MicroRNA-Dependent Regulation of Synaptic and Behavioral Plasticity in Drosophila

 描述(由申请人提供)：精确的基因表达的时空调控对神经系统发育、功能和可塑性的许多方面都是必不可少的。在几类基因调控因子中，非编码RNA已成为中枢神经系统丰富的潜在调控机制来源。特别是，microRNAs(MiRs)对靶mRNA的翻译和稳定性提供了序列特异性的控制，可以非常精确地调节下游蛋白质的水平，从而提高分子网络的稳定性和稳健性。然而，对完整神经系统内miR功能的全面分析一直是非常具有挑战性的，留下了一些关键问题，如：调节基本行为的神经回路的miR调控环境有多复杂？这些MIR主要在神经发育过程中起作用，还是被重复使用来管理正在进行的神经回路活动和对刺激的适应？在大脑的许多部位，miR机制在多大程度上被利用，或者它们在不同的细胞类型和/或发育阶段调节不同的靶基因集？为了解决这些问题，我们组建了一个由经验丰富的研究人员组成的团队，准备使用多种强大的行为和细胞分析作为综合计划的一部分进行协调工作。我们的团队包括哈佛医学院的大卫·范·维泰尔博士、布兰迪斯大学的莱斯利·格里菲斯博士、斯克里普斯研究所的罗纳德·戴维斯博士和斯坦福大学的丹尼斯·沃尔博士，他们将各自负责这一联合项目的关键部分。我们将使用果蝇作为模式生物，提供许多复杂的遗传工具，补充我们将开发的创新工具。果蝇已被证明在细胞和分子的鉴定和解剖方面特别有效 保守行为背后的机制。该模型还可用于确定特定途径中突变的详细细胞和生理表型的各种技术，从而提供了一个理想的系统，用于在综合范围内绘制miR功能图，然后进行机械解剖，从而有效地利用丰富的工具和知识。总之，我们将(I)建立和应用新的遗传工具，(Ii)应用这些工具来识别多个神经回路中所需的miR，(Iii)在每种情况下发现miR功能的机制和调控策略，然后(Iv)比较每个模型以区分一般和特定策略并检查它们的保守性。这将是第一次对神经系统进行此类分析。我们的初步发现已经确定了不同电路之间的融合，这将优先于我们对几个MIR-13、MIR-92、MIR-190和LET-7的详细研究。对miR-92的初步分析已经指出，一系列高度保守的下游基因参与了从昆虫到哺乳动物的神经回路发育和突触可塑性，提供了一组特定的机制假说，我们将在三个模型回路中进行测试，以定义每个有效靶点的调控逻辑。

 DESCRIPTION (provided by applicant): Precise temporal and spatial regulation of gene expression is essential to many aspects of nervous system development, function and plasticity. Among several classes of gene regulatory factors, non-coding RNAs have emerged as a rich potential source of regulatory mechanism in the central nervous system. In particular, microRNAs (miRs) provide sequence-specific control over target mRNA translation and stability that can tune the levels of downstream proteins quite precisely thus improving the stability and robustness of molecular networks. However, comprehensive analysis of miR function within the intact nervous system has been very challenging, leaving open key questions such as: How complex is the miR regulatory landscape for neural circuits that mediate essential behaviors? Are these miRs acting mainly during neural development or are they reused to manage ongoing neural circuit activity and adaptation to stimuli? To what extent are miR mechanisms utilized in many parts of the brain, or do they regulate distinct sets of target genes in different cell types and/or developmental stages? In order to address these questions, we have assembled a team of accomplished investigators prepared to work in unison using multiple robust behavioral and cellular assays as part of an integrated program. Our team includes Drs. David Van Vactor (Harvard Medical School), Leslie Griffith (Brandeis University), Ronald Davis (Scripps Institute), and Dennis Wall (Stanford University), who will each assume responsibility for key components of this joint program. We will use Drosophila as a model organism that offers many sophisticated genetic tools complementary to the innovative tools we will develop. Drosophila has proven to be particularly effective for identification and dissection of cellular and molecular mechanisms underlying well conserved behaviors. This model is also accessible to a full range of techniques for determining the detailed cellular and physiological phenotypes of mutants in specific pathways, thus offering a system ideal for mapping out miR functions on a comprehensive scale followed by mechanistic dissection that will effectively leverage a wealth of tools and knowledge. Together, we will (i) build and apply new genetic tools, (ii) apply these tools to identify miRs required in multiple neural circuits, (iii) discover the mechanisms and regulatory strategies for miR function in each context, and then (iv) compare each model to distinguish general and specific strategies and examine their conservation. This will be the first analysis of its kind in the nervous system. Our preliminary findings already identify convergence between different circuits that will prioritize our detailed studies of several miRs: miR-13, miR-92, miR-190 and let-7. Preliminary analysis of miR-92 already points to a series of highly conserved downstream genes implicated in both neural circuit development and synaptic plasticity from insects to mammals, providing a set of specific mechanistic hypotheses that we will test in the three model circuits to define the regulatory logic for each validated target.