Identification of Neuronal microRNA Targets

描述(申请人提供)：microRNAs在大脑中的关键和多样化的作用正在继续被发现。到目前为止，microRNAs与神经发生、神经发育、突触可塑性和神经精神障碍有关。为了充分了解microRNAs在正常和病理状态下是如何执行这些功能的，必须识别和表征它们的mRNA靶标。这个项目利用了RISC-TRAP试验，这是一个解决microRNAs在神经元中的作用的优秀工具。RISC-TRAP利用RNAi诱导的沉默复合体(RISC)的一个主要负成分--microRNA途径中的一个中心成分，即microRNA与其靶标之间的桥梁--来识别单个microRNAs的靶标。该项目将使用RISC-Trap来识别 由miR-132和miR-124靶向的一组全面的mRNAs参与神经元的成熟、功能成熟和疾病状态，被证明促进神经分化，最近被证明调节可塑性。神经元特异的microRNA靶标可能有两种类型。它们可以编码具有神经特定功能的转录本，如突触成分。或者，特别令人感兴趣的是在神经元和HEK细胞中都表达的转录本，但如果miR-132或miR-124相互作用仅发生在神经元中--这些相互作用可能需要特定RNA结合蛋白的活性。识别特定于神经元的靶点将促进对microRNAs在神经元中使用的机制的理解。这种全局靶点识别方法将通过研究一种新的miR-132靶点ARHGEF11的调节来补充，ARHGEF11是在HEK细胞的RISC-Trap筛选中发现的，也是已知对神经细胞信号转导有贡献的。这种含有PDZ的蛋白质很有趣，因为它已经被证明可以激活Rho信号并抑制轴突生长。此外，ARHGEF11和miR-132一样，也与精神分裂症有关。该项目利用miR-132基因敲除小鼠和条件基因敲除小鼠，它们提供了靶向切除条件基因敲除小鼠成年新生神经元中miR-132基因座的能力，以检查ARHGEF11是如何由miR-132在这些细胞中调节的。深入研究miR-132和miR-124靶点将有助于更深入地了解这些microRNAs的作用机制，并可能揭示治疗神经发育障碍和成人神经系统潜在障碍的药物靶点。

DESCRIPTION (provided by applicant): The critical and diverse roles of microRNAs in the brain are continuing to be discovered. To date, microRNAs have been implicated in neurogenesis, neurodevelopment, synaptic plasticity, and neuropsychiatric disorders. To fully understand how microRNAs carry out these functions, both in normal and pathological states, their mRNA targets must be identified and characterized. This project takes advantage of the RISC-trap assay, an excellent tool to address the roles of microRNAs in neurons. RISC-trap utilizes a dominant negative component of the RNAi-Induced Silencing Complex (RISC) - a central component in the microRNA pathway that is the bridge between microRNAs and their targets- to identify targets of individual microRNAs. This project will use RISC-trap to identify a comprehensive set of mRNAs targeted by miR-132, involved in neuronal maturation, mature function, and disease states, and miR-124, demonstrated to promote neural differentiation and recently shown to regulate plasticity. Neuron-specific microRNA targets may be in one of two categories. They may encode transcripts, such as synaptic components, that have neural-specific functions. Alternatively, and of particular interest, will be transcripts that are expressd in both neurons and HEK cells, but where the miR-132 or miR-124 interactions occur exclusively in neurons-these interactions may require the activities of specific RNA-binding proteins. Identifying targets specific to neurons will advance understanding of the mechanisms used by microRNAs in neurons. This global target-identification approach will be complemented by examining the regulation of a novel miR-132 target, ARHGEF11, which was identified in RISC-trap screen of HEK cells, and is also known to contribute to neuronal cell signaling. This PDZ-containing protein is interesting because it has been shown to activate Rho signaling and suppress neurite outgrowth. Additionally, ARHGEF11, like miR-132, has been linked to schizophrenia. This project takes advantage of both miR-132 knockout mice and conditional knockout mice, which provide the ability to target excision of the miR-132 locus in the adult newborn neurons of the conditional knockout mouse to examine how ARHGEF11 is regulated by miR-132 in these cells. A thorough investigation miR-132 and miR-124 targets will lead to a deeper understanding of the mechanism of action of these microRNAs and may reveal drug targets for neurodevelopmental disorders and potentially disorders of the adult nervous system.