Structural Interplay of Key Factors in the Human MicroRNA Pathway

Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. Summary Argonaute (Ago) proteins are viewed as key players in small RNA-guided gene silencing pathways in almost all eukaryotes. Small RNAs such as short interfering RNAs or microRNAs guide Ago proteins to specific target RNAs resulting in mRNA decay or translational silencing. In miRNA-mediated silencing, Ago proteins associate with a member of the GW182 protein family in order to achieve efficient repression. GW182 proteins comprise a N-terminal Ago-binding domain that is characterized by multiple GW-repeats and a C-terminal silencing domain with several globular domains. GW182 hooks via its GW-repeats into the PIWI domain of the Ago protein providing a binding platform for downstream silencing events. Although the general principles underlying Ago-GW interactions are well established, a detailed characterization of the GW-Ago association has not been performed. To elucidate the molecular basis for the interaction, we established a purification protocol to obtained the desired recombinant proteins and used NMR spectroscopy, biochemical and biophysical approaches as well as crosslinking to analyze affinities and binding properties of human Ago proteins to GW182 proteins. Hence, we found exciting new aspects: First, the tryptophan flanking glycines are not required and several other neighbors are accepted. Second, for efficient association only several specific Trps engage in binding, which reside in fully unstructured protein environments. Finally, we mapped the GW182 binding site on Ago and propose a model of the Ago-GW182 interaction. Thus, our results will contribute to the understanding of the mechanism of miRNA-mediated gene silencing. v Publications and Presentations Parts of this thesis are submitted for publication:ter. (2013). Structural insights into GW-Argonaute protein interactions, submitted.ing catalytically inactive human Argonaute proteins into active slicer enzymes. (2013).