The long term objectives of this project are to identify proteins of the nucleolus that regulate the synthesis of ribosomes, and to elucidate how such proteins couple this regulatory process to the control of cell growth. Among the first proteins to respond to changes in cell growth rate are nucleolar proteins known as proliferating cell nucleolar proteins (PCNPs). PCNPs have been implicated in the transmission of growth signals to the nucleolus and in the nucleolus and in the maintenance of elevated levels of growth. It is likely that PCNPs play a role in the growth-associated induction of ribosome synthesis, and that PCNPs mediate the coordination of nucleolar function with cell growth control. To investigate the regulation of nucleolar function, PCNPs will be identified and studied in the yeast Saccharomyces cerevisiae. We have isolated, cloned and sequenced NOP2, which encodes a protein similar to a human PCNP that is a significant prognostic marker for human breast carcinoma. The analysis of NOP2 in yeast offers an opportunity not available in higher cell types to explore the function of a putative PCNP, which may shed light on certain human diseases. This proposal will focus on the: 1. Expression of NOP2. The synthesis of the nop2 protein and the transcription of NOP2 mRNA will be analyzed. Expression during the G0-G1 transition, and during the cell cycle will be emphasized. 2. Characterization of the Nop2 Protein. The nop2 protein will be characterized by the investigation of phosphorylation, and other post- translational modifications. Interactions between the Nop2 protein and other nucleolar proteins, and/or small nucleolar RNAs, will be examined. 3. Nop2 Protein Function. Nop2 protein function will be explored by testing the effects of abolishing NOP2 expression on: growth rate; progression through the cell cycle; ribosome synthesis; pre-rRNA processing and modification; nuclear ultrastructure; and nucleolar ultrastructure. 4. Conditional Mutant Alleles of NOP2. NOP2 is an essential gene, which should allow the creation of thermal sensitive nop2 mutants. Phenotypes of nop2 cells held at the restrictive temperature will be studied. Effects on cell structures and functions mentioned in Specific Aim 3 will be evaluated. The ability of human PCNP120 to functionally substitute for NOP2 will be tested in vivo. 5. Identification of Putative Nucleolar Regulatory Proteins. Nucleolar proteins from cells in different growth states (e.g., G0 versus rapidly dividing) will be compared to identify candidate proteins. We have developed a method for the isolation of yeast nucleoli for this purpose. Extragenic suppressors of nop2 conditional mutations will be isolated and characterized. These studies address a long term goal of the proposal and will be initiated, but not necessarily completed, during this project.
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