Fig 2: UBE3A directly interacts with RARa. (A,B) The interaction of UBE3A and RARa in both endogenous and ectopically expressed HEK-293T cells. Co-immunoprecipitation (Co-IP) was performed for ectopically expressed (A) or endogenous (B) UBE3A and RARa in HEK-293T cells treated with 1 µM RA or DMSO for 8 h. (C) The directly interaction of UBE3A and RARa. GST pull-down assays were performed with GST or GST-RARa and UBE3A-His. (D,E) Endogenous RARa (D) or Rara (E) remained unchanged upon different dosages of UBE3A (D) in HEK-293T cells or Ube3a (E) protein levels in MEFs.

Fig 3: Gene expression profiling in the hippocampus of mice. (A) Volcano plot analysis from the hippocampus of Ube3am–/p+ mice and Ube3am+/p+ mice (Foldchange > 1.2; P < 0.05). (B) Venn analysis was used for enrichment between the most related to UBE3A and RA-regulated genes or differentially expressed genes. (C) Comparison of convergent differential RA-targeted genes expression between Ube3am+/p+ and Ube3am–/p+ mice. The mean numbers of reads were used. Mouse samples were divided into two groups: Ube3am+/p+ mice (n = 3) and Ube3am–/p+ mice (n = 3). (D) Enriched Biological Process pathway in GO analysis (P < 0.05).

Fig 4: Loss of UBE3A expression leads to the dysregulation of RA-targeted genes. (A) Endogenous RA-related genes were reduced in the Ube3a deficient hippocampus. The mRNA levels of target genes were quantitated by real-time qPCR in mouse hippocampal tissues. (B) Representative images of Erbb4 staining in hippocampal coronal slices. CA1 region was marked by the red dotted line. Scale bar, 50 µm. (C) Quantitated intensities of ERBB4 in the hippocampal region of wild-type and Ube3a deficient mice. (D) A working model depicting how UBE3A acts as a coactivator of RARa to participate in the regulation of RA signalling. The values are expressed as the means ± SEM from at least three independent repetitions. *P < 0.05, **P < 0.01, ***P < 0.001. (B,D) Unpaired two-tailed t test, (A) one-way ANOVA with Bonferroni post hoc test.

Fig 5: Enhancement of glycolysis activities in the cell as a result of PDHA1 degradation triggered by UBE3A. (A) UBE3A decreases PDH enzyme activity in HEK293 cells. Activities of the PDH enzyme in shUBE3A, blank HEK293, and cells overexpressing UBE3A were measured using a colorimetric kit from Biovision. In the assay, PDH converts pyruvate into an intermediate with the coproduction of NADH, which reduces the developer to a colored product with strong absorbance at 450 nm. Cells (1 × 106) were homogenized in 100 µL ice-cold PDH assay buffer. 5–50 µL of the sample was added to each well, and the volume in the well was adjusted to 50 µL with the PDH assay buffer. Standard samples of NADH were added into a series of wells in the 96-well plate. A reaction mixture containing PDH assay buffer, a PDH developer, and a PDH substrate was added to each well. The absorbance of 450 nm was measured in the kinetic mode for 100 min. PDH activity was calculated according to the NADH standard curve and plotted in the right panel. (B) UBE3A enhances glycolytic activity in HEK293 cells. ECAR (extracellular acidity rate) was measured using a Seahorse XFe96 Analyzer. Cells were inoculated into the XFe96 cell culture microplate at a density of 1 × 104 cells/well and then cultured overnight. Before analysis, the medium of the microplate was replaced with a new medium consisting of the Seahorse XF Base Medium, 10 mM glucose, 2 mM sodium pyruvate, and 2 mM glutamine. The microplate was placed in a CO2-free incubator for 1 h before it was transferred to the analyzer. Measurements were conducted using final concentrations of 10 mM glucose, 2 µM OM, or 50 mM 2-deoxyglucose (2-DG). Glycolysis and glycolytic capacity were calculated and plotted in the right panel.