MicroRNA-Mediated Translation Initiation Arrest In Ischemic Brain

描述(申请人提供)：脑缺血导致翻译停滞，而不能从这种状态恢复与缺血/再灌注损伤后神经细胞死亡有关。虽然翻译的每个阶段(开始、延长和终止)都是可以调控的，但有针对性的翻译启动是一种有效而快捷的翻译阻止手段。缺血减少了真核细胞起始因子2和4(eIF2和eIF4)的表达，这两个因子是形成翻译起始复合体所必需的；具体地说，eIF2B介导了与eIF2结合的GDP与GTP的交换，GTP是控制真核生物翻译起始的关键调控步骤，eIF4E具有结合mRNA帽并将其带到核糖体的功能。然而，缺血诱导抑制EIF蛋白的所有机制尚不清楚。MicroRNAs(MiRNAs)是一种小的、非编码的RNAs，通过抑制或降解mRNA靶标来调节转录后基因的表达。利用miRNAs的实时定量PCR图谱，我们发现了一个普遍存在的缺血诱导的miRNA图谱，旨在沉默男性和女性大脑中的EIF蛋白。我们的中心假设是，缺血诱导的miRNAs的这个共同子集抑制eIF2和eIF4，扰乱翻译起始复合体，导致翻译 逮捕。这一建议是创新的，因为它是第一次直接研究miRNA介导的EIF调节作为脑缺血后翻译停滞的机制所起的作用。目的1确定缺血对miRNA、eIF2B和eIF4E表达的影响，以及对翻译抑制的影响。我们假设，缺血诱导的miRNAs抑制eIF2B和eIF4E的表达，导致翻译停滞。将在小鼠体内进行研究，以确认缺血对(A)诱导大脑皮质候选miRNAs的表达和抑制eIF2B和eIF4E蛋白的影响，(B)候选miRNAs和eIF2B和eIF4E在大脑中的区域和细胞分布，以及(C)利用多聚体分析大脑皮质的翻译抑制。目的2阐明miRNAs在eIF2B和eIF4E表达、翻译停滞和神经细胞死亡中的作用。我们假设miRNAs直接抑制eIF2B和eIF4E，而eIF2B和eIF4E破坏了mRNA的翻译，最终导致细胞因缺血而死亡。我们将使用体外研究来(A)在荧光素酶报告分析系统中从功能上评估候选miRNAs调节eIF2B和eIF4E表达的能力，(B)检测miRNA模拟物和抑制剂对培养神经元内源性eIF2B和eIF4E表达和翻译抑制的影响，以及(C)检测miRNA模拟物和抑制剂以及直接操纵eIF2B和eIF4E表达对缺氧缺糖后神经细胞死亡的影响。这项拟议的工作具有重要意义，因为将获得关于相对于翻译停止的大脑对损伤的普遍反应中的miRNAs的新见解，以及这可能如何影响男性和女性的大脑健康和疾病。我们的发现也可能导致基于miRNAs的新中风疗法的开发，以及它们调节缺血脑中翻译启动的潜力。

DESCRIPTION (provided by applicant): Cerebral ischemia leads to translation arrest, and the inability to recover from this state correlates with neuronal cell death following ischemia/reperfusion injury. While each stage of translation (initiation, elongation, and termination) can be regulated, targeting translation initiation serves as an efficient and expeditious means of translation arrest. Ischemia reduces expression of eukaryotic initiation factors 2 and 4 (eIF2 and eIF4) which are essential for formation of the translation initiation complex; specifically, eIF2B mediates the exchange of GDP bound to eIF2 for GTP, a key regulatory step for the control of translation initiation in eukaryotes, and eIF4E functions to bin an mRNA cap and bring it to the ribosome. However, all of the mechanisms underlying ischemia-induced suppression of eIF proteins are not known. MicroRNAs (miRNAs) are small, non-coding RNAs which regulate post-transcriptional gene expression by repressing or degrading mRNA targets. Using real-time quantitative PCR profiling of miRNAs, we uncovered a universal ischemia-induced miRNA profile aimed at silencing eIF proteins in both male and female brain. Our central hypothesis is that this common subset of ischemia-induced miRNAs represses eIF2 and eIF4, disrupting the translation initiation complex and resulting in translation arrest. This proposal is innovative as it is the first to directly examine a role for miRNA- mediated regulation of eIFs as a mechanism underlying translation arrest following cerebral ischemia. Aim 1 will determine the effects of ischemia on miRNA and eIF2B and eIF4E expression, and on translation arrest. We hypothesize that ischemia-induced miRNAs suppress eIF2B and eIF4E expression, resulting in translation arrest. Studies will be conducted in mice in vivo to confirm the effects of ischemia on (a) induced expression of candidate miRNAs and suppression of eIF2B and eIF4E protein in cerebral cortex, (b) the regional and cellular distribution of candidate miRNAs and eIF2B and eIF4E mRNA and protein in brain, and (c) translation arrest in cerebral cortex using polysome profiling. Aim 2 will clarify the role of miRNAs on eIF2B and eIF4E expression, translation arrest and neuronal cell death. We hypothesize that miRNAs directly repress eIF2B and eIF4E, which disrupts mRNA translation and ultimately leads to cell death in response to ischemia. We will use in vitro studies to (a) functionally assess the ability of candidate miRNAs to regulate eIF2B and eIF4E expression in a luciferase reporter assay system, (b) examine effects of miRNA mimics and inhibitors on endogenous eIF2B and eIF4E expression and translation arrest in cultured neurons, and (c) examine effects of miRNA mimics and inhibitors, and of direct manipulation of eIF2B and eIF4E expression, on neuronal cell death following oxygen- glucose deprivation. The proposed work is significant as new insights will be gained about miRNAs in the brain's universal response to injury relative to translation arrest and how this might influence brain health and disease in men and women. Our findings could also lead to development of novel stroke therapies based on miRNAs and their potential for regulating translation initiation in ischemic brain.