Title

The role of Ube3a antisense RNA in the regulation of genomic imprinting of Ube3a, the gene associated with Angelman syndrome

Date of Completion

January 2006

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Angelman syndrome (AS) is a neurogenetic disorder characterized by severe neurodevelopmental abnormalities, impaired speech, ataxic gait, and inappropriate laughter. The majority of AS cases result from the failure to inherit a normal maternal allele of the imprinted gene encoding ubiquitin ligase 3A (UBE3A). In brain, Ube3a is expressed predominantly from the maternal allele, while a non-coding antisense RNA (Ube3a -ATS) is exclusively transcribed from the paternal allele. The current hypothesis is that the brain-specific Ube3a-ATS silences the paternal Ube3a in cis. ^ We tested this hypothesis in R1 murine embryonic stem cells (R1ESC), by studying the temporal patterns of total and allelic expression of Ube3a, and the expression of Ube3a-ATS. We found that, in undifferentiated R1ESC and early after retinoic acid neuronal induction, expression of Ube3a is predominantly from the maternal allele, while Ube3a-ATS transcription is detectable only seven days after induction. These data suggest that the mechanism of imprinting of Ube3a in R1ESC, and during early stages of differentiation is independent of Ube3a-ATS. We further support this conclusion by finding preferential expression of the paternal Ube3a allele in mouse spleen, an organ that does not express Ube3a-ATS. However, in the R1ESCderived neurons, which co-express Ube3a-ATS, we observed a more marked repression of Ube3a, and a decreased paternal contribution, consistent with roles of Ube3a-ATS in enhancing repression of the paternal Ube3a allele in fully differentiated neurons. These findings suggest the possibility of distinct imprinting mechanisms for Ube3a in R1ESC versus R1ESC-derived neuronal cells, and in mouse brain versus spleen. ^ Based on recent reports that several chicken orthologues of mammalian imprinted loci display DNA replication asynchrony, a property of imprinted genes, we tested parent-specific expression of avian Ube3a, in order to further investigate the mechanism of Ube3a imprinting. We find no evidence for avian Ube3a-ATS expression, and we observe biallelic UBE3A expression in embryonic chicken brain and limb, indicating that UBE3A is not subject to genomic imprinting in chicken. ^ These studies suggest that, while Ube3a imprinting is regulated by an antisense transcript in neurons, different mechanisms govern allele-specific expression in other tissues, and at other developmental stages. ^