RNA inteference and "in cellulo" reconstitution assays as tools to uncover the cellular functions of the ubiquitin-protein ligase E6-AP

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Genetic alterations of the UBE3A gene have been causally involved in the development of the Angelman syndrome (AS), a neurological disorder. The UBE3A gene encodes the ubiquitin-protein ligase E6-AP and all of the genetic alterations found in AS patients inactivate its ubiquitin-protein ligase function. However, only little is known about the cellular function(s) of E6-AP in general and how loss of E6-AP activity affects the functionality of neurons in particular. We have recently shown that modulation of E6-AP expression levels by RNA interference (RNAi) affects the viability of cells and have set up proteomic approaches to identify interaction partners and substrates of E6-AP. In this proposal, an RNAi-based screening system will be employed to identify proteins and processes that operate downstream of E6-AP and/or are required for E6-AP to exert its cellular functions. Furthermore, the interaction of E6-AP with putative binding partners identified by this screen will be characterized by biochemical and cell biological means. Finally, neuronal cell lines and primary neurons derived from Ube3a deficient mice and wild-type mice, respectively, will be used to obtain insight into the function of E6-AP in neurons. Taken together, the proposed studies will contribute to the identification of cellular processes that are controlled by E6-AP and, as a consequence of loss of E6-AP activity, are deregulated in AS patients.