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

Abstract

The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This mutation is shared with apes and Old-World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a single nucleotide in this non-coding exon, the exon became in frame with the remainder of the UBE3A protein. RNA-seq analysis of human brain samples showed that the human UBE3A isoforms arise by alternative splicing. Consistent with the predominant nuclear enrichment of UBE3A in human neurons, the two nuclear localized isoforms, hUBE3A-Iso1 and -Iso3, are the most abundantly expressed isoforms of UBE3A, while hUBE3A-Iso2 maintains a small pool of cytosolic UBE3A. Our findings provide new insight into UBE3A localization and evolution, and may have important implications for gene therapy approaches in Angelman Syndrome.

Evaluation of UBE3A antibodies in mice and human cerebral organoids

“…However, given the 100 million years of divergent evolution between rodents and primates, the patterns of UBE3A expression in humans may not be fully conserved. Recent studies support this notion where differences in the localization patterns and mechanisms of human and mouse isoforms were discovered 8 . This underscores the importance of investigating the subcellular distribution of UBE3A in human specific models.…”

“…3 B and S3 B). Previously it was shown that UBE3A localization is regulated by the N-terminal extension and the AZUL domain 8 , 13 . The hiPSC ASmut line was derived from a patient with a point mutation (F583S) on the N-terminal region of the N lobe of HECT domain, and previous reports indicate that this variant does not exhibit protein folding issues 20 .…”

“…Although basic local alignment search tool (BLAST) analysis did not yield another potential target, the comparison of the epitope sequence used to raise this antibody with the protein coding UBE3A isoform sequences showed that it does not have 100% sequence homology with the most common UBE3A variant (hUBE3A iso1 and mUbe3a iso3) (Figs. 1 A, B and S1 A) 8 , 9 . This may imply a reduced affinity for the majority of the UBE3A protein expressed in a cell.…”

“…In addition to its imprinted expression, one of the key molecular features of UBE3A is its distinct subcellular localization patterns, potentially regulated by its three isoforms 8 , 9 . Although it has a cytoplasmic component, UBE3A is predominantly nuclear in human and mouse neurons 10 – 13 ; however, its roles in these subcellular compartments in different cell types and developmental stages have not been fully uncovered.…”

Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations

“…Given that the loss of UBE3A affects synaptic function, most efforts have focused on its role in the synapse ( 10 , 11 ). However, there is also evidence for a role of UBE3A as a transcriptional co-activator ( 12 , 13 ), and we and others have recently reported that UBE3A staining is highly enriched in the nucleus of mouse and human neurons ( 6–8 , 14 , 15 ). The subcellular distribution of UBE3A in mouse and human is dictated by the expression of UBE3A isoforms that have distinct localizations.…”