A Novel Approach to Identify Neuronal mRNA Targets for Individual microRNAs

描述（由申请人提供）：微小RNA（miRNA）是小的非编码RNA，其前体由内源性miRNA基因转录。miRNA前体在细胞核中被drosha加工并在细胞质中被dicer加工以产生成熟的双链19-23 bp miRNA，然后将其加载到含有Argonaute的RNA诱导的沉默复合物（RISC）上，其进而通过与这些mRNA的不完全碱基配对靶向靶mRNA的3'UTR，从而主要抑制这些mRNA的翻译。一些miRNA可以靶向100个或更多个mRNA。新出现的证据表明，在正常发育和病理条件下，miRNA可能是基因表达的关键调节因子。然而，识别特定miRNA的mRNA靶标一直是一个主要问题。在这里，我们提出了一种新的方法来确定mRNA的目标在神经元中的个别miRNA。通过这样做，这种方法将显着推进我们对miRNAs调节神经元发育，突触功能和可塑性的机制的理解。我们的新方法提出了在RISC复合物中共免疫沉淀miRNA及其靶mRNA，并通过阵列和测序技术鉴定共免疫沉淀的靶点。这里的主要挑战是如何仅免疫沉淀给定的miRNA-RNP效应复合物。为了解决这个问题，我们建议使用条件性Dicer敲除小鼠（Dicer-/-），从这些小鼠中培养神经元，并使用慢病毒给药的cre重组酶（我们实验室的标准技术）删除这些神经元中的Dicer。因此，我们将有不表达加工过的miRNA的神经元，允许我们通过转染以成熟形式重新引入给定的miRNA。在这样的神经元中，唯一形成的miRNA-RNP复合物是我们特定miRNA的复合物，这使得我们有可能精确地定义这种特定miRNA的mRNA靶点。使用这种方法，一个接一个，我们将能够记录培养的皮层神经元中表达的所有单个microRNA的mRNA靶点。此外，这种新颖的方法可以很容易地适用于其他类型的神经元（例如，纹状体、小脑、海马或脊髓运动神经元）或其他系统，并将显著推动miRNA和神经科学研究领域向前发展。MicroRNA是基因组编码的基因表达的重要调节因子，在发育和多种疾病的发病机制中起着关键作用。单个microRNA靶向特定的信使RNA组以抑制蛋白质合成。然而，识别microRNA靶标非常困难。在这里，我们提出了一种新的方法来解决这个问题。如果成功的话，这种方法将彻底改变我们对microRNA的多种生物学功能的看法。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small non-coding RNAs whose precursors are transcribed from endogenous miRNA genes. miRNA precursors are processed by drosha in the nucleus and by dicer in the cytoplasm to generate mature double-stranded 19-23 bp miRNAs that are then loaded onto Argonaute-containing RNA-induced silencing complexes (RISC's), which in turn target the 3' UTRs of target mRNAs via imperfect base- pairing with these mRNAs, and thereby primarily inhibit translation of these mRNAs. Some miRNAs may target 100 or more mRNAs. Emerging evidence indicates that miRNAs may be key regulators of gene expression during normal development and under pathological conditions. However, it has been a major problem to identify the mRNA targets of a particular miRNA. Here, we propose a novel approach to identify mRNA targets for individual miRNAs in neurons. By doing so, this approach will significantly advance our understanding of the mechanisms by which miRNAs regulate neuronal development, synaptic function, and plasticity. Our novel approach proposes to co-immunoprecipitate a miRNA with its target mRNAs within the RISC complex, and to identify the co-immunoprecipitated targets by array and sequencing techniques. The main challenge here is how to immunoprecipitate only a given miRNA- RNP effector complex. To solve this problem, we propose to use conditional Dicer knockout mice (Dicer-/-), cultured neurons from these mice, and delete dicer in these neurons using lentivirally administered cre-recombinase, a standard technique in our laboratory. Thus we will have neurons that express no processed miRNAs, allowing us to reintroduce a given miRNA in mature form by transfection. In such neurons, the only miRNA-RNP complexes that are formed are those of our particular miRNA, making it possible for us to precisely define the mRNA targets for this particular miRNA. Using this method, one by one, we will be able to document mRNA targets of all of the individual microRNAs expressed in cultured cortical neurons. Moreover, this novel approach can easily be adapted to other types of neurons (e.g., striatal, cerebellum, hippocampal, or spinal motor neurons) or other systems and will significantly push both the miRNA and neuroscience research fields forward. Statement MicroRNAs are important regulators of gene expression that are encoded by our genome, and play a critical role in development and in the pathogenesis of several diseases. Individual microRNAs target specific sets of messenger RNAs to inhibit protein synthesis. However, it is extremely difficult to identify microRNA targets. Here we propose a novel approach to tackle this issue. If successful, this approach will revolutionize our view on the diverse biological functions of microRNAs.