Mechanisms for modulation of miRNA-mediated gene silencing

摘要 在真核细胞中，基因表达在多个水平上受到调节。转录后调节，在某种程度上， 由微小RNA（miRNA）介导，其在miRNA诱导的沉默复合物（miRISC）内起作用。 然而，mRNA对miRNA介导的沉默的敏感性似乎取决于细胞类型和细胞的分化程度。 mRNA靶向自身的特征。我们最近的研究表明，miRNA的稳定性和翻译效率- 靶向mRNA由miRISC和RNA结合蛋白（RBP）之间的相互作用决定。在这 我们试图阐明miRISC介导的翻译抑制的基本机制， 其通过RBP和靶mRNA以细胞类型特异性方式进行调节。在目标1中，我们打算 确定miRNAs作为miRISC的一部分如何在起始阶段抑制翻译。我们特别 将试图确定eIF 4A解旋酶和eIF 4F复合物在miRNA介导的翻译中的作用。 在翻译起始和43 S核糖体扫描期间的阻遏。为了实现这一目标，我们将大量使用 平行报告基因测定（MPRA）、CRISPR/Cas-9工程化细胞和翻译抑制剂。我们将使用 相同的方法来分析Pumilio和富含AU（ARE）的结合位点影响 miRNA介导的翻译抑制。在目标2中，我们将确定富含AU的基序如何调节miRNA- 介导的靶mRNA的抑制。具体而言，我们将确定战神和 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。 方式最后，我们将使用ARE-BP和靶mRNA的免疫沉淀，并测试ARE-BP和靶mRNA之间的相互作用。 miRISC、ARE-BPs和mRNA，以确定ARE-BPs如何以位置偏倚的方式与靶mRNA结合。 以干扰mRISC-mRNA靶相互作用的方式调节基因调控。在目标3中，我们 阐明RBP如何调节miRNA介导的基因沉默， 神经元，并导致区分成熟CNS中的神经元和星形胶质细胞的细胞特异性效应。 具体来说，我们将使用报告文库分析miRNA和RBP的单独和组合效应， 内源性mRNA、多核糖体谱分析和翻译核糖体亲和纯化（TRAP）， 评估小鼠脑细胞类型中基因表达的特化。我们的核心假设是细胞类型 靶mRNA、RBP和miRISC之间的特异性相互作用决定了miRNA介导的 翻译抑制和mRNA降解。我们的长期目标是确定miRNA介导的基因是如何在细胞内表达的。 通过调节RBP和靶细胞的特征，以细胞类型特异性方式建立表达控制。 mRNA。神经系统，具有精致的细胞异质性，是测试我们的神经系统的一个伟大结构。 假设总之，这些实验将揭示为什么特定的mRNA对miRISC反应强烈， 其他人没有，以及如何在细胞特异性表达中建立miRNA介导的基因沉默的调节。 通过RBP的活性和存在以及靶mRNA的序列特征来确定。

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.