Over the past decade, micro (mi) RNAs have emerged as key regulators of gene expression. Many miRNAs have been linked to diseases, such as cancer or diabetes. MiRNAs bind to specific sequences in the 3’-untranslated region (UTR) of mRNAs, thereby causing inhibition of translation and/or mRNA degradation. However, the detailed molecular mechanism and the factors involved in miRNA-mediated gene silencing are still incompletely understood. The primary research objective of the current proposal is the generation of a novel mammalian in-vitro system that will allow a detailed biochemical dissection of the molecular mechanisms underlying medical relevant miRNA-guided gene silencing. In particular, we will characterize repression mediated by the tumorsuppressor miRNA let-7. We will elucidate the role of cis- and trans-acting factors in miRNA mediated repression and use the system to purify native miRISC-RNP-complexes (Ribonucleoprotein-complexes) and subsequently identify and characterize the components of the complex. Thus, the knowledge gained from this proposal will enhance our understanding of miRNA function and provide novel insights into the role of RNA metabolism in disease, which will be applicable to the development of novel diagnostic and therapeutic strategies targeting miRNA-associated pathologies.