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A coding-independent function of an alternative Ube3a transcript during neuronal development

Nature Neuroscience volume 18, pages 666–673 (2015)Cite this article

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Abstract

The E3 ubiquitin ligase Ube3a is an important regulator of activity-dependent synapse development and plasticity. Ube3a mutations cause Angelman syndrome and have been associated with autism spectrum disorders (ASD). However, the biological significance of alternative Ube3a transcripts generated in mammalian neurons remains unknown. We report here that Ube3a1 RNA, a transcript that encodes a truncated Ube3a protein lacking catalytic activity, prevents exuberant dendrite growth and promotes spine maturation in rat hippocampal neurons. Surprisingly, Ube3a1 RNA function was independent of its coding sequence but instead required a unique 3′ untranslated region and an intact microRNA pathway. Ube3a1 RNA knockdown increased activity of the plasticity-regulating miR-134, suggesting that Ube3a1 RNA acts as a dendritic competing endogenous RNA. Accordingly, the dendrite-growth-promoting effect of Ube3a1 RNA knockdown in vivo is abolished in mice lacking miR-134. Taken together, our results define a noncoding function of an alternative Ube3a transcript in dendritic protein synthesis, with potential implications for Angelman syndrome and ASD.

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Figure 1: Ube3a1 RNA is an activity-dependent, dendritic transcript in primary hippocampal neurons.
Figure 2: Ube3a1 RNA knockdown increases dendrite complexity and reduces dendritic spine volume and mEPSC amplitudes.
Figure 3: Ube3a1 function in dendritogenesis is coding-independent.
Figure 4: Ube3a1 function in dendritogenesis requires an intact microRNA pathway.
Figure 5: Ube3a1 RNA is a competing endogenous RNA for the miR379–410 cluster.
Figure 6: Ube3a1 function in vivo depends on miR379–410 miRNAs.

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Acknowledgements

We acknowledge technical assistance of U. Beck, E. Becker, R. Gondrum, G. Jarosch, H. Kaiser and H. Rippberger. This work was funded by grants from the European Research Council (Starting Grant “Neuromir”), the European Union FP7 (“EpimiRNA”), the Deutsche Forschungsgemeinschaft (DFG) (SFB593, FOR2107: SCHR 1136/3-1) and the Universitätsklinikum Gießen-Marburg to G.S., the DFG (FOR2107) to R.S. (SCHW 559/14-1) and M.W. (WO 1732/4-1) and the Von Behring-Röntgen-Foundation (62-0004) to S.B.

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  1. Tatjana Wüst

    Present address: Present address: Sanquin Blood Supply, Amsterdam, the Netherlands.,

Authors and Affiliations

  1. Institute of Physiological Chemistry, Biochemical-Pharmacological Center Marburg, Philipps University Marburg, Marburg, Germany

    Jeremy Valluy, Silvia Bicker, Ayla Aksoy-Aksel, Martin Lackinger, Simon Sumer, Roberto Fiore & Gerhard Schratt

  2. Interdisciplinary Center for Neurosciences, SFB488 Junior Group, University Heidelberg, Heidelberg, Germany

    Tatjana Wüst

  3. Behavioral Neuroscience, Experimental and Biological Psychology, Philipps-University Marburg, Marburg, Germany

    Dominik Seffer, Markus Wöhr & Rainer Schwarting

  4. Max Planck Institute for Biology of Ageing, Computational RNA Biology Lab, Cologne, Germany

    Franziska Metge & Christoph Dieterich

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Contributions

J.V. performed most experiments (dendritogenesis, luciferase assays, western blots, rAAV injections, strand-specific qPCR) and analyzed the data. G.S. designed the study, supervised the project and wrote the manuscript. S.B. performed FISH, compartmentalized culture and synaptosome assays. A.A.-A. performed and analyzed patch-clamp recordings. M.L. and R.F. established and characterized the miR379–410 knockout colony. S.S. cloned and validated the Drosha shRNA construct. T.W. performed dendritogenesis assays and generated Ube3a constructs. R.S. and M.W. designed and supervised the rat behavioral studies. D.S. performed juvenile social isolation studies in rats. F.M. and C.D. analyzed deep sequencing data.

