Intellectual Merit: The objective of this research is to identify conserved nucleotide elements (CNEs) in ribosomal RNA (rRNA) and elucidate their functions in the ribosome. The CNE regions must be crucial for ribosome biogenesis, structure and/or function as they have been maintained throughout evolutionary time. Presumably, mutations in these regions would have been lethal to the organism and hence not perpetuated. This research project builds upon previous NSF-funded research in which a Complete Organismal rRNA Database (CORD) was developed and bioinformatic methods were designed to identify CNEs in the large ribosomal subunit rRNA. These methods were applied to eukaryotic 25-28S rRNAs and 42 CNEs were thus identified. Of these 42 CNEs, five are universally conserved in all Domains of life (eukaryotes, bacteria and archaea), and nine other CNEs are specific to eukaryotes with their sequences being degenerate in bacteria. This analysis will be expanded to identify CNEs in bacterial 23S rRNA and to discern which bacterial CNEs are universally conserved in all domains of life and which are specific to bacteria. Mutations will be made in each of the nine eukaryotic-specific CNEs to analyze their function in rRNA processing, ribosome export, ribosome function in protein synthesis and coordination with the cell cycle. Furthermore, an MS2-based pulldown strategy will be developed for isolation of ribosomes containing mutations in the rRNA. Development of this system will provide the foundation for future proteomic and structural studies on ribosome biogenesis. The results of the research described here will be of great value to the scientific community. rRNA is used as a yardstick for phylogenetic comparisons, so the derivation of CORD will be useful to evolutionary biologists. Moreover, the results will be very valuable to molecular biologists in their study of ribosomes. 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.
Broader Impacts: The research described here will be integrated with education, serving to train undergraduates through a postdoctoral associate who will help with this research. Graduate education and postdoctoral training are topics of great interest to the Principal Investigator who has played leadership roles at the national level in this area. The research described here will help to broaden participation in science by women and minorities, with students from these groups working on the research. 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 will be developed by the research described here, thus enhancing the infrastructure for scientific research. The results will be 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.
Intellectual Merit: our research has increased our information about ribosomes, which are the factories that produce proteins in cells of all forms of life. Ribosomes are composed of ribosomal RNA (rRNA) and ribosomal proteins. This NSF grant has funded our studies on rRNA of the large ribosomal subunit. We have developed a bioinformatic approach to identify regions within rRNA that are highly conserved among species, and we call these regions conserved nucleotide elements (CNEs). Sequence conservation is a hallmark of regions of functional importance, as mutation of a functionally important region will be lethal to the organism and hence not perpetuated in evolutionary time. Thus, evolution selects against mutation in functionally important regions. We have analyzed the three domains of life for CNEs in rRNA and have identified 57 CNEs in Eukaryotes, 49 CNEs in Bacteria and 47 CNEs in Archaea. Of these, 22 CNEs are universally conserved (uCNEs) in structural position and sequence in all three domains of life, with nine of these ≥ 90% conserved in sequence. Many uCNEs map to regions of rRNA with established functions for translation, thus validating our methodology. However, unexpectedly, some uCNEs reside in areas with no functions identified to date. This underscores the value of our approach to identify new areas in rRNA of potential functional importance. In contrast to uCNEs, domain-specific CNEs (dsCNEs) are conserved in just one phylogenetic domain and degenerate in the other two domains of life. Our results are the first report of conserved sequence in rRNA that are domain-specific. There are 9 dsCNEs in Eukarya, 2 dsCNEs in Bacteria, and 1 dsCNE in Archaea. Therefore, domain-specific motifs are largely a eukaryotic phenomenon. Thus, the identification of dsCNEs focuses attention on special features that may play unique roles for ribosome biogenesis and function in eukaryotes. The locations of the eukaryotic dsCNEs suggest that they may play roles in nascent polypeptide transit through the ribosome tunnel and in tRNA exit from the ribosome. In addition, two dsCNEs in bacteria offer targets for new antibiotics. Our findings provide insights and a resource for ribosome function studies. In addition, we have studied ribosomal protein L32, which is present in Eukaryotes but not in Bacteria. L32 binds to CNEs and is an essential protein in yeast. Genetic depletion of L32 blocks ribosomal RNA (rRNA) processing and the initial precursor (35S in yeast) accumulates. This suggested that L32 may bind to 35S pre-rRNA, and we have confirmed that this is the case by pull-down experiments. Not only is there an effect on rRNA processing, but L32 depletion also results in massive turnover of rRNA from the cytoplasmic ribosomes (possibly mimicking a starvation response). These events may be important for the sensing mechanism to coordinate ribosome biogenesis with the cell cycle, as we found that there is an arrest of the cell cycle at G1 phase after L32 depletion. This sensing mechanism could help to couple and co-regulate cell growth (viz., ribosome biogenesis) and cell division (viz., initiation of DNA synthesis). Broader Impact: the research funded by this NSF grant has been integrated with education, serving to train undergraduates through postdoctorals who were involved in this research. Graduate education and postdoctoral training are topics of great interest to the Principal Investigator who has played leadership roles at the national level in this area. The research described here has helped to broaden participation in science by women and minorities, with students from these groups working on the proposed research. 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 bioinformatic tools have been developed by the research described here, thus enhancing the infrastructure for scientific research. The results are disseminated 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. Some of the results provide new target sites for the development of new antibiotics, providing potential commercialization with an impact of medical significance.
