MicroRNAs (miRNAs) are a recently discovered class of evolutionary conserved noncoding RNAs that exist as short inverted repeats in the genomes of invertebrates and vertebrates. Unlike other small RNAs, many miRNAs exhibit highly specific and restricted spatial and temporal expression. MicroRNAs are believed to act by basepairing with the RNA of the 3'-untranslated sequence regions (3'-UTRs) of one or multiple target genes, which leads to post-transcriptional repression. The function of most miRNAs is currently unknown but several studies in worms and flies indicate that they regulate essential biological functions such as developmental timing, cell proliferation, cell death fat metabolism and stress resistance. We have recently identified several novel miRNAs are expressed in pancreatic beta-cells. These miRNAs are highly conserved between human and mouse, their expression is pancreatic islet-specific, and overexpression and repression in pancreatic beta-cells leads to inhibition and stimulation of insulin secretion, respectively. In this application we propose a series of molecular genetic, biochemical and bioinformatic studies to elucidate the molecular function of islet miRNAs. In aim 1 we will examine the role of islet-specific miRNAs in pancreatic beta-cell function in vitro by overexpression and inhibition using 2'-O-methyl RNAs that have the complimentary sequence to miRNAs expressed in beta-cells. In aim 2 we will examine the function of RNA silencing during development of the pancreas by studying a lox-P modified dicer conditional allele in pancreatic beta -cells, ngn3- and pdx-1-positive cells. We will also study the specific biological role of two novel islet miRNAs by generating mutant mice by targeted deletion and homologous recombination in ES cells. The pancreatic phenotype of these mice will be studied during development and in adult life. In aim 3 we propose to identify and validate target genes of pancreatic islet miRNAs using biochemical, bioinformatic and proteomic approaches. In aim 4 we will test the hypothesis if genetic variation in islet-enriched miRNAs or their respective target sequences predisposes to the development of type 2 diabetes. In summary, this research proposal will i.) establish the molecular function of miRNAs in the developing and mature pancreas, ii.) contribute to our basic understanding of regulatory networks in differentiating beta-cells, and iii.) provide the community with genetic information that can be used to test if RNA silencing in beta-cells contributes to genetic susceptibility in type 2 diabetes.