Fig. 3: (S)-PHA533533 and its analogs downregulate the Ube3a antisense (Ube3a-ATS) transcript and produce UBE3A protein from the paternal allele in mouse primary neurons derived from Angelman syndrome model mice.

a Simplified schematics of Ube3a and Ube3a-ATS in mature mouse neurons. Ube3a-ATS transcript, which together with Snurf/Snrpn, Snord107 (not depicted), Snord64 (not depicted), Snord116, and Snord115 is part of the Snhg14 host gene, is expressed only from the paternal allele (blue dashed line), where it represses the expression of Ube3a. Protein coding Snurf/Snrpn is expressed in all tissues (solid blue line), whereas other paternally expressed genes are exclusively expressed in neurons (blue dashed line). Maternal Ube3a-ATS expression is epigenetically silenced due to the methylation stretch of CpG islands at the Snrpn promoter region (red circle) that is unmethylated on the paternal allele (white circle), resulting in monoallelic expression of Ube3a/UBE3A in neurons (green arrow/ellipse). b–g Primary cortical neurons derived from wild-type (WT) or Angelman syndrome model (Ube3a^m–/p+) mice were treated either with 0.1% DMSO as a vehicle, 0.3 µM topotecan, 1 µM (S)- or (R)-PHA533533, 0.1 µM compound 2, 0.1 µM compound 3, 0.3 µM compound (±)-4, or 5 µM (S,R)-5 as indicated for 72 h at DIV7. Following the treatment, the relative quantities of (b) Ube3a-ATS, (c) Snord115, (d) Snord116, (e) Snrpn, and (f) Ube3a transcripts were determined by quantitative RT-PCR and the expression of (g) UBE3A protein was determined by western blotting. All data were normalized to GAPDH/Gapdh or β-ACTIN expression, log-transformed, mean-centered, and autoscaled for statistical analysis (n = 4 experiments; mean ± SEM, one-way ANOVA followed by Dunnett’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS non-significant). DMSO dimethyl sulfoxide, kDa kilodalton, Norm. normalized, me methylation. Source data and comprehensive statistics, including F-values, degrees of freedom, confidence intervals, and exact p values, are provided as a Source Data file.