Mechanisms for modulation of miRNA-mediated gene silencing

摘要 在真核细胞中，基因表达受到多种水平的调控。转录后调控在一定程度上是 由miRNAs(MiRNAs)介导，它在miRNA诱导的沉默复合体(MiRISC)内发挥作用。 然而，mRNAs对miRNA介导的沉默的敏感性似乎取决于细胞类型和 信使核糖核酸靶标本身的特点。我们最近的研究表明，miRNA-的稳定性和翻译效率 靶向mRNAs由miRISC和RNA结合蛋白(RBPs)之间的相互作用决定。在这 建议，我们试图阐明miRISC介导的翻译抑制和 限制性商业惯例和靶向mRNAs以特定细胞类型的方式对其进行调控。在目标1中，我们打算 确定miRNAs作为miRISC的一部分，如何在启动阶段抑制翻译。具体来说，我们 将寻求确定eIF4A解旋酶和eIF4F复合体在miRNA介导的翻译中的作用 翻译起始和43S核糖体扫描过程中的抑制。为了实现这一目标，我们将大量使用 平行报告分析(MPRA)、CRISPR/Cas-9工程细胞和翻译抑制物。我们将使用 用同样的方法分析Pumilio和Au富含的结合位点影响的机制 MiRNA介导的翻译抑制。在目标2中，我们将确定富含AU的基序如何调制miRNA- 介导的靶向mRNA的抑制。具体来说，我们将确定阿瑞斯和阿瑞斯 ARE结合蛋白(ARE-Bps)可以上调或下调miRNA介导的细胞特异性基因沉默 举止。最后，我们将使用ARE-BPS和靶mRNA的免疫沉淀，并测试两者之间的相互作用 MiRISC、ARE-Bps和mRNA，以确定-Bps如何与位置偏向的靶mRNAs结合 方式干扰mRISC-mRNA靶向相互作用来调节基因调控。在《目标3》中，我们将 阐明限制性商业惯例调节miRNA介导的基因沉默如何影响小鼠的分化 在成熟的中枢神经系统中，神经元和星形胶质细胞具有区分神经元和星形胶质细胞的特异性效应。 具体地说，我们将使用报告文库分析miRNAs和限制性商业惯例的单独和组合效应， 内源mRNAs，多聚体图谱分析，以及将核糖体亲和纯化(TRAP)翻译为 评估小鼠脑细胞类型中基因表达的特异化。我们的中心假设是细胞类型- 靶mRNAs、RBP和miRISC之间的特定相互作用决定了miRNA介导的程度 翻译抑制和信使核糖核酸的衰退。我们的长期目标是定义miRNA介导的基因如何 通过调节限制性商业惯例和靶标的特征，以特定细胞类型的方式建立表达控制 MRNAs。神经系统，具有精致的细胞异质性，代表了测试我们的 假设。总之，这些实验将揭示为什么特定的mRNAs对miRISC做出强有力的反应，而 其他的则没有，以及miRNA介导的基因沉默的调制是如何在细胞特异的 限制性商业惯例的活性和存在以及目标mRNAs的序列特征决定了这种方式。

Abstract In eukaryotic cells, gene expression is regulated at multiple levels. Post-transcriptional regulation is, in part, mediated by micro RNAs (miRNAs), which act within the miRNA-induced silencing complex (miRISC). However, susceptibility of mRNAs to miRNA-mediated silencing appears to depend on the cell type and on the features of mRNA target itself. Our recent studies indicate that the stability and translation efficiency of miRNA- targeted mRNAs are determined by interactions between miRISC and RNA-binding proteins (RBPs). In this proposal, we seek to elucidate the basic mechanism of miRISC-mediated translational repression and its modulation by RBPs and target mRNAs, in a cell-type-specific manner. In Aim 1, we intend to determine how miRNAs, as part of the miRISC, repress translation during the initiation phase. Specifically, we will seek to determine the role of eIF4A helicase and the eIF4F complex in miRNA-mediated translational repression during translation initiation and 43S ribosome scanning. To achieve this goal we will use massively parallel reporter assays (MPRA), CRISPR/Cas-9 engineered cells, and translation inhibitors. We will use the same approach to analyze the mechanism through which Pumilio and AU-rich (ARE) binding sites affect miRNA-mediated translational repression. In Aim 2, we will ascertain how AU-rich motifs modulate miRNA- mediated repression of target mRNAs. Specifically, we will determine the mechanism by which AREs and ARE-binding proteins (ARE-BPs) can up- or down-regulate miRNA-mediated gene silencing in a cell-specific manner. Lastly, we will use immunoprecipitation of ARE-BPs and target mRNA and test interaction between miRISC, ARE-BPs, and mRNA, to determine how ARE-BPs' binding to target mRNAs in a positionally biased manner interferes with mRISC-mRNA target interaction to modulate gene regulation. In Aim 3, we will elucidate how modulation of miRNA-mediated gene silencing by RBPs shapes the differentiation of mouse neurons and results in cell-specific effects distinguishing neurons and astrocytes in the mature CNS. Specifically, we will analyze individual and combinatorial effects of miRNAs and RBPs using reporter libraries, endogenous mRNAs, polysome profile analyses, and translating ribosome affinity purification (TRAP) to evaluate specialization of gene expression in murine brain cell types. Our central hypothesis is that cell-type- specific interactions between the target mRNAs, RBPs, and miRISC determine the extent of miRNA-mediated translational repression and mRNA decay. Our long-term goal is to define how miRNA-mediated gene expression control is established in a cell-type-specific manner by modulation of RBPs and features of target mRNAs. The nervous system, with an exquisite heterogeneity of cells, represents a great structure to test our hypotheses. Together, these experiments will reveal why specific mRNAs respond robustly to miRISC while others do not, and how the modulation of miRNA–mediated gene silencing is established in a cell-specific manner by the activity and presence of RBPs as well as sequence features of target mRNAs.