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 (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.
Storage proteins comprise much of the nutritional protein content of cereal grains. Rice has two major classes of storage proteins (prolamines and glutelins) that are targeted into different endomembrane compartments of the rice seed. This targeting is accomplished, in part, by the active transport of their RNAs to specific subdomains (protein body-ER and cisternal-ER) of the cortical endoplasmic reticulum, the entry site of the endomembrane system. The regulation of RNA transport and localization in the cytoplasm, an event initiated in the nucleus, and the relationship of this process to protein synthesis, RNA storage and/or RNA processing are poorly understood. The major goals of this project were to identify and characterize cytoskeletal-associated, cytoplasmic-localized RNA binding proteins associated with these cytoplasmic activities in developing rice seeds. We identified more than 250 RNA-binding proteins (RBPs) which are described in more detail in our online website (www.bioinformatics2.wsu.edu/RiceRBP). Fifteen of these RBPs bind to specific prolamine RNA cis-determinants, ‘zip-code’ sequences, which are required for targeted transport and localization of these RNAs to the protein body-ER. Available evidence indicates that these RBPs are present as multiple protein complexes, which share common RBPs. We also characterized another protein (RBP-P) that binds to the glutelin RNA zip-code sequence and to proteins that mediate membrane vesicle fusion. Genetic studies have demonstrated that Rab5, the small GTPase required for early endosome vesicle formation, is also required for glutelin RNA localization to the cisternal-ER. Collectively, these observations support the role of membrane vesicles as the vehicle mediating RNA transport on the cytoskeletal highways leading to the cortical ER. Ongoing studies using high-throughput sequencing methods are mapping the RNAs bound by five selected RBPs. Moreover, mutants, which mis-localize RNAs, have been identified in several of these RBP genes. Overall, our studies have provided a novel window into the broader function of these proteins in the process of RNA localization and associated downstream processes of protein synthesis and sorting of the protein products in different endomembrane compartments. This project generated 11 research articles, reviews, and book chapters with two manuscripts currently being prepared for submissions. Data generated by this project will also serve as the nucleus of several studies which will be published in the future and will acknowledge partial support with funds from this grant. In the course of this project, we provided broad interdisciplinary training in plant molecular biology, biochemistry, cell biology and/or bioinformatics for 5 postdoctoral and 36 undergraduate researchers. These trainees included 22 women and 13 individuals from groups under-represented in the sciences. A number of the undergraduates were summer interns from California State University Los Angeles or California State University Fullerton, who were exposed to new opportunities in careers in plant biology and university life in a rural setting. Approximately fifteen of the undergraduate students have gone on to pursue advanced degrees.
