The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption

<i>Drosophila S6 Kinase Like</i> Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

<div><p>Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In <i>Drosophila melanogaster</i>, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. <i>S6KL</i> null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of <i>tkv</i> in <i>S6KL</i> mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of <i>S6KL</i> mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv.</p></div

Genetic interactions between <i>S6KL</i> and components of the BMP signaling pathway in regulating NMJ growth.

<p>(A–H) Confocal images of NMJ 4 synapses of different genotypes co-stained with anti-HRP (green) and anti-CSP (magenta): WT (A), <i>S6KL</i>^<i>140</i> (B), <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/+</i> (C), <i>tkv</i>^<i>7</i><i>/tkv</i>^{<i>k16713</i>} (D), <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/tkv</i>^{<i>k16713</i>} (E), <i>S6KL</i>^<i>140</i><i>/+</i> (F), <i>dad</i>^<i>J1E4</i><i>/+</i> (G), and <i>S6KL</i>^<i>140</i><i>/+; dad</i>^<i>J1E4</i><i>/+</i> (H). Scale bar, 5 μm. (I–L) Confocal images of NMJ 4 terminals co-labeled with anti-pMad (green) and anti-HRP (magenta) in WT (I), <i>tkv</i>^<i>7</i><i>/+</i> (J), <i>S6KL</i>^<i>140</i> (K), and <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/+</i> (L) larvae. (M) Quantifications of synaptic bouton numbers in different genotypes including wild type (n = 17), <i>S6KL</i>^<i>140</i> (n = 19), <i>tkv</i>^<i>7</i><i>/tkv</i>^{<i>k16713</i>} (n = 17), <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/tkv</i>^{<i>k16713</i>} (n = 10), <i>tkv</i>^<i>7</i><i>/+</i> (n = 16), <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/+</i> (n = 19), <i>S6KL</i>^<i>140</i><i>/+</i> (n = 19), <i>dad</i>^<i>J1E4</i><i>/+</i> (n = 10), and <i>S6KL</i>^<i>140</i><i>/+; dad</i>^<i>J1E4</i><i>/+</i> (n = 22). (N) Quantification of the relative fluorescence intensities of pMad in NMJ terminals of different genotypes. n = 8, 9, 8, and 8 for wild type, <i>tkv</i>^<i>7</i><i>/+</i>, <i>S6KL</i>^<i>140</i>, and <i>S6KL</i>^<i>140</i><i>; tkv</i>^<i>7</i><i>/+</i>, respectively. **<i>p</i><0.01 by one-way ANOVA with Tukey post hoc test; error bars indicate SEM. (O) Western results of larval brains from wild-type control and <i>S6KL</i>^<i>140</i> mutants. Actin was used as a loading control. (P) Quantification of the relative protein levels of pMad and Mad in the larval brains of wild type and <i>S6KL</i>^<i>140</i> mutants. The level of pMad but not Mad was increased in <i>S6KL</i> mutants. <i>n</i> = 3, **<i>p</i><0.01 by Student’s <i>t</i>-tests; error bars indicate SEM.</p

Overgrown NMJs in <i>S6KL</i> mutants.

<p>(A–F) Representative NMJ 4 synapses from different genotypes double-stained with anti-HRP recognizing neuronal plasma membrane (green) and an antibody against CSP (magenta), a synaptic vesicle protein. Insets show higher-magnification images of terminal boutons. The genotypes are: (A) Wild type, (B) homozygous <i>S6KL</i>^<i>140</i> mutants, (C) hemizygous <i>S6KL</i>^<i>140</i><i>/Df(1)ED741</i> mutants, (D) neuronal rescue of <i>S6KL</i>^<i>140</i> by overexpression of S6KL under the control of <i>elav-Gal4</i> (<i>S6KL</i>^<i>140</i><i>elav-Gal4/S6KL</i>^<i>140</i><i>; UAS-S6KL/+</i>), (E) Muscular rescue of <i>S6KL</i>^<i>140</i> by overexpression of S6KL under the control of <i>Mhc-Gal4</i> (<i>S6KL</i>^<i>140</i>; <i>UAS-S6KL/Mhc-Gal4</i>), and (F) Neuronal rescue of <i>S6KL</i>^<i>140</i> by overexpression of the kinase dead S6KL (S6KL^K193Q) under the control of <i>elav-Gal4</i> (<i>S6KL</i>^<i>140</i><i>elav-Gal4/ S6KL</i>^<i>140</i><i>; UAS-S6KL</i>^<i>K193Q</i><i>/+</i>). Satellite boutons are indicated by arrows in B. Insets D and F show Western results of larvae brain extracts using anti-S6KL and anti-actin antibodies. Scale bar, 5 μm. (G, H) Statistical results of the number of total boutons (G) and satellite boutons (H) in different genotypes. <i>n</i> = 17, 19, 15, 16, 19 and 18 NMJs for wild type, <i>S6KL</i>^<i>140</i>, <i>S6KL</i>^<i>140</i><i>/Df(1)ED7413</i>, <i>S6KL</i>^<i>140</i><i>elav-Gal4/S6KL</i>^<i>140</i><i>; UAS-S6KL/+</i>, <i>S6KL</i>^<i>140</i>; <i>UAS-S6KL/Mhc-Gal4</i>, and <i>S6KL</i>^<i>140</i><i>elav-Gal4/S6KL</i>^<i>140</i><i>; UAS-S6KL</i>^<i>K193Q</i><i>/+</i>, respectively. ***<i>p</i><0.001 by one-way ANOVA with Tukey post hoc test; error bars indicate SEM.</p

