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

Summary

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.

Abstract

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.

Introduction

Recent technological advances have made the sequencing of exomes and genomes routine in clinical settings^1,2. 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 interpretation^1,3.

A prominent example of this problem is Ube3a, a gene that encodes an E3 ....

Protocol

1. Mutagenesis cloning to generate  Ube3a variants

1. Clone all Ube3a variants into the pCIG2 plasmid (Figure 1A), a bicistronic vector that contains a chicken-β-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' SacI site and a 3' 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 ....

Results

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.......

Discussion

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 β-catenin activated reporter (BAR)²¹, a reporter that contains a concatemer of 12 T-cell factor (TCF) response elements separated by specifically.......

Materials

List of materials used in this article

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