MECHANISMS OF MRNA TRANSPORT INTO NEURONAL DENDRITES

描述(摘自申请者的摘要)：神经元接收各种各样的 通常被隔离在其细胞外的离散域中的输入 浮出水面。这种分布的不均一性反映在数量和 以细胞周围不同突触为目标的各种蛋白质。 突触的异质性也反映在神经元的不同分布上 被运输到树突中的选定的mRNA。据认为，一些人 定位于树突内的mRNAs在突触可塑性中发挥作用。这个 本提案中所述实验的目的是为 下面的一般假设是：局部电信号影响 顺式作用mRNAs在神经元树突中的分布和利用 序列，并且需要这种级别的控制来提供一种适应 细胞对不断变化的模式和水平的电刺激。这 提案将利用浦肯野的独特性质 细胞特异性mRNA，PCP-2(L7)，以及小脑系统的 MRNAs树突路由的分子和细胞机制。A合并在一起 体内(转基因小鼠)和体外(将标记的mRNAs直接注射到 小脑切片)方法将被用来定位RNA路由信号(目标1)。在……里面 此外，mRNA转运的动态方面以及由电引起的影响 将研究信使核糖核酸路由、本地化和利用方面的活动，以及 这些研究将通过一些新的分析方法来促进 合成PCP-2(L7)信使核糖核酸(目标2)。测试的功能相关性 树突状mRNA是编码PCP-2(L7)蛋白的一种变异形式的mRNA，但不是 将被路由到树突中的mRNA与行为中正常路由的mRNA进行比较 PCP-2(L7)缺失突变小鼠的抢救实验(目标3)。因此，分子 目标1中的分析将提供工具来研究 AIM 3基因的信使核糖核酸路径及其直接注射实验 影响AIM 2树突状细胞内定位的细胞外信号。 同样，L7细胞中L7基因和蛋白诱导表达的建立 AIM 3中的零突变背景将使另一个工具能够用于 目的2研究对新合成的信使核糖核酸和蛋白质的刺激作用。

DESCRIPTION (from applicant's abstract): Neurons receive an enormous variety of inputs that are often segregated in discrete domains on their extracellular surface. This heterogeneity of distribution is reflected in the quantities and kinds of proteins that are targeted to different synapses around the cell. Synaptic heterogeneity is also reflected in the differential distributions of selected mRNAs that are transported into dendrites. It is thought that some mRNAs localized within dendrites play a role in synaptic plasticity. The purpose of the experiments described in this proposal is to provide support for the following general hypothesis: Local electrical signals affect the distribution and utilization of mRNAs in neuronal dendrites via cis-acting mRNA sequences, and this level of control is required to provide a means of adapting the cell to changing patterns and levels of electrical stimulation. This proposal will take advantage of the unique properties of a Purkinje cell-specific mRNA, Pcp-2(L7), and of the cerebellar system to investigate the molecular and cellular mechanisms of dendritic routing of mRNAs. A combined in vivo (transgenic mice) and in vitro (direct injection of labeled mRNAs in cerebellar slices) approach will be used to map RNA routing signals (Aim 1). In addition, dynamic aspects of mRNA trafficking and effects due to electrical activity on mRNA routing, localization and utilization will be examined, and these studies will be facilitated by some novel means of analyzing newly synthesized Pcp-2(L7) mRNA (Aim 2). To test the functional relevance of dendritic mRNA a variant form of mRNA that encodes Pcp-2(L7) protein but is not routed into dendrites will be compared to a normally routed mRNA in behavioral rescue experiments in Pcp-2(L7) null mutant mice (Aim 3). Thus, the molecular analysis in Aim 1 will provide tools to study the functional significance of mRNA routing in Aim 3 as well as an assay (direct mRNA injection) for studying extracellular signals that influence intradendritic localization in Aim 2. Likewise, establishment of inducible expression of L7 mRNA and protein in an L7 null mutant background in Aim 3 will enable an additional tool to be used to study stimulatory effects on newly synthesized mRNA and protein in Aim 2.