Oweis, W. | bioRxiv Science

bioRxiv2020

Pluripotent stem cell derived models of neurological diseases reveal early transcriptional heterogeneity

BackgroundMany neurodegenerative diseases (NDs) develop only later in life, when cells in the nervous system lose their structure or function. In genetic forms of NDs, this late onset phenomenon remains largely unexplained. ResultsAnalyzing single cell RNA sequencing (scRNA-seq) from Alzheimers disease (AD) patients, we find increased transcriptional heterogeneity in AD excitatory neurons. We hypothesized that transcriptional heterogeneity precedes ND pathologies. To test this idea experimentally, we used juvenile forms (72Q; 180Q) of Huntingtons disease (HD) iPSCs, differentiated them into committed neuronal progenitors, and obtained single cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking-down several differentially-variable genes resulted in increased aggregate formation, a pathology associated with HD. We further validated the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder. ConclusionsOverall, our results suggest that although NDs develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.

molecular biology↗

bioRxiv2017

Conformational locking of Ufm1 upon binding to the Ufm1-interacting sequence of Uba5

Ubiquitin fold modifier 1 (Ufm1) is a ubiquitin-like protein (UBL) found in eukaryotic organisms which plays a crucial role in ER stress management and signal transduction. The crystal structure of UFM1 and its E1 (Uba5) in complex shows that Ufm1 binds to the adenylation domain of UBA5 and interacts with a separate Ufm1-interacting sequence (UIS) in the C-terminus of UBA5. The UIS interacts with Ufm1 on the opposite side of Ufm1 protein from the adenylation domain of Uba5 and the reason for this second interaction site is unclear. We analyzed Ufm1 bound to the UIS sequence through molecular dynamics simulations in order to identify additional functions for this interaction. We found that the residues in the adenylation interaction site of Ufm1 have less movement when the UIS peptide was bound to Ufm1 and formed a structure that aligns well with Ufm1 bound to the Uba5 adenylation domain. We further identified an amino acid that connects the UIS to the adenylation domain interacting site. Mutation of this amino acid decreases charging activity and shifts the Ufm1 conformation population toward the unlocked configuration even in the presence of the UIS peptide. These data suggest a role for the Uba5 UIS in stimulating activation of Ufm1.

biochemistry↗