Dysregulated pre-mRNA Splicing by RBM25 in Endothelial Injury Under Hypoxia Stress
Abstract Under physiological conditions, the endothelial monolayer of vessel maintains the balance between vasodilation and vasoconstriction, inhibits leukocyte and platelet adhesion, platelet aggregation, exerts anticoagulant and profibrinolytic effects. Apoptosis is believed to play pivotal role in endothelial injury. Endothelial cells (ECs) can upregulate expression of anti-apoptotic genes to protect ECs from injury. A recent study suggests that Bcl-x(L) overexpression exerts an anti-apoptotic and protective effect on EC function. Therefore, protection of ECs from apoptosis should prove to be a beneficial strategy for maintaining endothelial homeostasis and restoring endothelial function. It is well documented that aberrant alternative pre-mRNA splicing plays pivotal role in apoptosis. We have previously shown that splicing factor RBM25 promotes apoptosis through regulating alternative pre-mRNA splicing of Bcl-X. In conjunction with hLuc7A, another spliceosome-associated factor, it shifts the ratio of Bcl-X long (Bcl-X(L) and short (Bcl-X(s) alternatively spliced mRNA forms leading to an increase in apoptosis when RBM25 is upregulated. Our preliminary data show that RBM25 expression increases under hypoxia stress. Based on the above, we hypothesized that the overexpressed RBM25 contributes to endothelial injury under hypoxia stress by increasing apoptosis through regulating alternative pre-mRNA splicing. In this proposal, we will delineate the pathophysiological roles of RBM25 in endothelial cell injury and the regulated alternative pre-mRNA splicing network by RBM25 in endothelial cells. The information we gain from this study may help protect endothelial cells from apoptosis due to cellular stress or promote endothelial repair by manipulation of RBM25 expression. We shall characterize in detail the expression of RBM25 in endothelial cells under normal and hypoxia stress condition using Human Umbilical Vein Endothelial Cells (HUVEC) and Human Coronary Artery Endo Cells (HCAEC) by quantitative real-time PCR (RT-qPCR) and Westernblot. We shall also determine the functional importance of RBM25 by assessing the effects of its overexpression or repression on endothelial injury by manipulation the expression of RBM25 using inducible tet-On oeverexpression and repression systems. Apoptotic ECs will be quantified by flow cytometry with Annixin V, propidium iodide (PI) and active caspase 3 staining. Furthermore, we shall determine the functional importance of RBM25 in the endothelial cell apoptotic program. We shall analyze the effect of RBM25 expression levels on the alternatively spliced isoforms of predicted targets other than Bcl-X using PCR and protein analysis with which we are experienced. At last, we shall decipher the global impact of RBM25 on the alternative pre-mRNA splicing program in endothelial cells under hypoxia stress using PAR-CLIP assay. We expect that hypoxia will result in increased RBM25 expression, increased pro-apoptotic splice variant abundance, decreased anti-apoptotic splice variant abundance and increased endothelial cell apoptosis. A reduction in the expression of RBM25 to SCN5a will protect endothelial cells from injury. Our comprehensive understanding of the role of alternative pre-mRNA splicing regulation in the onset and progression of endothelial injury gained from this project would help us to find druggable targets for treatments of human diseases related to endothelial injury and insufficiency of repair.