Disease modeling of core pre-mRNA splicing factor haploinsufficiency

The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.

BCL-Xl Contributes to Decreased Cellular Respiration During Erythroid Differentiation

Abstract 3201 The BCL2 family of proteins are well known for their ability to both positively and negatively regulate mitochondrial mechanisms of apoptosis. The anti-apoptotic members of this family can decrease mitochondrial outer membrane permeability and cytochrome c release. By stabilizing the cell against apoptosis, these proteins allow cell survival even in states of low energy. However, despite this intimate link between the BCL2 family proteins and mitochondria, their direct effect on metabolism is less clearly understood. It is generally expected that metabolic changes induced by the BCL2 family of proteins will further impact cell survival as murine hepatoma and cancer cells overexpressing Bcl-xL are sensitive to Bcl-xL inhibition1 but BCL-xL is also known to be essential for erythroid differentiation and more recently was linked specifically to heme synthesis2. We therefore set out to investigate whether there was a connection between BCL-xL induced changes in cellular respiration and erythroid differentiation. Murine erythroleukemia (MEL) cells were differentiated by exposure to 2% DMSO for 5 days and then real time oxygen consumption was measured on the Seahorse extracellular flux analyzer (XFA). DMSO induced differentiation yielded a 4-fold decrease in oxygen consumption. Western blot analysis revealed that BCL-xL was induced during differentiation. We then generated cell lines in which BCL-xL was knocked down with small hairpin RNA (shRNA). As differentiation has previously been reported to be fatal in MEL cells without BCL-xL activity, both parental cells and BCL-xL knockdowns were infected with a vector over expressing BCL2. Differentiation over 5 days with 2% DMSO was performed on these new cell lines. Erythroid differentiation was confirmed using Benzidine staining. While the control cell line showed high rates of Benzidine staining after exposure to DMSO, the BCL-xL knockdown cell line consistently showed <5% benzidine positivity. Western blot analysis confirmed the absence of BCL-xL induction by DMSO exposure in the knockdown cell line. Using the Seahorse XFA the control cell line was shown to have significant decrease in oxygen consumption when exposed to DMSO, while DMSO exposed BCL-xL knockdown cells showed less than half this drop in oxygen consumption. However, both control and BCL-xL knockdowns have limited respiratory reserve as the response to CCCP, an uncoupler of electron transport, is diminished after DMSO exposure as compared to their undifferentiated counterparts. Our results suggest that erythroid differentiation is associated with a significant decrease in cellular respiration. Although, not the only contributor to the decreased dependence on oxidative phosphorylation of cells undergoing erythroid differentiation, BCL-xL expression is clearly a necessary factor. Our data is able to connect BCL-xL expression to both erythroid differentiation and this distinct metabolic phenotype. As BCL-xL's role in erythroid differentiation has previously been reported to be associated with heme synthesis, future work will focus on identifying oxidative metabolic pathways associated with BCL-xL expression and heme synthesis. Disclosures: No relevant conflicts of interest to declare.