Distel, B. | bioRxiv Science

bioRxiv2024

Localization of Human UBE3A Isoform 3 is Highly Sensitive to Amino Acid Substitutions at p.Met21 Position

UBE3A encodes three isoforms of Ubiquitin E3 ligase A, which differ in their N-terminal sequence, abundance, and localization. Recently, three individuals diagnosed with Angelman Syndrome have been described who carry a variant that abrogates the start codon of the predominant nuclear isoform 1 (hUBE3A-Iso1p.Met1Thr) and concomitantly results in a missense variant in isoform 3 (hUBE3A-Iso1p.Met21Thr), which we previously reported to be nuclear enriched as well. Here, we studied the effect of the p.Met21Thr variant on hUBE3A-Iso3 localization. Recombinant expression of hUBE3A-Iso3p.Met21Thr in U2-OS and mouse neurons revealed similar UBE3A labelling in the nucleus and cytosol, indicating hUBE3A-Iso3 localization is sensitive to amino acid changes at this position. This finding prompted us to revisit hUBE3A-Iso3 localization, since we previously introduced a p.Met21Ala/p.Met22Ala amino acid substitution in hUBE3A-Iso3 and its mouse orthologue mUBE3A-Iso2 to prevent translation of the shorter hUBE3A-Iso1 and mUBE3A-Iso3 nuclear isoforms. Introduction of silent mutations to disfavour translation of the short UBE3A isoforms enabled us to determine the localization hUBE3A-Iso3 and mUBE3A-Iso2 in the absence of amino acid changes at the p.Met21/p.Met22 position, respectively. Surprisingly, hUBE3A-Iso3 localization shifted from predominant nuclear localization for hUBE3A-Iso3p.Met21Ala to a predominant cytosolic localization of the Kozak optimized hUBE3A-Iso3KOZAK, while their mouse orthologues mUBE3A-Iso2p.Met22Ala and mUBE3A-Iso2KOZAK both localized predominantly to the cytosol. Taken together, these experiments indicate that the localization of human UBE3A-Iso3 is highly sensitive to amino acid substitutions at the p.Met21 position and that variants at this position not only abrogate the translation of hUBE3A-Iso1, but can also change the localization of hUBE3A-Iso3.

molecular biology↗

bioRxiv2020

Mono-ubiquitination of Rabphilin 3A by UBE3A serves a non-degradative function

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function.

neuroscience↗