Angelman Syndrome is caused by loss of expression of the UBE3A gene in the brain. In most cases this is due to a large deletion on the maternal copy of chromosomal region 15q11-13, where the UBE3A gene is located. The paternal copy of UBE3A remains intact, but is epigenetically silenced in the brain by transcription of the UBE3A antisense transcript (UBE3A-ATS). The exact mechanism by which the UBE3A-ATS causes silencing of UBE3A is unknown, but the two main possible routes are that: (i) transcription of UBE3A ends prematurely when the RNA polymerases transcribing sense and antisense UBE3A collide, and (ii) UBE3A-ATS recruits proteins that act to silence UBE3A. Since the latter mechanism implies that the sequence of the UBE3A-ATS is important for its silencing activity (whereas the former collision model is sequence-independent), our project is focused on determining whether UBE3A-ATS activity is sequence-dependent, thereby distinguishing between these two potential mechanisms of action. To address this question, we will make a series of deletions in the UBE3A-ATS (using the CRISPR/Cas9 system), without affecting the UBE3A gene, then measuring expression of UBE3A in differentiated neurons. In order to test the hypothesis that UBE3A-ATS binds epigenetic repression complexes in order to silence UBE3A, we will also test for such RNA-protein interactions in a neuronal nuclear extract by using the UBE3A-ATS as bait.  Any proteins pulled down by UBE3A-ATS will be identified by mass spectrometry.