Investigating the role of autism gene Ube3a in the transcriptional regulation in neurons
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UBE3A is a protein with dual functions as an E3 ubiquitin ligase and as a Steroid Hormone Receptor (SHR) transcriptional coactivator. It is expressed ubiquitously in tissues and has significant importance in neurons, where it is expressed exclusively from the maternal allele. Thus, while UBE3A has a wide variety of targets in various tissues, it is especially important in the brain as UBE3A regulates several aspects of neuronal growth, function, and maintenance. Therefore deficits of Ube3a cause Angelman Syndrome (AS) and increased dosage causes Autism Spectrum Disorder (ASD), two neurological disorders. The pathological phenotype of both diseases involves behavioral dysfunctions in learning, motor skills, and sociability. Through microarray studies, our laboratory has found that Ube3a is involved in the regulation of neuronal proteins such as CBLN1, which has been found to have significant importance in parallel fiber synapse formation onto Purkinje cells. To determine how regulation of CBLN1 occurs, mutant variants of human Ube3a isoform III were then generated. E3 ligase-dead, substrate-binding defective, nonphosphorylatable mutant, and phosphor-mimetic mutants were produced and inserted into a pLVX-IRES-mCherry vector. A quantitative transcriptional analysis demonstrated that increasing wild-type (WT) UBE3A decreased Cbln1 expression. The ligase dead mutant mimicked the WT suggesting that E3 ligase activity is not required in the regulation of Cbln1. The nonphosphorylatable mutant demonstrated an increase in Cbln1 expression, which may be due to a dominant negative effect on native UBE3A causing its degradation. The phosphor-mimetic mutant had no statistical effect. This may be due to its inability to enter the nucleus and affect transcription. The substrate-binding mutant also showed no statistical effect possibly because of its inability to bind to any substrate and that may be necessary to regulate transcription. These preliminary results demonstrate that UBE3A may be regulating CBLN1 at the transcriptional level independent of its E3 ubiquitin ligase function. Future studies will be required to more precisely determine the mechanisms involved in UBE3A’s regulation of CBLN1.