A Neural Development mRNA Decay Network

描述(由申请人提供)：发育过程中的基因表达通常只在转录水平上进行研究。然而，一个额外的和基本的控制水平发生在转录后mRNA的衰退。在神经系统发育过程中，对mRNA衰变的调控尤为重要，神经元的结构需要选择性地稳定远离其合成部位的mRNAs，而细胞多样性的产生需要调节增殖和分化的mRNAs的程序性衰变。有缺陷的mRNA降解与许多神经出生缺陷有关，包括脆性X综合征(最常见的遗传性智力障碍形式)和脊髓性肌萎缩(婴儿死亡的主要遗传原因)。之前，由于缺乏允许在体内、特定细胞类型的转录本稳定性测量的方法，对胚胎发育过程中的mRNA衰退的研究一直受到阻碍。我们已经开发了一种技术(基于TU标记方法)，可以克服这一技术挑战，并允许在完整的果蝇胚胎中进行神经特异性的、全基因组范围的mRNA衰变测量。这项技术为我们构建神经发育信使核糖核酸衰变网络的系统级方法奠定了基础。这个网络将包含以下信息：在胚胎神经系统中表达的所有mRNAs的衰减率，针对这些mRNAs进行衰变的顺式元件，结合这些顺式元件的反式RNA结合蛋白和miRNAs，以及mRNA-RNA结合蛋白相互作用的空间动力学。这些信息将通过系统水平的方法获得，该方法结合了对野生型和rbp突变胚胎中mrna衰减率的全基因组分析，候选线性和结构顺式元件的计算鉴定，顺式的分子遗传学定义。 元素对体内转录稳定性的影响，体外和体内RBP-mRNA相互作用的生化分析，以及神经元内荧光mRNA-RBP相互作用的成像。这项工作将生成一个全面的和可预测的神经mRNA衰变动力学网络图，从而填补目前神经发育过程中基因表达模型的一个重大空白。

DESCRIPTION (provided by applicant): Gene expression during development is often exclusively studied at the level of transcription. However, an additional and essential level of control occurs via post-transcriptional mRNA decay. Regulation of mRNA decay is particularly important during nervous system development, where the structure of neurons requires selective stabilization of mRNAs far from their site of synthesis and the generation of cellular diversity requires the programmed decay of mRNAs that regulate proliferation and differentiation. Defective mRNA decay has been implicated in many neurological birth defects, including fragile X-syndrome (the most common form of hereditary mental impairment) and spinal muscular atrophy (a leading genetic cause of infant mortality). The study of mRNA decay during embryonic development has previously been hindered by the lack of methods allowing in vivo, cell type-specific measurements of transcript stability. We have developed a technique (based on TU-tagging methods) that overcomes this technical challenge and allows neural-specific, genome-wide measurements of mRNA decay in intact Drosophila embryos. This technique provides the foundation for a systems-level approach that will allow us to construct a Neural Development mRNA Decay Network. This network will contain the following information: the decay rates of all mRNAs expressed in the embryonic nervous system, the cis-elements that target these mRNAs for decay, the trans-acting RNA-binding proteins and miRNAs that bind these cis-elements, and the spatial dynamics of mRNA - RNA-binding protein interactions. This information will be obtained using a systems-level approach that combines genome-wide analysis of mRNA decay rates in wildtype and RBP mutant embryos, computational identification of candidate linear and structural cis-elements, molecular genetic definition of cis element effects on transcript stability in vivo, biochemical analysis of RBP-mRNA interactions in vitro and in vivo, and imaging of fluorescent mRNA - RBP interactions within neurons. This work will generate a comprehensive and predictive network map of neural mRNA decay dynamics, thus filling a significant gap in current models of gene expression during neural development.