2008 Annual Report 1a.Objectives (from AD-416) The existence of a class of genes whose products are small RNA molecules apparently having major roles in gene regulation has very recently been discovered. These genes produce untranslated RNA molecules that are processed into short (17-23 nucleotides in length) “micro-RNAs” forming part of RNA-protein (“RISC”) complexes. Specific RISC complexes have been shown to regulate diverse processes in growth, development, and immune response. The micro-RNA (“miRNA”) genes have been shown to be expressed in tightly regulated, tissue and temporal-specific patterns in humans, mice, flies, worms, and even plant species. They appear to work mainly by targeting the RISC complex to complementary sequences in the untranslated sequences of RNAs that code for proteins (“mRNA”), preventing them from being translated into their protein products and thus providing a means to regulate gene expression after the messenger RNA has been produced. MicroRNA (miRNA) expression and the potential effects on physiology in bovine tissues is currently unknown. The current project has three objectives: (i) High-resolution discovery of bovine miRNA genes using in-silico analysis of the emerging draft genome sequence, validated by in-depth sequencing of small RNA fractions from a pool of diverse cattle tissues. (ii) Examination of the tissue specificity of cattle miRNA expression by characterization of miRNA expression profiles from 50 individual tissues. (iii) Prediction of the mRNA targets of miRNA regulation in each tissue examined. 1b.Approach (from AD-416) Combined in silico and laboratory methods will be applied to predict, verify, and discover bovine miRNA genes and characterize their expression in cattle tissues. These results will be used in combination with data from bovine EST libraries and the bovine genome draft sequence to predict mRNA targets. First, characteristic signatures of miRNA genes will be searched for in bovine genome sequence to predict putative bovine miRNA genes. Validation of these results will be performed by pooling small RNA fractions of many tissues and subjecting the pool to high-density sequencing using “next generation” sequencing technology. This will reveal a large number of true miRNA for comparison with the predicted set to test the prediction, which may be modified and redone if the validation indicates a problem with the prediction set. Then, individual tissues will be examined by creation of libraries from small RNA fractions and lower-density sequencing to determine relative expression levels. Finally, the individual tissue data will be merged with EST data from the NCBI database to identify putative targets for regulation by the microRNAs present in the same tissue as the target. 3.Progress Report This project is aligned with the National Program 101 Action Plan Component 1 (Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources) and Component 2 (Enhancing Animal Adaptation, Well-being and Efficiency in Diverse Production Systems). MicroRNA clone libraries have been produced from over 50 tissues of fetal, calf, and adult bovine for evaluation utilizing “next generation” sequencing technology. In addition, methods for creating cDNA libraries have been developed to evaluate the transcriptome for microRNA gene targets. These libraries are an important tool for identification of microRNA and gene targets that have a role in growth and development of livestock.