Mechanisms for modulation of miRNA-mediated gene silencing

miRNA 介导的基因沉默的调节机制

• 批准号: 10674772
• 负责人: Sergej Djuranovic
• 金额: $ 34.07万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674772
• 关键词: 3' Untranslated Regions; Adult; Affect; Affinity Chromatography; Astrocytes; Binding; Binding Proteins; Binding Sites; Biological Assay; Brain; Cell Differentiation process; Cell Line; Cell Shape; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Development; Disease; Elements; Engineering; Eukaryotic Cell; Exhibits; Gene Expression; Gene Expression Regulation; Gene Silencing; Goals; Heterogeneity; Homeostasis; Human; Immunoprecipitation; In Vitro; Individual; Investigation; Knowledge; Libraries; Measures; Mediating; Messenger RNA; MicroRNAs; Modeling; Molecular; Mus; Mutate; Nervous System; Neuronal Differentiation; Neurons; Organism; Outcome; Peptide Initiation Factors; Phase; Polyribosomes; Positioning Attribute; Post-Transcriptional Regulation; Predisposition; RNA Binding; RNA Helicase; RNA-Binding Proteins; RNA-Induced Silencing Complex; RNA-Protein Interaction; Reporter; Repression; Ribosomes; Role; Scanning; Shapes; Structure; System; Testing; Transcript; Translating; Translation Initiation; Translational Repression; Translations; Untranslated RNA; Variant; Work; brain cell; cell type; combinatorial; experimental study; helicase; human disease; in vivo; inhibitor; mRNA Decay; nerve stem cell; postnatal; tissue culture

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.

抽象的 在真核细胞中，基因表达在多个水平上受到调节。转录后调控部分是 由微小 RNA (miRNA) 介导，在 miRNA 诱导的沉默复合物 (miRISC) 内发挥作用。 然而，mRNA 对 miRNA 介导的沉默的敏感性似乎取决于细胞类型和 mRNA靶标本身的特征。我们最近的研究表明 miRNA 的稳定性和翻译效率 目标 mRNA 由 miRISC 和 RNA 结合蛋白 (RBP) 之间的相互作用决定。在这个 提案中，我们试图阐明 miRISC 介导的翻译抑制的基本机制和 它通过 RBP 和目标 mRNA 以细胞类型特异性的方式进行调节。在目标 1 中，我们打算 确定 miRNA 作为 miRISC 的一部分如何在起始阶段抑制翻译。具体来说，我们 将寻求确定 eIF4A 解旋酶和 eIF4F 复合物在 miRNA 介导的翻译中的作用 翻译起始和 43S 核糖体扫描期间的抑制。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剂。我们将使用 使用相同的方法来分析 Pumilio 和富含 AU (ARE) 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 的基序如何调节 miRNA- 介导的靶 mRNA 抑制。具体来说，我们将确定 ARE 和 ARE 结合蛋白 (ARE-BP) 可以在细胞特异性中上调或下调 miRNA 介导的基因沉默 方式。最后，我们将使用 ARE-BP 和靶标 mRNA 的免疫沉淀来测试两者之间的相互作用 miRISC、ARE-BP 和 mRNA，以确定 ARE-BP 如何以位置偏向的方式与目标 mRNA 结合 方式干扰 mRISC-mRNA 靶点相互作用来调节基因调控。在目标 3 中，我们将 阐明 RBP 对 miRNA 介导的基因沉默的调节如何塑造小鼠的分化 神经元并导致细胞特异性效应，区分成熟中枢神经系统中的神经元和星形胶质细胞。 具体来说，我们将使用报告库分析 miRNA 和 RBP 的个体和组合效应， 内源 mRNA、多核糖体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专业化。我们的中心假设是细胞类型- 目标 mRNA、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 mRNA 衰减。我们的长期目标是定义 miRNA 介导的基因如何 通过调节 RBP 和靶标特征，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重要结构。 假设。总之，这些实验将揭示为什么特定的 mRNA 对 miRISC 反应强烈，而 其他人则不然，以及 miRNA 介导的基因沉默的调节是如何在细胞特异性中建立的 RBP 的活性和存在以及目标 mRNA 的序列特征。

项目成果

Urb-RIP - An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins.

• DOI: 10.1371/journal.pone.0167877
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Cottrell KA;Djuranovic S
• 通讯作者: Djuranovic S

PolyA tracks, polybasic peptides, poly-translational hurdles.

• DOI: 10.1002/wrna.1486
• 发表时间: 2018-09
• 期刊: Wiley interdisciplinary reviews. RNA
• 作者: Arthur LL;Djuranovic S
• 通讯作者: Djuranovic S

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

• DOI: 10.3390/pathogens12010095
• 发表时间: 2023-01-06
• 期刊: Pathogens (Basel, Switzerland)

Rapid generation of hypomorphic mutations.

快速产生型肌差突变。

• DOI: 10.1038/ncomms14112
• 发表时间: 2017-01-20
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Arthur LL;Chung JJ;Jankirama P;Keefer KM;Kolotilin I;Pavlovic-Djuranovic S;Chalker DL;Grbic V;Green R;Menassa R;True HL;Skeath JB;Djuranovic S
• 通讯作者: Djuranovic S

Association of the receptor for activated C-kinase 1 with ribosomes in Plasmodium falciparum.

• DOI: 10.1016/j.jbc.2022.101954
• 发表时间: 2022-06
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Erath, Jessey;Djuranovic, Sergej
• 通讯作者: Djuranovic, Sergej