Identification of Neuronal microRNA Targets

描述（由申请人提供）：microRNA在大脑中的关键和多样性作用正在继续被发现。到目前为止，microRNA已涉及神经发生，神经发育，突触可塑性和神经精神疾病。为了充分理解microRNA在正常和病理状态下如何执行这些功能，必须识别和表征它们的mRNA靶点。该项目利用RISC陷阱分析，这是一种解决microRNA在神经元中作用的优秀工具。 RISC陷阱利用RNAi诱导沉默复合物（RISC）的显性负性组分-microRNA途径中的中心组分，是microRNA与其靶标之间的桥梁-来鉴定单个microRNA的靶标。这个项目将使用RISC陷阱来识别一个 miR-132和miR-124靶向的一组全面的mRNA，miR-132参与神经元成熟、成熟功能和疾病状态，miR-124被证明可促进神经分化，最近还显示可调节可塑性。 神经元特异性microRNA靶标可以是两类之一。它们可以编码具有神经特异性功能的转录物，如突触成分。或者，特别感兴趣的是在神经元和HEK细胞中表达的转录物，但是当miR-132或miR-124相互作用仅发生在神经元中时，这些相互作用可能需要特异性RNA结合蛋白的活性。识别神经元特异性靶点将促进对神经元中microRNA使用机制的理解。这种全局靶点识别方法将通过检查一种新的miR-132靶点ARHGEF 11的调节来补充，ARHGEF 11在HEK细胞的RISC陷阱筛选中被鉴定，并且也已知有助于神经元细胞信号传导。这种含PDZ的蛋白是有趣的，因为它已被证明可以激活Rho信号并抑制神经突生长。此外，ARHGEF 11与miR-132一样，与精神分裂症有关。该项目利用miR-132敲除小鼠和条件性敲除小鼠，其提供了靶向切除条件性敲除小鼠的成年新生神经元中的miR-132位点的能力，以检查这些细胞中miR-132如何调控ARHGEF 11。对miR-132和miR-124靶点的深入研究将有助于更深入地了解这些microRNA的作用机制，并可能揭示神经发育障碍和潜在成人神经系统疾病的药物靶点。

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.