Arabidopsis 2010: Global Analysis of Translational Regulons

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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, http://www.ncbi.nlm.nih.gov/geo/).