Name: A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants (Video) | JoVE Journal
Uploaded: 2022-10-10T15:00:00.000+00:00
Duration: 6 min 35 s
Description: Learn a scalable cell-based method to assess functional impacts of Ube3a variants linked to Angelman syndrome and autism. This rapid assay enables researchers to differentiate gain and loss of function mutations efficiently.

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5 minutes

This work was supported by a Simons Foundation Bridge to Independence Award (SFARI Award #387972; J.J.Y.), a NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (J.J.Y.), a Research Fellowship from the Alfred P. Sloan Foundation (J.J.Y.), and research grants from the Angelman Syndrome Foundation (J.J.Y.), the Whitehall Foundation (J.J.Y.), and the NIMH (R01MH122786; J.J.Y.).

1. Mutagenesis cloning to generate&#160;&#160;Ube3a variants
<ol>
	<li>Clone all Ube3a variants into the pCIG2 plasmid (Figure 1A), a bicistronic vector that contains a chicken-&#946;-actin promoter and an internal ribosome entry site (IRES) for EGFP expression19. Full-length Ube3a constructs contain an N-terminal Myc-tag sequence and are cloned into pCIG2 using a 5&#39; SacI site and a 3&#39; XmaI site. In addition, naturally occurring EcoRI, EcoRV, and PstI sites within the Ube3a coding sequence are also used to subclone fragments. 
	NOTE: De-identified variants for Ube3a can be obtained through the literature and in publicly accessible repositories such as the ClinVar database1. Additional unreported variants can be obtained through personal communication with medical centers.</li>
	<li>Use an adapted two-step megaprimer mutagenesis method20 to introduce mutations into the Ube3a reading frame. In the first step, design the mutagenic oligonucleotide that contains the desired mutation (the example shown in this protocol is to generate a UBE3A Q588E variant) flanked by at least 30 base pairs (bp) of homology 5&#8242; and 3&#8242; to the mutation (Figure 1B and Table 1).</li>
	<li>Use the mutagenic oligonucleotide along with a complementary oligonucleotide for the first round PCR to generate a 200-400 bp megaprimer containing the mutation (Figure 1C; Table 2 and Table 3). Use the entire 50 &#956;L of the PCR reaction volume for agarose gel electrophoresis using a 1% gel and subsequent gel purification (Table of Materials). Elute the PCR product in 30 &#956;L of deionized H2O (diH2O) for use in the second round PCR step below.</li>
	<li>Set up the second round PCR as indicated (Figure 1C; Table 2 and Table 3). This step will generate larger DNA fragments (typically ~1-1.5 kb in length) containing the mutation and terminal restriction sites suitable for sub-cloning (Figure 1C).</li>
	<li>After completion of the PCR reaction, purify the resulting product using a PCR purification kit (Table of Materials). Elute in 30 &#956;L and digest all of the purified product and the pCIG2 Ube3a WT construct with the appropriate restriction enzymes (the example shows digestion with EcoRI and XmaI).</li>
	<li>After 2 h of digestion at 37 &#176;C, resolve the digestion products through agarose gel electrophoresis (1% gel), and gel-purify the appropriate products. Set up ligations according to the manufacturer&#39;s protocol (Table of Materials), transform the ligation products into a DH10B bacterial strain (Table of Materials), and then plate with ampicillin (100 &#956;g/mL) selection.</li>
	<li>The next day, pick two to four colonies to inoculate 3 mL cultures containing Luria Broth (LB) and 100 &#956;g/mL ampicillin. Let the cultures grow overnight at 37 &#176;C in an orbital incubator with shaking (225 rpm).</li>
	<li>The following day, use 1-1.5 mL of the bacterial culture to miniprep the plasmid DNA (Table of Materials). Save the remaining culture by placing them at 4 &#176;C. Screen the miniprepped plasmids by Sanger sequencing using an oligonucleotide that binds ~200 bp from the desired mutation.</li>
	<li>Use the bacterial cultures saved at 4 &#176;C to re-inoculate 50 mL cultures to midiprep the correct plasmid (Table of Materials). Fully sequence the Ube3a coding sequence to validate the correct sequence of the DNA.</li>
