Abundance and localization of human UBE3A protein isoforms

Abstract

Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman Syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.Angelman Syndrome (AS) is a rare neurodevelopmental disorder that affects 1 in 15,000 individuals, and is characterized by severe seizures, intellectual disability, absent speech, ataxia, and happy affect 1 . AS is caused by loss of function from the maternally-inherited copy of UBE3A. UBE3A is regulated by tissue-specific genomic imprintingit is expressed exclusively from the maternal allele in neurons and is expressed biallelically in other cell types 2,3 . UBE3A encodes an E3 ubiquitin ligase that forms polyubiquitin chains to substrates, targeting them for degradation by the 26S proteasome 4 . In humans, there are three known UBE3A protein isoforms 5 , all of which include the Homologous to E6AP Carboxy Terminus (HECT) domain and are thus capable of functioning as an E3 ligase. Human isoforms 2 and 3 only differ from human isoform 1 by 23 and 20 amino acids, respectively, at their N termini 6 (Fig 1a). While there has been little published research examining the human protein isoforms in human cells, human isoforms ectopically expressed in mouse indicate that the isoforms likely have differences in localization and function 7-9 . Furthermore, it is currently unknown whether the different human isoforms have different abundances or functions. This knowledge is important as some therapeutic avenues currently being explored for AS involve delivery and expression of exogenous UBE3A transgenes using vector-based delivery 10 . For these approaches, knowledge of which of the protein isoforms need to be replaced in AS is essential.Recently, Sadhwani and colleagues published three cases of AS in two different families caused by missense mutations at the isoform 1 translational start site 11 . Interestingly, these patients present with milder phenotypes than AS patients with 15q11-q13 deletions or other UBE3A loss of function mutations, including normal gait and use of syntactic speech. This data, coupled with the fact that the human and mouse isoforms are not entirely conserved, illustrates the need to study the human UBE3A protein isoforms specifically, as they may play a role in both normal neuronal function and in disease. Here, we have used human embryonic stem cells (h...

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

“…Considering the very good match of the transcriptome data (this study) and proteome data ( 9 , 26 ), the expression of distinct human UBE3A isoforms appears to be entirely governed by alternative splicing. But unlike human UBE3A isoforms, our data suggest that mouse UBE3A protein isoforms arise to a large extent by alternative translation.…”

“…In an elegant study published alongside our own, Chamberlain and co-workers successively deleted each of the three UBE3A isoforms from human iPS-derived neurons, in turn enabling the focused study of the remaining UBE3A proteins ( 26 ). Despite our different experimental approaches (i.e.…”

Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A

“…AS hiPSCs were developed in the Chamberlain and Lalande groups and obtained from Kerafast ( Chamberlain et al., 2010 ). UBE3A double-knockout H9 cells (H9 UBE3A m-/p- ) with a 66 kb deletion (chr15: 25,338,949–25,405,676) were provided by Dr. Stormy Chamberlain (UCONN) ( Sirois et al, 2020 ). Cells were maintained in tissue culture dishes (Fisher Scientific Corning Costar) coated with 0.5 mg/cm 2 vitronectin (VTN-N; Thermo Fisher Scientific) in E8 medium (Thermo Fisher Scientific) and passaged using standard protocols.…”

“…In addition to its imprinted expression, one of the salient molecular features of UBE3A is its nuclear localization in neurons, which also occurs perinatally and in the first couple postnatal weeks of murine neurodevelopment ( Burette et al., 2017 ; Judson et al., 2014 ). This localization may be regulated by shifts in the expression levels of UBE3A isoforms ( Sirois et al, 2020 ). It was recently shown that mice lacking a nuclear UBE3A isoform exhibited electrophysiological and behavioral deficits similar to those in other AS model mice ( Avagliano Trezza et al., 2019 ).…”

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases

“…In contrast to AS neurons, UBE3A-overexpressing neurons do not exhibit polarization problems [ 69 ]. Another report provides evidence that isoform 1 accounts for the majority of UBE3A in neurons [ 70 ]. Moreover, knockdown of alternatively spliced Ube3a1 devoid of its catalytic activity prompts robust dendritic growth, reduces the volume of spines and leads to decreased synaptic transmission.…”