A novel method for identifying microRNA targets

描述（由申请人提供）：microRNA信号传导的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靶点，就无法进一步理解这些关联。由于microRNA与其靶点的不完全互补性，预测真正的microRNA：mRNA相互作用可能是困难的。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沮丧。因此，表征microRNA：mRNA相互作用的经验方法受到越来越多的关注。我们已经开发了一种方法，利用表位标记的，显性负性版本的GW 182，RNA诱导的沉默复合物（RISC）的一个组成部分，以纯化microRNA：mRNA复合物之前microRNA介导的mRNA降解。这种方法，我们称之为RISC-Trap，比以前报道的方法更加强大，使我们能够解决microRNA生物学中的基本问题，例如，mRNA靶点的比例与翻译停滞相比，哪些靶点是直接或间接的，以及一些相互作用是否专门发生在神经细胞与非神经细胞中。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陷阱方法来确定HEK 293细胞中哪些miR-124靶点受mRNA降解与翻译停滞的调控。这些研究将提供一个重要的大脑microRNA的调控作用的全面图片和一个简单，易于应用的方法来识别microRNA的目标一般。我们的第二个目标将是确定miR-124对神经元细胞的特异性靶点，并确定microRNA的作用模式是否不同。虽然许多重要的miR-124靶点在神经和非神经细胞中表达，但也可能存在特定的神经元靶点。我们将 通过用dnGW 182慢病毒感染SH-SY 5 Y神经母细胞瘤和原代海马神经元并如上所述分析靶标来鉴定这些神经特异性靶标。一些靶标仅在神经元细胞中检测到，因为它们的表达与这种细胞类型有关。涉及在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的RNA结合蛋白的参与。我们相信RISC陷阱检测可能会揭示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.