A novel method for identifying microRNA targets

一种识别 microRNA 靶标的新方法

• 批准号: 8492900
• 负责人: RICHARD H. GOODMAN
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8492900
• 关键词: Address; Aftercare; Algorithms; Attention; Bioinformatics; Biological Assay; Biological Models; Biology; Brain; Cells; Complex; Data; Data Set; Development; Dominant-Negative Mutation; Epitopes; Hippocampus (Brain); Human; Individual; Laboratories; Link; Luciferases; Mediating; Mental disorders; Messenger RNA; Methods; MicroRNAs; Neuroblastoma; Neuroglia; Neurologic; Neurons; Population; Process; RNA-Binding Proteins; RNA-Induced Silencing Complex; Regulation; Reporter; Reporting; Repression; Signal Transduction; Subfamily lentivirinae; System; Testing; Transcript; Western Blotting; cell type; genome-wide; interest; mRNA Transcript Degradation; mRNA tagging; nervous system disorder; neuroblastoma cell; novel; novel strategies; public health relevance; relating to nervous system

DESCRIPTION (provided by applicant): Abnormalities in microRNA signaling have been associated with multiple neurological and psychiatric diseases. Understanding these associations cannot proceed further, however, without better methods for determining microRNA targets. Because of the incomplete complementarity of microRNAs and their targets, predicting authentic microRNA:mRNA interactions can be difficult. Predictions typically rely on bioinformatics, but it is well known that the concurrence of different algorithms is distressingly low. Thus, empirical approaches for characterizing microRNA:mRNA interactions have received increasing attention. We have developed an approach that utilizes an epitope-tagged, dominant negative version of GW182, a component of the RNA-induced silencing complex (RISC), to purify microRNA:mRNA complexes prior to microRNA-mediated mRNA degradation. This method, which we term RISC-Trap, is considerably more robust than previously reported approaches and allows us to address fundamental problems in microRNA biology, such as, what proportion of mRNA targets undergo degradation versus translational arrest, which targets are direct or indirect, and whether some interactions occur specifically in neural versus non-neural cells. To begin to address these questions, we will use RNASeq to examine complexes containing miR-124, an abundant, neural-specific microRNA with a large data set of previously characterized targets. Our approach should be generally applicable to other systems, however, and should significantly increase understanding of the contributions of microRNAs to neurodevelopmental processes, plasticity, and certain neurological and psychiatric diseases. Our first specific aim is to use the RISC-trap approach to determine which miR-124 targets in HEK293 cells are regulated by mRNA degradation versus translational arrest. These studies will provide a comprehensive picture of the regulatory effects of an important brain microRNA and a straightforward and easily applicable method for identifying microRNA targets in general. Our second aim will be to identify miR-124 targets specific to neuronal cells and determine whether the mode of microRNA action differs. Although many important miR-124 targets are expressed in both neural and non-neural cells, specific neuronal targets are likely to exist as well. We will identify these neural-specific targets by infecting SH-SY5Y neuroblastomas and primary hippocampal neurons with a dnGW182 lentivirus and analyzing the targets as described above. Some targets will be detected only in neuronal cells because their expression is linked to this cell type. Neural-specific microRNA interactions involving mRNAs expressed in both cell types may indicate the involvement of essential RNA binding proteins. We believe that the RISC-trap assay may uncover new aspects of microRNA function.

描述（由申请人提供）：microRNA 信号传导异常与多种神经和精神疾病相关。然而，如果没有更好的方法来确定 microRNA 靶标，就无法进一步了解这些关联。由于 microRNA 及其靶标的不完全互补性，预测真实的 microRNA:mRNA 相互作用可能很困难。预测通常依赖于生物信息学，但众所周知，不同算法的并发性低得令人痛苦。因此，表征 microRNA:mRNA 相互作用的经验方法受到越来越多的关注。我们开发了一种方法，利用 GW182（RNA 诱导沉默复合物 (RISC) 的一个组成部分）的表位标记、显性失活版本，在 microRNA 介导的 mRNA 降解之前纯化 microRNA:mRNA 复合物。这种方法，我们称之为 RISC-Trap，比以前报道的方法要强大得多，使我们能够解决 microRNA 生物学中的基本问题，例如，相对于翻译停滞，mRNA 目标的比例有多大比例经历降解，哪些目标是直接或间接的，以及某些相互作用是否专门发生在神经细胞和非神经细胞中。为了开始解决这些问题，我们将使用 RNASeq 来检查含有 miR-124 的复合物，miR-124 是一种丰富的神经特异性 microRNA，具有大量先前表征的靶标数据集。然而，我们的方法应该普遍适用于其他系统，并且应该显着增加对 microRNA 对神经发育过程、可塑性以及某些神经和精神疾病的贡献的理解。我们的第一个具体目标是使用 RISC-trap 方法来确定 HEK293 细胞中哪些 miR-124 靶标受到 mRNA 降解与翻译停滞的调节。这些研究将全面介绍一种重要的大脑 microRNA 的调节作用，并提供一种简单易行的方法来识别一般的 microRNA 靶点。我们的第二个目标是识别神经元细胞特异的 miR-124 靶标，并确定 microRNA 作用模式是否不同。尽管许多重要的 miR-124 靶标在神经细胞和非神经细胞中都有表达，但特定的神经元靶标也可能存在。我们将 通过用 dnGW182 慢病毒感染 SH-SY5Y 神经母细胞瘤和原代海马神经元并如上所述分析目标来识别这些神经特异性目标。一些靶标只能在神经元细胞中检测到，因为它们的表达与这种细胞类型相关。涉及两种细胞类型中表达的 mRNA 的神经特异性 microRNA 相互作用可能表明必需 RNA 结合蛋白的参与。我们相信 RISC-trap 检测可能会揭示 microRNA 功能的新方面。