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Integrated supplementary information

Supplementary Figure 1 Ube3a1 RNA expression analysis by RNA sequencing.

a) Genomic view (UCSC genome browser) of the murine Ube3a locus including alternative Ube3a transcripts. The region surrounding the Ube3a1 3’UTR is boxed. Note the presence of sequence reads in the aggregate RNA-seq exon coverage panel at the genomic location of the Ube3a1 3’UTR. b) Representation of sequence reads at the location of the Ube3a1 3’UTR (boxed region in a) from rat hippocampal neurons according to ribo-minus RNA sequencing. The position of Ube3a exons 9-11, intron 11 and the UTR1 is indicated. c) Strand-specific qPCR analysis of 3’UTR1 or intron11 containing transcripts in either antisense (as) or sense orientation.

Supplementary Figure 2 Developmental expression of Ube3a1 RNA.

qPCR analysis of Ube3a1 RNA (a), Limk1 mRNA (b) Ube3a2/3 mRNA (c) expression from developing primary rat hippocampal neurons. Values are presented relative to expression at 3 DIV. N=3-5. *p<0.05, **p<0.01, ***p<0.001 (T-test).

Supplementary Figure 3 Subcellular expression of Ube3a1 RNA

a) Representative immunofluorescence analysis of cell body (upper panel) or process (lower panel) compartments from standard (left panel) or FUDR-treated (right panel) compartmentalized hippocampal neuron cultures, stained with anti-GFAP (green), anti-MAP2 (red) or Hoechst (blue). Scale bar = 50 μm. b) qPCR analysis of Ube3a1 and Ube3a2/3 with RNA from cell bodies and processes of compartmentalized hippocampal neuron cultures. Bar graphs represent the average ratio of Ube3a1 to Ube3a2/3 RNA levels from three independent preparations ± SD. c) qPCR analysis of indicated RNAs in rat P15 forebrain synaptosomes. Values are presented relative to a whole forebrain control sample. d) Conventional RT-PCR analysis of P15 whole rat forebrain (WB) and synaptosomes (SY) with primers directed against the indicated transcripts.

Supplementary Figure 4 Ube3a protein expression and knockdown validation.

a) Different fractions from a rat P15 forebrain synaptosome preparation were used for Western blotting with a monoclonal mouse anti-Ube3a antibody (Becton Dickinson) which recognizes aa. 501-712 of mouse Ube3a. Marker lane indicates 100 kD. Sup: supernatant; Pel: pellet; Syn: synaptosomes. b) HEK293 cells were transfected with the indicated constructs and analyzed for the expression of GFP-Ube3a-fusion proteins by Western blotting using the anti-Ube3a antibody described in a). c) Primary rat hippocampal neurons (13-18 DIV) were co-transfected with GFP-Ube3a-fusion constructs and dsRed and analyzed by confocal microscopy. Scale bar = 50 μm. d) HEK293 cells were co-transfected with GFP-Ube3a-fusion and indicated shRNA constructs. Expression was assessed by Western blotting using an anti-GFP antibody and an anti-Actin antibody as a loading control. e) Primary rat hippocampal neurons (10-18 DIV) were infected with rAAV expressing the indicated shRNA constructs. Expression of endogenous Ube3a protein was measured by Western blotting using an anti-Ube3a antibody that recognizes the common Ube3a N-terminus. anti-ß-actin Western served as a loading control. f) qRT-PCR analysis of indicated RNAs from hippocampal neurons infected with rAAV-Ube3a1-shRNA or rAAV-control-shRNA. Values were normalized to Gapdh expression and are presented as the ratio of Ube3a1 vs. control shRNA infected neurons ± SD. N=4 (ANOVA p=0.015323; *p<0.05 (post-hoc T-test).

Supplementary Figure 5 Regulation of dendritogenesis by GFP-Ube3a1 and GFP-Ube3a2.

a) qPCR analysis of GFP RNA expression in primary cortical neurons that had been nucleofected with the indicated GFP-Ube3a-fusion constructs. Values are presented as 40-ct and are representative for multiple independent experiments. b) Representative images of primary hippocampal neurons (DIV 13 - 18) transfected with the indicated shRNA and Ube3a1 expression constructs. c) Representative images of primary hippocampal neurons (DIV 7 - 10) transfected with the indicated expression vectors and treated with BDNF (40 ng/ml) for 48 hours prior to fixation. Scale bar = 50 μm. d) Quantification of dendrite complexity in neurons transfected with the indicated GFP expression plasmids. Values are presented relative to GFP-only transfected neurons. n=3.

