Mohan Sruthi Priya, Nagarajan Hemavathy, Vetrivel Umashankar, Ramasubramanyan Sharada
R.S. Mehta Jain Department of Biochemistry and Cell Biology, KBIRVO, Vision Research Foundation, Chennai, India.
School of Chemical and Biotechnology, SASTRA Deemed to be University, Thanjavur, India.
Biochem Biophys Rep. 2024 Aug 12;39:101807. doi: 10.1016/j.bbrep.2024.101807. eCollection 2024 Sep.
The fate and stability of messenger RNA (mRNA), from transcription to degradation is regulated by a dynamic shuttle of epigenetic modifications and RNA binding proteins in maintaining healthy cellular homeostasis and disease development. While Transforming Growth Factor Beta 1 (TGFβ1) has been implicated as a key regulator for diabetic retinopathy, a microvascular complication of diabetes, the RNA binding proteins post-transcriptionally regulating its expression remain unreported in the ocular context. Further, dysfunction of TGFβ1 signalling is also strongly associated with angiogenesis, inflammatory responses and tissue fibrosis in many eye conditions leading to vision loss. In this study, computational and molecular simulations were initially carried out to identify Human Antigen R (HuR) binding sites in mRNA and predict the structural stability of these RNA-protein interactions. These findings were further validated through experiments utilizing Cobalt Chloride (CoCl) as a hypoxia mimetic agent in human retinal microvascular endothelial cells (HRMVEC). analysis revealed that HuR preferentially binds to the 5'-UTR of and displayed more stable interaction than the 3'UTR. Consistent with analysis, RNA immunoprecipitation demonstrated a robust association between HuR and mRNA specifically under hypoxic conditions. Further, silencing of HuR significantly reduced TGFβ1 protein expression upon CoCl treatment. Thus, for the first time in ocular pathological milieu, direct evidence of HuR- mRNA interaction under conditions of hypoxia has been reported in this study providing valuable insights into RNA binding proteins as therapeutic targets for ocular diseases associated with TGFβ1 dysregulation.
从转录到降解，信使核糖核酸（mRNA）的命运和稳定性受到表观遗传修饰和RNA结合蛋白动态穿梭的调控，以维持健康的细胞内稳态和疾病发展。虽然转化生长因子β1（TGFβ1）被认为是糖尿病视网膜病变（糖尿病的一种微血管并发症）的关键调节因子，但在眼部环境中，转录后调节其表达的RNA结合蛋白尚未见报道。此外，TGFβ1信号传导功能障碍在许多导致视力丧失的眼部疾病中也与血管生成、炎症反应和组织纤维化密切相关。在本研究中，最初进行了计算和分子模拟，以确定mRNA中的人类抗原R（HuR）结合位点，并预测这些RNA-蛋白质相互作用的结构稳定性。这些发现通过在人视网膜微血管内皮细胞（HRMVEC）中使用氯化钴（CoCl）作为缺氧模拟剂的实验得到进一步验证。分析表明，HuR优先结合TGFβ1的5'-非翻译区（UTR），并且与3'-UTR相比显示出更稳定的相互作用。与分析一致，RNA免疫沉淀证明在缺氧条件下HuR与TGFβ1 mRNA之间存在强烈关联。此外，沉默HuR可显著降低CoCl处理后的TGFβ1蛋白表达。因此，本研究首次在眼部病理环境中报道了缺氧条件下HuR与TGFβ1 mRNA相互作用的直接证据，为将RNA结合蛋白作为与TGFβ1失调相关眼部疾病的治疗靶点提供了有价值的见解。
