A Link between RNAi and Fragile X Mental Retardation

描述（由申请人提供）：脆性X染色体是男性中最常见的遗传性智力低下。目前还没有治疗方法。病理结果是Fmr1基因表达缺失。Fmr1的蛋白产物FMRP是一种RNA结合蛋白，已被证明与有限的神经元mrna亚群相互作用。人们认为FMRP的主要功能是对这些基因的翻译调控。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的存在。基于这一发现，我们提出了以下假设：FMRP作为RNAi机制的一部分起作用，而这种共享的生物机制的功能障碍是导致脆性X智力迟钝的原因。这一假设将在本提案中得到验证。在第一个目标中，我们将定义哺乳动物细胞中共享的FMRP/RNAi机制的组成。这将使用我们开发的分馏方法和分析方法来完成。在第二个目标中，我们将确定受该活性调节的mRNA靶标。在第三个目标中，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。我们已经提出了翻译在延伸阶段被中断的假设。我们将采用细胞提取物和体外提取物的多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靶点的功能验证将提高我们对脆性X智力低下的认识，并为人类疾病的治疗提供新的药物靶点。这项工作还将确定RNAi在哺乳动物基因调控中的作用，这是目前完全未知的生物学途径。虽然该项目侧重于FMRP对神经元基因的调控，但它将为RNAi调控哺乳动物细胞通路的各个方面的研究提供一个总体框架。

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.