A Link between RNAi and Fragile X Mental Retardation

描述（由申请人提供）：脆性X是男性中最常见的遗传性智力低下。目前没有治疗。病理结果从Fmr 1基因表达的损失。Fmr 1的蛋白质产物FMRP是一种RNA结合蛋白，已被证明与有限的神经元mRNA子集相互作用。据信这些基因的翻译调节是FMRP的主要功能。我们最近报道了果蝇脆性X同源物dFxr作为RNA干扰（RNAi）机制的一个组成部分。基于这一发现，我们提出了以下假设：FMRP作为RNAi机制的一部分发挥作用，这种共享的生物机制的功能障碍是脆性X智力低下的原因。本提案将检验这一假设。在第一个目标中，我们将定义哺乳动物细胞中这种共享的FMRP/RNAi机制的组成。这将使用我们开发的分馏方法和测定法来完成。在第二个目标中，我们将确定由这种活性调节的mRNA靶点。在第三个目的中，我们研究了FMRP/RNAi机制的基因调控机制。已知该途径调节mRNA翻译。我们已经发展了这样的假设，即在延伸步骤中翻译被中断。我们将采用细胞提取物和体外提取物的多聚核糖体分析来验证这一假设。这项工作将有几个重要的好处。对脆性X基因靶点的功能验证将有助于加深我们对脆性X基因智力低下的认识，并为人类脆性X基因智力低下的治疗提供新的药物靶标。这项工作还将确定RNAi在哺乳动物基因调控中的作用，这是一种目前完全未知的生物途径。虽然这个项目的重点是FMRP调控神经元基因，它将提供一个总体框架的研究在哺乳动物细胞通路的RNAi调控的各个方面。

DESCRIPTION (provided by applicant): Fragile X is the most common form of inherited mental retardation in males. It currently has no treatment. The pathology results from loss of expression of the Fmr1 gene. The protein product of Fmr1, FMRP, is an RNA binding protein that has been shown to interact with a limited subset of neuronal mRNAs. It is believed that translational regulation of these genes is the primary function of FMRP. We recently reported the presence of the Drosophila Fragile X homolog, dFxr, as a component of the RNA interference (RNAi) machinery. Based on this finding we developed the following hypothesis: FMRP functions as part of the RNAi machinery, and dysfunction of this shared biological machinery is responsible for Fragile X mental retardation. This hypothesis will be tested in this proposal. In the first Aim we will define the composition of this shared FMRP/RNAi machinery in mammalian cells. This will be done using fractionation methods and assays we have developed. In the second Aim we will identify mRNA targets that are regulated by this activity. In the third Aim we investigate the mechanism of gene regulation by the FMRP/RNAi machinery. This pathway is known to regulate mRNA translation. We have developed the hypothesis that translation is interrupted during the elongation step. We will employ polyribosome analysis of cell extracts and in vitro extracts to test the hypothesis. This work will have several important benefits. Functional validation of Fragile X targets will improve our understanding of Fragile X mental retardation, as well as providing new drug targets for the therapy of the human disease. This work will also define a role for RNAi in the regulation of mammalian genes, a biological pathway that is presently totally unknown. While this project focuses on FMRP regulation of neuronal genes, it will provide a general framework for studies on all aspects of RNAi regulation of mammalian cellular pathways.