Vascular endothelial growth factor (VEGF) A is a master regulator of neovascularization and angiogenesis. VEGFA is potently induced by hypoxia and by pathological conditions including diabetic retinopathy and tumorigenesis. Fine-tuning of VEGFA expression by different stimuli is important for maintaining tissue vascularization and organ homeostasis. Here, we tested the effect of the hypoxia mimetic cobalt chloride (CoCl2) on VEGFA expression in human cervical carcinoma HeLa cells. We found that CoCl2 increased the levels of VEGFA mRNA and VEGFA protein without affecting VEGFA mRNA stability. Biotin pulldown analysis to capture the RNA-binding proteins (RBPs) bound to VEGFA mRNA followed by mass spectrometry analysis revealed that the RBP HuR [human antigen R, a member of the embryonic lethal abnormal vision (ELAV) family of proteins], interacts with VEGFA mRNA. VEGFA mRNA-tagging experiments showed that exposure to CoCl2 increases the interaction of HuR with VEGFA mRNA and promoted the colocalization of HuR and the distal part of the VEGFA 3'-untranslated region (UTR) in the cytoplasm. We propose that under hypoxia-like conditions, HuR enhances VEGFA mRNA translation.
Keywords: RNA-binding proteins; biotin pulldown; mass spectrometry; polysome profiling; post-transcriptional gene regulation; ribonucleoprotein complex.