Human Argonaute proteins: Analysis of endonucleolytic activity and endogenous phosphorylation sites

Argonaute (Ago) proteins interact with small non-coding RNAs, which guide them to complementary target RNAs to mediate gene silencing. The domain organization of Ago proteins clearly reflects their function. The ends of the small RNA are bound by the Mid and PAZ domains and the structure of the PIWI domain closely resembles that of the endonuclease RNase H. Indeed, some Ago proteins can cleave perfectly complementary target RNAs. Here, the endonucleolytically inactive human Ago proteins Ago1, Ago3, and Ago4 were mutated to yield minimally changed, catalytically active proteins. The generation of these mutants revealed several features that are important for Ago cleavage activity, in particular two regions in the N-terminal domain and two structural elements in the catalytic domain. The identification of these structural requirements contributes essentially to our understanding of Ago-mediated silencing activity. Ago proteins not only cleave their targets. In case of partial complementarity between small RNA and target RNA, they recruit additional factors to mediate gene silencing. Here, predominant mechanisms are translational repression and reduction of target stability. Considering the wide spectrum of gene regulation emanating from Ago proteins, it is important to understand how the proteins are regulated themselves. Here, post-translational modifications, in particular phosphorylations, of Ago proteins were analyzed by mass spectrometry. Multiple endogenous phosphorylation sites of Ago2 and its paralogues were detected. Among them are sites, which are organized in phosphorylation clusters. The data generated in this thesis represents a valuable resource for future analyses of Ago proteins and their modifying enzymes, which will provide insights into the interplay of small RNA-guided gene silencing and signaling pathways. The phosphorylation sites analyzed here were identified on endogenous Ago proteins. To provide large amounts of endogenous protein, a novel, peptide-based purification strategy was ...