A Link between RNAi and Fragile X Mental Retardation

描述(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。它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，已被证明与神经元mRNAs的有限子集相互作用。这些基因的翻译调控被认为是FMRP的主要功能。我们最近报道了果蝇脆性X同源基因dFxr的存在，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，这一共享生物机制的功能障碍是脆性X智力低下的原因。这一假设将在这项提案中得到检验。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分析方法来完成。在第二个目标中，我们将确定受该活动调控的mRNA靶点。在第三个目的中，我们研究了FMRP/RNAi机制对基因调控的机制。这一途径是已知的，调节信使核糖核酸的翻译。我们提出了一个假设，即翻译在伸长步骤中被打断。我们将使用细胞提取物和体外提取物的多聚核糖体分析来检验这一假设。这项工作将有几个重要的好处。脆性X靶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，这是一种目前完全未知的生物途径。虽然这个项目的重点是FMRP对神经元基因的调控，但它将为研究哺乳动物细胞通路的RNAi调控的各个方面提供一个总体框架。

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.