bioRxiv ScienceSearch

bioRxiv · 10.1101/535443

Saccharomyces cerevisiae strains display robust phenotypes in the presence of Dyskeratosis congenita mutations in the Cbf5 gene

Abstract

Dyskeratosis congenita is a rare, congenital disorder affecting the skin, nails and oral mucosa of patients that often progresses to bone marrow failure and an increased predisposition for a variety of carcinomas. Mutations in the human dyskerin gene have been identified as the most prevalent cause of the disease. Dyskerin is a pseudouridine synthase and the catalytic subunit of H/ACA ribonucleoproteins (RNPs) responsible for the modification of uridines to pseudouridine in ribosomal RNA (rRNA), but dyskerin also binds to the telomerase RNA component (TERC). Accordingly, Dyskeratosis congenita mutations have been reported to affect both telomerase function as well as ribosome biogenesis, but the relative contribution of each pathway to the diseases is under debate. As the yeast homolog of dyskerin, Cbf5, does not interact with telomerase RNA, Saccharomyces cerevisiae is an ideal model to identify the selective impact of Dyskeratosis congenita mutations on ribosome biogenesis. Therefore, chromosomal mutations in the yeast homologue of dyskerin, Cbf5, were introduced at positions corresponding to the mutations in human dyskerin that result in Dyskeratosis congenita. To determine if the mutations affect cellular fitness, we screened for growth defects in yeast. Growth curves at different temperatures and yeast spot assays under several stress conditions revealed that the mutations in cbf5 did not impair growth compared to wild type. These findings suggest that in the yeast cell, Dyskeratosis congenita mutations do not significantly affect ribosome biogenesis, and we discuss the implications for understanding the molecular cause of Dyskeratosis congenita.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kothe, U., Ogailan, A. A., Rintala-Dempsey, A. C.. 2019-01-30. Saccharomyces cerevisiae strains display robust phenotypes in the presence of Dyskeratosis congenita mutations in the Cbf5 gene. https://doi.org/10.1101/535443

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology

Impaired proteostasis is an early feature of the diabetic heart in humans and mice

Diabetes and obesity increase cardiac lipid levels leading to cardiomyopathy and heart failure. We hypothesized that intermittent fasting would reduce cardiac lipid levels. Surprisingly, intermittent fasting increased myocardial triglyceride content, but rescued mortality and attenuated cardiomyopathy in mice overexpressing cardiomyocyte acyl-CoA synthetase 1 (MHC-ACSL1). Lipid overload caused cardiomyocyte accumulation of polyubiquitinated protein aggregates containing desmin, a scaffolding intermediate filament protein, which intermittent fasting prevented. Furthermore, intermittent fasting reversed elevated myocardial C16:0 ceramide content, and knockdown of ceramide synthase CerS5 and CerS6 reduced palmitate-induced protein aggregation, highlighting a role for C16:0 ceramides in this pathology. Conversely, impairing aggrephagy with cardiomyocyte-specific p62 ablation induced heart failure in mice fed a high-fat diet, with paradoxically reduced cardiac lipid content. Crucially, non-failing diabetic human hearts also exhibited protein aggregate pathology. Taken together, these results demonstrate that impaired proteostasis characterizes cardiomyopathy from cardiac lipid overload and identify a promising new therapeutic target for this condition.

molecular biology

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology