycle is unclear. In this proposal, we will apply quantitative analysis of ribosome biogenesis using mass spectrometry with yeast genetic analysis to address both of these key questions. This will be accomplished with three Specific Aims: 1) Implement Isotope Pulse-Quantitative Mass Spectrometry (IP-QMS) for monitoring ribosome biogenesis in yeast. 2) Elaborate the mechanism of ribosome biogenesis by defining the pathway(s) for assembly of ribosomal proteins. 3) To apply IP-QMS to analyze the roles of ribosome biogenesis factors and place them on the assembly pathway map developed in Aim 2 and to elucidate the pathways coordinating regulated ribosome biogenesis with growth and the cell cycle. The main goal of this proposal is to combine yeast genetics and QMS to interrogate the intermediates in ribosome biogenesis involving ribosomal proteins and biogenesis factors and to identify connections between those intermediates and the regulation of cell growth and the cell cycle.
|Haase, Steven B; Wittenberg, Curt (2014) Topology and control of the cell-cycle-regulated transcriptional circuitry. Genetics 196:65-90|