An Integrated Genomic, Proteomic and Cytological Approach to Understand the Function of Cytoplasmic Localized, Cytoskeletal-Associated RNA-Binding Proteins in Rice

Washington State University, Pullman WA

Investigators

Abstract

PI: Thomas W. Okita (Washington State University) CoPI: John Wyrick (Washington State University) Plant RNAs are transported from the nucleus to the cell's peripheral endoplasmic reticulum (cortical ER) membrane complex where they are stored, processed or translated into protein. In developing rice endosperm, RNAs are transported as particles along the cytoskeleton to the cortical ER. Cytoplasmic particles have also been suggested to be sites for storage and processing of RNAs. To identify RNA binding proteins involved in these cytoplasmic processes, more than 25 RNA-binding proteins (RBPs) from a cytoskeleton-enriched fraction have been identified using mass spectrometry and subsequent bioinformatics analysis of web-based rice genomic and EST databases. By using an integrated, broad-based approach, the project goal is to elucidate the roles of selected cytoplasmic localized, cytoskeletal-associated RBPs in the events governing cytoplasmic gene expression during rice endosperm development. Specifically, (i) the interacting protein partners of these RBPs will be identified by TAP technology (ii) their intracellular locations and cellular structure determined by immunofluorescence microscopy; (iii) the RNAs recognized by these RBPs identified by coupled immunoprecipitation and microarray technology; and (iv) insight on the roles in RNA metabolism gained by the study of rice genetic mutants or RNAi lines; in this regard, the project will benefit from an international collaboration with scientists at Kyushu University (Japan). Expected outcomes will include the development of a comprehensive, publicly accessible relational database (http://www.bioinformatics2.wsu.edu/RiceRBP) of rice cytoplasmic RBPs containing (i) information on their organization into peptide domains, their relationship with orthologous proteins in other plant species, their interacting proteins, their predicted and actual intracellular locations, and their temporal expression patterns, (ii) raw data including microarrays, 2-D gel maps and mass spectrometry, (iii) lists of molecular tools for biochemical, cytology and reverse genetics, and (iv) detailed laboratory protocols including those for facile rice transformation. The project will serve to train young scientists at the postdoctoral, graduate and undergraduate levels with special focus on those under-represented in the sciences. It will provide these young scientists the unique opportunity to conduct research in Japan and will expose them to the career prospects in international agricultural research.