MicroRNA-Mediated Translation Initiation Arrest In Ischemic Brain

描述（由申请人提供）：脑缺血导致翻译停滞，并且不能从这种状态恢复与缺血/再灌注损伤后的神经元细胞死亡相关。虽然翻译的每个阶段（起始、延伸和终止）都可以调节，但靶向翻译起始是一种有效和快速的翻译停滞手段。缺血降低了真核起始因子2和4（eIF 2和eIF 4）的表达，这是形成翻译起始复合物所必需的;具体而言，eIF 2B介导与eIF 2结合的GDP交换为GTP，这是真核生物中控制翻译起始的关键调控步骤，而eIF 4 E的功能是结合mRNA帽并将其带到核糖体。然而，缺血诱导的eIF蛋白抑制的所有潜在机制尚不清楚。microRNA（miRNAs）是一类小的非编码RNA，通过抑制或降解mRNA靶点来调控转录后基因的表达。使用实时定量PCR分析miRNA，我们发现了一个通用的缺血诱导的miRNA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，这种常见的缺血诱导的miRNAs抑制eIF 2和eIF 4，破坏翻译起始复合物，导致翻译 逮捕了该提议是创新的，因为它是第一个直接研究miRNA介导的eIF调节作为脑缺血后翻译停滞的潜在机制的作用。目的1将确定缺血对miRNA和eIF 2B和eIF 4 E表达的影响，以及对翻译停滞的影响。我们推测缺血诱导的miRNAs抑制eIF 2B和eIF 4 E的表达，导致翻译停滞。将在小鼠体内进行研究，以确认缺血对以下的影响：（a）大脑皮层中候选miRNA的诱导表达和eIF 2 B和eIF 4 E蛋白的抑制，（B）脑中候选miRNA和eIF 2 B和eIF 4 E mRNA和蛋白的区域和细胞分布，以及（c）使用多核糖体分析的大脑皮层中的翻译停滞。目的2阐明miRNAs在eIF 2B和eIF 4 E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eIF 2B和eIF 4 E，从而破坏mRNA翻译并最终导致细胞响应缺血而死亡。我们将使用体外研究来（a）在荧光素酶报告分析系统中功能性地评估候选miRNA调节eIF 2 B和eIF 4 E表达的能力，（B）检查miRNA模拟物和抑制剂对培养的神经元中内源性eIF 2 B和eIF 4 E表达和翻译停滞的影响，以及（c）检查miRNA模拟物和抑制剂以及直接操纵eIF 2 B和eIF 4 E表达的影响，对缺氧缺糖后神经细胞死亡的影响。这项工作意义重大，因为将获得关于大脑对损伤的普遍反应中的miRNAs的新见解，相对于翻译停滞，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及其调节缺血性脑中翻译起始的潜力的新型中风疗法的开发。

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.