Small silencing RNAs regulate gene expression in nearly all eukaryotes and have enormous biotechnological and therapeutic potential. MicroRNAs belong to the larges family of trans-acting gene regulatory molecules in multicellular organisms. In flies and mammals, they control more than half of the protein-coding transcriptome, and act as key regulators of organismal development, physiology, and disease.Here, we propose to study the molecular mechanisms that regulate microRNA homeostasis. We aim to understand how distinct small RNA profiles are established and maintained to coordinate the expression of more than half of all protein coding genes in flies and mammals. Our studies will provide insight into the processes that regulate the function of miRNAs, determine possible causes for aberrant miRNA levels, that have been associated with human diseases, and provide guidelines how to efficiently inhibit miRNA function for analytical and therapeutic purposes.We aim to identify and characterize the molecular determinants of microRNA stability, to dissect the pathways that promote the sequence-specific degradation of microRNAs, and to understand the biological consequences and therapeutic potential of small RNA decay. We will develop novel tools to obtain a view on the intracellular dynamics of RNA silencing pathways, in order to determine the molecular features associated with small RNA biogenesis and decay.Because of its genetic and biochemical tools, we will use Drosophila melanogaster as a model organism. We will employ a combination of bioinformatics, cell-free biochemical experiments, cell culture methods, and in vivo genetics. What we learn in flies we will test in vitro in mammalian cell extracts, in cultured human cell lines and in vivo in mice to identify where these processes are conserved and where they diverge.Overall, our goal is to determine fundamental biological mechanisms of RNA silencing, a phenomenon with enormous biological and biomedical impact.