	<li>Create working stocks of Ube3a variant plasmids, as well as the &#946;-catenin activated reporter (BAR)21, TK-Renilla luciferase, ligase-dead Ube3a (UBE3A C820A mutation), and pCIG2 empty vector at a concentration of 100 ng/&#956;L using sterile H2O for the subsequent transfection steps.</li>
</ol>
2. Preparation and transfection of human embryonic kidney 293T (HEK293T) cells
<ol>
	<li>Perform the assays in HEK293T cells grown in Dulbecco&#39;s modified Eagle medium (DMEM) pre-supplemented with glutamine, 10% fetal bovine serum (FBS), and antibiotic-antimycotic agent as described previously17. Grow the cells in a sterile, humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Using a biosafety cabinet, first plate the suspended HEK293T cells onto tissue culture-treated 96-well flat-bottomed plates at a density of 2.2 &#215; 104 cells per well in 100 &#956;L of culture media and allow them to grow overnight.</li>
	<li>On the second day, transfect the cells in a biosafety cabinet with Firefly and Renilla luciferase reporter plasmids (Table 4) and Ube3a plasmids in triplicate. Perform the transfections in 10 &#956;L of total volume per well using a 10:1:8 ratio of plasmids (50 ng of BAR, 5 ng of TK-Renilla, and 40 ng of Ube3a plasmid per well, respectively), as previously described18, and 0.4 &#956;L of transfection reagent (Table 4). 
	NOTE: For each experiment, an empty vector (GFP only) and a plasmid encoding ligase-dead Ube3a are also transfected to serve as negative controls.</li>
	<li>Create a master mix for the transfections for each variant in a 1.5 mL microcentrifuge tube (Table 4) and incubate at room temperature (RT) for 15 min. After the incubation, gently agitate the transfection mixture by tapping the tube, and then add 10 &#956;L of the transfection mixture directly to the existing growth media in the wells.</li>
	<li>Afterward, return the cells to the incubator and allow the plasmids to express for 48 h. Replacement of the growth media is not necessary.</li>
	<li>Monitor the transfection efficiency by GFP fluorescence using a fluorescence microscope. HEK293T cells are efficiently transfected by this method, and &#62;80% of cells should express GFP after 48 h.</li>
</ol>
3. Measurement of luciferase activity
NOTE: Luciferase activity is assessed using a commercially available system that assays both Firefly and Renilla luciferase (Table of Materials) according to the manufacturer&#39;s protocol.
<ol>
	<li>Carefully aspirate the culture media from transfected HEK293T cells, and then wash the cells with cold phosphate-buffered saline (PBS). Aspirate the PBS and lyse the cells by adding 25 &#956;L of non-denaturing lysis buffer and incubate for 15 min with gentle rocking on ice.</li>
	<li>Afterward, add 20 &#956;L of the resulting lysate (no centrifugation is necessary) into a new 96-well assay plate containing 100 &#956;L of a Firefly luciferase substrate reagent. Mix the plate gently by tapping.</li>
	<li>Load the plate onto a plate reader and measure the luminescence using a top detector with a read height of 1 mm and an integration time of 1 s. 
	NOTE: The gain of the detector may need to be adjusted based on the strength of the signal.</li>
	<li>Immediately after reading the Firefly luciferase luminescence, add 100 &#181;L of Renilla luciferase substrate to the wells. This step simultaneously quenches the Firefly luciferase activity while assessing Renilla luciferase activity. Mix the samples by gentle agitation of the plate and measure the luminescence again to assess the Renilla luciferase activity. 
	NOTE: While other plates can be used for this assay, using plates with black frames and white wells will provide the most sensitive results as this amplifies the luciferase signal while minimizing noise.</li>
	<li>Quantify the reporter responses as the Firefly luciferase to Renilla luciferase ratio (Firefly/Renilla), and average the values of the triplicate measurements. Afterward, normalize the Firefly/Renilla value for each variant against the values for wild-type (WT) Ube3a expressing cells to compare the relative activity of variant proteins.</li>
</ol>

