PI: Alice Barkan, University of Oregon coPI: Maureen Hanson, Cornell University
Interactions between proteins and RNAs underlie every aspect of plant development and function. Accordingly, plant genomes encode >700 proteins that harbor predicted RNA binding motifs, but few of these proteins have been studied. To dissect RNA-protein interaction networks in plants, it is necessary to catalog plant RNA-binding proteins, to identify the RNAs with which they interact, and to determine how those interactions influence the fate of the RNA and downstream processes. This project develops tools to address each of these issues, and applies them to a set of 40 RNA binding proteins. The experimental set of 40 proteins emphasizes the CRM and PPR protein families, which are largely specific to plants and which together contain ~500 members, most of which are predicted to be targeted to chloroplasts or mitochondria. Project goals are to: (i) Develop a relational database of predicted plant RNA binding proteins that will integrate data from rice, maize, and Arabidopsis. The database will feature cross-referenced orthologs and paralogs, annotated with gene models, intracellular targeting predictions, experimental data and literature citations; (ii) Use an approach that couples coimmunoprecipitation with microarray technology to identify RNAs with which each of the 40 proteins in the test set is associated in vivo; (iii) Pinpoint aspects of RNA metabolism that are influenced by these 40 proteins, through the analysis of molecular defects resulting from reduced expression of their genes in maize and rice; (iv) Test an approach to increase the efficiency of cloning maize mutations caused by insertions of Mu transposons and, if successful, apply it to an existing set of Mu-induced mutants with defects in chloroplast RNA metabolism; (v) Increase community awareness of plant research through outreach programs at both sites. Expected outcomes include (i) a comprehensive relational database of RNA-binding proteins in plants, which will be of value for a broad range of plant biology researchers; (ii) elucidation of the general properties of two recently recognized plant protein families, the PPR and CRM families, through the identification of specific RNA substrates and physiological roles for representative family members; (iii) continuation of a reverse-genetic screening service for chloroplast biogenesis genes; (iv) tutorials on rice transformation and genetics for college and high school students, respectively, and K-12 outreach programs in Ithaca and Eugene. Access to tools and data is available through the project's Photosynthetic Mutant Library and database at http://chloroplast.uoregon.edu/.
