Investigating the interaction between UBE3A and the UBE3A antisense

Date of Completion

January 2006


Biology, Molecular|Biology, Genetics




Failure to inherit a normal active maternal copy of the gene encoding ubiquitin protein ligase E3A (UBE3A) results in the neurogenetic disorder, Angelman syndrome. UBE3A is transcribed predominantly from the maternal allele in brain but is expressed from both alleles in most other tissues. A number of studies suggest that brain-specific silencing of the paternal UBE3A allele is mediated by a large (>500 kb) paternal noncoding antisense transcript (UBE3A-ATS). In order to investigate the interaction between Ube3a and Ube3a-ATS, a new mouse model of Ube3a inactivation that more closely mimics mutations observed in AS patients was proposed. ES cells harboring an Ube3a two base pair deletion were isolated, but attempts to create mice with the correctly targeted ES cells were unsuccessful. Concurrently, we performed shRNA-mediated knockdown of Ube3a in the murine P19 embryonic carcinoma (EC) cell line, a widely accepted model of in vitro neuronal differentiation. We have achieved stable knockdown of Ube3a mRNA and protein in P19 cells by transfection of a 7SK polymerase III promoter vector system to express shRNAs against Ube3a. Knockdown of Ube3a does not appear to affect either gross neuronal morphology during P19 cell differentiation or the expression of some neuronal markers. Cell fractionation experiments reveal that all Ube3a isoforms are efficiently knocked down by shRNA expression including two large (≥10kb) transcripts that are localized exclusively in the nucleus. While knockdown of the large nuclear transcripts could suggest that the shRNAs against Ube3a function in the nucleus, a marked increase of the Ube3a-ATS transcript is also detected in the nuclear fraction. Up-regulation of Ube3a-ATS could mediate repression of Ube3a in cis and offer an alternative explanation for the observed decrease of Ube3a transcripts in the nucleus. Increased expression of Ube3a-ATS is also observed in P19 cells treated with a histone deacetylase inhibitor, leading us to hypothesize that Ube3a regulates Ube3a-ATS via a chromatin-mediated mechanism. In relation to the molecular pathogenesis of Angelman syndrome, our results raise the interesting possibility that abnormal Ube3a-ATS expression resulting from loss of Ube3a may contribute to the phenotypic manifestations of this neurogenetic disorder. ^