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<li>Albrecht, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imprinted+expression+of+the+murine+Angelman+syndrome+gene%2C+Ube3a%2C+in+hippocampal+and+Purkinje+neurons%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.</a> Nature Genetics. 17 (1), 75-78 (1997).</li>
<li>Rougeulle, C., Glatt, H., Lalande, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Angelman+syndrome+candidate+gene%2C+UBE3A%2FE6-AP%2C+is+imprinted+in+brain%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain.</a> Nature Genetics. 17 (1), 14-15 (1997).</li>
<li>Vu, T. H., Hoffman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imprinting+of+the+Angelman+syndrome+gene%2C+UBE3A%2C+is+restricted+to+brain%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain.</a> Nature Genetics. 17 (1), 12-13 (1997).</li>
<li>Kishino, T., Lalande, M., Wagstaff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UBE3A%2FE6-AP+mutations+cause+Angelman+syndrome%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">UBE3A/E6-AP mutations cause Angelman syndrome.</a> Nature Genetics. 15 (1), 70-73 (1997).</li>
<li>Jiang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutation+of+the+Angelman+ubiquitin+ligase+in+mice+causes+increased+cytoplasmic+p53+and+deficits+of+contextual+learning+and+long-term+potentiation%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.</a> Neuron. 21 (4), 799-811 (1998).</li>
<li>Mabb, A. M., Judson, M. C., Zylka, M. J., Philpot, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angelman+syndrome%3A+Insights+into+genomic+imprinting+and+neurodevelopmental+phenotypes%5BTitle%5D)+AND+%22Trends+in+Neuroscience%22%5BJournal%5D)">Angelman syndrome: Insights into genomic imprinting and neurodevelopmental phenotypes.</a> Trends in Neuroscience. 34 (6), 293-303 (2011).</li>
<li>Hogart, A., Wu, D., LaSalle, J. M., Schanen, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+comorbidity+of+autism+with+the+genomic+disorders+of+chromosome+15q11.2-q13%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13.</a> Neurobiology of Disease. 38 (2), 181-191 (2010).</li>
<li>Urraca, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interstitial+duplication+15q11.2-q13+syndrome+includes+autism%2C+mild+facial+anomalies+and+a+characteristic+EEG+signature%5BTitle%5D)+AND+%22Autism+Research%22%5BJournal%5D)">The interstitial duplication 15q11.2-q13 syndrome includes autism, mild facial anomalies and a characteristic EEG signature.</a> Autism Research. 6 (4), 268-279 (2013).</li>
<li>de la Torre-Ubieta, L., Won, H., Stein, J. L., Geschwind, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advancing+the+understanding+of+autism+disease+mechanisms+through+genetics%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Advancing the understanding of autism disease mechanisms through genetics.</a> Nature Medicine. 22 (4), 345-361 (2016).</li>
<li>Scheffner, M., Staub, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HECT+E3s+and+human+disease%5BTitle%5D)+AND+%22BMC+Biochemistry%22%5BJournal%5D)">HECT E3s and human disease.</a> BMC Biochemistry. 8, Suppl 1 (2007).</li>
<li>Cooper, E. M., Hudson, A. W., Amos, J., Wagstaff, J., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+analysis+of+Angelman+syndrome-associated+mutations+in+the+E3+ubiquitin+ligase+E6-associated+protein%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein.</a> Journal of Biological Chemistry. 279 (39), 41208-41217 (2004).</li>
<li>Yi, J. J., Barnes, A. P., Hand, R., Polleux, F., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta+signaling+specifies+axons+during+brain+development%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">TGF-beta signaling specifies axons during brain development.</a> Cell. 142 (1), 144-157 (2010).</li>
<li>Yi, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+autism-linked+UBE3A+T485A+mutant+E3+ubiquitin+ligase+activates+the+Wnt%2Fbeta-catenin+pathway+by+inhibiting+the+proteasome%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/beta-catenin pathway by inhibiting the proteasome.</a> Journal of Biological Chemistry. 292 (30), 12503-12515 (2017).</li>
<li>Weston, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+disease-linked+hyperactivating+mutations+in+UBE3A+through+large-scale+functional+variant+analysis%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis.</a> Nature Communications. 12 (1), 6809(2021).</li>
<li>Hand, R., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenin2+regulates+the+initial+axon+guidance+of+cortical+pyramidal+neurons+projecting+medially+to+the+corpus+callosum%5BTitle%5D)+AND+%22Neural+Development%22%5BJournal%5D)">Neurogenin2 regulates the initial axon guidance of cortical pyramidal neurons projecting medially to the corpus callosum.</a> Neural Development. 6, 30(2011).</li>
<li>Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., Hahn, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+allosteric+activation+of+kinases+in+living+cells%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Engineered allosteric activation of kinases in living cells.</a> Nature Biotechnology. 28 (7), 743-747 (2010).</li>
<li>Biechele, T. L., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assaying+beta-catenin%2FTCF+transcription+with+beta-catenin%2FTCF+transcription-based+reporter+constructs%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Assaying beta-catenin/TCF transcription with beta-catenin/TCF transcription-based reporter constructs.</a> Methods in Molecular Biology. , 99-110 (2008).</li>
<li>Yi, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Autism-linked+mutation+disables+phosphorylation+control+of+UBE3A%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">An Autism-linked mutation disables phosphorylation control of UBE3A.</a> Cell. 162 (4), 795-807 (2015).</li>
<li>Kuhnle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angelman+syndrome-associated+point+mutations+in+the+Zn%282%2B%29-binding+N-terminal+%28AZUL%29+domain+of+UBE3A+ubiquitin+ligase+inhibit+binding+to+the+proteasome%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Angelman syndrome-associated point mutations in the Zn(2+)-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome.</a> Journal of Biological Chemistry. 293 (47), 18387-18399 (2018).</li>
<li>Yamamoto, Y., Huibregtse, J. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+E6-AP+gene+%28UBE3A%29+encodes+three+potential+protein+isoforms+generated+by+differential+splicing%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing.</a> Genomics. 41 (2), 263-266 (1997).</li>
<li>Avagliano Trezza, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+nuclear+UBE3A+causes+electrophysiological+and+behavioral+deficits+in+mice+and+is+associated+with+Angelman+syndrome%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.</a> Nature Neuroscience. 22 (8), 1235-1247 (2019).</li>
<li>Bossuyt, S. N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+nuclear+UBE3A+activity+is+the+predominant+cause+of+Angelman+syndrome+in+individuals+carrying+UBE3A+missense+mutations%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations.</a> Human Molecular Genetics. 30 (6), 430-442 (2021).</li>
</ol>

