Biology subjects

Verhagen, R. F. M.

Publications and source records attributed to Verhagen, R. F. M..

2 recordsLinked to original sources

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

Inducible expression of human C9ORF72 36x G4C2 hexanucleotide repeats is sufficient to cause RAN translation and rapid muscular atrophy in mice

The hexanucleotide G4C2 repeat expansion in the first intron of the C9ORF72 gene explains the majority of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) cases. Numerous studies have indicated the toxicity of dipeptide repeats (DPRs) which are produced via repeat-associated non-AUG (RAN) translation from the repeat expansion and accumulate in the brain of C9FTD/ALS patients. Mouse models expressing the human C9ORF72 repeat and/or DPRs show variable pathological, functional and behavioral characteristics of FTD and ALS. Here, we report a new Tet-on inducible mouse model that expresses 36x pure G4C2 repeats with 100bp upstream and downstream human flanking regions. Brain specific expression causes the formation of sporadic sense DPRs aggregates upon 6 months dox induction but no apparent neurodegeneration. Expression in the rest of the body evokes abundant sense DPRs in multiple organs, leading to weight loss, neuromuscular junction disruption, myopathy and a locomotor phenotype within the time frame of four weeks. We did not observe any RNA foci or pTDP-43 pathology. Accumulation of DPRs and the myopathy phenotype could be prevented when 36x G4C2 repeat expression was stopped after 1 week. After 2 weeks of expression, the phenotype could not be reversed, even though DPR levels were reduced. In conclusion, expression of 36x pure G4C2 repeats including 100bp human flanking regions is sufficient for RAN translation of sense DPRs and evokes a functional locomotor phenotype. Our inducible mouse model highlights the importance of early diagnosis and treatment for C9FTD/ALS patients. Summary statementOnly 36 C9ORF72 repeats are sufficient for RAN translation in a new mouse model for ALS and FTD. Reducing toxic dipeptides can prevent but not reverse the phenotype.

neuroscience↗