Inactivation of Ube3a in P19 embryonic carcinoma cells by RNA interference
Date of Completion
Biology, Molecular|Biology, Genetics
UBE3A, the gene associated with the neurogenetic disorder Angelman syndrome (AS), is imprinted in certain regions of normal brain, where it is expressed from the maternal allele only, while in most other tissues, the gene is biallelically expressed. One current hypothesis for the silencing of paternal UBE3A is based on mouse data showing that the large, noncoding, brain-specific, paternally expressed antisense (Ube3a-ATS) transcript overlaps most of the coding sequence of Ube3a. This suggests that the sense and antisense transcripts might regulate each other, possibly via RNA-RNA interaction. ^ The murine P19 embryonic carcinoma (EC) cell line, a widely used model of in vitro neuronal differentiation, is a suitable system for studying certain aspects of the interaction between Ube3a and Ube3a-ATS. We optimized the conditions for P19 cell culture and differentiation and studied gene-specific knockdown generated by siRNAs. Due to the transient nature of siRNA-induced silencing in the rapidly dividing P19 cells, we used a 7SK polymerase III promoter vector including a matrix-associated region (MAR) sequence to express shRNAs targeting Ube3a and achieved stable knockdown of Ube3a RNA and protein levels in P19 cells. Although gross neuronal morphology was not affected by shRNA-mediated knockdown of Ube3a in P19 cells, increased expression of Ube3a-ATS was observed. The levels of the Ube3a-ATS transcript are augmented in the nuclear fraction of P19 cells upon Ube3a knockdown, a result consistent with the predominant nuclear localization of Ube3a-ATS in P19 cells. Cell fractionation experiments reveal efficient knockdown of all Ube3a isoforms by shRNA expression, including two large (≥10kb) transcripts that are localized exclusively in the nucleus. Although knockdown of the large nuclear transcripts could suggest that the shRNAs against Ube3a act in the nucleus, the marked increase of the Ube3a-ATS transcript in the nuclear fraction suggests an alternative mechanism whereby Ube3a-ATS up-regulation represses Ube3a in cis. We propose that normally the Ube3a protein represses Ube3a-ATS expression via chromatin silencing or ubiquitin-mediated pathway. Upon Ube3a knockdown, the Ube3a-ATS transcript is up-regulated and could induce silencing of the large Ube3a isoforms in the nucleus. The observed up-regulation of Ube3a-ATS might contribute to the AS phenotype. ^
Sotirova, Vihra Nikolova, "Inactivation of Ube3a in P19 embryonic carcinoma cells by RNA interference" (2006). Doctoral Dissertations. AAI3217050.