S6KL functions presynaptically in regulating NMJ growth.

<p>(A–E) Representative NMJ4 synapses from different genotypes doubly stained with anti-HRP (green) and anti-CSP (magenta). (A) wild type, (B) <i>elav-Gal4/+; UAS-S6KL/+</i>, (C) <i>C57-Gal4/UAS-S6KL</i>, (D) <i>elav-Gal4/+; S6KL RNAi/+</i>, and (E) <i>S6KL RNAi/+; C57-Gal4/+</i>. Scale bar, 5 μm. (F, G) Statistical results of the number of boutons (F) and satellite boutons (G) in different genotypes. <i>n</i> >16 for each genotype; ***<i>p</i> < 0.001 by one-way ANOVA with Tukey post hoc test; error bars indicate SEM.</p

Fewer but larger synaptic vesicles at the active zone of <i>S6KL</i> mutant boutons.

<p>(A–D) Ultrastructure of wild type (A and C) and <i>S6KL</i>^<i>140</i> mutant (B and D) NMJ boutons. (A and B) Transmission electron micrographs of synaptic boutons. Arrows indicate active zones with T bars; SSR, sub-synaptic reticulum. Scale bar, 500 nm. (C and D) Higher-magnification images of active zones. Fewer and larger synaptic vesicles are present in <i>S6KL</i>^<i>140</i> mutant active zones compared with wild type. Large synaptic vesicles are indicated by asterisks. Scale bar, 200 nm. (E–G) Quantification of the ultrastructural features: synaptic vesicle density in the whole presynaptic bouton area (E), the number (F) and diameter (G) of synaptic vesicles in a 200 nm radius around the T-bar. For the analysis of SV density, <i>n</i> = 73 and 46 boutons for wild type and <i>S6KL</i>^<i>140</i>, respectively. For the analysis of SV in a 200 nm radius around T-bar, <i>n</i> = 40 and 34 boutons for wild type and <i>S6KL</i>^<i>140</i>, respectively. ***<i>p</i><0.001 by Student’s <i>t</i>-test; error bars indicate SEM. (H) Cumulative histograms of synaptic vesicle diameter indicating larger vesicles in <i>S6KL</i>^<i>140</i> mutants.</p

Negative regulation of Tkv protein level by S6KL via proteasomal degradation pathway.

