MicroRNA-Dependent Regulation of Synaptic and Behavioral Plasticity in Drosophila

 描述（由申请人提供）：基因表达的精确时间和空间调节对于神经系统发育、功能和可塑性的许多方面都是必不可少的。在几类基因调节因子中，非编码RNA已成为中枢神经系统调节机制的丰富潜在来源。特别地，microRNA（miR）提供对靶mRNA翻译和稳定性的序列特异性控制，其可以相当精确地调节下游蛋白质的水平，从而改善分子网络的稳定性和鲁棒性。然而，完整的神经系统内的miR功能的全面分析一直是非常具有挑战性的，留下开放的关键问题，如：如何复杂的miR调控景观的神经回路，介导的基本行为？这些miR主要在神经发育过程中起作用，还是被重新用于管理正在进行的神经回路活动和对刺激的适应？miR机制在大脑的许多部分中被利用到什么程度，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组？为了解决这些问题，我们已经组建了一个有成就的研究人员团队，他们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。我们的团队包括大卫货车·瓦克特博士（哈佛医学院）、莱斯利·格里菲斯博士（布兰迪斯大学）、罗纳德·戴维斯博士（斯克里普斯研究所）和丹尼斯·沃尔博士（斯坦福大学），他们将分别负责这一联合项目的关键组成部分。我们将使用果蝇作为模式生物，它提供了许多复杂的遗传工具，以补充我们将开发的创新工具。果蝇已被证明是特别有效的识别和解剖的细胞和分子 保守行为背后的机制。该模型也可用于确定特定途径中突变体的详细细胞和生理表型的全方位技术，从而提供了一个理想的系统，用于全面绘制miR功能，然后进行机械解剖，有效地利用丰富的工具和知识。我们将共同（i）构建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）发现miR在每个环境中的功能机制和调控策略，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。这将是第一次在神经系统中进行此类分析。我们的初步发现已经确定了不同回路之间的收敛性，这将优先考虑我们对几种miR的详细研究：miR-13，miR-92，miR-190和let-7。对miR-92的初步分析已经指出了一系列高度保守的下游基因，这些基因涉及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设，我们将在三个模型回路中进行测试，以定义每个验证目标的调控逻辑。

 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.