The authors have nothing to disclose.

The protocol described here provides an efficient and scalable method to assess the enzymatic activity of Ube3a variants. There are several technical details that warrant careful consideration when using this assay. One consideration is the choice of Wnt reporter plasmids used in this assay. The protocol described here specifically uses the &#946;-catenin activated reporter (BAR)21, a reporter that contains a concatemer of 12 T-cell factor (TCF) response elements separated by specifically...

Recent technological advances have made the sequencing of exomes and genomes routine in clinical settings1,2. This has led to the discovery of a large number of genetic variants, including millions of missense variants that typically change one amino acid in a protein. Understanding the functional significance of these variants remains a challenge, and only a small fraction (~2%) of the known missense variants have a clinical interpretation1,3.
A prominent example of this problem is Ube3a, a gene that encodes an E3 ubiquitin ligase that targets substrate proteins for degradation4. Ube3a resides within chromosome 15q11-13 and is expressed exclusively from the maternally inherited allele5,6,7. Observations from disease genetics strongly suggest that insufficient or excessive activity of the UBE3A enzyme causes distinct neurodevelopmental disorders. Deletion of maternal chromosome 15q11-13 causes Angelman syndrome (AS)8, a disorder characterized by severe intellectual disability, motor impairments, seizures, a happy demeanor with frequent smiling, and dysmorphic facial features8,9,10. In contrast, duplication or triplication of maternal chromosome 15q11-13 causes Dup15q syndrome, a heterogeneous condition recognized as one of the most prevalent syndromic forms of autism11,12,13. In addition, there are hundreds of missense variants identified in Ube3a, the majority of which are considered variants of uncertain significance (VUS) as their functional and clinical significance are unknown. Thus, there is considerable interest in developing methods that can empirically assess Ube3a variants to determine whether they contribute to neurodevelopmental disease.
The UBE3A enzyme belongs to the HECT (homologous to E6-AP C-terminus) domain family of E3 ubiquitin ligases that all possess the eponymous HECT domain, which contains the biochemical machinery necessary to accept activated ubiquitin from E2 enzymes and transfer it to substrate proteins14. Historically, the characterization of E3 enzymes has relied on reconstituted in vitro systems that require an ensemble of purified proteins4,15,16. Such methods are slow and labor-intensive and not amenable to assessing the activity of a large number of variants. In previous work, UBE3A was identified to activate the Wnt pathway in HEK293T cells by modulating the function of the proteasome to slow the degradation of &#946;-catenin17. This insight allows the use of Wnt pathway reporters as an efficient and rapid method to identify both loss- and gain-of-function variants of Ube3a18. The protocol below describes in detail a method to generate Ube3a variants as well as a luciferase-based reporter to assess changes in the activity of Ube3a variants.

