Fig 2: UBE3A acts as a coactivator regulating RARE-luciferase expression. (A) Schematic structure of UBE3A protein. UBE3A is an 862-amino acid protein that mainly includes five LxxLL domains, a zinc finger domain and a HECT domain. (B) Endogenous UBE3A protein was knocked down upon siRNA treatment. siRNA-UBE3A1, siRNA-UBE3A2 and negative control were transfected into H1299 lung cancer cells, and the protein expression of UBE3A was assessed by western blotting. (C) UBE3A knockdown decreased luciferase activities. H1299 cells were transfected with siUBE3As (the indicated siRNA sequences are listed in Supplementary Table 5), and RARE luciferase activities were tested after stimulation with 1 μM ATRA for 8 h. (D) All isoforms of UBE3A showed activation of RARE-luciferase activities. H1299-KO-UBE3A cells were transfected with vector or different isoforms of UBE3A and then treated with 1 μM ATRA for 8 h. (E) UBE3A could activate luciferase activities in a dose-dependent manner. SH-SY5Y cells were transfected with the indicated dosages of UBE3A followed by 1 μM ATRA treatment for 8 h, and the luciferase ratios were detected. (F) UBE3A and RARα showed coactivation of RARE-luciferase activities. H1299-KO-UBE3A cells were transfected with vector, UBE3A, RARα or UBE3A + RARα and then treated with 1 μM ATRA for 8 h. (G) Mutant in the LxxLL domain compromised the activation of UBE3A. H1299-KO-UBE3A cells were transfected with the indicated LxxLL mutation (L1A or L2A or L3A or L4A or L5A or LA, labelled in method 2.2) followed by 1 μM ATRA treatment for 8 h. (H) The ligase mutant C843A mutant did not affect luciferase activity. Wild-type UBE3A and ligase mutants were overexpressed in H1299-KO-UBE3A cell lines followed by 1 μM ATRA treatment for 8 h. The normalized luciferase activities represent ratios of firefly to Renilla values. The values are expressed as the means ± SEM from at least three independent repetitions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C–H) one-way ANOVA with Bonferroni post hoc test.

Fig 3: 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 RARα 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 4: UBE3A directly interacts with RARα. (A,B) The interaction of UBE3A and RARα in both endogenous and ectopically expressed HEK-293T cells. Co-immunoprecipitation (Co-IP) was performed for ectopically expressed (A) or endogenous (B) UBE3A and RARα in HEK-293T cells treated with 1 μM RA or DMSO for 8 h. (C) The directly interaction of UBE3A and RARα. GST pull-down assays were performed with GST or GST-RARα and UBE3A-His. (D,E) Endogenous RARα (D) or Rarα (E) remained unchanged upon different dosages of UBE3A (D) in HEK-293T cells or Ube3a (E) protein levels in MEFs.