One of the most important discoveries in molecular biology in recent years was that small RNA molecules are very important for regulating genes. Plants have two main types of small RNA, the microRNAs (miRNAs) and the small interfering RNAs (siRNAs), that can be distinguished by how they are made in the cell. These small RNAs range in size from about 21-24 nucleotides, and were missed for many years because they are so tiny. Despite their late discovery, small RNAs are very important in both plants and animals because they function to shut off genes. Small RNAs recognize the target genes they silence by base-pairing interactions. This project will investigate small RNA molecules of the model plant Arabidopsis on a genome-wide scale and investigate the functional significance of those associated with how plants respond to environmental stress. Deep sequencing of a small number of collections made from key small RNA metabolism mutants and plants subjected to stress will result in more than 200,000 sequences per sample. Analysis of the data will provide insight about which small RNAs are miRNAs and which are siRNAs. Assembly of a DNA microarray containing many small RNA sequences will provide the means to test additional mutants and stresses for their regulatory effect on the levels of various small RNAs. These data as well as the incorporation of miRNA/target RNA information into a computer model will lead to new hypotheses about the roles of small RNAs in environmental stress. Many tools to examine these roles are well-established, such as miRNA sensor genes, and overexpression and knockout lines. If time permits, it will be straightforward to begin testing major predictions of some of these hypotheses within the funding period. This project will identify key examples of miRNA involvement in environmental stress and lay the foundation for studying the mechanistic details in the future. Because many Arabidopsis miRNAs are conserved in rice and other plants, this work should lead to the discovery of new functions of general significance.
