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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048586-05
Application #
2634710
Study Section
Molecular Biology Study Section (MBY)
Project Start
1994-01-01
Project End
2000-06-30
Budget Start
1998-01-01
Budget End
2000-06-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Florida
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Bhabhra, Ruchi; Richie, Daryl L; Kim, H Stanley et al. (2008) Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus. Eukaryot Cell 7:575-83
Hong, B; Wu, K; Brockenbrough, J S et al. (2001) Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae. Nucleic Acids Res 29:2927-37
Wu, K; Wu, P; Aris, J P (2001) Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae. Nucleic Acids Res 29:2938-49
Fahrenkrog, B; Aris, J P; Hurt, E C et al. (2000) Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy. J Struct Biol 129:295-305
Wu, K; Dawe, J H; Aris, J P (2000) Expression and subcellular localization of a membrane protein related to Hsp30p in Saccharomyces cerevisiae. Biochim Biophys Acta 1463:477-82
Nelson, S A; Aris, J P; Patel, B K et al. (2000) Multiple growth factor induction of a murine early response gene that complements a lethal defect in yeast ribosome biogenesis. J Biol Chem 275:13835-41
Oakes, M; Siddiqi, I; Vu, L et al. (1999) Transcription factor UAF, expansion and contraction of ribosomal DNA (rDNA) repeats, and RNA polymerase switch in transcription of yeast rDNA. Mol Cell Biol 19:8559-69
Tolerico, L H; Benko, A L; Aris, J P et al. (1999) Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations. Genetics 151:57-75
Wu, P; Brockenbrough, J S; Metcalfe, A C et al. (1998) Nop5p is a small nucleolar ribonucleoprotein component required for pre-18 S rRNA processing in yeast. J Biol Chem 273:16453-63
Wu, P; Brockenbrough, J S; Paddy, M R et al. (1998) NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae. Gene 220:109-17

Showing the most recent 10 out of 16 publications