A novel method for identifying microRNA targets

描述(申请人提供)：microRNA信号的异常与多种神经和精神疾病有关。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，不同算法的重合率低得令人担忧。因此，表征microRNA：mRNA相互作用的经验方法受到了越来越多的关注。我们开发了一种方法，利用RNA诱导沉默复合体(RISC)的一个成分GW182的表位标记的显性负版本，在microRNA介导的mRNA降解之前纯化microRNA：mRNA复合体。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，有多大比例的mRNA靶经历了降解和翻译停滞，哪些靶是直接的或间接的，以及一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来检查包含miR-124的复合体，miR-124是一种丰富的神经特异性microRNA，具有先前表征的靶标的大量数据集。然而，我们的方法应该普遍适用于其他系统，并应该显著增加对microRNAs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。我们的第一个特定目标是使用RISC-TRAP方法来确定HEK293细胞中哪些miR-124靶点受到mRNA降解与翻译停滞的调节。这些研究将全面了解一种重要的大脑microRNA的调控作用，并提供一种简单易用的方法来识别一般的microRNA靶标。我们的第二个目标将是识别神经细胞特异性的miR-124靶点，并确定microRNA的作用模式是否不同。虽然许多重要的miR-124靶点在神经细胞和非神经细胞中都有表达，但也可能存在特定的神经元靶点。我们会 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，并如上所述分析这些靶点，来识别这些神经特异性靶点。有些靶点只能在神经细胞中检测到，因为它们的表达与这种细胞类型有关。神经特异性的microRNA相互作用涉及两种细胞中表达的mRNAs，这可能表明必要的RNA结合蛋白参与其中。我们相信，RISC-TRAP试验可能会发现microRNA功能的新方面。

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.