Supplementary Figure 6 The Ube3a1 coding sequence (cds) is not involved in dendritogenesis.

a) anti-GFP Western blot with lysates from HEK293 cells transfected with the indicated GFP-Ube3a-fusion constructs. b) Quantification of dendrite complexity in neurons transfected with the indicated shRNA and Ube3a1 expression constructs. Values are presented relative to control shRNA transfected neurons. N=3. n.s.: not significant.

Supplementary Figure 7 Full-length blots related to Figure 4a.

a) GFP-Drosha. b) GFP. c) beta-Actin. Boxes indicate regions of the blot presented in Fig. 4a. Additional band in a) represents overexposed GFP signal.

Supplementary Figure 8 Ube3a1 ceRNA function.

a) Schematic representation of rat 3’UTRs for known miR-134 target mRNAs. Putative binding sites predicted by TargetScan (www.targetscan.org) for miR-134, miR-485 and miR-758 are marked by colored circles. The total number of additional miR379-410 seed matches is indicated. UTRs are not drawn to scale. b) Luciferase assay in primary hippocampal neurons co-transfected with Creb1-luc reporters and indicated shRNAs. Values are relative to reporter expression under basal conditions. N=7 (Creb1-luc). N=4 (Creb1-m134-luc). **p<0.01 (T-test).

Supplementary Figure 9 Quantitative PCR of miR-134 target mRNAs in neuronal cell bodies and dendrites.

a-c) Standard curves with indicated amounts of plasmids containing Pum2 (a), Limk1 (b) and Ube3a1 (c) sequence. The slope of a regression curve based on measured Ct-values is indicated in each diagram. d) Calculated RNA molecules per ng total RNA isolated from either the cell body or dendrite compartment of FUDR-treated hippocampal neurons (DIV 18) cultured on filter insets. Results from three independent experiments ± SD are shown.

Supplementary Figure 10 Full-length blots related to Figure 5f.

a) Limk1. b) Creb1. c) Pum2. d) Tubulin. Boxes indicate regions of blot presented in Fig. 5f. Membranes were cut after blotting to simultaneously probe for proteins running at different molecular weights.

Supplementary Figure 11 Genotyping and validation of miR379-410−/− mice.

a) Genotyping PCR of miR379/410−/− (KO) and wild-type (WT) mice using the indicated primer pairs. b) qPCR analysis of pre-miR-134 expression in wild-type (wt; n=7) and miR379/410−/− mice (ko; n=7). **p<0.01.

Supplementary Figure 12 Normal cortical layering in miR379-410−/– (ko) mice.

Coronal brain sections (80 mm, single hemisphere) of 8-weeks old wildtype (a), miR379-410+/− (b) or miR379-410−/− (ko; c, d) mice stained with nuclear Hoechst dye. The positions of cortical layers I-VI are indicated in the higher magnification images (right panels). No differences were observed in the general organization of the cerebral cortex or hippocampus.

Supplementary Figure 13 Model for the ceRNA function of Ube3a1 during dendritic development.

During normal activity-dependent neuronal development, Ube3a1 RNA levels steadily increase, resulting in the sequestration of dendritic miRNAs from the miR379-410 cluster, including miR-134 and miR-485. This releases protein-coding targets of these miRNAs, such as Limk1 and Pum2 mRNA, from translational repression, thereby leading to enhanced local synthesis of Limk1 and Pum2, which in turn promotes spine growth and inhibits dendrite elaboration, respectively. In conditions of Ube3a1 RNA deficiency, spine growth is suppressed whereas the brake on dendrite elaboration is loosened.

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Valluy, J., Bicker, S., Aksoy-Aksel, A. et al. A coding-independent function of an alternative Ube3a transcript during neuronal development. Nat Neurosci 18, 666–673 (2015). https://doi.org/10.1038/nn.3996

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