The ribosome is a cellular organelle that is required for protein synthesis and is essential for life in all kingdoms. It is composed of structural RNAs (ribosomal RNAs - rRNAs) complexed with a set of ribosomal proteins. Because of the integral role of the ribosome in basic cellular mechanisms, it is important to understand how this crucial molecular machine functions. Recent work has identified conserved nucleotide elements (CNEs) within rRNAs that are perfectly conserved in all eukaryotic rRNAs but not in those from bacteria. CNEs are likely to carry out important eukaryotic-specific functions for the ribosome, such as nuclear export. Preliminary data support the idea that CNE-1 contains nuclear export information, which may be mediated by the proteins that are bound there. This project has two specific aims. The first aim focuses on characterization of CNEs in 28S rRNA and their role in mediating ribosome export. Nuclear export will be assayed for yeast ribosomes that are mutated in CNE-1; these mutant ribosomes will contain an MS2 coat protein (cp) binding site that will bind a green fluorescent protein (GFP)-cp fusion protein to facilitate visualization of the export process. The eight CNEs in large subunit (LSU) rRNA will be screened for nuclear export information, using GFP for live cell imaging in budding yeast. The data will indicate which CNEs in addition to CNE-1 contain sufficient information for nuclear export. Leptomycin B (LMB) inhibition in yeast will test Crm1/Xpo1 as the transport receptor for nuclear export of the CNE chimera. The second aim of the project focuses on CNE binding proteins, which will be identified by three complementary approaches. One involves streptavidin-bead pull-down of biotinylated CNE after incubation with yeast extracts, with subsequent identification of the bound proteins by gel electrophoresis and mass spectrometry. In a second approach, a yeast three-hybrid screen will identify genes which encode proteins that interact with CNE-1. As a third approach, a high copy suppressor screen of rDNA with mutated CNE-1 will be used to identify plasmids with genes that are high copy suppressors. In any remaining time of the three year project period, experiments will be initiated to explore the function of proteins that bind to CNE-1.
The intellectual significance of this project is the elucidation of a nuclear export function for a eukaryotic-specific conserved nucleotide element. The project also has broader significance for science and society. It will advance discovery and understanding while promoting teaching, training and learning, by involvement of undergraduates in the research. It will broaden participation by underrepresented groups, as some of the undergraduates working on this project will be women and others will be minority students from Brown or from minority-serving institutions that participate in the Leadership Alliance which is housed at Brown. The data from these experiments will be published in widely read journals so that it is readily accessible to the scientific community.
") Ribosomes are essential for all Domains of life (eukaryotes, bacteria, archaea), as the ribosomes are the factories where proteins are made in the cell. Ribosomes are composed of ribosomal RNA (rRNA) and ribosomal proteins. Decades of experiments by many groups culminated in the x-ray crystal structure of the ribosome and demonstrated that the catalytic activity of the ribosome for peptide bond formation resides in the rRNA component. We sought to identify which regions within rRNA are crucial for ribosome function. We reasoned that these regions would be evolutionarily conserved in all organisms, as a mutation in such an essential region would be lethal and not perpetuated. We developed computational methods for bioinformatic analysis of rRNA from hundreds or organisms spanning the three Domains of life. For each Domain of life, we identified Conserved Nucleotide Elements (CNEs) that are more than 90% conserved in all species in that Domain. When the CNEs were compared between the three Domains, we found that some were universally conserved (uCNEs). These highlight the regions most crucial for ribosome function. Some of the uCNEs correspond to areas of known function (e.g., peptidyl transferase domain), but others map to areas where the function still needs to be explored. Some other CNEs are fully conserved within eukaryotes but degenerate in the other two Domains of life. The functions of these eukaryotic-specific CNEs remain to be elucidated. Proteins may mediate the functions of CNEs. We have characterized one such protein in budding yeast--- ribosomal protein L32. It binds to several sites in rRNA, and we demonstrated by site directed mutagenesis of L32 that its binding to a particular CNE is the most important of its contacts. We found that L32 is an essential gene. Genetic depletion of L32 blocks rRNA processing and the 35S rRNA precursor accumulates. The immature ribosomes are not exported to the cytoplasm. Moreover, the cytoplasm senses that ribosome biogenesis is impaired, and the mature ribosomes begin to turn over by two hours after L32 depletion, with degradation of the mature rRNA. Subsequently, by four hours after L32 depletion, the cell cycle is arrested at G1 and DNA synthesis (S phase) is not initiated. This reveals coordinate regulation of cell growth and cell division. There is much intellectual merit in our data. We have developed new methodology for the field of Bioinformatics, allowing comparison of hundreds to thousands of ribosomal RNA (rRNA) sequences that are aligned with regard to secondary structure. We have developed methods (i) to identify Conserved Nucleotide Elements in the aligned rRNAs, (ii) to identify CNEs that are universally conserved in all three Domains of Life, and (iii) to identify CNEs that are Domain-specific. As a foundation for our analyses, we created a refined database (CORD = Complete Organismal rRNA Database) that will be of use for scientists who study ribosomes as well as for evolutionists who will use it for phylogenetic relationships. Moreover, our map of conserved nucleotide elements in rRNA will be a valuable resource, identifying the most important regions of the molecule for future study by the scientific community. In addition, identification of conserved nucleotide elements in rRNA will provide new targets for antibiotic drug design. The next frontiers of ribosome research are (i) a study of eukaryotic ribosomes, (ii) a full description and understanding of ribosome biogenesis and (iii) elucidation of conformational changes that occur in ribosomes during elongation in protein synthesis. The data from the research described here will highlight the regions in rRNA of great functional importance for these processes, thus helping to focus studies on structure and function by the scientific community. Our research also has a broader impact beyond its intrinsic scientific merit. Our research has been integrated with education, serving to train undergraduates through postdocs who have helped with this research. Three undergraduate women are co-authors on the publications resulting from this research. One of the postdocs is an African-American and is co-author on several of the resulting publications. Thus, the research described here is helping to broaden participation in science by women and minorities. Furthering the opportunities in science for women and minorities is a topic in which the Principal Investigator has been actively involved at Brown University and at the national level. New methods and tools have been developed by the research described here, thus enhancing the infrastructure for scientific research. The results are disseminated to the scientific community through publications and talks. The Principal Investigator has a track record of bringing the benefits of biological research to the attention of the lay public and to Congress.
