The goal of this project is to determine the biological functions of a newly recognized class of Arabidopsis regulatory molecules - the small RNAs. The small RNAs typically contain 20-25 nucleotides and are likely generated by a dsRNA-specific, RNaseIII-like mechanism. Recently, 125 small RNAs were identified in Arabidopsis. Several arise from sequences in known or predicted protein-coding genes and transposons. The vast majority, however, arise from intergenic sequences between conventional genes. The Arabidopsis small RNAs resemble small interfering RNAs (siRNAs) associated with RNA silencing and small temporal RNAs (stRNAs) associated with translational control in C. elegans. These Arabidopsis RNAs are proposed to function in posttranscriptional regulation of gene and mRNA activity by several mechanisms, including RNA silencing, translational interference and epigenetic mechanisms in the nucleus. New libraries of small RNAs will be prepared using different tissues, a database of small RNA information will be assembled, and the small RNA sequences will be deposited into GenBank (www.ncbi.nlm.nih.gov). The function of small RNAs from protein-coding genes in posttranscriptional RNA silencing, and the function of small RNAs from intergenic regions in translational control and modification of nuclear DNA will be determined. Finally, the roles of each of nine RNaseIII-like proteins in small RNA biosynthesis will be analyzed. Results will be shared in peer-reviewed journals, at the International Conference on Arabidopsis Research, and on a database of small RNA information. The database, which will include capabilities for searching small RNA map positions, information about nearest genes, whether or not a given gene is near a small RNA, and other interactive features and results, will be made web-accessible to the scientific community (www.cgrb.orst.edu/carrington/smallRNAs). The publicly accessible database of small RNA information will have broad impact by providing researchers with information about small RNAs near or within genes of interest and allowing subsequent experiments on the roles of small RNA-based regulation of the Arabidopsis genome. Genes are controlled by many different mechanisms. Recently, a new type of regulatory system involving an unusual class of small RNA molecules was discovered. The small RNAs are proposed to function by binding to messenger RNA from specific gene targets. The small RNA would then trigger either cleavage of messenger RNA or inhibit the function of the messenger RNA to which it is bound. This project will result in discovery of new types of small RNA molecules and determine how these regulators affect gene expression in the model plant, Arabidopsis thaliana.