The goal of this project is to develop and test methods to isolate, clone and sequence small RNA molecules (21 to 24 nucleotides) on a larger scale than previously possible. RNAs of this size have specific biological activities in many eukaryotes. Preliminary data suggest that most of these tiny RNA molecules are small interfering RNAs (siRNAs) or microRNAs (miRNAs). These small RNAs can turn off genes by inhibiting the synthesis or function of longer RNAs, like mRNAs, that they can base pair with. They do this by guiding protein complexes to the RNA or the DNA that encodes it. In this manner, siRNAs can inhibit the synthesis or cause degradation of mRNAs, whereas miRNAs can inhibit the ability of mRNAs to be translated into protein. miRNAs can also cause accelerated degradation of the mRNAs, similar to siRNAs. The identification and/or prediction of siRNA and miRNA molecules have been hampered by limited experimental data that characterize these molecules in depth. The application of high-throughput approaches in this project will facilitate the identification of these molecules and provide quantitative estimates of abundance for specific small RNA molecules in a given tissue. Comparisons of the small RNA sequences to genomic DNA may identify the source and/or target of these RNA fragments. The methodology will be broadly applicable in many eukaryotes and can be used to identify and measure small RNAs that are regulated in response to developmental, biotic or abiotic cues. In this project, methods for the isolation, cloning and sequencing by MPSS of these small RNA molecules will be developed and implemented, and bioinformatics methods for the characterization and analysis of the data will be initiated. These methods will be tested in two plant species under multiple treatment conditions. For the genomic analysis of the data, an existing customized database and web interface for the visualization of MPSS data will be used (http://mpss.udel.edu/at). The data will be made public via this web interface.