<table><tbody><tr><td>0.05% Trypsin-EDTA (1x), phenol red</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>1 Kb DNA ladder</td><td>Lambda Biotech</td><td>M108-S</td><td></td></tr><tr><td>100 bp DNA Ladder</td><td>Lambda Biotech</td><td>M107</td><td></td></tr><tr><td>10x Buffer for T4 DNA Ligase with 10 mM ATP</td><td>New England BioLabs</td><td>B0202A</td><td></td></tr><tr><td>5x Phusion HF Reaction Buffer</td><td>New England BioLabs</td><td>B0518S</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution</td><td>Corning</td><td>30004CI</td><td></td></tr><tr><td>Black/White Isoplate-96 Black Frame White Well plate</td><td>PerkinElmer</td><td>6005030</td><td></td></tr><tr><td>Carbenicillin Disodium Salt</td><td>Midwest Scientific</td><td>KCC46000-5</td><td></td></tr><tr><td>Countess cell counting chamber&amp;nbsp; slides</td><td>Invitrogen by Thermo Fisher Scientific</td><td>C10283</td><td></td></tr><tr><td>Countess II Automated Cell Counter&amp;nbsp;</td><td>life technologies</td><td></td><td>Cell counting machine</td></tr><tr><td>Custom DNA oligos</td><td>Integrated DNA Technologies (IDT)</td><td></td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix</td><td>New England BioLabs</td><td>N0447S</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td><td>Gibco</td><td>10569044</td><td>Basal medium for supporting the growth of HEK293T cell line</td></tr><tr><td>DPBS (1x)</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>EcoRI-HF&amp;nbsp;</td><td>New England BioLabs</td><td>R3101S</td><td>Restriction enzyme</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Gibco</td><td>16140071</td><td>Fetal bovine serum</td></tr><tr><td>Fisherbrand Surface Treated Tissue Culture Dishes</td><td>Fisherbrand</td><td>FB012924</td><td></td></tr><tr><td>FuGENE 6 Transfection Reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Gel Loading Dye Purple (6x)</td><td>New England BioLabs</td><td>B7024A</td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>High Efficiency ig 10B Chemically Competent Cells</td><td>Intact Genomics</td><td>1011-12</td><td>E. coli DH10B cells</td></tr><tr><td>HiSpeed Plasmid Midi Kit</td><td>Qiagen</td><td>12643</td><td>Midi prep</td></tr><tr><td>pCIG2 plasmid</td><td></td><td></td><td></td></tr><tr><td>pGL3 BAR plasmid</td><td></td><td></td><td></td></tr><tr><td>Phusion HF DNA Polymerase</td><td>New England BioLabs</td><td>M0530L</td><td>DNA polymerase</td></tr><tr><td>ProFlex 3 x 32 well PCR System</td><td>Applied biosystems by life technologies</td><td></td><td>Thermocycler</td></tr><tr><td>pTK Renilla plasmid</td><td></td><td></td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>Qiagen</td><td>27106</td><td>Mini prep</td></tr><tr><td>QIAquick Gel Extraction Kit (250)</td><td>Qiagen</td><td>28706</td><td>Gel purification</td></tr><tr><td>QIAquick PCR Purification Kit (250)</td><td>Qiagen</td><td>28106</td><td>PCR purification</td></tr><tr><td>rCutSmart Buffer</td><td>New England BioLabs</td><td>B6004S</td><td></td></tr><tr><td>SacI-HF</td><td>New England BioLabs</td><td>R3156S</td><td>Restriction enzyme</td></tr><tr><td>Synergy HTX Multi-Mode Reader</td><td>BioTek</td><td></td><td>&amp;nbsp;Plate reader runs Gen5 software v3.08 (BioTek)</td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202L</td><td>Ligase</td></tr><tr><td>TAE Buffer, Tris-Acetate-EDTA, 50x Solution, Electrophoresis</td><td>Fisher Scientific</td><td>BP13324</td><td></td></tr><tr><td>Tissue Culture Plate 96 wells, Flat Bottom</td><td>Fisherbrand</td><td>FB012931</td><td></td></tr><tr><td>UltraPure Ethidium Bromide Solution</td><td>Invitrogen by Thermo Fisher Scientific</td><td>15585011</td><td></td></tr><tr><td>XmaI</td><td>New England BioLabs</td><td>R0180S</td><td>Restriction enzyme</td></tr></tbody></table>

a scalable, cell-based method for the functional assessment of ube3a variants

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

Large-scale functional screening of Ube3a missense variants identifies a broad landscape of loss- and gain-of-function mutations 
Previous work with Ube3a mutants suggested that the Wnt response can serve as a reporter of cellular UBE3A protein activity. These observations were expanded, and additional validation experiments were performed to investigate whether the BAR assay is suitable to report a range of UBE3A activities in the cell. First, HEK293T cells were transfected with var...

A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

a-scalable-cell-based-method-for-functional-assessment-ube3a

Research

JoVE Journal

Neuroscience

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 µm) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Learn a scalable cell-based method to assess functional impacts of Ube3a variants linked to Angelman syndrome and autism. This rapid assay enables researchers to differentiate gain and loss of function mutations efficiently.

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

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