<p>(A–B′) Confocal images of NMJ 4 branches double-labeled with anti-GFP (green) and anti-HRP (magenta) in control (<i>elav-Gal4/+; UAS-Tkv-GFP/+</i>) and mutants (<i>S6KL</i>^<i>140</i><i>elav-Gal4/S6KL</i>^<i>140</i><i>; UAS-Tkv-GFP/+</i>) showing elevated and punctate Tkv staining signals in <i>S6KL</i>^<i>140</i> mutants. Scale bar, 5 μm. (C) Western results of total larval brain extracts probed with anti-GFP and anti-Wit antibodies. Actin was used as a loading control. (D) Tkv protein level was increased in S2 cells expressing a reduced level of S6KL. S2 cells were transfected with expression vector for Tkv-GFP and dsRNAs targeting two different sequences of the <i>S6KL</i> transcript. (E) Overexpression of S6KL suppresses Tkv protein level. S2 cells co-transfected with plasmids encoding Flag-S6KL and Tkv-GFP were untreated or treated with the proteasome inhibitor MG132 and subjected to western analysis with different antibodies. (F) Wild-type and kinase dead K193Q mutant (S6KL^KD) S6KL interact with Tkv in S2 cells. S2 cells were co-transfected with expression vectors for Myc-Tkv or Myc-GFP and Flag-S6KL or Flag-S6KL^KD. Cell lysates were subjected to immunoprecipitation with anti-IgG or anti-Flag and subsequently analyzed by western analysis. (G) Poly-ubiquitinated Tkv is decreased in S6KL—knockdown S2 cells. S2 cells were co-transfected with expression vectors for Myc-Tkv, HA-ubiquitin, and dsRNAs targeting two different sequences of the <i>S6KL</i> transcript. Asterisk denotes IgG. (H) S6KL and Tkv co-localize in the soma of motor neurons in the ventral nerve cord. Tkv-GFP alone (upper panels) or Tkv-GFP and S6KL (lower panels) were expressed under the control of the motoneuron specific <i>OK6-Gal4</i>. Scale bar, 5 μm.</p

Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in <i>Drosophila</i>

<div><p>Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the <i>in vivo</i> function of UBE3A in the nervous system, we generated multiple mutations of <i>ube3a</i>, the <i>Drosophila</i> ortholog of <i>UBE3A</i>. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of <i>ube3a</i> mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of <i>ube3a</i> mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in <i>ube3a</i> mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.</p></div

Synaptic endocytosis is impaired in <i>ube3a</i> mutants.

<p>(A–C) Statistical results of EJP amplitudes (A), mEJP amplitudes (B), and mEJP frequencies (C) of wild type and <i>ube3a</i>^<i>35</i> mutants. (D) Relative EJP amplitudes (%) during high-frequency stimulation (10 Hz) for 10 min in wild type and <i>ube3a</i>^<i>35</i> mutants. <i>n</i> ≥ 11 larvae; error bars indicate SEM. (E) Loss of <i>ube3a</i> causes synaptic endocytic defects. FM1-43 dye uptake in wild type, <i>ube3a</i>^<i>35</i> mutants, and rescued animals (<i>gube3a</i>^<i>+</i><i>/+; ube3a</i>^<i>35</i>). Scale bar: 10 μm. (F) Normalized fluorescence intensity (%) of endocytosed FM1-43 for wild type, <i>ube3a</i>^<i>35</i> and rescued mutants (<i>gube3a</i>^<i>+</i><i>/+; ube3a</i>^<i>35</i>). <i>n</i> ≥ 20 NMJs, error bars indicate SEM, *** indicates <i>p</i> < 0.001.</p

<i>ube3a</i> interacts genetically with BMP signaling pathway components <i>tkv</i> and <i>mad</i> in regulating NMJ growth.

<p>(A) Confocal images of muscle 4 NMJ synapses co-stained with anti-CSP (magenta) and anti-HRP (green) to reveal synaptic vesicles and presynaptic membrane, respectively. In <i>ube3a tkv</i> double mutants, bouton numbers were similar to those of <i>tkv</i>^<i>8</i><i>/tkv</i>^{<i>K16713</i>} single mutants. One copy of <i>tkv</i>^<i>8</i> rescued the synapse overgrowth in <i>ube3a</i>^<i>35</i> mutants. Scale bar = 10 μm. (B, C) Quantification of total bouton number (B) and satellite bouton number (C) of various genotypes including wild type, <i>ube3a</i>^<i>35</i>, <i>tkv</i>^<i>8</i><i>/tkv</i>^{<i>K16713</i>}, <i>tkv</i>^<i>8</i><i>/ tkv</i>^{<i>K16713</i>}; <i>ube3a</i>^<i>35</i>, <i>tkv</i>^<i>8</i><i>/+</i>, <i>tkv</i>^<i>8</i><i>/+; ube3a</i>^<i>35</i>, <i>mad</i>^<i>12</i><i>/+</i>, <i>mad</i>^<i>12</i><i>/+; ube3a</i>^<i>35</i>. <i>n</i> ≥ 18 NMJ terminals, one-way ANOVA test, ***<i>P</i> < 0.001, ns, not significant,. Error bars represent SEM.</p