This project will make the first comprehensive effort, for any eukaryote, toward defining genes whose expression is regulated by the general translation initiation machinery. Evidence indicates that gene-specific regulation of translation initiation is widespread, phylogenetically conserved, and occurs under a variety of environmental and developmental conditions. Gene-specific pathways of translational control are disrupted by mutations in individual subunits of the eukaryotic initiation factor (eIF) complexes, but there is no comprehensive information about the extent of translational regulation and its dependence on specific eIFs. Building on our preliminary data and complementary expertise, this project will define groups of genes whose mRNAs are co-dependent on specific translation factors, i.e. "translational regulons". This work will provide the first genome-wide overview of the network of translational regulatory pathways through the pursuit of three specific aims:
1) Determine the developmental, physiological, and stress-related phenotypes of eif mutants. The metabolic and developmental pathways impacted by the loss of a specific translation factor will be assessed at the level of gross morphology and development, responses to an abiotic stress (i.e., heat), light, and photosynthesis.
2) Identify candidate client mRNAs whose translation is controlled by specific eIFs. DNA microarray analysis on polysomal RNA from eif mutants will be performed to identify client mRNAs for each eIF. Detailed bioinformatic analyses of the client mRNAs will be used to define translational regulons and to predict mRNA structural and/or sequence features that may influence translational regulation.
3) Validation of eIF and client mRNA translational regulation. The requirement of an eIF in the translation of a subset of client mRNAs will be validated using in vivo mRNA-reporter constructs. An in vitro translation lysate will be developed from Arabidopsis to be used in the analysis of the eIF-dependence of client mRNAs in a homologous in vitro translation assay.
With respect to broader impacts, this project will dramatically increase our understanding of translational control in plants and as the first genome-wide effort for any eukaryote and will make a considerable contribution to plant science and to the greater scientific community. Taking advantage of the fact that UC-Riverside, UT-Austin, UT-Knoxville, and U-Arizona have substantial representation of minority students, the project will involve a robust educational component emphasizing the involvement of minority graduate and undergraduate students in scientific research, acting to encourage minority students to pursue a career in scientific research, education, or policy. Graduate and post-doctoral students will be engaged in full-time research on the project. Undergraduate students will be involved in cross-disciplinary training through the NSF-supported Freshman Research Initiative (UT-Austin), which provides freshmen a significant research experience, or the Undergraduate Biology Research Program (U-Arizona) that places undergraduate students into research laboratories. The project will also partner with federally-funded programs that promote science training for minorities including the NSF-funded California Alliance for Minority Participation in Science, Engineering and Mathematics (CAMP-UCR) that works to double the number of minority students receiving a degree in science; the Copernicus Project, a U.S. Department of Education program that seeks to increase substantially the number, quality and diversity of science teachers (UC-Riverside); and the NSF-funded Louise Stokes Alliance for Minority Participation, which brings minority students from other U-Tennessee system schools to do summer research. In addition, each laboratory will use summer REU supplements to provide research experience to additional undergraduate students. The proposed research includes research approaches that are suitable for the involvement of students at every level (high school, undergraduate, intern, graduate and post-doctoral), thus providing a positive research experience while producing scientifically literate citizens as well as generating extensive information about the role of translational machinery in regulating gene expression. All project data and biological resources can be accessed through FIAT, the project website (http://research.cm.utexas.edu/kbrowning/fiat/). Mutants and associated phenotypic data and images will also be available through the Arabidopsis Biological Resource Center while microarray data will be deposited and available through the NCBI Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo/).
For cells and organisms to function properly, the biological information contained within the genome must be translated into thousands of different proteins, at the right time and in the correct amounts. All proteins are synthesized on a macromolecular machine called the ribosome, using as an informational template a molecule of RNA that was copied from genomic DNA. This project set out to improve our understanding of how the synthesis of different proteins by ribosomes is regulated. The work under this award was performed using the reference plant species, Arabidopsis thaliana, with the rationale that findings made in this species are likely to apply more broadly to other flowering plants including crops. Moreover, the findings may well have relevance for equivalent processes in other organisms including humans. For example, in humans, the regulation of protein synthesis is implicated in growth and development, in aging, cancer, and infectious disease, as well as in learning and memory. The work extended our knowledge of an intricate and complex system that tunes the translation of individual proteins according to external factors such as the daily light-dark cycle, and certain environmental conditions. Not all proteins are affected in the same way by each condition. Instead, proteins are organized into cohorts whose synthesis is regulated in a similar way. The work also made use of genetic perturbations in order to gain unprecedented insights into the functioning of the molecular apparatus for protein synthesis. A central mission of modern biological science is to predict with accuracy the behavior and properties of cells upon genetic manipulation, and to analyze which genetic change is needed to achieve a desirable outcome. Comprehensive data on the efficiency of protein synthesis as directed by the genetic code are needed to make these predictions. Such data were collected here and are being made available to the scientific community and the general public. Although data on protein synthesis will not be sufficient on their own, they are needed for integration with other datasets. In addition to its innate scientific merit, the project furthered the training and career development of postdoctoral scientists, predoctoral students, undergraduate students, and high school students.
